SOMATIC GENE THERAPY 'MEDIA and NEWS 2004' a collection of Gene Therapy-related news updated Dec 27, 2004

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Catalogue of Entries 2004

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Nr	Title of the message	place&source
0.	The Lab Animal...	New York Times ) (18.1.2004)
1.	Gene Therapy Could Lead to Super Athletes...	Associated Press (Internet)) (15.2.2004)
2.	One Dose Of 'Designer' Gene Therapy May Target Specific Body Area...	American Heart Association (Internet) (20.1.2004)
3.	Gene therapy to treat deadly cancer...	new scientist (Internet)) (2.6.2004)
4.	Major Advance Made in Gene Therapy...	health day news (Internet)) (3.6.2004)
5.	Side Effects Sideline Hemophilia Trial...	science magazine (Internet)) (4.6.2004)
6.	Researchers report major advance in gene therapy technique...	university of winsconsin-madison (In) ()
7.	Gene Therapy Successful in Treating Neurodegenerative Disease ...	American Society of Gene Therapy (In) (4.6.2004)
8.	Regulatable gene therapy may advance treatment of Parkinson's disease ...	northwestern university (Internet)) (6.6.2004)
9.	Gene therapy tested to protect bone marrow during chemotherapy...	ASGT (Internet)) (6.6.2004)
10.	Gene therapy to improve extremity blood flow...	ivanhoe broadcast news (Internet)) (9.6.2004)
11.	Gene therapy fights neurodegeneration ...	Drugresearcher.com (Internet)) (7.6.2004)
12.	Scientists test growth factors in fight against brain diseases ...	Milwaukee Journal Sentinel (Internet) (25.6.2004)
13.	Gene Therapy Restores Wasted Muscles Cures mice with muscular dystrophy, corrects defects in human cells...	nature magazine (Internet)) (11.6.2004)
14.	Gene therapy reaches muscles throughout the body and reverses muscular dystrophy in animal model...	eurekalert (Internet)) (25.7.2004)
15.	Improving a life Rare disorder responds to gene therapy ...	Web MD (Internet)) (25.7.2004)
16.	Workaholic gene therapy holds promise for mentally ill...	The Star (Internet)) (14.8.2004)
17.	Viral Targeting Could Make Gene Therapy Safer Different vectors prefer different parts of the human genome ...	Betterhumans (Internet)) (17.8.2004)
18.	Virus Surfs Nerves to Deliver Brain-healing Genes New approach can access remote brain cells to treat diseases such as P...	betterhumans (Internet)) (30.8.2004)
19.	Muscle building gene therapy might build super athletes, scientist warns...	SF gate (Internet)) (16.2.2004)
20.	Muscle-bound Rats Prompt Sporting Debate ...	betterhumans (Internet)) (16.2.2004)
21.	Gene doping is 'new frontier' for sports cheats ...	Times UK (Internet)) (16.2.2004)
22.	Study raises fears of genetically modified athletes ...	NewScientist (Internet)) (17.2.2004)
23.	Mighty Mice Are Less Susceptible To Muscular Dystrophy Gene's Effects ...	Science daily (Internet)) (26.11.2002)
24.	"Mighty Mice" Gene Is Mutated In Beefy Bovines ...	sciernce daily (Internet)) (13.11.1997)
25.	Gene therapy on rats could possibly lead to 'superathletes' ...	Iowa state daily (Internet)) (3.3.2004)
26.	Genetic engineering is next doping threat ...	Stuff Co (Internet)) (16.3.2004)
27.	Muscles, maladies and mischief ...	radio netherlands 2 (Internet)) (22.3.2004)
28.	Genetic Research Boosts Athlete Cheats ...	Daily Champion (Internet)) (26.3.2004)
29.	...	SF Gate (Internet)) (17.4.2004)
30.	Steroids Make Stronger Gene Therapy Wrapping DNA in a common steroid increases treatment's effectiveness ...	betterhumans (Internet)) (13.2.2004)
31.	Loss of Skeletal Muscle HIF-1? Results in Altered Exercise Endurance ...	Plos Biology (Internet)) ()
32.	Mice flex muscles in genetic studies ...	AP , Union Tribune (Internet)) (24.8.2004)
33.	Gene therapy for rapid weight loss ...	PNAS online 9th February 2004 (Inter) ()
34.	Gene therapy that melts away the fat ...	BBC (Internet)) (10.2.2004)
35.	Enrolment in Clinical Studies for Angiogenic Gene Therapy Candidate Stopped ...	biz yahoo (Internet)) (30.1.2004)
36.	Designer Virus Delivers Long-lasting Gene Therapy ...	Betterhumans Staff (Internet)) ()
37.	Gene Therapy in Salivary Glands Could Lead to Promising Applications in Oral Diseases ...	NIH.gov (Internet)) (22.1.2004)
38.	Tracing the Cause of Leukemia in Gene Therapy Trial ...	Genome News Network (Internet)) (23.1.2004)
39.	Unsportsmanlike behavior ...	Washington Times (Internet) ) (23.1.2004)
40.	Drugs in sports a fact of life ...	Sun Times (Internet)) (21.1.2004)
41.	Gene therapy promising for muscular dystrophy Quebec scientists pioneer research Cells transplanted into young patients ...	Toronto Star (Internet)) (19.2.2004)
42.	Gene Therapy Shows Promise for Cystic Fibrosis ...	Reuters (Internet)) (19.2.2004)
43.	Give gene therapy time to deliver on promise ...	Straight Times (Internet)) (20.2.2004)
44.	Study May Improve Gene Therapy Safety GENE THERAPY, VECTOR, MODIFIED VIRUS, GENE EXPRESSION...	UNC Chapel Hill (Internet)) (23.2.2004)
45.	World's first gene therapeutic medicine approved for market in China ...	people daily (Internet)) (4.3.2004)
46.	Good Gene Therapy Viruses Sorted from Bad Studying their impact on gene activity could improve genetic treatments ...	Molecular Therapy journal (Internet)) (24.2.2004)
47.	Swedish committee stirs debate Recommendations supporting cloning are not universally welcomed ...	biomed central (Internet)) (25.3.2004)
48.	Tenth Annual Report of the Gene Therapy Advisory Committee published ...	medical news today (Internet)) (26.3.2004)
49.	NIH and FDA Launch New Human Gene Transfer Research Data System ...	NIH, FDA (Internet)) (26.3.2004)
50.	Focus: What was cancer, great grandad? ...	The Sunday Times (Internet)) (28.3.2004)
51.	Signs of progress in gene therapies ...	Drugresearcher (Internet)) (19.4.2004)
52.	MELANOMA,TERAPIA GENICA A MILANO: UN «VACCINO» DA PROVARE SU 22 MALATI (Corsera) ...	Luca Coscioni (Internet)) (24.5.2003)
53.	Italia e Francia a confronto...	UIDM (Internet)) (1.9.1999)
54.	...	pedriatria uni pd (Internet)) ()
55.	Cos'è la terapia genica?...	www.ica-net (Internet)) ()
56.	TERAPIA GENICA UMANA ...	utenti lycos (Internet)) ()
57.	Terapia genica con cellule somatiche nei bambini ...	bionet online (Internet)) ()
58.	La terapia genica delle patologie ereditarie ...	torino scienza (Internet)) ()
59.	L’ADA ...	torino scienza (Internet)) ()
60.	I problemi tecnici della terapia genica...	torino scienza (Internet)) ()
61.	Gentherapie bei Mukoviszidose . Suche nach dem Genvektor...	thieme verlag (Internet)) ()
62.	Gentherapie - Taxi für Gene...	life science (De) ((Internet)) ()
63.	Doppelter Erfolg mit neuartiger Diabetestherapie...	Ärzte Zeitung (Internet)) (8.9.2004)
64.	Gentherapeuten haben die Hoffnung noch nicht aufgegeben ...	Ärzte Zeitung (Internet)) (7.4.2004)
65.	Gentherapeutisch veränderte Haut zieht Cholesterin aus dem Blut ...	Ärzte Zeitung (Internet)) (18.4.2000)
66.	Somatische Gentherapie bei Patienten mit Fanconi-Anämie (FA) ...	uni klinik duesseldorf (Internet)) ()
67.	Gene Therapy Reduces Drinking in Rats with Genetic Predisposition to "Alcoholism" Finding confirms earlier result using ...	bnl.gov (Internet)) (5.5.2004)
68.	Parkinson: Gentherapie, Wachstumsfaktoren (GDNF, NGF) ...	med knowledge (Internet)) (26.6.2004)
69.	Gentherapie bei Parkinson Die Therapie mit menschlichen Genen zur Behandlung von Parkinson macht Hoffnung...	aerztliche praxis (Internet)) (6.9.2004)
70.	Gentherapie lässt Fettpolster schwinden ...	netz zeitung (Internet)) (10.2.2004)
71.	...	home t-online (Internet)) ()
72.	Ex-vivo-Gentherapie der Epilepsie...	uni zuerich (Internet)) ()
74.	Thérapie génique de la drépanocytose - Guérison de la souris...	Science, n°5550, vol. 294 (Internet)) (1.1.2002)
76.	THERAPIE GENIQUE POUR TRAITER LE DIABETE ...	Nature Medecine Vol 9(5):596-603, ma) (1.9.2003)
78.	MODELE EXPERIMENTAL DE THERAPIE GENIQUE DES METASTASES HEPATIQUES DU CANCER PANCREATIQUE PAR TRANSFERT IN VIVO DU GENE S...	SNF GE .org (Internet)) (26.3.2002)
79.	De nouvelles perspectives sur les rétinopathies pigmentaires. ...	Le Rétino n° 42, juin 2002. (Intern) (1.6.2002)
80.	Treatment hope for nerve disease ...	bbc news (Internet)) (27.5.2004)
81.	Genetic "smart bomb" knocks out hepatitis ...	Nature Biotechnology (Internet)) ()
82.	New cancer case halts US gene therapy trials ...	NewScientist.com news service (Inter) (15.1.2003)
83.	Undercover genes slip into the brain ...	New Scientist Online News (Internet)) (20.3.2003)
84.	"Human Gene Therapy: Present and Future"...	The Institute for Human Gene Therapy) (1.2.1999)
85.	Gene Therapy for Thyroid Cancer: Current Status and Future Prospects ...	medscape.com (Internet)) (26.7.2004)

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0. The Lab Animal

New York Times , 18.1.2004

On a brisk day last month, I was led through a warren of red brick buildings on the campus of the University of Pennsylvania in West Philadelphia and then up to a fifth-floor molecular physiology laboratory. I had come to visit some mice -- and to get a peek at the future of sport. I had heard about these mice, heard them called ''mighty mice,'' but I was still shocked at the sight of them. There they were in several small cages, grouped with normal mice, all of them nibbling on mouse chow pellets. The mighty mice looked like a different animal. They were built like cattle, with thick necks and big haunches. They belonged in some kind of mouse rodeo. The Penn researchers have used gene therapy on these mice to produce increased levels of IGF-1, or insulinlike growth factor-1, a protein that promotes muscle growth and repair. They have done this with mice before birth and with mice at four weeks of age. A result has been a sort of rodent fountain of youth. The mice show greater than normal muscle size and strength and do not lose it as they age. Rats altered in the same fashion and then put into physical training -- they climb little ladders with weights strapped to their backs -- have experienced a 35 percent strength gain in the targeted muscles and have not lost any of it ''detraining,'' as a human being will when he quits going to the gym. To the scientists, H. Lee Sweeney, chairman of Penn's department of physiology, and Elisabeth Barton, an assistant professor, the bizarre musculature of their lab specimens is exciting. This research could eventually be of immense benefit to the elderly and those with various ''muscle wasting'' diseases. ''Our impetus, going back to 1988, was to develop a therapy to stop people from getting weak when they get old,'' Sweeney, 50, explained. ''They fall and injure themselves. We wanted to do something about that.'' Barton, 39, has the broad shoulders and athletic build of the competitve cyclist and triathlete she once was. ''You see children with muscular dystrophy, and their parents are just so broken up because it's so sad,'' she said. ''You see grandparents who can't get out of bed. These are the people this is for.'' But the Penn team has become acutely aware of a population impatient to see its research put into practice -- the already strong, seeking to get stronger still. Sweeney gets their e-mail messages. One came from a high-school football coach in western Pennsylvania not long after Sweeney first presented his findings at a meeting of the American Society for Cell Biology. ''This coach wanted me to treat his whole team,'' he said. ''I told him it was not available for humans, and it may not be safe, and if I helped him we would all go to jail. I can only assume he didn't understand how investigational this is. Or maybe he wasn't winning, and his job was on the line.'' Other calls and e-mail messages have come from weight lifters and bodybuilders. This kind of thing happens often after researchers publish in even the most arcane medical and scientific journals. A whole subculture of athletes and the coaches and chemists who are in the business of improving their performances is eager for the latest medical advances. Sweeney knows that what he is doing works. The remaining question, the one that will require years of further research to answer, is how safe his methods are. But many athletes don't care about that. They want an edge now. They want money and acclaim. They want a payoff for their years of sweat and sacrifice, at whatever the cost. ''This was serious science, not sports science,'' Dr. Gary Wadler, a United States representative to the World Anti-Doping Agency, said when I spoke to him about the Penn experiments. ''As soon as it gets into any legitimate publication, bango, these people get ahold of it and want to know how they can abuse it.'' Sweeney's research will probably be appropriated before it is ever put to its intended medical purpose. Someone will use it to build a better sprinter or shot-putter. There is a murky, ''Casablanca''-like quality to sport at the moment. We are in a time of flux. No one is entirely clean. No one is entirely dirty. The rules are ambiguous. Everyone, and everything, is a little suspect. Months before the great slugger Barry Bonds was summoned before a grand jury in December to answer questions about his association with the Bay Area Laboratory Co-Operative, known as Balco, which has been at the center of a spreading drug scandal after the discovery of a new ''designer steroid,'' tetrahydrogestrinone (THG), a veteran American sprinter named Kelli White ran the track meet of her dreams at the World Championships in Paris. She captured the gold medal in the 100-meter and 200-meter races, the first American woman ever to win those sprints in tandem at an outdoor world championship. In both events, the 5-foot-4, 135-pound White, a tightly coiled ball of power and speed, exploded to career-best times. On a celebratory shopping trip on the Champs-Elysees, White, 26, glimpsed her name in a newspaper headline and asked a Parisian to translate. She learned that she had flunked a postrace drug test and that her medals and $120,000 in prize money were in jeopardy. Later, she acknowledged that she had taken the stimulant modafinil, claiming that she needed it to treat narcolepsy but had failed to list it on a disclosure form. What she added after that was revealing, perhaps more so than she intended. ''After a competition,'' she told reporters in Europe, ''it's kind of hard to remember everything that you take during the day.'' The THG scandal and the attention focused on Balco, which has advised dozens of top athletes (including Kelli White) on the use of dietary supplements, has opened the curtain on a seamy side of sport and on the fascinating cat-and-mouse game played between rogue chemists and the laboratory sleuths who try to police them. But White's statement exposed another, deeper truth: elite athletes in many different sports routinely consume cocktails of vitamins, extracts and supplements, dozens of pills a day -- the only people who routinely ingest more pills are AIDS patients -- in the hope that their mixes of accepted drugs will replicate the effects of the banned substances taken by the cheaters. The cheaters and the noncheaters alike are science projects. They are the sum total of their innate athletic abilities and their dedication -- and all the compounds and powders they ingest and inject. A narrow tunnel leads to success at the very top levels of sport. This is especially so in Olympic nonteam events. An athlete who has devoted his life to sprinting, for example, must qualify for one of a handful of slots on his Olympic team. And to become widely known and make real money, he probably has to win one of the gold medals that is available every four years. The temptation to cheat is human. In the realm of elite international sport, it can be irresistible. After Kelli White failed her drug test, the United States Olympic Committee revealed that five other American athletes in track and field had tested positive this summer for modafinil. Did they all suffer from narcolepsy? That would be hard to believe. More likely, word of modafinil and its supposed performance-enhancing qualities (perhaps along with the erroneous information that it was not detectable) went out on the circuit. It became the substance du jour. For athletes, performance-enhancing drugs and techniques raise issues of health, fair play and, in some cases, legality. For sports audiences, the fans, the issues are largely philosophical and aesthetic. On the most basic level, what are we watching, and why? If we equate achievement with determination and character, and that, after all, has always been part of our attachment to sport -- to celebrate the physical expression of the human spirit -- how do we recalibrate our thinking about sport when laboratories are partners in athletic success? Major League Baseball, which came late to drug testing and then instituted a lenient program, seems to have decided that the power generated by bulked-up players is good for the game in the entertainment marketplace. The record-breaking sluggers Mark McGwire and Sammy Sosa have been virtual folk heroes and huge draws at the gate. Their runs at the record books became the dominant narratives of individual seasons. (Barry Bonds has been less popular only because of a sour public persona.) But the sport is much changed. Muscle Baseball is the near opposite of what I and many other fans over 30 were raised on, a game that involved strategy, bunting, stolen bases, the hit-and-run play -- what is called Little Ball. Professional basketball is not generally suspected of being drenched in steroids and other performance enhancers. But anyone who has seen even a few minutes of old games on the ESPN Classic network from, say, 20 years ago, is immediately struck by the evolution of players' physiques. Regardless of how it happened, today's N.B.A. players are heavier and markedly more muscled, and the game is tailored to their strengths. It is played according to a steroid aesthetic. What was once a sport of grace and geometry -- athletes moving to open spaces on the floor, thinking in terms of passing angles -- is now one primarily of power and aggression: players gravitate to the same space and try to go over or through one another. But it is sports that have fixed standards and cherished records that present fans with the greatest conundrum. If what's exciting is to see someone pole vault to a new, unimaginable height -- or become the ''world's fastest human'' or the first big-leaguer since Ted Williams to hit .400 -- how do we respond when our historical frame of reference is knocked askew by the suspicion, or known fact, that an athlete is powered by a banned substance? In elite sport, the associations of competitors who have never been sanctioned for drug use or known to fail a drug test can still raise questions. Marion Jones, the breathtaking sprinter and featured American performer of the 2000 Sydney Olympics, was married to the shot-putter C.J. Hunter -- who was banned from those games after testing positive for the steroid nandralone. Jones later divorced Hunter, but then trained (briefly) with Charlie Francis, the disgraced ex-coach of Ben Johnson, the disgraced Canadian sprinter who was stripped of an Olympic gold medal. Carl Lewis, the greatest U.S. Olympian in history and a longtime crusader against performance-enhancing drugs -- it was Lewis who was outsprinted by the steroid-fueled Ben Johnson at the 1988 Games in Seoul -- has been accused of flunking a drug test of his own before the 1988 U.S. Olympic Trials. Lance Armstrong, brave cancer survivor, fierce and inspiring competitor, has kept up a long association with an Italian doctor in the thick of a sprawling drug scandal in Europe, although Armstrong himself has never come up positive on a drug test. Even the substances themselves are murky. Because the $18-billion-a-year dietary-supplement industry is (at best) loosely regulated, some of the potions in the vitamin store at your local mall could well be tainted by steroids or growth hormones. The Food and Drug Administration just got around to banning the sale of ephedra last month, long after the herbal stimulant was blamed for numerous serious health problems, along with the sudden death last year of Steve Bechler, a Baltimore Orioles pitcher. The whole situation cries out for a dose of clarity, but the closer you look, the fuzzier the picture. Start with the line between what's legal and illegal when it comes to enhancing performance. The line, already blurry, is likely over time to disappear entirely. I visited a U.S. swimmer last September as technicians sealed up his bedroom, after which they installed equipment that reduced the amount of oxygen in his room and turned it into a high-altitude chamber. This is a common and legal training method that Ed Moses, America's best male breaststroker, said he hoped would increase his count of oxygen-carrying red blood cells. A whole team of long-distance runners sponsored by Nike lives in a much more elaborate simulated high-altitude dwelling in Portland, Ore. The desired effect of the so-called ''live high, train low'' method -- sleep at altitude, train at sea level -- is the same as you would get from taking erythropoietin, or EPO, which increases red-blood-cell production and is banned in sports. Two other U.S. swimmers, in the lead-up to the Olympic Games in Sydney, were on a regimen of 25 pills a day, including minerals, proteins, amino acids and the nutritional supplement creatine, an effective but not necessarily safe builder of muscle mass. Much of the mix may well have been useless, but athletes tend to take what's put in front of them for fear of passing up the one magic pill. ''I like to think we're on the cutting edge of what can be done nutritionally and with supplements,'' the swimmers' coach, Richard Quick, said then as his athletes prepared for the 2000 games. ''If you work hard consistently, with a high level of commitment, you can do steroidlike performances.'' One of his swimmers, Dara Torres, who increased her bench press from 105 pounds to 205 pounds and swam career-best times at the age of 33, said at the time that her goal was to ''keep up with the people who are cheating without cheating.'' And who are the cheaters? Everyone else. One primary motivation to cheat is the conviction that everyone else is cheating. To draw the often arbitrary lines between performance enhancing and performance neutral, between health endangering and dicey but take it at your own risk -- to ensure that sport remains ''pure'' -- a vast worldwide bureaucracy has been enlisted. At the lowest level are those who knock on the doors of athletes in their homes and apartments in the United States and Europe and in the mountain villages of Kenya and at the training sites in China and demand ''out of competition'' urine samples. Higher up on the pyramid are the laboratories around the world chosen to scan the urine (and blood) of elite athletes for the molecular signatures of any of hundreds of banned substances. At the top of the drug-fighting pyramid are the titans of international sport -- the same people who cannot see to it that a figure skating competition is fairly judged. The titans created the World Anti-Doping Agency, which works with governments and designated national organizations, including the United States Anti-Doping Agency. In combination with the urine-sample collectors, the various couriers in the chain of custody and the laboratories, W.A.D.A. is charged with making sure that the world's premier athletes are clean -- and additionally that they have not concealed drug use through the use of various ''masking agents.'' (The latest U.S.A.D.A. list specifically prohibits the following brand names: Defend, Test Free, Test Clean, UrinAid and Jamaica Me Clean.) It is all an immensely complicated endeavor, one that requires W.A.D.A. to keep up with the onrushing science, to disseminate information to thousands of athletes, to navigate in different legal systems so that accused competitors get due process and, lastly, to manage the worldwide trafficking of urine samples. And it is all, in the end, quite possibly pointless. Despite the hundreds of people and tens of millions of dollars devoted to the effort, international and national sports organizations may just lack the will to catch and sanction cheaters. The United States, specifically, has been singled out as negligent in its oversight. ''The real issue is that USA Track and Field has become a complete and utter scofflaw,'' the W.A.D.A. president, Richard Pound, a Canadian, told me. ''They have gone to extraordinary lengths to hide identities and data and to exonerate athletes who have tested positive.'' Can you really have a serious antidoping effort without the full cooperation of the world's most powerful nation -- and most powerful sports nation? It's hard to see how. The tougher question is whether it will be scientifically possible to stay ahead of the cheaters. The rogue scientists and coach-gurus have been winning for years, and they have ever more tools available to them. THG, which set off the Balco inquiry, is only a slightly more clever version of an old thing: an anabolic steroid -- the kind of blunt builder of muscle mass and strength prevalent in sports since the 1950's. But its discovery required an insider tip, and THG is child's play compared with what's coming in the near future (if, in fact, it is not here already): genetic manipulation in order to improve athletic performance. Ultimately, the debate over athletic doping extends beyond sport. ''The current doping agony,'' says John Hoberman, a University of Texas at Austin professor who has written extensively on performance drugs, ''is a kind of very confused referendum on the future of human enhancement.'' Pete Rose was the prototypical ''self-made'' athlete, which is code for a sort of seeming nonathlete who makes the most of his meager abilities. But fans overlooked important genetic traits that made him baseball's all-time hits leader -- chiefly, uncommon durability that allowed him to play 24 seasons virtually injury free. And what did Rose do to attain that? Nothing, really. As the son of a semipro athlete who played sandlot baseball and football into his early 40's, he came by that blocky, unbreakable body by way of genetic inheritance. In the off-season, Rose maintained himself by playing casual basketball a couple of times a week and eating greasy food and heaping bowls of potato chips. When it comes to elite sport, there is no such thing as self-made. No amount of dedication can turn someone of average ability into a world-class sprinter, an N.B.A. player or a champion marathoner. You can't be an Olympic pistol shooter without some innate steadiness of hand or a Tour de France cyclist without a far-above-average efficiency at moving oxygen to muscles. Even a humdrum, physically unimpressive player on a major-league baseball team has something -- usually extraordinary hand-eye coordination -- that is not apparent to those who regard athletic gifts only in terms of great size, speed, endurance or power. The former Olympic track coach Brooks Johnson once told me that sport at its highest level should be viewed as a competition waged among ''genetic freaks.'' He mentioned Carl Lewis and Michael Jordan. But anyone who reaches the top echelon of Olympic competition or draws a paycheck for playing sports professionally should be considered in the same category. You cannot will yourself into an elite athlete, or get there through punishing workouts, without starting out way ahead of the rest of the human race. You may, through pure dedication, be able to jump one level -- from a middle-of-the-pack Olympic sprinter to the final heat, from a marginal N.F.L. prospect to a midround draft pick. Chemical enhancement can produce more significant improvements, but the principle is the same. You've got to start out as a member of the athletic elite. At the 1996 Summer Olympics in Atlanta, a middling Irish swimmer named Michelle Smith de Bruin raised suspicions when she won three gold medals. She later flunked drug tests. But before the presumed cheating, she was already a competitor on the international swim scene, not a lap swimmer at the Dublin Y. The use and abuse of performance-enhancing drugs in elite sport, or doping, as it has been called since around 1900, is a mutant form of an exclusive competition. It is an effort by individuals who are already part of a thin slice of humanity -- the genetic freaks -- to gain an edge against one another, to exceed their physiological limits in a way that they could not through pure training. (The word itself is believed to derive from the Dutch word dop, an alcoholic beverage consumed by Zulu warriors before battle.) While systematic doping -- with the collaboration of chemists, doctors, coaches and trainers -- is a modern phenomenon, scientific interest in athletes is not new. The medical establishment once viewed athletes with curiosity and occasionally with alarm. The act of training and pushing yourself to physical limits was considered dangerous or even a form of sickness. Sports science was observational, an opportunity to study the body in motion by looking at individuals at the extremes of human capacity. The British physiologist A.V. Hill, a Nobel laureate in 1922, went to Cornell to study sprinters because, as he wrote, ''matters of very great scientific interest can be found in the performances of that extraordinary machine, the human athlete.'' John Hoberman, the historian of sports doping, has written that scientists and doctors viewed the high-performance athlete as ''a wonder of nature -- a marvelous phenomenon that did not require improvement.'' Certainly, athletes have long sought their own chemical and nutritional means to enhance performance. The ancient Greeks ran and wrestled in the nude because nothing, not even fabric, was supposed to interfere with the purity of sport, yet they ate mushrooms, sesame seeds, dried figs and herbs that were believed to give a precompetition energy boost. Marathoners and cyclists as recently as a century ago competed under the influence of strychnine, which is both a stimulant and a poison. Cyclists also used caffeine, cocaine, alcohol and even heroin. What changed everything -- what transformed performance-enhancing efforts from the realm of superstition into a true science -- was the isolation of the male hormone testosterone in 1935. That led to the development by the late 30's of synthesized testosterone variants, or anabolic steroids. The difference between steroids and all previous performance enhancers was that steroids demonstrably worked -- and they worked really well. Nearly every drug used by athletes to boost performance started out as a therapeutic miracle. Steroids are still prescribed for men with serious testosterone deficiencies. AIDS patients and others with muscle-wasting conditions are dosed with steroids. Until the mid-80's, people suffering from severe anemia, as a result of chronic renal failure or other causes, had to undergo frequent blood transfusions. The development of recombinant human erythropoietin was a godsend. Instead of transfusions, anemics could get injections to boost their red-blood-cell count. But what would the effect of EPO be on a person with a normal or better than normal red blood count? What could it do for an already genetically gifted, highly trained endurance athlete? Just what you would expect: make a superendurance athlete. EPO swept the professional cycling circuit in Europe like a plague, nearly wrecking the sport. There were police raids, huge stockpiles of EPO confiscated from cyclists' hotel rooms, arrests, trials, wholesale suspensions of competitors. ''Each racer had his little suitcase with dopes and syringes,'' a former doctor for European professional cycling teams told a British newspaper. ''They did their own injections.'' EPO migrated to other endurance sports, including cross-country skiing, marathoning and orienteering. Inevitably, it showed its fatal flip side. ''In simplest terms, EPO turns on the bone marrow to make more red blood cells,'' says Gary Wadler, the American delegate to W.A.D.A. ''But there's a very delicate balance. You can have too much EPO. The body is a finely tuned instrument. It has feedback mechanisms to keep it in balance. What these athletes are often trying to do is get around the feedback, to trick their own bodies.'' Between 1989 and 1992, seven Swedish competitors in orienteering -- a mix of running and hiking that is sometimes called ''cross country with brains'' -- died, apparently from heart attacks. Nearly all were in their 20's. As many as 18 Dutch and Belgian cyclists died under similarly mysterious circumstances between 1987 and 1990. ''At first they said it was some kind of virus, a respiratory virus,'' Wadler says. ''But what kind of virus only knocks off the most fit individuals in their country? The autopsies were private. All the deaths were not definitively linked. But it was EPO. That was obvious to a lot of people.'' For weight lifters and competitors in the ''throwing'' sports of shot-put, javelin, discus and hammer, the performance enhancer of choice has long been steroids. Anabolic steroids (anabolic means tissue building) increase muscle mass and enhance the explosiveness needed for a wide range of other athletic endeavors: sprinting, jumping, swimming, serving a tennis ball, swinging a baseball bat, delivering a hit on the football field. They afford an additional benefit in a violent sport like football because one of their side effects is aggressiveness or, in extreme cases, so-called roid rage. Their use is starkly high risk, high reward. Other side effects include liver tumors, impotence, breast enlargement and shrunken testicles in men and male sexual characteristics in women. (Some of the side effects for women include enlargement of the clitoris, deepening of the voice, facial hair and male-pattern baldness.) If you want a peek at the future of performance-enhanced sport -- at what drug-laced athletes can accomplish -- look back to the mid-80's, the apex of East Germany's shameful and ruthlessly effective doping program. The East Germans were not the only practitioners of extreme pharmacological sport, only the most flagrant and well organized. (East Germany is the only nation known to have systematically doped athletes, often minors, without their knowledge.) ''Things really got out of hand in the 1970's, 80's and 90's,'' Richard Pound of the W.A.D.A. says. Even as the science of detection improved, the International Olympic Committee and other global sports bodies were constrained, he says, by a ''hesitancy to offend'' either side while the world was still divided between East and West. ''We looked away, and it snowballed.'' Steroid usage works particularly well for women athletes, because they naturally make only a fraction of the testosterone that men produce. John Hoberman says: ''In the 80's, what we saw was this new breed of monster athletes, particularly on the female side.'' Certain records from this heyday of unpoliced steroid abuse -- particularly in sports in which raw strength is a primary requirement -- suggest that performances were achieved then that are unlikely to be matched by a clean competitor. The top 14 men's hammer throws in history occurred between 1984 and 1988. In the women's shot-put, you must go all the way down to the 35th farthest throw in history to find one that occurred after 1988. Until last April, the top 10 men's shot-put throws in history occurred between 1975 and 1990. Then, at a competition in Kansas, the American shot-putter Kevin Toth finally broke into that elite group. His distance, 22.67 meters, was the farthest that anyone had put the shot in 13 years. Six months later, Toth's name was among the first to surface in the Balco scandal. Published reports said he had tested positive for THG, the new designer steroid. In women's sprinting in the 80's, the star -- and still the world-record holder in the 100- and 200-meter dashes -- was Florence Griffith Joyner, FloJo. Americans loved her style, her body-hugging track suits, her long and fabulously decorated nails, her ebullience. Elsewhere in the world, and even in the United States among those with a knowledge of track and field, FloJo's exploits were viewed with more skepticism. After Joyner died in 1998, at 38 (the cause was related to a seizure), a strange hybrid of a column appeared in the New York Times sports section. Written by Pat Connolly, who had coached Evelyn Ashford, the woman whose 100-meter record Joyner smashed, it was partly a tribute and partly a posthumous indictment. ''Then, almost overnight, Florence's face changed -- hardened along with her muscles that now bulged as if she had been born with a barbell in her crib,'' Connolly wrote. ''It was difficult not to wonder if she had found herself an East German coach and was taking some kind of performance-enhancing drugs.'' FloJo had been a very good, but never a champion, world-class sprinter. Her 1988 performance in Seoul was -- in the damning parlance of international sport -- anomalous. We don't normally think of baseball in the context of hammer throwing, shot-putting or women's sprinting. But in terms of anomalous performance, baseball is East Germany in the 1980's: a frontier. Just as in the steroid-drenched days of Olympic sport, a deep suspicion has attached itself to some of the latest records in baseball. This accompanies the grotesqueness of the appearance of some of the players. Curt Schilling, the All-Star pitcher, memorably told Sports Illustrated in 2002, ''Guys out there look like Mr. Potato Head, with a head and arms and six or seven body parts that just don't look right.'' I'm not sure whom, exactly, Schilling had in mind, but for me, his comment recalls a particular photograph taken in the 2002 season. The subjects are the home-run kings Barry Bonds and Sammy Sosa, sitting together, both of them with thick necks and bloated-looking faces. They look, well, freakish -- as well as starkly different from their appearance as young players. Bonds entered baseball lean and wiry strong, much like his late father, the All-Star outfielder Bobby Bonds. Sosa, early in his career, was not particularly big and showed little power at the plate. The question of how many home runs it is possible to hit in one season is more open-ended than, say, the fastest possible time a person can achieve in the 100-meter dash. Factors like the size of the ballpark, liveliness of the ball and skill of opposing pitchers affect the outcome. Nevertheless, a century's worth of experience amounted to a pretty persuasive case that around 60 home runs, for whatever combination of reasons, was about the limit. In 1927, Babe Ruth slugged 60, which remained the record until 1961, when Roger Maris (in a slightly longer season) hit 61. But in 1998 Mark McGwire of the St. Louis Cardinals obliterated Maris's record by hitting 70 home runs. Late in that season, a reporter snooping around McGwire's locker spotted a bottle of androstenedione, or andro, a substance usually described as a steroid ''precursor'' that provides a steroidlike effect (and that is still unregulated in the major leagues). McGwire was forced to acknowledge that his strength was neither entirely ''God given'' nor acquired solely in the weight room. But at least McGwire entered baseball already big and as a prodigious home-run hitter; he hit 49 in his first big-league season, a record for rookies. Contrast that with the career arcs of Bonds and Sosa, which are unlike any in the game's long history. Bonds had never hit more than 46 home runs until the 2000 season, and in most years his total was in the 30's. But at age 35, when players normally are on the downside of their production, he hit 49 home runs. The following season he turned into superman, breaking McGwire's record by hitting 73. Bonds's totals in the next two seasons, 46 and 45, were artificially low because pitchers walked him a staggering 346 times. His new capabilities had thrown the balance between pitcher and hitter completely out of whack: the new Barry Bonds was too good for the game. He needed a league all his own. Sosa's progression was even more unusual. In his first eight major-league seasons he averaged 22 home runs, although his totals did steadily increase and he hit 40 in 1996, then a career high. He was selected an All-Star exactly once. Unlike Bonds, he was not considered among baseball's elite players. Then in 1998, McGwire's record-breaking year, Sammy Sosa hit 66 home runs -- 6 more than the great Babe Ruth had hit in his best season. Sosa wasn't done. The following year he hit 63, followed by seasons of 50, 64 and 49 -- the best five-year total in baseball history. That there is rampant steroid use in baseball, at all levels, is undeniable. Ken Caminiti, the 1996 National League M.V.P., admitted his own use in a Sports Illustrated article in 2002 and estimated that at least half the players in the big leagues built strength with steroids. The former slugger Jose Canseco has acknowledged steroid use. In a 2002 USA Today survey of 556 big-league players, 44 percent said they felt pressure to take steroids. Last year, The Washington Post published a sad series of stories revealing that teenage prospects in the baseball-rich Dominican Republic, the source of nearly one-fourth of all players signed to U.S. pro contracts, are taking veterinary steroids to try to get strong enough to attract the interest of scouts. Whether Sosa and Bonds have built home-run power chemically cannot be known definitively. Nobody has presented evidence that they have, and both vehemently deny it. Sosa's name has not surfaced in the Balco case, and he has not testified before the grand jury. Bonds did testify in December. The home of his personal trainer and boyhood friend, Greg Anderson, has been searched by federal agents. Bonds has acknowledged patronizing Balco, which under Victor Conte, its founder, has specialized in testing athletes' blood to determine the levels of elements like copper, chromium and magnesium and then recommending supplements. Experts I talked to say they consider Conte's theories medical mumbo jumbo, but he consulted with dozens of top athletes, including Marion Jones; Amy Van Dyken, an Olympic champion swimmer; and Bill Romanowski, a linebacker in the N.F.L. Jason Giambi of the Yankees was also a client and also testified before the grand jury. In an article that appeared last June, Bonds told Muscle and Fitness magazine: ''I visit Balco every three to six months. They check my blood to make sure my levels are where they should be. Maybe I need to eat more broccoli than I normally do. Maybe my zinc and magnesium intakes need to increase.'' Bob Ryan, a veteran Boston Globe sports columnist, is among the baseball devotees who want to believe all Bonds is taking is broccoli and vitamins. But with both Bonds and Sosa, the presumption of innocence he would like to grant them clashes with the accumulation of circumstantial evidence and his own common sense. ''I knew every baseball benchmark from the time I was 10 or 11 years old,'' Ryan says. ''I knew 60, and I knew 61. I knew 714 (the former career home-run mark held by Babe Ruth). Stats frame who a player is. They're part of the romance of the game, the enjoyment.'' Bonds, with 658 career home runs, could surpass Hank Aaron's all-time total of 755 in just two or three more seasons. If he does, what will it mean? Will it carry the romance of other cherished baseball records? ''Bonds was a leadoff man who could run early in his career, and now he is this hulking slugger,'' Ryan says. ''Sammy, same thing. You want to believe it's all due to weight training and nutrition, but you have these guys hitting 40 home runs, maximum, and then well into their careers, they're in the 60's and 70's. It doesn't happen.'' But Ryan is not seeking much new information on this subject. ''I'm afraid of what you're going to tell me next,'' he says at one point in our conversation. ''I'm living in some sort of denial. I'm afraid to look under the rock.'' The world Anti-Doping Agency, imperfect as it may be, is generally considered an improvement over the patchwork approach to drug enforcement that preceded it. Created in 1999 at the World Conference on Doping in Sport in Lausanne, Switzerland, the agency was intended to bring coherence to antidoping regulations and ''harmonization'' among all the different nations and sports bodies expected to enforce them. In theory, it is the ultimate authority on matters of drugs and sport -- looming over national Olympic committees and the national and international federations of all the individual sports and making it more difficult for those parochial interests to protect athletes caught doping. W.A.D.A.'s medical committee devoted several years to compiling an impressively voluminous list of banned substances. But the role of W.A.D.A. and its president, Richard Pound, is mainly bureaucratic and political. W.A.D.A. can't slow science down -- or influence a culture that hungrily pursues human enhancements of all kinds. ''All of these issues are going to be moot in 20 or 30 years,'' says Paul Root Wolpe, a professor of psychiatry at Penn and the chief of bioethics at NASA. ''We already are seeing a blurring of the line between foods and drugs, so-called nutraceuticals. In the future, it will be more common, accepted. We'll eat certain engineered foods to be sharp for a business meeting, to increase confidence, to enhance endurance before a race or competition.'' Currently, in determining whether to put something on its banned list, W.A.D.A. considers whether a substance is performance enhancing, contrary to the spirit of sport or potentially dangerous to health. ''If it meets two of the three criteria, we are likely to put it on the list,'' Pound says. But the first two criteria are ambiguous. Steroids and EPO are clearly performance enhancing. But so might Gatorade be, if you believe its advertising and all the data on the ''science of hydration'' disseminated by the Gatorade Sports Science Institute. And plenty of sports drinks claim to do more than Gatorade. ''You identify a line and draw it somewhere,'' Pound says. ''Why is it the 100-meter dash and not the 97-meter dash? It just is.'' Between Gatorade and anabolic steroids lie all those powders and pills and injectibles that elite athletes put into their bodies, in quantities and combinations that may enhance performance or may prove innocuous. In most cases, no one is quite sure. Less open to interpretation is ''potentially dangerous to health.'' Any medical or pseudo-medical activity that takes place underground or in the black market is, by definition, dangerous. Nearly everyone, regardless of how they feel about abortion, will agree that it's more dangerous when it occurs in a back alley. Steroid use, dicey in most situations, is certainly more so when it takes place in the dark. So issues of health are the strongest rationale for W.A.D.A. and the whole antidoping effort: to protect athletes from their own worst instincts. (Though the sports world is selective about its concerns for athletes' health. Offensive lineman in the N.F.L. just keep on getting fatter. The typical career of a major-league pitcher usually involves the gradual deterioration of shoulder and elbow.) But safety is going to become less of an issue. ''Right now we have a crude way of enhancing muscle mass,'' Wolpe says. ''Years from now we'll look back on it, and it will seem low tech. When it's all on the dining-room table, there will not be the same kind of health issues we are seeing now with the unregulated and illicit supplements and drugs.'' What I learned during my visit to Lee Sweeney's lab at the University of Pennsylvania is that lifting his research for purposes of athletic enhancement is not from some sci-fi future. It's possible -- now. Sweeney and his team know for sure they can build muscle mass and strength. Their next step as they try to determine if their methods are safe for humans will be to experiment on larger animals, most likely dogs with muscular dystrophy. I asked Elisabeth Barton what would happen if some rogue nation or outlaw conglomerate of athletes asked her to disregard scientific prudence and create a human version of the mighty mice. Could she do it? ''Could I?'' she answered. ''Oh, yeah, it's easy. It's doable. It's a routine method that's published. Anyone who can clone a gene and work with cells could do it. It's not a mystery.'' Behind her, Sweeney nodded his head in agreement. ''It's not like growing a third arm or something,'' he said. ''You could get there if you worked at it.'' Sweeney said that once someone decided to use gene therapy to enhance performance, ''you would not be limited to what I'm doing. You could change the endurance of the muscle or modulate the speed -- all the performance characteristics. All the biology is there. If someone said, 'Here's $10 million -- I want you to do everything you can think of in terms of sports,' you could get pretty imaginative.'' To strengthen leg muscles for a sprinter, Sweeney said, he would ''put the whole leg on bypass. I would isolate the leg and put in the virus through the blood. It would be more efficient than injections, which you would need a lot of because you're dealing with large muscles. But this is nothing a vascular surgeon couldn't do.'' Could one already be doing it? ''I don't know that it's not happening.'' IGF-1 is already available on the Internet in ingestible form. It is advertised as a component of various powders and pills, and in this form it falls somewhere in that vast, murky area of legal, quasi-legal, black-market and plainly illegal substances for sale in the semiregulated supplement industry. But Sweeney says that any nongenetic transfer of the protein would be ineffective -- it would not circulate in the blood in levels high enough to build muscle -- and unsafe, because to the extent that it does circulate, it would target nonskeletal muscle, including the heart. (The mighty mice have shown no signs of enlarged hearts or other organs and no sign at all that the IGF-1 is circulating in their bloodstreams.) For the elite athlete, that would be one of the benefits of genetic IGF-1. It wouldn't circulate in the blood. It would be detectable only through a muscle biopsy. It took a long time for the world's athletes to agree to submit to blood tests; it's difficult to imagine them consenting to having investigatory needles stuck in their muscles. W.A.D.A. invited geneticists and others involved in the latest medical research to a conference in 2002 on Long Island. The antidoping officials were (and still are) focused on the IGF-1 research at Penn, so Lee Sweeney was there. He listened as Richard Pound tried a very tough sell. The W.A.D.A. president told the scientists that he certainly appreciated the work they were doing, knew that they approached it with single-minded dedication and understood full well that nothing was more important than seeking cures for dread diseases. He then talked about another ''humanistic activity'' that he said was already threatened by science of a certain kind -- the current science of performance enhancement -- and could be ruined by the misuse of their research. As they moved forward, Pound asked, could they somewhere keep in mind the interests of sport? As Pound recalls, the initial responses he got were somewhat dismissive: ''They said we work at the gene level. You can't really tell what was altered from what was there naturally.'' Pound, a lawyer, then asked rhetorically: ''What if I could assure the Nobel Prize in Medicine would be awarded to the person in this room who figured out how to make a test to determine if a competitor had been genetically enhanced? You could do it, right?'' Pound got an acknowledgment that detection might be possible with enough resources devoted to it. Lee Sweeney generously consults with W.A.D.A. and other antidoping officials. He's sympathetic to their cause. He just says it's hopeless. ''There will come a day when they just have to give up,'' he says. ''It's maybe 20 years away, but it's coming.'' There is a parallel from the past for the entire issue of performance-enhancing drugs, one tied to what was once another unwelcome substance in sports: money. Some casual followers of the Olympic movement may still not fully realize that nearly all of the participants are now paid professionals. There never was any big announcement that the cherished concept of amateurism -- athletes competing for the pure love of sport -- had been discarded. But over time, the changed reality has been accepted. Top athletes profiting from under-the-table payments? The public didn't care, and the ideal of amateurism expired, outdated and unenforceable. One of the last things Pound said to me indicated that he knows, too, that W.A.D.A.'s mission has an expiration date pending. Maybe genetic enhancements really won't work for athletes, he speculated. ''If you strengthen the muscle to three times its normal strength, what happens when you break out of the starting blocks? Do you rip the muscle right off the bone?'' Pound seemed to like the thought of this gruesome image. He paused, then extended the thought. ''That would be nice if that happened,'' he said. ''It would be self-regulating.'' Michael Sokolove, a contributing writer for the magazine, is the author of ''The Ticket Out: Darryl Strawberry and the Boys of Crenshaw,'' to be published in April. Copyright 2004 The New York Times Company | Home | Privacy Policy | Search | Corrections | Help | Back to Top

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1. Gene Therapy Could Lead to Super Athletes

Associated Press (Internet), 15.2.2004

- A gene therapy that has been shown in rats to double muscle strength and power could illegally be used to build super athletes, a researcher said Monday. Sports officials are looking for ways to detect the genetic manipulation. Lee Sweeney of the University of Pennsylvania said that laboratory studies show that injecting a virus carrying the gene for insulin-like growth factor 1 into lab rats caused their target muscles to grow in size and strength by 15 to 30 percent. When the technique was used on rats that were also put through an exercise program, the animals doubled their muscle strength. "The things we are developing with diseases in mind could one day be used for genetic enhancement of athletic performance," Sweeney said at the national meeting of the American Association for the Advancement of Science (news - web sites). Richard Pound of McGill University and the World Anti-Doping Agency, an organization that polices performance-enhancing drugs in international athletics, said his agency already has passed regulations forbidding genetic manipulation in athletes. But he is concerned that the new muscle-building therapy may not be easily detected. "We would like to be there early (in the research) and to help regulate it," said Pound. "We'll find a way." There are blood and urine tests to detect most performance-enhancing drugs, but the gene therapy detection would be much more difficult. Sweeney said that the presence of added genes in muscle could be detected now only through a muscle biopsy, a severely invasive procedure. The gene therapy is being developed to increase the strength of muscles for the elderly and for treatment of muscular dystrophy, a muscle wasting disorder. Sweeney said that as people age, muscles weaken and his lab is trying to determine if gene therapy would slow or reverse this decline. "The same approaches could be used in a normal person's muscles to make a them stronger and better able to repair themselves," said Sweeney. It would also keep the muscle at its peak performance for a longer period of time, he said. The treatment has not been tried on humans because of safety concerns and Sweeney said it may be years before it is ready for human clinical trials. But word of the research has reached the sports community and Sweeney said that half of the e-mails he receives now come from athletes or trainers wanting to get information about the muscle-building therapy.

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2. One Dose Of 'Designer' Gene Therapy May Target Specific Body Area

American Heart Association (Internet), 20.1.2004
http://www.sciencedaily.com/releases/2004/01/040120034706.htm

, Jan. 20 &endash; Doctors may soon be able to inject gene therapy intravenously that travels to a specific part of the body, according to a study published in today's rapid access issue of Circulation: Journal of the American Heart Association. Gene Therapy Reverses Heart Disease In Mice Putting Genes In A Pill Study Shows Combining Gene Therapy And Radiation Holds Promise "It may be possible to design and construct genetically engineered 'designer' gene therapy for selectively delivering genes to any part of the body," said Andrew H. Baker, Ph.D., lead investigator of the study and a reader in molecular medicine at the University of Glasgow in Scotland. "We can't do that now because much of what's injected would be sequestered by the liver." The liver cleanses the blood of foreign material, among other functions. Gene therapy involves inserting the treatment genes into a virus that is either harmless to humans or has had its disease-causing component removed. The virus is then injected or inserted into the body where it "infects" an area with gene therapy. Baker's team redesigned a virus called adeno-associated virus (AAV) so that it is not quarantined by the liver, but rather remained in the bloodstream long enough to "infect" specific cells in the body -- in this case, the vascular endothelial cells (ECs). The new therapy targets vascular endothelial cells, which line the inside of blood vessels. "Vascular endothelial cells, which are in continuous contact with the bloodstream and integrally involved in cardiovascular abnormalities, are appropriate targets for gene therapy," Baker said. Baker said that AAV is important because it has the potential for long-term gene expression from a single dose. This is based on results from hemophilia studies from other laboratories in which the virus was used to deliver gene therapy. In these studies, Baker said that a single dose could last up to five years, possibly longer as the studies are ongoing. AAV is also a good choice because it does not cause disease in humans. "The concept of developing systematically injectable gene transfer vehicles is important for a number of potential cardiovascular gene therapies, particularly in those conditions where access to the target site can only realistically be achieved via the bloodstream" he said. "This work shows for the first time that this is possible using cell-specific peptides to modify AAV vehicles for systemic gene delivery. Researchers modified the virus with two new peptides, or small proteins, that bind specifically to ECs. They inserted these peptides into the virus to create endothelial cell-selective proteins to make, essentially, a designer virus, Baker said. The peptides are called msl and mtp. The unmodified form of AAV is called the "wild type," or AAVwt. The modified forms of the virus are called AAVmsl and AAVmtp. Baker and his colleagues studied how the modified viruses interacted with endothelial cells in the laboratory as well as in mice. In laboratory studies, researchers showed that the unmodified virus was 100 times more infective in liver cells than in ECs. The modified virus, however, had more favorable infectivity for vascular ECs, Baker said. In mouse studies, the modified viruses accumulated at lower levels than the wild type in the major organs, predominantly the liver. Secondly, the modified AAV remained in the blood circulation longer than wild-type AAV, presumably because of reduced liver sequestration. Also, the modified virus accumulated at the target vascular endothelial site. Baker said that his results could improve the selectivity and efficiency of gene delivery to the cardiovascular system, which could also improve the safety of using it. Co-authors are Steve D. White, D. Phil.; Stuart A. Nicklin, Ph.D.; Hildegard Büning, Ph.D.; M. Julia Brosnan, Ph.D.; Kristen Leike; Emmanuel D. Papadakis, B.Sc.; and Michael Hallek, M.D.

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3. Gene therapy to treat deadly cancer

new scientist (Internet), 2.6.2004
http://www.newscientist.com/news/news.jsp?id=ns99995067

The world's first gene therapy trial to treat patients with pancreatic cancer is being launched in the UK. The therapy uses a "Trojan horse" technique to hit cancer cells with large doses of toxic drugs. MetXia, developed by UK company Oxford BioMedica, consists of a retrovirus which has been modified to carry a gene for an enzyme that normally occurs in the liver. Retroviruses only replicate in dividing cells, so the treatment mainly targets cancer cells. Once in the cell, the virus inserts the gene for the enzyme and the cell starts producing it. The enzyme then converts an inactive drug into a toxic form. This obliterates the tumour cells, without harming normal ones. It also means a hugely increased dose of chemotherapy can be delivered to tumours, without patients suffering greater harmful effects. "In the industrialised world there are at least a quarter of a million people a year dying from pancreatic cancer," says John Neoptolemos, professor of surgery at the University of Liverpool, UK, who is leading the trial. "The horrible thing about pancreatic cancer is that if you have advanced disease, and you usually do, you can predict the date of death within a matter of a few weeks." Alan Kingsman, chief executive of Oxford BioMedica, says: "We are really excited about the pancreatic cancer trial. We have reason to believe that MetXia may be quite potent in that type of cancer." Source of destruction Work in animal models of pancreatic cancer and in the test tube have shown promising results for MetXia. And early clinical trials to test its safety in patients with breast cancer and melanoma have also been encouraging. The modified retrovirus carries a gene for an enzyme known as cytochrome P450 which is essentially a detoxifying gene, says Neoptolemos. The enzyme converts an inactive chemotherapy drug called cyclophosphamide into toxic phosphoramide mustard and acrolein. Neoptolemos and his team are currently recruiting nine patients for a "proof of principle" trial - to test that the virus is indeed taken up by cancer cells when injected either locally or systemically. After a day or two, the patients will then be given cyclophosphamide, which should be converted into the toxic form in the cancer cells only. If the first trial is successful, the team will then run a safety trial in 20 to 40 patients. Bubble boys Progress in gene therapy has been faltering in the past few years. Safety concerns over inserting new genes were raised when some boys being treated for X-SCID - "bubble boy" syndrome - unexpectedly developed leukaemias during a French trial in 2003. And on 28 May, a hemophilia gene therapy trial by Avigen was halted because of safety issues. But Neoptolemos says that MetXia is a different type of gene therapy. "This is a different type of gene therapy in the sense that we are not replacing a lost gene." "We are actually killing the cell," he says. "Normally you can't get enough of the drug into the cell because of the drug-resistant mechanisms of cancer cells - but here we are able to deal with that."

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4. Major Advance Made in Gene Therapy

health day news (Internet), 3.6.2004
http://www.forbes.com/lifestyle/health/feeds/hscout/2004/06/03/hscout519308.html

A major advance in solving a stumbling block of gene therapy -- how to safely and effectively deliver therapeutic DNA inside cells -- has been reported University of Wisconsin scientists. In what's described as a remarkably simple solution, they used a system similar to that used for intravenous injection (IV) to inject genes and proteins into the limb veins of laboratory animals. The injected genetic material made its way to the animals' muscle cells and functioned properly for an extended period. "I think this is going to change everything relating to gene therapy for muscle problems and other disorders," gene expert Jon Wolff said in a prepared statement. "Our non-viral, vein method is a clinically viable procedure that lets us safely, effectively and repeatedly deliver DNA to muscle cells. We hope that the next step will be a clinical trial in humans," Wolff said. Many other scientists have experimented with using harmless viruses to deliver DNA to cells. The UW research was presented June 3 at the annual meeting of the American Society for Gene Therapy.

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5. Side Effects Sideline Hemophilia Trial

science magazine (Internet), 4.6.2004
http://www.sciencemag.org/cgi/content/full/304/5676/1423b

GENE THERAPY: One of the most promising gene therapy trials to date has been halted because two patients developed mild side effects. Over the past 3 years, the biotech company Avigen of Alameda, California, had been conducting clinical safety tests with a therapy shown to partially cure hemophilia in dogs. But after the latest of seven patients developed minor signs of toxicity, Avigen last week stopped the trial. The study built on the work of Stanford's Mark Kay and the University of Pennsylvania's Katherine High, who showed in 1999 that dogs with hemophilia B that were injected with an adeno-associated virus (AAV) vector carrying a gene for Factor IX, a blood-clotting protein, improved significantly (Science, 2 March 2001, p. 1692 ). Since 2001, Avigen has been conducting safety tests on people in which the vector carrying Factor IX is injected into a liver artery. But in October 2002, a patient given a higher dose of vector construct developed slightly elevated levels of liver enzymes, while the patient's Factor IX levels went down. Liver enzymes also rose in another patient this spring, suggesting that their bodies were mounting an immune response to the vector. "It really didn't make sense to pursue it" further, says Avigen research vice president Glenn Pierce. Kay and High hope they can modify the trial and continue testing the therapy, which they and others believe shows promise. "In terms of the science, I think things are going pretty well," says High. The first step, Kay says, is to confirm with ongoing tests that the AAV vector triggered the immune response. Then, they would like to conduct a trial that briefly treats patients with immunosuppressive drugs at the same time as the gene therapy. Avigen and co-sponsor Bayer are testing this approach in animals and haven't ruled out another human trial in the future, says Pierce. But if they can't come up with the funding, another possibility is a trial with academic support, High says. Avigen's decision isn't a major setback to the field, researchers say, because the safety problem was minor and seems to be specific to this trial. This is in contrast to the death of a patient in Pennsylvania in 1999, apparently caused by reaction to a vector, and two cases of leukemia that developed in children in a trial in France, most likely because a vector inserted near an oncogene. Some other gene therapy trials are using the AAV vector, and more plan to do so. But most are injecting small doses locally into tissues, not the liver artery--into the eye or brain, for example --a practice that is less likely to provoke side effects, notes gene therapy expert Savio Woo of Mount Sinai Medical Center in New York City. Also, "there are improved [AAV] vectors on the horizon" that are more efficient and can likely be used in smaller doses, says Woo. But there is one broader lesson from the hemophilia trial, Kay says: "Until you go into humans, you just don't know" if it will work

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6. Researchers report major advance in gene therapy technique

university of winsconsin-madison (Internet),
http://www.innovations-report.com/html/reports/medicine_health/report-29935.html

Despite a roller-coaster ride of ups and downs during the past 15 years, gene therapy has continued to attract many of the world’s brightest scientists. They are tantalized by the enormous potential that replacing missing genes or disabling defective ones offers for curing diseases of many kinds. One group, consisting of researchers from the University of Wisconsin Medical School, the Waisman Center at UW-Madison and Mirus Bio Corporation of Madison, Wis., now reports a critical advance relating to one of the most fundamental and challenging problems of gene therapy: how to safely and effectively get therapeutic DNA inside cells. The Wisconsin scientists have discovered a remarkably simple solution. They used a system that is virtually the same as administering an IV (intravenous injection) to inject genes and proteins into the limb veins of laboratory animals of varying sizes. The genetic material easily found its way to muscle cells, where it functioned as it should for an extended period of time. "I think this is going to change everything relating to gene therapy for muscle problems and other disorders," says Jon Wolff, a gene therapy expert who is a UW Medical School pediatrics and medical genetics professor based at the Waisman Center. "Our non-viral, vein method is a clinically viable procedure that lets us safely, effectively and repeatedly deliver DNA to muscle cells. We hope that the next step will be a clinical trial in humans." Wolff conducted the research with colleagues at Mirus, a biotechnology company he created to investigate the gene delivery problem. He will be describing the work on June 3 at the annual meeting of the American Society for Gene Therapy in Minneapolis, and a report will appear in a coming issue of Molecular Therapy. The research has exciting near-term implications for muscle and blood vessel disorders in particular. Duchenne’s muscular dystrophy, for example, is a genetic disease characterized by a lack of muscle-maintaining protein called dystrophin. Inserting genes that produce dystrophin into muscle cells could override the defect, scientists theorize, ensuring that the muscles with the normal gene would not succumb to wasting. Similarly, the vein technique can be useful in treating peripheral arterial occlusive disease, often a complication of diabetes. The disorder results in damaged arteries and, frequently, the subsequent amputation of toes. What’s more, Wolff says, with refinements the technique has the potential to be used for liver diseases such as hepatitis, cirrhosis and PKU (phenylketonuria). In the experiments, the scientists did not use viruses to carry genes inside cells, a path many other groups have taken. Instead, they used "naked" DNA, an approach Wolff has pioneered. Naked DNA poses fewer immune issues because, unlike viruses, it does not contain a protein coat (hence the term "naked"), which means it cannot move freely from cell to cell and integrate into the chromosome. As a result, naked DNA does not cause antibody responses or genetic reactions that can render the procedure harmful. Researchers rapidly injected "reporter genes" into a vein in laboratory animals. Under a microscope, these genes brightly indicate gene expression. A tourniquet high on the leg helped keep the injected solution from leaving the limb. "Delivering genes through the vascular system lets us take advantage of the access blood vessels have - through the capillaries that sprout from them - to tissue cells," Wolff says, adding that muscle tissue is rich with capillaries. Rapid injection forced the solution out of the veins into capillaries and then muscle tissue. The injections yielded substantial, stable levels of gene activity throughout the leg muscles in healthy animals, with minimal side effects. "We detected gene expression in all leg muscle groups, and the DNA stayed in muscle cells indefinitely," notes Wolff. In addition, the scientists were able to perform multiple injections without damaging the veins. "The ability to do repeated injections has important implications for muscle diseases since to cure them, a high percentage of therapeutic cells must be introduced," he says. The researchers also found that they could use the technique to successfully administer therapeutically important genes and proteins. When they injected dystrophin into mice that lacked it, the protein remained in muscle cells for at least six months. Similar lasting power occurred with the injection of erythropoietin, which stimulates red blood cell production. Furthermore, in an ancillary study, the researchers learned that the technique could be used effectively to introduce molecules that inhibit - rather than promote - gene expression, a powerful new procedure called RNA interference. "This could be very useful if you want to down-regulate a protein that’s causing a muscle disorder, such as with myotonic dystrophy," says Wolff. In the late 1980s, Wolff and his UW-Madison colleagues surprised the scientific world with their discovery that they could get genes to express in muscle cells simply by injecting naked DNA into rodent muscle. The Wisconsin Alumni Research Foundation (WARF) licensed the technology to Vical, a California biotechnology company. Once Wolff created Mirus, a local company, he and his colleagues turned their attention to the vascular system, a conduit to multiple leg and arm muscles they felt would work more efficiently than direct injection into muscle. WARF licensed the vascular technique to Mirus, which now holds the patent and continues to commercialize the technique. In their first studies, the researchers focused on arteries, but then began to concentrate on veins. "Injecting any substance into arteries carries a degree of risk since, unlike veins, only one artery feeds a whole limb," notes Wolff. In a related procedure, they experienced excellent results with high-pressure injection of genes into the tail veins of rodents, a technique that yielded extensive gene expression in the animals’ livers. "We think the genes traveled from the capillaries through the relatively large holes that exist in liver cells," Wolff says, adding that the technique has become a successful research tool for many laboratories around the world. "For delivering genes to limb muscles, the vein approach is so simple," he says. "We never expected it to work so well." Collaborating on the study were James Hagstrom, Julia Hegge, Mark Nobel, David Lewis and Hans Herweijer, from Mirus Bio; and Guofeng Zhang and Vladimir Budker, from the Waisman Center.

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7. Gene Therapy Successful in Treating Neurodegenerative Disease

American Society of Gene Therapy (Internet), 4.6.2004
http://biz.yahoo.com/prnews/040604/cgf012a_1.html

-- For the first time, a dominant neurodegenerative disease has been successfully treated using gene therapy, according to a study presented today at the 7th Annual Meeting of the American Society of Gene Therapy (ASGT). A research team led by Beverly Davidson, University of Iowa, investigated gene silencing by RNA interference (RNAi) as a potential therapy for Spinocerebella ataxia type 1 (SCA1), a dominant neurodegenerative disease caused by the expansion of the polyglutamine tract within the gene called ataxin-1. These are the same mechanisms underlying Huntington's disease, an inherited degenerative neuropsychiatric disorder which affects both body and mind. Using RNAi expressed from within Adeno Associated Virus (AAV) vectors, researchers showed anatomical, pathological and functional protection from the inherited neurodegeneration in SCA1 transgenic animals. AAV vectors are present in many humans, but have never been associated with any disease, making them an excellent gene transfer vehicle. The research provides hope for rapidly progressing towards a clinical trial for inherited dominant neurodegenerative diseases such as SCA1 and Huntington's disease. Researchers explore effective gene transfer method Researchers have found that a single IV injection of a new gene delivery vehicle will effectively deliver genes to the majority of muscle cells in the body, including those within the heart muscle. These findings were presented today at the 7th Annual Meeting of the American Society of Gene Therapy (ASGT). The results may provide a practical solution to the problem of how one might deliver gene therapy for the most common form of muscular dystrophy, a disease that causes progressive weakness of most of the muscles, including the heart. Paul Gregorevic, PhD, and colleagues from the Muscular Dystrophy Co- operative Research Center at the University of Washington used a newly characterized harmless virus, called adeno-associated virus type 6 (vAAV6) in a new manner. Instead of doing a large number of injections into individual muscles, researchers were able to get a similar result by injecting a single bolus of the rAAV6 into a vein of a mouse. The virus was then able to spread through the bloodstream to deliver genes to the heart and most of the other muscles throughout the body. Previous attempts at such a gene therapy in mice approached the problem by doing scores or even hundreds of injections into all of the various muscle groups, including the diaphragm and muscles in the trunk and limbs. Studies are currently underway to demonstrate the corrective effects in mice with muscular dystrophy and could potentially improve the prospects that a practical treatment for the disease will emerge within the next several years. Researchers develop novel vaccine concept for treating HIV A group of researchers have developed a novel vaccine concept for HIV using cytolytic T lymphocytes, according to a study presented today at the 7th Annual Meeting of the American Society of Gene Therapy (ASGT). Cytolytic T lymphocytes (CTL) are capable of killing cells that are infected with a virus by recognizing pieces of the virus that are displayed on the surface of the infected cell. Hildegund C.J. Ertl, MD, and colleagues from the Wistar Institute in Philadelphia, used an adenovirus vector from a chimpanzee to deliver the gene encoding of HIV protein gag in a mouse. A single administration of the vector resulted in the generation of CTL, which initially declined, then increased. This suggested that the CTL had encountered additional or continued expression of antigen that caused their number to increase. The American Society of Gene Therapy is the largest medical professional organization representing researchers and scientists dedicated to discovering new gene therapies. ASGT was established in 1996, and has grown to nearly 3,000 members. It is committed to promoting and fostering the general field of research involving gene therapy and to promoting professional and public education in all areas of gene therapy.

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8. Regulatable gene therapy may advance treatment of Parkinson's disease

northwestern university (Internet), 6.6.2004
http://www.eurekalert.org/pub_releases/2004-06/nu-rgt060404.php

Regulatable gene therapy may advance treatment of Parkinson's disease Northwestern University neuroscientists have overcome a major obstacle in gene therapy research. They've devised a method that will safely deliver and regulate expression of therapeutic genes introduced into the central nervous system to treat Parkinson's disease and other neurodegenerative diseases. The method, developed by Martha C. Bohn and colleagues, is described in the June issue of the journal Gene Therapy. Bohn is Medical Research Institute Council Professor of Pediatrics at the Children's Memorial Institute for Education and Research and professor of pediatrics and of molecular pharmacology and biological chemistry at Northwestern University Feinberg School of Medicine. Jiang Lixin, a post-doctoral fellow in Bohn's laboratory, created three different viral vectors -- carrier molecules -- that used human fluorescent green protein to track gene delivery and expression in cells. The vectors, made with the harmless adeno-associated virus (AAV), carried the "tet-off" system, in which the introduced gene is continually expressed or "on" but can be temporarily "turned off" when a small dose of the tetracycline antibiotic derivative doxycycline is administered. One vector, known as rAAVS3, displayed particularly tighter regulation in neurons when gene expression was measured at the protein and molecular RNA levels. To assess regulation in the brain, the researchers injected the vector into the striatum of rats, the area in the brain where the neurotransmitter dopamine activates the nerve cells that control motor coordination. In their experiments, Bohn and co-researchers found that up to 99 percent of the vector-introduced gene was turned off when the rats were given small doses of doxycycline. In Parkinson's disease, dopamine-producing neurons degenerate, resulting in gait problems, muscle rigidity and tremors . Several years ago Bohn's laboratory group discovered that glial cells in the embryonic brain stem secrete factors, or proteins, that promote survival and differentiation of dopamine neurons. One of these proteins, called glial cell line-derived neurotrophic factor (GDNF), is a potent factor that promotes growth of not only dopamine neurons, but also motor neurons and several other types of neurons. GDNF may have therapeutic potential for several neurodegenerative diseases, including Parkinson's disease and Lou Gehrig's disease. Bohn's laboratory was the first to show that introduction of a GDNF gene in a rodent model of Parkinson's disease halts the disease process. "GDNF gene therapy has exciting potential to 'cure' Parkinson's disease, but since putting a gene into the brain may lead to expression and increased levels of GDNF protein for years, it will be important to have some way to turn off gene expression to arrest unanticipated side effects," Bohn said. Bohn and her colleagues have been developing viral vectors that offer a safe means to deliver GDNF, as well as other therapeutic genes. The AAV vector that the researchers used in these experiments is safe and approved for use in several clinical trials in the brain of humans; however, no vector in which the gene can be turned off is yet approved for use in clinical trials. "A crucial piece of our research is related to safety," Bohn said. "We were excited to find the right mechanism to deliver the gene into the nervous system and tightly control its expression using doxycycline, a drug already approved by the Food and Drug Administration and found to have no side effects." Bohn cautioned that thorough safety and toxicity studies of the new vector are needed and that her laboratory group is not ready to assess its use in humans. ### This research was conducted as part of the Parkinson's Disease Gene Therapy Study Group, a consortium formed by the National Institute of Neurological Disorders and Stroke. Howard J. Federoff, University of Rochester, is the principal investigator of the study group. The research was sponsored by grants from the National Institutes of Health, the Walden W. and Jean Y. Shaw Foundation and the Medical Research Institute Council of Children's Memorial Hospital.

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9. Gene therapy tested to protect bone marrow during chemotherapy

ASGT (Internet), 6.6.2004
http://www.eurekalert.org/pub_releases/2004-06/cwru-gtt060304.php

Researchers at the Center for Stem Cell and Regenerative Medicine and the Case Comprehensive Cancer Center at Case Western Reserve University and the Ireland Cancer Center at University Hospitals of Cleveland report progress toward the goal of employing gene therapy to help protect the bone marrow cells of cancer patients undergoing chemotherapy. Stanton Gerson, M.D., professor of medicine, has been leading the effort to introduce a gene into bone marrow cells that would protect the cells against the debilitating effects of chemo, thereby helping the patients maintain greater strength following chemotherapy. June 6, at the American Society of Gene Therapy meeting in Minneapolis, Gerson and colleagues will present preliminary results of a Phase I clinical trial to test the safety of the method in humans. The study found no complications in five patients who were tested thus far, and found up to 41 percent transfer of the protective gene to the bone marrow, or blood stem cells. Gerson, who also directs the Center for Stem Cell and Regenerative Medicine, said, "The results are encouraging and will help move this novel approach into new therapies." Gerson's group, which includes Jane Reese and Omer Koc, M.D., has studied the gene mutant MGMT, that is able to protect stem cells from chemotherapy. In animal studies, they have found that this gene can provide stem cells with very high levels of survival advantage [more than 500 fold] compared to normal stem cells not carrying the gene. Based on those preclinical animal results, they have begun this clinical trial in patients with advanced cancer. Blood stem cells are collected from patients and exposed to a retrovirus containing the gene, which inserts the gene into the cells. Patients are then infused with their own genetically-modified cells. Patients are then treated with combination chemotherapy. Because stem cells have the new gene, they are resistant to these chemotherapy agents. This trial is unique because the patients do not undergo treatment to empty the bone marrow prior to cell infusion, which is the standard procedure. Instead, the intent is to "select" for the genetically altered cells with intermittent outpatient chemotherapy treatments, said Gerson. So far, five patients have entered the trial at the Ireland Cancer Center at University Hospitals of Cleveland, all with advanced malignancies. Only one patient has been able to receive more than one dose of chemotherapy because the others had evidence of tumor growth and were switched to other therapies. In one patient, evidence of genetically altered cells was documented by molecular analysis in both the blood and marrow six weeks after the infusion. Accrual for this study continues so that different levels of cell infusion and the impact of more doses of chemotherapy on the ability to select for the genetically altered stem cells can be assessed. Future clinical trails with this stem cell gene may be used to improve treatments for patients with specific cancers and with inherited stem cell diseases.

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10. Gene therapy to improve extremity blood flow

ivanhoe broadcast news (Internet), 9.6.2004
http://www.news8austin.com/content/headlines/?ArID=109341&SecID=2

According to the Vascular Disease Foundation, Critical Limb Ischemia, or CLI, is a severe obstruction of the arteries that seriously decreases blood flow to the extremities (hands, feet and legs) and has progressed to the point of severe pain and even skin ulcers or sores. CLI is often present in individuals with severe peripheral arterial disease (PAD). The pain caused by CLI can wake an individual up at night. This pain, also called "rest pain," can be relieved temporarily by hanging the leg over the bed or getting up to walk around. CLI is a very severe condition of PAD and needs comprehensive treatment by a vascular surgeon or vascular specialist. This condition will not improve on its own. CLI can be attributed to a number of factors, including genetics, smoking, diet, cholesterol, diabetes and hypertension. "All of those factors that promote blood vessel disease anywhere in the body including specifically the disease of the heart will cause this," Dr. Stanley Rockson, of Stanford University, said. An injection of genetic material can instruct new tissue to grow and ward off amputation. Patients with CLI don't get enough blood supply to the muscle and the skin in the legs. "These patients face nearly certain amputation. If you don't have enough blood supply, you can't carry oxygen to the tissues. You can't carry disease-fighting cells to the tissues. Nothing gets down there," Rockson said. Not only is the condition extremely painful, but it also provokes extreme anxiety, he said. "I can't stress enough how odious the concept is to a potential amputee, to think that they will lose the leg, even if we can guarantee them a well-fitting prosthesis. Just having a part of your body cut away forever is something that most people almost cannot appreciate as a realistic option," he said. Rockson and his team at Stanford are involved in a trial of a novel gene therapy technique to treat CLI. The research is the first placebo-controlled study of its kind. They're testing the use of hepatocyte growth factor to stimulate vessel formation and re-establish blood flow in these limbs with blocked arteries. Unlike other gene therapies, which rely on partly disabled viruses to carry genes into human cells, the growth factor will be injected directly into the damaged tissue of the legs. This unusual method should reduce some adverse effects sometimes caused by other gene therapy techniques. Rockson said past studies have suggested the delivery of growth factors can help improve blood flow, heal ulcers, minimize pain, and reduce the need for amputation. "This represents a new potential avenue for patients with little recourse besides amputation and death," he said. A small study in Japan actually showed the growth of new blood vessels with this technique, as well as an improvement in patients' symptoms. These results are what prompted the larger multicenter study. "It's like coming up with a cure for somebody who has widespread cancer, to use an analogy. Somebody that was previously considered untreatable is now treatable, and perhaps even curable," Rockson said.

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11. Gene therapy fights neurodegeneration

Drugresearcher.com (Internet), 7.6.2004
http://www.drugresearcher.com/news/news-NG.asp?id=53354

- Researchers have demonstrated for the first time that physical symptoms and neurological damage caused by an inherited neurodegenerative disease that is similar to Huntingdon’s disease (HD) may be prevented by gene therapy. This therapeutic approach may provide a treatment for a group of incurable, progressive neurological diseases called polyglutamine-repeat diseases, which include HD and several spinocerebellar ataxias (loss of coordinated movement caused by disease of nervous system). Beverly Davidson of the University of Iowa in the US, a professor of internal medicine and neurology and a lead investigator in the study, said: "This is the first example of targeted gene silencing of a disease gene in the brains of live animals and it suggests that this approach may eventually be useful for human therapies," "We have had success in tissue culture, but translating those ideas to animal models of disease has been a barrier. We seem to have broken through that barrier." The results of the study are also significant because they show that it is feasible to deliver therapeutics based on the concept of RNA interference (RNAi) - a means of silencing the activity of genes - using a gene therapy approach. The successful delivery method of the RNAi via a viral vector (a stripped-down virus) was used to deliver small interfering RNA (siRNA) fragments to critical brain cells of mice with a disorder that mimics the human neurodegenerative disease spinocerebellar ataxia 1 (SCA1). The siRNA material was designed to bind to and suppress the disease-causing SCA1 gene. Mice with the SCA1 gene that were treated with the gene therapy had normal movement and coordination and their brain cells were protected from destruction. The active treatment also prevented the build up of protein clumps within the cells, a marker for cellular dysfunction. Meanwhile, SCA1 mice that were not treated developed movement problems and lost brain cells in a manner similar to humans with this condition. Both SCA1 and Huntingdon’s are inherited diseases caused by a particular type of genetic flaw in which a single mutated gene inherited from either parent produces a protein that is toxic to cells. Any successful therapy must remove or suppress the disease-gene as opposed to simply adding a corrected version. Davidson explained. "With our approach we can marry our gene therapy research using viral vectors with RNA interference." "Although we know how to put genes into cells, the difficulty we face in treating dominant diseases is how to remove or silence genes." Inhibiting the SCA1 gene with RNA interference causing the non-production of a neurotoxic protein provides the foundation for a treatment against other neurological degenerative diseases caused by neurotoxic proteins, such as Alzheimer’s disease. An additional finding by the researchers was that RNA interference in and of itself does not appear to be toxic to normal brain cells. The study demonstrated that neither animal behaviour nor brain structures were affected by RNA interference gene therapy. Davidson said: "As yet, we have noted no unwanted side effects. RNAi in normal mice was not detrimental, it did not induce behavioural or morphological problems using the assays described in the paper." The study additionally revealed the specific vectors used to target those cells that are most involved in causing the disease symptoms. The vector used in this study, specifically targeted Purkinje cells, essential for movement and coordination. Davidson explained: "Choosing the right vector for the right cells could help us limit gene expression to those cells where altering expression will have a beneficial effect." "This viral vector is currently being used for human brain disease therapy and we and others are investigating lentivirus vectors also." Davidson told DrugResearcher.com that currently there was no date set for human trials as the research was only at a basic research stage. However they were looking toward initiating studies in larger mammals. Currently there is no cure for Huntington's disease and treatment options are generally aimed at controlling symptoms. Antidepressants such as fluoxetine (Eli Lilly's Prozac) can be helpful with depression, and mood stabilisers and antipsychotic drugs can help with some of the emotional disturbances. In addition, Aventis’ Rilutek (riluzole) is a compound that diminishes the glutamatergic signals sent between brain cells, already approved for the treatment of motor neuron disease. Since glutamate excitotoxicity is believed to contribute to HD pathogenesis, it seemed a strong candidate for therapeutic treatment of HD. Early studies have demonstrated that HD patients tolerated riluzole well and some decreases in the uncoordinated movements characteristic of the disease were seen. The study, which was conducted by scientists at the UI Roy and Lucille Carver College of Medicine and colleagues at the University of Minnesota and the National Institutes of Health (NIH), appears in the August issue of Nature Medicine and in the journal's advanced online publication 4 July.

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12. Scientists test growth factors in fight against brain diseases

Milwaukee Journal Sentinel (Internet), 25.6.2004

MILWAUKEE - (KRT) - Twice a day, Martha Stolar takes a vial out of the refrigerator and injects herself in the stomach with a clear liquid. The syringe contains either a placebo or a cutting-edge drug that may delay the death sentence she received when she was diagnosed with amyotrophic lateral sclerosis, also known as Lou Gehrig's disease. To buy herself some time from Alzheimer's disease that's slowly destroying her brain, Lola Crosswhite endured a considerably more invasive treatment: Two small bore holes were drilled into her head. Long needles, inserted deep in her brain, injected millions of cells engineered to produce a substance crudely described as plant food for ailing brains. These are two examples of experiments, using various growth factors to treat diseases such as Alzheimer's, Parkinson's and ALS that are sprouting up around the country. Short of a cure, these trials are designed with the hope of buying time - more than that offered by the best available medicines and possibly enough to keep the diseases at bay for a few years. Stolar, 69, a retired high school teacher, was enrolled in the study on ALS at the Medical College of Wisconsin. The college is one of several centers around the nation that are taking part in the trial. The results probably won't be known for a couple of years. Crosswhite, 75, was one of eight, early-stage Alzheimer's patients treated at the University of California, San Diego, the first patients to get a gene therapy treatment for the incurable disease. Promising results from that trial were reported at the American Academy of Neurology conference in April. And this month, researchers in Chicago will begin a follow-up study with as many as 12 more patients. The expectations of scientists in the field range from highly optimistic to calmly practical. If growth factors continue to prove to be safe, they may someday even be administered to older people who don't have brain disease but who may be forgetful or a little slow in moving around, said Clive Svendsen, a University of Wisconsin-Madison scientist. "It won't cure aging," he said. "It would just give you a boost. You may get your tennis stroke back." Svendsen is part of an experiment in which a type of growth factor is being pumped directly into the brains of Parkinson's patients, an approach that seems to be fending off their decline. He also has engineered immature brain cells to produce their own growth factor and wants to transplant those into people. However, Jeffrey Rothstein, an ALS researcher and professor of neurology and neuroscience at Johns Hopkins University, said growth factors most likely will offer only incremental improvement in the treatment of brain diseases, not a magic bullet. Either way, he said, growth factors represent a reversal of the conventional approach to fighting disease. With diseases such as cancer, the goal is to kill cells through processes like chemotherapy. With growth factors, the goal is to keep them alive. "We are trying to slow a process where cells are dying," he said. Growth factors, also known as neurotrophic factors, are a family of proteins that help brain cells develop and maintain their function. They also may be able to repair damaged brain cells. "It's very clear that these neurotrophic factors can protect nerve cells," said Mark Mattson, chief of the laboratory of neurosciences at the National Institute on Aging, part of the National Institutes of Health. "What's not clear is to what extent they can delay the progression of disease. We'll see what happens." Much of the optimism with growth factors stems from animal research showing that the substances do indeed keep cells alive and healthy for long periods of time. A microscopic view of brain tissue from an old monkey shows an atrophied network of brain cells and the root-like appendages known as axons. But an aged monkey brain that has been treated with a substance known as nerve growth factor shows a restored network of cells and dendrites as extensive as that found in a young monkey. Interest in growth factors waned in the mid-1990s after a couple of trials failed to produce positive results. Researchers now say those failures likely stemmed from flawed methods of delivering the drugs. The latest experiments target specific brain regions or types of nerve cells. And they use novel delivery systems: _Parkinson's UW's Svendsen is part of team of scientists in Britain and Madison, Wisc., who are conducting a small trial in which a type of growth factor is pumped directly into the brains of Parkinson's patients. The approach uses a pump implanted in the abdomen and a catheter to deliver the drug GDNF to the putamen area of the brain, which is important to movement control. It is believed that the GDNF stimulates dopamine release by brain cells, and it may also protect against brain cell death. In April, researchers announced that after two years, the five patients who were treated experienced more than 40 percent improvement in their motor function. Brain scans also showed a significant increase in dopamine, a neurotransmitter, storage at the tip of the catheter. Svendsen said the drug firm that makes GDNF is expected to announce the results of a similar U.S. study involving 32 patients at the end of July. Ultimately, he said, the best approach will be to use neural stem cells that have been genetically engineered to make growth factor and then transplant them into the brains of patients. Using stem cells obtained from fetal tissue rather than engineered viruses or implanted pumps has several advantages, he said. "Once they get to the brain, they swim out," he said. "Each of the stem cells is a little pump. We can engineer them to make anything." In addition, Svendsen said, his team has been able to control growth factor production in those cells by turning it on or off by using a common antibiotic. Svendsen has received a five-year, $1.8 million grant to bring his research from the lab, to rodents, to monkeys and to people. "The beauty is you don't have to take a drug every day," he said. "It's just minor surgery, a drill hole in the head and a few stem cells." Ironically, the simplicity of such therapies could hinder their development. Drugs companies normally develop new medicines that can be taken daily during the course of many years. How will they make money on a one-time treatment? "It won't be a viable company if it charges you $10," said Rothstein, of Johns Hopkins. "Will it be $20,000 or $100,000?" _ALS About a year and a half ago, Stolar was diagnosed with ALS and told she had two to five years to live. ALS is a disease that attacks nerve cells in the spinal column and brain that control voluntary movement. While thinking, vision and hearing remain relatively intact, muscles waste away. Stolar, who lives in Brookfield, Wisc., said the disease primarily affects her speaking and also causes some weakness in her right hand. But she still lives by herself and drives. In February, she was offered the chance to take part in a clinical trial of a substance known as IGF-1 or insulin-like growth factor 1. The study, which is being coordinated by the Mayo Clinic, involves 330 ALS patients from 16 medical centers around the nation. For up to two years, patients will get small vials of a clear liquid, either IGF-1 or a placebo, and must inject themselves in the abdomen twice a day. It is believed that the drug improves the health of nerve endings and that it may also travel back to nerve cells in the spinal column from nerve endings in the abdomen. In a European trial, IGF-1 failed to show effectiveness. But in an earlier U.S trial, it slowed progression of the disease by 26 percent. This third trial will determine whether the U.S. Food and Drug Administration approve the drug, said Eric Sorenson, a neurologist at the Mayo Clinic. "I'm hoping we'll have people engaging in a higher quality of life and that we can extend survival by 25 percent," said Paul Barkhaus, a professor of neurology at the Medical College of Wisconsin and Froedtert Memorial Lutheran Hospital, outside of Milwaukee. Stolar said she enrolled in the study for herself and others. "If it helps me survive, fine," she said. "If it helps other people, maybe I won't be around, but that's all right." _Alzheimer's The gene therapy approach that was taken with Crosswhite and seven other early-stage Alzheimer's patients offers at least a glimmer of hope that a new treatment could be available in a few years. After bore holes were drilled into their heads, about 5 million genetically engineered cells were injected slowly into the nucleus basalis, a region deep in the front of the brain that is associated with cognitive decline in the early and middle stages of Alzheimer's. While the study was only intended to look at safety, patients showed about a 50 percent reduction in the expected decline over a period of up to two years. Brain imaging also showed increased levels of healthy metabolic activity at the site of the gene therapy. The initial results show the approach is safe, said Mark Tuszynski, the study's lead author and a professor of neurosciences at the University of California, San Diego School of Medicine. "They (the engineered cells) act as biological pumps," he said. "They don't divide. They don't form tumors. They don't cause disease. "You're not flooding the brain with gene therapy. You're using precision-guided therapy." Because the brain cell death in Alzheimer's occurs during a period of many years, it's best to intervene early, he said. "This will not be a cure for Alzheimer's," he said. But the approach may significantly slow the disease, he said. A follow-up to the trial will begin this month at Rush University Medical Center in Chicago. Initially, six to 12 Alzheimer's patients and, eventually about 32 patients, are expected to undergo the treatment. "I don't want to use therapy because we don't know if it will be of benefit," said Zoe Arvanitakis, a neurologist at Rush. "Ultimately, if we could prevent and not only treat, we'd be a lot better off." However, for patients like Crosswhite, prevention and cure are not possible. Crosswhite, who lives by herself and still drives, said she first noticed something was wrong about five years before she was diagnosed. Since then, she has tried to stay active by walking and doing photography. Crosswhite and her daughter Diana Shaw say they both believe the treatment has helped. "We are starting to see some decline again," Shaw said. "I think she got two years of very cognitive thinking. I definitely think there have been benefits." Crosswhite said that even if the treatment has not helped, it was worth it to be a part of the experiment. "Somebody has to be in these things," she said. "Plus, there is the hope that there will be help, if not for me, then someone along the line."

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13. Gene Therapy Restores Wasted Muscles Cures mice with muscular dystrophy, corrects defects in human cells

nature magazine (Internet), 11.6.2004
http://www.betterhumans.com/News/news.aspx?articleID=2004-06-11-3

Researcher Kevin Campbell and colleagues at the University of Iowa College of Medicine in Iowa City found similar benefits when they caused the protein, called LARGE, to be expressed in cells from people with muscular dystrophy. "What's nice about this is that cells from patients, which we can grow up in the laboratory, actually showed that we can correct the defect in their cells with LARGE," says Campbell. Irreparable damage Muscular dystrophy refers to diseases characterized by progressive skeletal muscle weakness, defects in muscle proteins and the death of muscle cells and tissue. A group of muscular dystrophies has recently been linked to mutations in enzymes that add sugars to the muscle protein alpha-dystrogylcan. Without the attached sugars, alpha-dystroglycan is unable to provide structural support that protects muscles from contraction-induced damage. "In most cases, if a normal person went out and ran 20 miles, the muscle is going to be damaged. But the muscle is actually able to repair itself," says Campbell. "However, patients with muscular dystrophy undergo this process much more quickly, and they eventually lose the ability to repair. That's when they get weak." Enzyme defect Defects in enzymes that transfer sugar molecules, known as glycosyltransferases, have been implicated in at least six different types of muscular dystrophy, says Campbell. The researchers therefore set out to determine whether restoring glycosyltransferase activity would correct the defects. To find out, they used mice in which the gene for LARGE, a type of glycosyltransferase, is mutated. LARGE is key to maintaining healthy muscle, and in mice that lack it alpha-dystroglycan lacks attached sugars and cannot support muscle. LARGE increase To increase levels of LARGE in the deficient mice, the researchers engineered a virus expressing the LARGE gene. When they injected the virus directly into the muscle of mice that were a few days old, their muscle cells produced the LARGE protein. Examining alpha-dystroglycan, they found that sugars had been properly attached and that it was able to properly link between muscle cells and its surroundings. The researchers next tested the mice to see whether the changes had functional benefits. Mice with and without the transferred gene were put on a treadmill and examined after their workout. The mice that received the gene therapy had significantly less muscle damage. Cell restoration The researchers then tested whether LARGE had similar effects in the cells of people with muscular dystrophy. They found that, indeed, adding LARGE had an effect similar to that seen in mice&emdash;glycosylation was restored when the LARGE gene was expressed. "At least for all the glycosylation-related muscular dystrophies, we think that LARGE may be able to restore the function of alpha-dystroglycan," says Campbell. "If we could come up with a drug that would stimulate LARGE activity, then we could possibly bypass the defect that's seen in these different forms of muscular dystrophy."

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14. Gene therapy reaches muscles throughout the body and reverses muscular dystrophy in animal model

eurekalert (Internet), 25.7.2004
http://www.eurekalert.org/pub_releases/2004-07/uow-gtr072204.php

The gene therapy was able to perform in all muscles in the mouse, and would not necessarily have to carry the dystrophy gene Researchers have found a delivery method for gene therapy that reaches all the voluntary muscles of a mouse &endash; including heart, diaphragm and limbs &endash; and reverses the process of muscle-wasting found in muscular dystrophy. "We have a clear 'proof of principle' that it is possible to deliver new genes body-wide to all the striated muscles of an adult animal. Finding a delivery method for the whole body has been a major obstacle limiting the development of gene therapy for the muscular dystrophies. Our new work identifies for the first time a method where a new dystrophin gene can be delivered, using a safe and simple method, to all of the affected muscles of a mouse with muscular dystrophy," said Dr. Jeffrey S. Chamberlain, professor of neurology and director of the Muscular Dystrophy Cooperative Research Center at the University of Washington School of Medicine in Seattle. He also has joint appointments in the departments of medicine and biochemistry. Chamberlain is the senior author of the paper describing the results, which will be published in the August edition of Nature Medicine. The paper describes a type of viral vector, a specific type of an adeno-associated virus (AAV), which is able to 'home-in' on muscle cells and does not trigger an immune system response. The delivery system also includes use of a growth factor, VEGF, that appears to increase penetration into muscles of the gene therapy agent. Chamberlain said the formula was the result of about a year of trying different methods. Duchenne muscular dystrophy is an X-linked genetic disorder that strikes one of every 3,500 newborn boys. The genetic disorder eliminates production of the dystrophin protein, which is necessary for the structural support of muscle. Without this protein, muscles weaken to the point where the victim cannot survive. "By giving one single injection of this AAV vector carrying a mini-dystrophin gene into the bloodstream, we are able to deliver therapeutic levels of dystrophin to every skeletal and cardiac muscle of an adult, dystrophic mouse," Chamberlain said. "These muscles include the heart, the muscles used during breathing, and all the limb muscles. The mice show a whole body effect, with a dramatic improvement of their dystrophy." The findings hint that it may be possible someday to introduce other genes into adult muscle to address conditions besides muscular dystrophy. The gene therapy developed at the UW was able to perform in all muscles in the mouse, and would not necessarily have to carry the dystrophy gene. Muscle represents about 40 percent of the human body, and there are a number of ailments that involve muscle. Gene therapy could someday reinforce muscles weakened by cancer or normal aging, or treat cardiac disease. But Chamberlain stressed that the paper represents one discovery on the long path to any clinical applications in people. The results involved mice, so researchers do not know if the method will work in larger animals or people. Chamberlain and colleagues in the Muscular Dystrophy Cooperative Research Center are gathering data to seek regulatory approval for a limited trial in humans to determine the safety of a very small amount of the vector in human muscle. If the experiments take place &emdash; and if results are encouraging &emdash; researchers would continue to test the method in larger animals and hopefully eventually humans. But Chamberlain stressed that there are a number of scientific challenges and regulatory requirements along the way, so any tests on humans are many years in the future. ### The research was funded by grants from the National Institutes of Health, the Muscular Dystrophy Association, and Bruce and Jolene McCaw. Other authors of the paper, all at UW, include co-first authors Drs. Paul Gregorevic and Michael Blankenship, Department of Neurology; James M. Allen, Robert W. Crawford, and Leonard Meuse, also of Neurology; and Daniel G. Miller, Department of Medicine, as well as David W. Russell of the Departments of Medicine and Biochemistry, who identified the particular adeno-associated virus, AAV6, used in the experiments.

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15. Improving a life Rare disorder responds to gene therapy

Web MD (Internet), 25.7.2004
http://www.jsonline.com/alive/well/jul04/245963.asp

Four-year-old Ashtyn Fellenz cannot sit up or raise her head or talk. And she "eats" only through a tube attached to her stomach. The Menomonee Falls girl has a rare genetic brain disorder known as Canavan disease. Speech pathologist Rona Alexander works with Ashtyn while her mother, Arlo Clark-Fellenz holds a mirror to stimulate Ashtyn's vision. Ashtyn Fellenz also takes nourishment through a tube implanted in her abdomen. Ashtyn's condition, which results in a deterioration of the brain, has improved since she began participating in a gene-therapy program. Canavan Disease Canavan disease is a rare inherited neurological disorder characterized by spongy degeneration of the brain and spinal cord. Symptoms that appear in early infancy may include progressive mental decline accompanied by the loss of muscle tone, poor head control, an abnormally large head, and/or irritability. Ashtyn is one of only 21 participants from the United States, Europe and South America in an enterprising clinical trial where researchers pump genes into the brain in hopes of improving motor skills and brain function. A floppy head and stiff limbs are trademarks of the disorder. Other symptoms include mental retardation, blindness, deafness and paralysis. Chemical builds up In Canavan disease, a malfunctioning gene causes a buildup in the brain of a chemical called N-acetyl aspartate. The gene fails to produce a protein responsible for breaking this substance down into the building blocks of the brain's white matter. Also called myelin, white matter is a fatty layer that coats nerves in the brain and spinal cord. It transmits nerve impulses from the brain to other cells in the body. In Canavan disease, the white matter becomes spongy and full of tiny fluid-filled holes. Children with the disease usually die by age 4, but in rare cases could live into their 20s, according to the National Institute of Neurological Disorders and Stroke. The clinical trial - which began in April 2003 and will run through summer 2005 - is testing how gene therapy can improve the odds for children with Canavan disease. "This is a first step; the potential down the road for a cure is a real one," said Christopher Janson, principal investigator of gene therapy for Canavan disease at Cooper Hospital, a branch of the University of Medicine and Dentistry of New Jersey. Researchers from Robert Wood Johnson Medical School - another branch of the same university - and Children's Hospital of Pennsylvania are also part of the team. The group plans to extend its methods to treating more common neurodegenerative disorders such as Alzheimer's, Parkinson's and Tay-Sachs. Eight years ago, in New Zealand, the same group performed the first gene therapy treatment of a genetic brain disorder on children with Canavan disease. This work ruffled some feathers within the gene-therapy field because the investigators tested the method on humans without first showing that it was effective in animals. At the time, they delivered genes surgically, using a fat-based molecule to shuttle genes to the brain. These days, the team delivers genes using a different kind of brain surgery as well as a new vehicle - an adeno-associated virus. But some gene-therapy experts remain cautious because no researchers have successfully used that kind of delivery method in humans. Adding to the uncertainty, in late May, California-based Avigen Inc. - a company that develops gene-therapy products - yanked its support from a hemophilia gene therapy trial that used this type of delivery to supply genes to the liver. Two of seven patients in the study - conducted at Stanford University, Children's Hospital of Pennsylvania and University of Pittsburgh - developed mild liver trouble. The researchers feared the toxicity might interfere with the treatment, said Mark Kay, one of the trial's scientific advisers based at Stanford. Test animals had not shown any liver damage. "You can't always predict what will happen in humans even if you have the best animal studies," Kay said. The Canavan disease researchers are unfazed. Immune responses to a virus used in the liver do not necessarily forecast what will happen when the virus is used in the brain. Indeed, in the Canavan disease trial, tests of blood serum and spinal fluid show that there is little immune response to the virus or to the gene's protein products, Janson said. Furthermore, the difference in gene delivery method between the two trials will also affect the results, said Paola Leone, co-principal investigator of the Canavan disease trial. In the hemophilia trial, the virus was administered to an artery and transported through the bloodstream to the liver. In the Canavan disease trial, the virus was injected directly into the brain. "If they demonstrate that it is effective in Canavan, it would be a remarkable result," said Roscoe Brady, chief of the developmental and metabolic neurology branch of the National Institute of Neurological Disorders and Stroke. "Now we're comfortable that the risk is minimal, and we can think more about the benefit side of the equation," said Janson. "We want to move a little more aggressively." How gene is delivered To try to arrest deterioration of the brain's white matter, the researchers open a patient's skull and drill six holes into the brain. Into each hole, they introduce a catheter through which a pump slowly delivers a few drops of solution with about 900 billion viral particles containing the gene. The viral particles make their way inside the brain's nerve cells, where they disassemble, releasing the gene. The genes make copies of themselves and produce the protein vital for white matter formation. After the two- to three-hour surgery, the young patients in the trial typically stay in the hospital for three days. Afterward, for at least 24 months, patients go for frequent follow-up visits at Children's Hospital of Philadelphia. There, researchers image the patients' brains to see if - over time - the harmful chemicals and water levels in the brain decrease and some white matter is restored. They also monitor responses such as changes in muscle tone and awareness level. "It's a huge effort," said Leone. Janson said he has seen a wide range of outcomes in patients, depending on their age and the stage of the disease. While some patients did not show marked improvement after treatment, others gained better control of their head and limbs, and had improved sleep patterns, bowel movements and awareness levels. "What we are seeing is the best effect that we can have," said Leone, considering that the patients have already lost up to 40% of their brain mass. Seeing progress Since her gene therapy treatment one year ago, Ashtyn can now move her head from side to side and even lift her arms and legs in ways she was unable to do before. And her facial expressions have a brightness that wasn't there before, said her nurse, Jacalyn Anderson. Ashtyn has come a long way. "She used to be just a rag doll," said Ashtyn's mother, Arlo Clark-Fellenz. Ashtyn receives physical, occupational and music therapy for up to four days a week at the School for Blind and Visually Impaired Children on Brown Deer Road. Twice a month, she also has sessions with speech pathologist Rona Alexander who treats children with feeding and swallowing difficulties. First - with her hands and a small, gently vibrating rod - Alexander stimulates Ashtyn's face, hands and mouth. This prepares the muscles to take on the task of eating. Alexander waves tiny spoonfuls of chocolate pudding and yogurt under Ashtyn's nose then places the food inside her mouth, while applying slight pressure to Ashtyn's chin and lower lip. Ashtyn smiled after swallowing her pudding. Then she took a small spoonful of water. "You did a great job today," Alexander told Ashtyn. The simple act of swallowing is a chore for children such as Ashtyn who have Canavan disease, a disorder that affects mainly Ashkenazi Jews and people of Saudi Arabian descent. Neither of Ashtyn's parents has a known Jewish or Saudi Arabian heritage. Both parents must carry the culprit gene mutation for offspring to have the disease. There is then a 1-in-4 chance a child will be born with the disease. A simple prenatal blood test can reveal if a child is affected. Parents can be screened to see if they are carriers of the defective gene that causes the disease. In the long term, Leone hopes to be able to do more than just stop the progress of Canavan disease: She wants to reverse it. She is exploring the possibility of replacing damaged brain cells with stem cells. Although Ashtyn may not benefit from the proposed stem cell work, her physicians and family are doing their best. Clark-Fellenz said: "We all know that we have done our part to give her a full life."

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16. Workaholic gene therapy holds promise for mentally ill

The Star (Internet), 14.8.2004
http://www.thestar.com/NASApp/cs/ContentServer?pagename=thestar/Layout/Article_Type1&c=Article&cid=1092262212626&call_pageid=991479973472&col=991929131147

&emdash;Procrastinating monkeys turned into workaholics when a gene treatment prevented them from knowing how much work they had to do to get a reward. U.S. government researchers found monkeys work harder at a task &emdash; and were better at it &emdash; when a key brain compound, dopamine, was blocked. Like humans, monkeys tend to wait until the last possible minute to finish up the work, and become very adept at estimating how long they have. Dopamine, a message-carrying chemical associated with rewards, movement and a variety of other important functions, is the key to this kind of perception. Barry Richmond and colleagues at the National Institute of Mental Health used a new genetic technique to block the D2 gene, which makes a receptor for dopamine. "The gene knockdown triggered a remarkable transformation in the simian work ethic," Richmond said in a statement Wednesday. "Like many of us, monkeys normally slack off initially in working toward a distant goal." The NIMH team said they are hoping to understand mental illness. "In this case, it's worth noting that the ability to associate work with reward is disturbed in mental disorders, including schizophrenia, mood disorders and obsessive-compulsive disorder, so our finding of the pivotal role played by this gene and circuit may be of clinical interest," Richmond said. "For example, people who are depressed often feel nothing is worth the work. People with obsessive-compulsive disorder work incessantly; even when they get rewarded they feel they must repeat the task. In mania, people will work feverishly for rewards that aren't worth the trouble to most of us." For their study, Richmond and colleague used seven rhesus monkeys who had to push a lever in response to visual cues on a projection screen, and got a drop of water as a reward. "They work more efficiently &emdash; make fewer errors &emdash; as they get closer to being rewarded," Richmond said. "But without the dopamine receptor, they consistently stayed on-task and made few errors, because they could no longer learn to use visual cues to predict how their work was going to get them a reward." Results were published in the Proceedings Of The National Academy Of Sciences.

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17. Viral Targeting Could Make Gene Therapy Safer Different vectors prefer different parts of the human genome

Betterhumans (Internet), 17.8.2004
http://www.betterhumans.com/News/news.aspx?articleID=2004-08-17-1

Different viruses have been found to target different parts of the human genome, suggesting that the safety of gene therapy could be improved through the selection of the most appropriate viral integration system. Researchers at the University of Pennsylvania School of Medicine in Philadelphia have completed a whole genome survey of the locations where three commonly used retroviruses integrate into human DNA, and have found 3,127 sites where the viruses typically integrate as well as different target preferences. "There's a picture forming of where different retroviruses integrate in human cells, and it seems to be quite different from virus to virus, which is not something anyone would have ever suspected," says Frederic Bushman, professor of microbiology. Viral vectors In gene therapy, researchers are trying to treat disease by replacing or augmenting defective genes with health-promoting genes. Engineered viruses&emdash;so-called viral vectors&emdash;are often used to deliver therapeutic genes because they naturally insert themselves into DNA so that they can be replicated throughout the body. Gene therapy using viruses, however, has had safety issues because researchers could not tell exactly where viruses would insert themselves. Researchers discovered , for example, that two young boys who received gene therapy for the immunodeficiency condition known as "bubble boy disease" developed leukemia-like symptoms 30 months after treatment because the virus used for their treatment integrated near a region that can promote the initiation of cancer. Better integration For their study, Bushman and colleagues compared vectors derived from HIV, murine lukemia virus (MLV) and avian sarcoma-leukosis virus (ASLV). They found that each virus preferred a different integration pattern. HIV integrated near active genes, MLV integrated near points on the chromosome where protein translation starts and ASLV integrated more randomly throughout the genome. The findings suggest that viruses target chromosomal features for inserting themselves, and the researchers speculate that integration is guided by a system of biochemical recognition between proteins bound on human chromosomes and viral proteins. "There's a prospect of modulating or engineering that kind of system, once we understand it better to direct integration to different locations," says Bushman. The research is reported in PLoS Biology (read full text

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18. Virus Surfs Nerves to Deliver Brain-healing Genes New approach can access remote brain cells to treat diseases such as Parkinson's and Alzheimer's

betterhumans (Internet), 30.8.2004
http://www.betterhumans.com/News/news.aspx?articleID=2004-08-30-4

The approach, being developed by researcher Wang Shu and colleagues at the Institute of Bioengineering & Nanotechnology in Singapore, uses the body's own axons&emdash;cable-like structures that neurons use to communicate&emdash;to transport therapeutic genes to damaged areas located deep within the nervous system. "The use of axonal transport for neuronal gene transfection allows viral vectors to target neurons in remote parts of the circuit," says Shu. "By injecting the virus into the body in a more accessible region, we can target other neurons deep within the central nervous system or minimize the damage to sensitive regions, which may be caused by the injection procedure." Difficult delivery Gene therapy is a technique that involves replacing, silencing or otherwise altering disease-causing genes with therapeutic genetic material. A carrier molecule called a vector&emdash;usually a disabled virus&emdash;is used to ferry therapeutic cargo to target cells. Gene therapy has shown promise for treating many diseases. The widespread distribution of affected neurons and their remote location, however, have been obstacles to the use of gene therapy for such diseases as Alzheimer's and Parkinson's. Several viruses, such as the herpes virus , are capable of overcoming such problems by using axonal transport, by which viruses travel along axons to target cells located deep within the nervous system. These viruses, however, can elicit an immune response&emdash;a defensive measure taken by the immune system to defend against foreign and potentially harmful substances. Axonal transport Recently, Shu and colleagues came up with the idea of using a type of virus called a baculovirus to target cells in the central nervous system by axonal transport. Primarily infecting insects, baculoviruses can also enter cells of other organisms but do not become infectious in them. Unlike the herpes virus and other viruses, baculoviruses are relatively safe in humans and don't cause as much damage to the body, says Shu. Besides their promise for treating neurological diseases, baculoviruses could also be used to trace neuronal pathways. By using a baculovirus that has been genetically modified for imaging purposes, for example, researchers could track the virus as it traveled a subject's central nervous system. The researchers have already begun testing their new method on animal models of Parkinson's disease. The research will be reported in an upcoming issue of the journal Molecular Therapy.

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19. Muscle building gene therapy might build super athletes, scientist warns

SF gate (Internet), 16.2.2004
http://www.sfgate.com/cgi-bin/article.cgi?f=/news/archive/2004/02/16/sports1620EST0295.DTL

Gene injections in rats can double muscle strength and speed, researchers have found, raising concerns that the virtually undetectable technology could be used illegally to build super athletes. A University of Pennsylvania researcher seeking ways to treat illness said studies in rats show muscle mass, strength and endurance can be increased by injections of a gene-manipulated virus that goes to muscle tissue and causes a rapid growth of cells. "The things we are developing with diseases in mind could one day be used for genetic enhancement of athletic performance," Lee Sweeney said Monday at the national meeting of the American Association for the Advancement of Science. Sports officials said the gene therapy has the potential of betraying the very essence of sport -- athletes using their natural talents and training to compete. Tom Murray of the Hastings Center, a research organization, said it would be like allowing an athlete to compete in the Boston Marathon wearing roller blades. "Performance enhancing drugs have been a concern in sports and gene therapy has the potential to kick it up a notch," said Murray, who has studied the issue of doping in sports. "It makes the challenges greater (of controlling performance-enhancing measures)." Murray said he "has no doubt athletes will be in touch with Sweeney" when they learn of his research. Sweeney said that already half the e-mails he receives are from athletes or sports trainers. Richard Pound of McGill University and the World Anti-Doping Agency, said the sports community lost control of drugs for performance-enhancement in the 1960s to 1990s and "we've been playing catch-up ever since." Now gene therapy looms as an even more serious threat, he said. "Sport is and should be an effort to see how far you can go with your natural talents honed by exercise and skill perfection," he said, and not by manipulating genes to build muscle. He said international sports already has regulations forbidding gene therapy for performance improvement and his agency hopes to be active in efforts to control use of the technique as the science develops. Sweeney said that his laboratory studies show that injecting into muscles a manipulated virus that carries a gene for insulin-like growth factor 1 (IGF1) causes target muscles in rats to grow in size and strength by 15 to 30 percent. The inserted gene causes formation of extra IGF1 which, in turn, prompts the growth of muscle cells. When the technique was used on rats that were also put through an exercise program, the animals doubled their muscle strength, he said. "If a normal person would inject this, their muscles would get stronger without them doing anything," Sweeney said. "If they are athletes in training, the rat study indicates that their training would be much more effective, injury would be overcome more easily and the effect of the training would last a much longer time." The effect appeared to last throughout the life of the rats. He said the technique was designed so that the IGF1 gene stays in the target muscle and does not move into the blood stream where it could cause damage to other organs. Sweeney said the gene therapy was being developed to treat muscular dystrophy and the natural decline in muscle strength associated with aging. Unlike performance-enhancing drugs, Sweeney said the gene therapy could not be detected by blood or urine tests. He said it would require a biopsy of specific muscles followed by a sophisticated DNA laboratory study to detect the use of gene therapy in an athlete. Sweeney said because of the potential of cancer and other side effects, it may be years before the muscle-strengthening gene therapy is ready for human trials. "There are issues of safety," he said. "It is not going to be as trivial as taking a drug." Sweeney said the gene therapy technique is highly complex and requires expert laboratory preparation. "This is not something an athlete could do in his garage," he said. "The athlete couldn't do this without a lot of help." He said that some countries, in a drive for athlete glory, could allow the gene therapy, just as earlier in history Olympic programs in some countries tolerated the use of performance-enhancing drugs. "That is the short-term fear," Sweeney said.

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20. Muscle-bound Rats Prompt Sporting Debate

betterhumans (Internet), 16.2.2004
http://www.betterhumans.com/News/news.aspx?articleID=2004-02-16-2

Engineered to have larger leg muscles, animals spark discussion of genetic doping Rats engineered to grow bigger leg muscles following weight training have prompted discussion of genetic enhancement in sports. Lee Sweeney of the University of Pennsylvania in Philadelphia and colleagues have used gene therapy to make rats that build and retain muscle better after exercise. Such genetic enhancement of skeletal muscles could help athletes, patients rehabilitating from injury-induced muscle wasting and elderly people with muscular weakness, the researchers say. Growth factor To conduct their study, the researchers used a virus carrying a gene for IGF-I. IGF-I is a growth factor that promotes gains in muscle strength and mass. The virus was injected into the hind-leg muscles of rats that were then put through ladder-climbing exercises. The rats bulked up more than rats given only exercise and rats given only the gene therapy. They also retained more muscle mass after they stopped working out. Strength satellites The researchers think that muscle-precursor stem cells called "satellite cells" were responsible for the increased muscle mass and strength. Their findings support the theory that exercise "primes" satellite cells: Exercised satellite cells expressed IGF-I receptors that made them more responsive to the increased levels of IGF-I. While the application of the gene therapy approach to humans is still hypothetical, the experiment has prompted discussion of such genetic engineering in sports. It was presented in Seattle, Washington at the annual meeting of the American Association for the Advancement of Science, where Richard Pound from the World Anti-Doping Agency compared the situation with new doping techniques such as genetic doping to the period 30 to 40 years ago when drug detection techniques and regulatory mechanisms were not in place for sports competition. The rat research will be published in the March 2004 issue of the Journal of Applied Physiology

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21. Gene doping is 'new frontier' for sports cheats

Times UK (Internet), 16.2.2004
http://www.timesonline.co.uk/article/0,,1-1004273,00.html

A "gene doping" technique that makes the muscles of mice grow up to 30 per cent stronger will be the next frontier of cheating in professional sport, scientists gave warning today. The new form of gene therapy, which is being developed to treat patients with muscular dystrophy and other wasting diseases, will inevitably be abused by athletes seeking the ultimate illegal edge, according to the researchers behind it and anti-doping experts. Experiments at the University of Pennsylvania have revealed that when normal mice are injected with a modified virus that adds a vital growth gene to their cells, they develop into heavily muscled, super-strong animals that the scientists have nicknamed "mighty mice". The effects are almost doubled when the gene therapy is coupled with a weight-training regime. Such genetic enhancements would deliver vast improvements in speed, power and strength if given to an athlete, said Lee Sweeney, who led the Pennsylvania research. Gene doping has the potential to boost an unscrupulous sportsman's performance more steeply even than existing drugs such as anabolic steroids and erythropoeitin (EPO). It would also be extremely difficult to detect, as it mimics the quirks of inheritance that give some athletes a natural genetic advantage. Dr Sweeney told the American Association for the Advancement of Science conference in Seattle that while the technology was designed for bona fide medical purposes, it was "inevitable" that it would eventually be abused "off label" in sport. He has already been approached by coaches and athletes, mainly bodybuilders, seeking to try gene therapy, even though the technique is barely ready for patient trials. "This is but one example of a number of potential gene therapies that are being developed with disease treatment as the goal, but if given to a healthy individual would provide genetic enhancement of some trait," Dr Sweeney said. "As these developments go forward, they inevitably will find their way into the healthy population. "The prospects are especially high that muscle-directed gene transfer will be used by the athletic community for performance enhancement, just as many drugs are used and abused today. "It is unclear what the risks are that are associated with such use. In many cases, policing such abuse in the sports community will be much more difficult that in the case of drugs, since detection will be difficult." In the experiments, Dr Sweeney's team genetically engineered a harmless virus to carry a gene known as insulin-like growth factor I (IGF-I), which is critical to muscle growth and repair. The gene is often missing or faulty in patients with wasting diseases such as muscular dystrophy, and the goal of the work is to design a means of fixing this genetic mistake and curing these conditions. When the virus was injected into normal, healthy mice, their muscles grew in size and strength by between 15 and 30 per cent. The genetically-enhanced muscles were more durable, and repaired themselves much more quickly when damaged. The added IGF-I gene also stopped the ageing process in its tracks: when middle-aged mice were injected with it, and then grew old, they suffered none of the muscle wastage typical of old age. The benefits of gene doping were greater still when combined with a training regime. The team injected IGF-I into the muscle of one leg of a rat, which was then kept in a cage in which it had to climb ladders regularly to reach food. At the end of this training, the gene-doped leg was nearly twice as powerful as the untreated one, and retained its strength for longer when training was stopped. The findings, which will be published next month in the Journal of Applied Physiology, indicate that gene doping in human athletes would provide benefits even greater than those of anabolic steroids. The therapy would enhance lean muscle mass, allow athletes to train for longer and to recover more quickly from injury, and would prolong their careers by slowing down the muscle wastage that takes place with age. Dick Pound, chairman of the World Anti-Doping Agency, said he was concerned that gene doping would become a major problem for sport. "I'm certainly worried about the potential," he said. "I don't think we're going to see it in Athens [at the 2004 Olympics], and I rather doubt Beijing [in 2008], but 2012 is certainly realistic. "The thought that we might be able to cure muscular dystrophy is wonderful, but what's not so good is the idea you'd have an 8ft shot-putter who could throw the shot 100 metres into the crowd." Many existing performance-enhancing drugs used in sport were originally developed for medical use. Synthetic EPO, for instance, is used for treating anaemia, and human growth hormone for treating growth deficiencies and wasting diseases. Thomas Murphy, president of the Hastings Centre, a US bioethics group, said athletes were certain to start using gene enhancement, even before it is available for therapeutic use. "Knowing what I know about athletes, long before it's perfected people will be peddling gene enhancement technologies," he said. "I'm sure somebody out there is formulating a business plan about how they're going to do this." He said gene therapy would pose great challenges for testing regimes, as it would be extremely difficult to detect. One possibility would be to test athletes for an immune response to the viral vector involved. "I'm not completely pessimistic about the prospects of detection, but neither am I optimistic," he said. Dr Pound said another answer would be to insist on the development of a test to show whether gene therapy has been used before potential techniques are allowed to begin clinical trials. Any test would have to be sensitive enough to discriminate between artificial and natural performance-enhancing genetic oddities. "If God did it to you, your competitors have to live with it," Dr Pound said. "If your genetic consultant did it, that's different."

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22. Study raises fears of genetically modified athletes

NewScientist (Internet), 17.2.2004
http://www.newscientist.com/news/news.jsp?id=ns99994688

A study showing that gene therapy can make muscles respond much better to exercise has raised the prospect of genetically modified athletes. "Half of the emails I get are from patients," says Lee Sweeney, at the University of Pennsylvania, and leader of the research. "And the other half are from athletes." The researchers injected rats with a modified virus that transported a gene to their hind leg muscles. The gene triggered increased production of a growth hormone called IGF-I. Combined with an intensive exercise regime of ladder climbing, this caused the rats' muscles become 15 to 30 percent stronger than would be expected with exercise alone. Even without exercise, the genetically modified rats' muscles grew by 15 to 20 per cent, Sweeney says. The research is aimed at developing treatments for diseases such as muscular dystrophy. Such therapies are not yet ready for use in humans and such genetic enhancement is likely to remain beyond the reach of athletes for some time. But the prospect of genetically modified athletes is already alarming drug testers. Muscle biopsy "It's a matter of some concern," says Dick Pound, chairman of the World Anti-Doping Agency. "What's most disturbing is that some of the first inquiries have come from trainers." Genetic enhancements are already banned under international sporting rules. But, unlike many of the drugs used to enhance performance, genetic modifications would leave no trace in the blood or urine. A muscle biopsy would be the only means of detection. Sweeney says genetic researchers may therefore need to design their treatments to be susceptible to discovery. "Given current testing, athletes would be able to get away with it," he says. "They would have to change the testing mechanism." There is also concern that athletes will put their health at risk by using untested genetic technologies. Gene therapies used to correct illnesses have had only limited success and two patients treated with gene therapy for "bubble boy syndrome" in France developed leukaemia as a result. It is also possible that genetic modifications targeting IGF-I could make muscles so strong that they could damage the recipient's bones, Sweeney says. The research was presented at the annual meeting of the American Association for the Advancement of Science in Seattle, Washington. Journal reference: Journal of Applied Physiology (vol 96 p 1)

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23. Mighty Mice Are Less Susceptible To Muscular Dystrophy Gene's Effects

Science daily (Internet), 26.11.2002
http://www.sciencedaily.com/releases/2002/11/021126072241.htm

Date: 2002-11-26 The Johns Hopkins scientists who first discovered that knocking out a particular muscle gene results in "mighty mice" now report that it also softens the effects of a genetic mutation that causes muscular dystrophy. The findings, scheduled for the December issue of the Annals of Neurology and currently online, build support for the idea that blocking the activity of that gene, known as myostatin, may one day help treat humans with degenerative muscle diseases. Working with mice carrying the genetic mutation that causes Duchenne muscular dystrophy in humans, the scientists discovered that mice without the gene for myostatin had less physical damage to their muscles and were stronger than other mice with the Duchenne mutation. "'Knocking out' the myostatin gene isn't possible for treating patients, but blocking the myostatin protein might be," says senior investigator Se-Jin Lee, M.D., Ph.D., professor of molecular biology and genetics at Johns Hopkins School of Medicine. "However, myostatin still needs to be studied in people to see if it has the same role in our muscles as it has in mice." The researchers caution that, even if myostatin does limit muscle growth in people, blocking it would not cure muscular dystrophy or any other degenerative muscle condition because the underlying cause of disease would be unchanged. "However, increasing muscle mass and strength by blocking myostatin could conceivably delay progression or improve quality of life," notes first author Kathryn Wagner, M.D., Ph.D., assistant professor of neurology at Hopkins. The Hopkins team bred mice without the myostatin gene with mice carrying the genetic mutation that causes Duchenne muscular dystrophy in humans. Muscular dystrophy mice completely lacking myostatin were more muscular and stronger than those with myostatin at 3, 6 and 9 months of age, the researchers report. Perhaps most importantly, their muscle tissue appeared to be healthier. Duchenne muscular dystrophy is the most common muscular dystrophy and the most common inherited lethal disease of childhood, affecting 1 in 3,500 live male births. (The genetic mutation that causes it is found on the X chromosome, and so is "covered up" in girls, who have two X chromosomes.) There's no good treatment at this time, and few patients survive into adulthood. Early in the disease in humans, the regenerative capacity of stem cells in muscle, known as satellite cells, keep up with the damage, but eventually the damaging factors win. The result is not just loss of muscle, but also its replacement with non-muscle tissues, essentially scar tissue and fat. This scarring process, called fibrosis, is also seen in mice with the muscular dystrophy-causing mutation. The Hopkins team reports that loss of myostatin function significantly reduced the amount of fibrosis, suggesting that the muscle regenerative process was improved. The Hopkins scientists hope to unravel the mechanism of muscle regeneration in mice with and without myostatin, possibly revealing even better targets for improving the process. They also plan to use special genetic manipulations to turn off the myostatin gene in adult mice, rather than at conception, to see if losing myostatin later in the course of muscular dystrophy is also beneficial. Authors on the study are Wagner, Lee, Alexandra McPherron and Nicole Winik, all of The Johns Hopkins University School of Medicine. Funding was provided by the National Institutes of Health, the Duchenne Parent Project, and the Muscular Dystrophy Association. Myostatin was licensed by The Johns Hopkins University to MetaMorphix, Inc., and sublicensed to Wyeth Pharmaceuticals, Inc. Lee and McPherron are entitled to a share of sales royalty received by the University from sales of this factor. Lee, McPherron and the University own MetaMorphix stock, which is subject to certain restrictions under University policy. Lee is a paid consultant to MetaMorphix. The terms of these arrangements are being managed by the University in accordance with its conflict of interest policies.

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24. "Mighty Mice" Gene Is Mutated In Beefy Bovines

sciernce daily (Internet), 13.11.1997
http://www.sciencedaily.com/releases/1997/11/971113074953.htm

Date: 1997-11-13 Gene's ability to slow muscle growth extends beyond mice Courtesy of the University of Whatever The same genetic "secret formula" that gave unusually large muscles to the "mighty mice" engineered by Johns Hopkins is also at work naturally in specially bred cattle that have extra muscle, according to a new report from the researchers. "Mutations in the myostatin gene in two different species produced the same result," says Se-Jin Lee, M.D., Ph.D., an assistant professor of molecular biology and genetics. "This strongly suggests that the normal human form of the gene, which we've already identified, helps suppress muscle growth. If we can find a drug that blocks myostatin activity, patients with muscular dystrophy or muscle wasting due to AIDS or cancer may really benefit." Results of the study, which was supported by grants from the Edward Mallinckrodt, Jr. Foundation and MetaMorphix, Inc. are published in the Nov. 11 issue of the Proceedings of the National Academy of Science. Cattle breeders knew nothing of myostatin when they succeeded in developing more muscular cattle breeds like the Belgian Blue and the Piedmontese. Hopkins researchers went searching for mutant forms of myostatin in these cattle after discovering what eliminating it could do to mice. "We wondered right away if interfering with the gene in livestock could give us animals with more meat and relatively less fat," says Alexandra McPherron, Ph.D., a Hopkins postdoctoral fellow. "We first became aware that there might be some breeds of livestock that already have mutated myostatin when someone described a large-muscled breed of sheep to us." Through literature and Internet searches, researchers learned of the Belgian Blue breed of cattle. From genetic information available online, they could see that the cattle's altered gene appeared to be in the same spot on the genetic code as human and mouse myostatin. To confirm their suspicions, Lee and McPherron then analyzed DNA from cattle blood samples supplied by a ranch in Missouri. They also detailed the DNA blueprint of the myostatin gene from 12 non-double-muscled breeds of cattle and found that their copies of myostatin were all normal. Scientists also sequenced the myostatin gene in humans, chickens, pigs, turkeys, sheep, baboons, zebrafish and rats, and found that there were relatively few differences among the species. Rights to myostatin are owned by The Johns Hopkins University and exclusively licensed to MetaMorphix Inc. MetaMorphix was established in 1995 to capitalize on work by Hopkins and Genetics Institute, a private pharmaceutical company, in the field of growth and differentiation factors. Lee is a shareholder in and scientific founder of the company. Under an agreement between MetaMorphix and The Johns Hopkins University, McPherron and Lee are entitled to shares of royalty received by the University from MetaMorphix. The University, McPherron and Lee also own MetaMorphix stock, which is subject to certain restrictions under University policy. Lee is also a consultant to MetaMorphix. The terms of this arrangement are being managed by the University in accordance with its conflict-of-interest policies.

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25. Gene therapy on rats could possibly lead to 'superathletes'

Iowa state daily (Internet), 3.3.2004
http://www.iowastatedaily.com/vnews/display.v/ART/2004/03/03/404554e5652c0

A muscle-growing drug designed to fight muscle-wasting illness may have implications for athletic departments across the country if it is made available in the medicinal market. There is concern among American sports officials that the new drug, a gene for insulin-like growth factor-1 (IGF-1) -- originally designed to help people with muscle-wasting illnesses such as AIDS or muscular dystrophy -- could be used illegally to build "superathletes." The drug is virtually undetectable and could make users' muscles larger and stronger without much effort. The drug is being tested at the University of Pennsylvania, where trials showed a 15 to 30 percent increase in the mass, strength and endurance of the muscles of lab rats after they were injected with a gene-manipulated virus. Officials in the ISU athletic department said they had not heard about this study and were surprised to hear what it could do. They said they were concerned that, if the drug does work on humans the way it is supposed to, it could have damaging physical side effects on anybody who takes it for performance-enhancing purposes. "Everyone in athletics, period, would be concerned," said Mark Coberley, head football athletic trainer. "To level playing fields, you would like people to perform with their own natural abilities. That's why they have drug testing." The NCAA has strict regulations against the usage of performance-enhancing drugs and gene therapy. "It's very closely monitored anymore, with drug testing by the NCAA and our own drug tests," said Terry Allen, associate head football coach. "[The NCAA] can come in at any time, at least three times a year." Allen said members of Iowa State and the Big 12 Conference conduct additional random drug tests on student-athletes throughout the year. Coberley said if the gene therapy is released for human usage, it will not go unnoticed. "I'm sure that the appropriate governing bodies will take whatever action is necessary, if needed," Coberley said. Marc Shulman, team physician at the Thielen Student Health Center, said there may not be cause to worry because results in lab rats may differ from results in humans. "It takes a lot of research to see if it works the same in humans as it does in lab animals," Shulman said. Douglas King, professor of health and human performance, agrees. "In terms of some aspects of physiology, there are some differences in rats and humans," King said. "The bodies may not deal with it in the same way." Although there is concern physical damages could occur from using this muscle-building gene therapy, good results are possible for those who need to take it. "There are a lot of good things that come from gene therapy," Shulman said. "It has to be in the right patients at the right time for the right reasons." He said a good example of the appropriate use for this type of gene therapy would be in patients with muscular dystrophy, who experience significant loss of muscle mass. A bad use of such therapy would be for the purposes of enhancing physical performance. "I don't think athletes here would take the risk of losing their scholarships and the [possibility of experiencing the] unknown medical risk of these things," Shulman said. Coberley said he wasn't aware of any abuse by ISU athletes. Shulman said for now, he expects more concern for non-athletes who aren't sanctioned by any organizations and would take gene therapy drugs to enhance their appearance. He said they could be at greater risk of physical harm due to side effects including liver damage, testicle and ovary changes and mental disorders. -- The Associated Press contributed to this article.

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26. Genetic engineering is next doping threat

Stuff Co (Internet), 16.3.2004
http://www.stuff.co.nz/stuff/0,2106,2847465a1823,00.html

LONDON: Back in the depths of time athletes used ginseng, opium and steroids from sheep testicles to enhance their performance. Anabolic steroids made their debut in sport in the 1940s and 50s and chemical agents followed. Now the big fear is that advances in biotechnology and gene therapy could result in genetically modified athletes with the bodies of Greek gods and the prowess of Superman overwhelming ordinary mortals at future Olympics. Gene therapy, to treat or prevent disease, has not developed with the speed scientists had initially hoped but it is moving forward and it could be just a matter of time before it infiltrates sport. "If the science develops and the regulatory and ethical frameworks are not properly established, I think there is a danger. We've seen it with the use of drugs that were developed for therapeutic purposes," said Dick Pound, president of the Montreal-based World Anti-Doping Agency (WADA). "The science could probably be misapplied." Genetic doping is unlikely to be an issue at the Athens Olympics in August or the Turin Winter Games in 2006, but it could be a problem come Beijing in 2008. "It's a realistic problem which we may have to face, but not today," said Dr Bengt Saltin, director of the Centre for Muscle Research at Copenhagen University and a member of the International Olympic Committee (IOC) science committee. "The Beijing Olympics would be the earliest possible occasion." Researchers have identified the gene for erythropoietin, or EPO, which stimulates the production of red blood cells - important for endurance sports such as the marathon and cycling. Synthetic EPO, which is used to treat anaemia and is banned by the IOC, was at the centre of a doping scandal that rocked the Tour de France cycling classic in 1998. Scientists have already injected bits of the EPO gene, using a weakened virus, into the leg muscles of monkeys in research that may one day help kidney patients awaiting an organ transplant to maintain a steady supply of red blood cells. "There were quite positive results but they can't control it enough," Saltin said, referring to the research. "When they find the control of the gene then they will definitely use it on kidney patients and I don't think the road to the sporting world is very far." One of the chief doping problems for sport is anabolic steroids. Androstenedione, nandrolone and stanozolol bulk up muscle mass and increase strength. "In so many sports the muscle mass and the strength is the critical factor," said Saltin. Steroids are non-specific and as researchers learn more about local growth factors, improving individual muscles with injections or genetic modification could become a possibility. "There is very good research in the field because there are muscular dystrophy patients with selective loss of muscles. If you could find how to counteract that with these local factors it will help many patients around the world," Saltin said. Dr Lee Sweeney of the University of Pennsylvania and his team have already coaxed muscles in mice to grow up to 30 per cent stronger. They injected mice with a growth gene known as insulin-like growth factor I (IGF-I). "The prospects are high that muscle-directed gene transfer will be used for performance enhancement," Sweeney told a science conference where he presented the research. At the moment, scientists believe genetic engineering is too dangerous and too little is known about the technology and its impact to pose an immediate problem. However, they believe it could be just a matter of time before it reaches sport. In the meantime, WADA plans to keep up with advances in the technology and the with people likely to bend or break the rules. "In the case of drugs, I think the genie was allowed to get out of the bottle early on, before people realised what the full implications would be and before the science got developed to the point where you could detect these sort of things," said Pound. "In genetics, our objective is to try and be there as the policy framework is developed, to be part of that process." Saltin believes doping controls have never been as good and this trend will continue. "We have seen through the years that there are always people willing to use their knowledge, experience and technology to improve the athlete's performance. If those people were not around the problem would be so much easier to handle," he said.

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27. Muscles, maladies and mischief

radio netherlands 2 (Internet), 22.3.2004
http://www.rnw.nl/science/html/040322rf.html

[ratmuscles] Untreated rat muscles are smaller than those of rats given gene therapy (Courtesy of Prof. Sweeney) A gene therapy approach intended to help the ill and the elderly could aid athletes aiming to augment their natural abilities. Research published in this month's edition of the Journal of Applied Physiology shows that gene enhancement increases the strength of leg muscles in 'weight-trained' rats. But there are growing concerns that the technique could be abused to give a secret boost to competitive athletic performances. [Click to hear the full programme] This report was featured in Research File. Listen to the programme in full. (29:30) By taking advantage of rats' innate liking for climbing ladders, scientists from the University of Pennsylvania Medical School in the USA were able to implement an ‘exercise programme' in which the rodents repeatedly climbed small ladders in order to receive a food reward. Over time, extra weight was added to a small ‘backpack' that each of the creatures wore, so mimicking the effects of weight-training. Some of these animals received ‘gene therapy', others did not. A third group was genetically enhanced but did no extra exercise. [Professor Sweeney] Trojan training Professor Lee Sweeney, who's Chairman of the Department of Physiology, says gene therapy alone produced muscles that were "about 15 percent bigger and stronger", which was equivalent to the gains from weight-training alone. "But if we did both, train them and give them genetic enhancement, they got twice as strong, they got 30% stronger and much healthier looking muscle." The gene enhanced animals also gained the extra strength faster and maintained the effects for longer after weight-training ended, as compared with their non-enhanced counterparts. In this form of gene therapy the scientists use a ‘Trojan horse' approach. A virus known to specialise in entering skeletal muscles is adapted so that it carries a synthetic gene into the muscle cells. There the gene produces a protein &endash; in this case a growth factor called ‘IGF-1' which promotes muscle growth and repair. "In fact the weight training was severe enough that there had actually been some scarring in the muscle of the non-genetically enhanced animals," adds Professor Sweeney, "whereas the genetically enhanced ones had repaired so rapidly, there was no scar tissue and the muscles looked much healthier." Athletic Advantage Professor Sweeney and his team conducted the study because they had already "had so many enquiries as to whether it really would be an enhancement" if athletes were to use gene therapy techniques in order to boost their muscle power and performance. He [80b_rat] says the study findings "showed that it would actually provide an athlete with marked enhancement." While some members of the athletics community have already begun to ask how they may be able to make use of these methods, yet others are becoming increasingly concerned about this potential new ‘doping' threat. They believe gene enhancement could become an issue as early as the 2008 Olympic Games in Beijing. And there is currently no regulatory mechanism or detection technique in place to keep a check on potential abuse. Detection difficulty Professor Sweeney says one problem in devising ways to detect this sort of gene enhancement is that only urine and blood can be taken from an athlete for testing, meaning that some indirect sign of the gene's presence would have to be identified: "you'd only find the gene if you could get a biopsy of the muscle, so you'd have to look for something that might show up in either the blood or the urine. In some cases that would be easy to do and in other cases it might be nearly impossible." Medical muscle While the prospect of genetically enhanced athletes is a valid concern, it's perhaps as well not to lose sight of the fact that a large number of other people could benefit from these techniques. Professor Sweeney's primary aim is to alleviate the kind of metabolic and stability problems that occur as a result of muscle loss in old age or in diseases such as muscular dystrophy. "We've shown that producing, or over-producing in this case, [Rat muscle] IGF-1 in muscles can prevent almost all of ageing-related changes," he says, although safety concerns about gene enhancement will mean that therapeutic use in the elderly is not likely in the immediate future. However, he envisages applications for muscular dystrophy, where "you might not be able to genetically correct the problem itself, but by allowing the muscle to repair more rapidly and by trying to make the muscle bigger, it would actually improve the condition in these patients, hopefully slowing down the progression of the disease. That's probably where we'll take this first."

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28. Genetic Research Boosts Athlete Cheats

Daily Champion (Internet), 26.3.2004
http://allafrica.com/stories/200403260218.html

The world of sports seems to be in trouble. This is informed more by the quanta of drug related sports based cheats either testing positive to one banned substance or the other. The population of those in the centre have increased especially in the last five years to date. Tennis with its stained hands was diplomatic enough in clearing one of its biggest stars, Greg Rusedski. Athletics, swimming, weightlifting, football, the martial arts etc have had a fair share of the drug hangover. [Subscribe to AllAfrica] Or how else do we describe the situation where a new revelation that athletes will soon try to enhance their bodies with gene technology is raised by the results of a new study to boost muscles in rats? The scientist behind the research says his intention was to find new ways of treating muscle-wasting diseases. (Whatever that means.) Lee Sweeney, from the University of Pennsylvania, says trainers are already making inquiries about his technology. "I would say half the e-mails I get now are from athletes," he told an American science conference in Washington State. The other half is from patients with muscular dystrophy." Sweeney and colleagues injected their rats with a virus which carried a gene into muscle cells to produce a growth hormone called IGF-I. The rodents given the therapy and put on an exercise programme developed bigger and stronger muscles. The scientists also found the rats with genetically elevated levels of IGF-I retained more of their muscle mass after they stopped exercising. All this could be of considerable help to patients with muscular disorders and muscle loss associated with either disuse or ageing. But the benefits are also those that athletics cheats have sought in the past from standard drug technologies. Hijacking research While the leap from laboratory animals to human beings is still hypothetical, Lee Sweeney and other speakers here at the annual meeting of the American Association for the Advancement of Science said it was inevitable that athletes and their trainers would attempt to hijack the rat research. "The world anti-doping code at the moment includes gene transfer technology as a banned practice," said Dick Pound, the chairman of the World Anti-Doping Agency (WADA). This is where the fears of the world of sports is planted when Pound said, "We're not sure of course we can detect it. Today, in fact, it would be quite difficult." The speakers suggested that any clinical trials of new gene technologies to treat disease might include a requirement on the part of scientists to develop a test that could also be used on sports people to check there is no abuse of the research. "The only way to detect this would be through a muscle biopsy," said Sweeney, an associate professor of physiology at Penn. Thomas Murray, a bioethicist and president of The Hastings Center in New York, has looked closely at the issue of drugs in sport. He said sports administrators would have to raise their game yet again. "Performance enhancing drugs in sport have posed a challenge to what we care about in sport - what gives it meaning," he told the meeting. "Gene transfer technologies have the potential to kick it up a notch and make the challenge even greater." Gene therapy has had a chequered history. It has long promised to revolutionise medicine by correcting or replacing faulty genes in patients - to cure diseases for which current drugs will only treat symptoms. Haphazard technique But the process of getting corrected genes into patients' cells can be haphazard, and clinical trials have resulted in at least one death. Two boys in France treated for X-Scid, popularly known as "bubble boy disease", developed leukaemia. Relevant Links West Africa Athletics Crime and Corruption Nigeria Sport Pound said it was essential that sports governing bodies worked with science to make sure the fruits of the genomic age went solely to diseased patients and not to gold medal frauds. "In terms of these genetic applications, we're at the beginning of the cycle," he said. "In the 60s, 70s, 80s and even the 90s, sport rather lost control of drug use. We've been playing catch-up ever since - with some success. What we'd like to do with this new branch of science is be there early to help in the formation of the regulation of it." The rat research will appear in the March 2004 issue of the Journal of Applied Physiology.

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29.

SF Gate (Internet), 17.4.2004
http://sfgate.com/cgi-bin/article.cgi?f=/news/archive/2004/04/17/sports1444EDT0356.DTL

Experts believe athletes and coaches will soon turn to gene doping in an attempt to gain a performance edge. Authorities told a workshop on gene doping Saturday that recent discoveries have made it inevitable that athletes and coaches will try to abuse gene therapy to gain an edge in speed, strength or endurance -- despite huge health risks. "I'm very pessimistic -- I think it won't take very long," said Hidde Haisma, a professor of gene therapy at the University of Groningen. The needed tools "are available at labs around the world," he said. Though the idea of manipulating genes to enhance performance has been around for more than a decade, it gained attention this year after a University of Pennsylvania study showed that muscle mass, strength and endurance in rats can be increased by altering their genes. Scientists have treated roughly 3,000 humans suffering from life-threatening illnesses with gene therapy, but few cases have been successful and some have been fatal. In one non-human study where monkeys' genes were manipulated to produce an extra protein called erythropoietin, some of the monkeys developed the disease anemia. Given the risks involved, the first gene doping in the sports world may be in an animal sport like dog racing, Haisma said. But Olivier Rabin, science director at the World Anti-Doping Agency, said human athletes won't wait long. He pointed to instances when athletes began using new steroids "straight from the test tube, before they were even tested on animals." Current blood and urine tests cannot detect gene doping, and Haisma said the most promising technique of detection involved analyzing the proteins in blood samples, looking for a suspicious spike. "There is no doubt in the minds of people working in the sports community that gene doping is coming," he said.

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30. Steroids Make Stronger Gene Therapy Wrapping DNA in a common steroid increases treatment's effectiveness

betterhumans (Internet), 13.2.2004
http://www.betterhumans.com/News/news.aspx?articleID=2004-02-13-3

Simply coating DNA with a common topical steroid could make gene therapy more effective. According to researchers at the University of Pennsylvania in Philadelphia, wrapping DNA in a common steroid solves some problems associated with current gene-delivery techniques. "The steroid coating not only allows the gene to be taken up into a cell more easily, but the steroid itself also prevents the sort of inflammatory immune response seen in gene transfer therapy," says Penn researcher Scott Diamond. Delivering health Gene therapy is a technique that involves inserting genes into a person's DNA, usually to replace existing abnormal or defective genes. There are two approaches to delivering the therapeutic genes: Viral and nonviral. The viral approach uses viruses that are genetically altered to deliver the genes. The nonviral approach introduces the genes directly into target cells, usually with the help of tiny particles. Both methods are imperfect and still in their infancy for treating living organisms. One big problem, the researchers say, is that DNA is a large, negatively charged molecule, which inhibits cells from taking up introduced genes. Another problem is an inflammatory immune system response that reduces the action of introduced genes. Reduced inflammation To address the problems, Diamond and colleagues attached a steroid to DNA to suppress inflammatory cytokines created by the immune system after gene delivery. To counter DNA's negative charge, they modified a common steroid, dexamethasone, for the job by adding a nitrogen-rich, positively charged tail that "glues" the steroid to the naked DNA. "The steroid is a fatty lipid so, in essence, we have greased up DNA for cellular uptake," says Diamond. "Plus the cells get a big dose of steroid." According to studies in cell cultures and animals, the steroid-coated DNA caused less inflammation and greater gene expression. Cells also seemed more able to use foreign DNA effectively. "In humans, especially in inflammatory diseases, a steroid coating would greatly enhance the chances of successful gene transfer," says Diamond. "As an alternative, I could foresee the use of this coating technique to tailor therapies by choosing drugs that would amplify the benefit of a particular therapeutic gene." The research is reported in the journal Gene Therapy

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31. Loss of Skeletal Muscle HIF-1? Results in Altered Exercise Endurance

Plos Biology (Internet),
http://www.plosbiology.org/plosonline/?request=get-document&doi=10.1371/journal.pbio.0020288

1 Molecular Biology Section, Division of Biology, School of Medicine, University of California, San Diego, California, United States of America, 2 Division of Physiology, School of Medicine, University of California, San Diego, California, United States of America, 3 Cardiology Division, Yale University Medical School, New Haven, Connecticut, United States of America, 4 Joslin Diabetes Foundation, Harvard Medical School, Boston, Massachusetts, United States of America The physiological flux of oxygen is extreme in exercising skeletal muscle. Hypoxia is thus a critical parameter in muscle function, influencing production of ATP, utilization of energy-producing substrates, and manufacture of exhaustion-inducing metabolites. Glycolysis is the central source of anaerobic energy in animals, and this metabolic pathway is regulated under low-oxygen conditions by the transcription factor hypoxia-inducible factor 1? (HIF-1?). To determine the role of HIF-1? in regulating skeletal muscle function, we tissue-specifically deleted the gene encoding the factor in skeletal muscle. Significant exercise-induced changes in expression of genes are decreased or absent in the skeletal-muscle HIF-1? knockout mice (HIF-1? KOs); changes in activities of glycolytic enzymes are seen as well. There is an increase in activity of rate-limiting enzymes of the mitochondria in the muscles of HIF-1? KOs, indicating that the citric acid cycle and increased fatty acid oxidation may be compensating for decreased flow through the glycolytic pathway. This is corroborated by a finding of no significant decreases in muscle ATP, but significantly decreased amounts of lactate in the serum of exercising HIF-1? KOs. This metabolic shift away from glycolysis and toward oxidation has the consequence of increasing exercise times in the HIF-1? KOs. However, repeated exercise trials give rise to extensive muscle damage in HIF-1? KOs, ultimately resulting in greatly reduced exercise times relative to wild-type animals. The muscle damage seen is similar to that detected in humans in diseases caused by deficiencies in skeletal muscle glycogenolysis and glycolysis. Thus, these results demonstrate an important role for the HIF-1 pathway in the metabolic control of muscle function. Citation: Mason SD, Howlett RA, Kim MJ, Olfert IM, Hogan MC, et al. (2004) Loss of skeletal muscle HIF-1? results in altered exercise endurance. PLoS Biol 2(10): e288. Received February 9, 2004; Accepted June 29, 2004; Published August 24, 2004

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32. Mice flex muscles in genetic studies

AP , Union Tribune (Internet), 24.8.2004
http://www.signonsandiego.com/uniontrib/20040824/news_1n24mice.html

Genetic changes made on mice in a UCSD study improved their running and swimming ability. Autumn Cruz / Union-Tribune They might be considered the Olympians of the mouse world. Genetically engineered mice developed by Salk Institute and UCSD scientists in separate studies have greater performance abilities when running and swimming. In one case, the genetic enhancements seemed to prevent obesity, even when the mice were inactive and fed a high-fat diet. The gene engineered in these mice essentially mimics exercise: Researchers say it conferred endurance and prevented the modified mice from becoming obese. "This is a real breakthrough in our understanding of exercise and diet and their effects on obesity," said Ronald Evans, lead researcher in the Salk study published today by the online journal, PLoS Biology. "The practical use of this discovery is the implication in controlling weight," he said. "This gives us a real lever on metabolism." The study also describes how engineered mice, even the couch-potato variety, were able to run farther and longer if their "fat switch" genes remained on continuously. Evans found the gene he dubbed the "fat switch" more than 10 years ago, and now its broad implications are being understood. Evans believes his work has implications for just about every disease of the metabolism, from obesity to heart disease. "It's a bit ironic that we developed these marathon mice at the same time of the Olympics," Evans said. Nobody cares more about the intricacies of the human metabolism than Olympic athletes. Many predict that steroids, growth hormones and other drugs and chemicals that cheating athletes use to gain the smallest sliver of an advantage will soon be replaced by hard-to-detect genetic engineering, which could become commonplace as soon as the Beijing Olympics four years from now. Evans said he is bracing for a flood of inquiries from athletic trainers now that his research paper has been published. A second study in the same journal by UCSD professor Randall Johnson found that mice engineered without a gene called HIF-1 were able to run and swim longer than their natural counterparts that have the gene. Johnson believes the finding may reveal information about how athletes train, tire and recover to perform again. HIF-1, or hypoxia inducible transcription factor-1, allows mammals to switch from aerobic metabolism to anaerobic metabolism when oxygen levels in muscle run low. Two mice ran on treadmills as part of a study into oxygen regulation and exercise conducted at the University of California San Diego. Without that ability, the animals in the UCSD study relied solely on aerobic metabolism &endash; gaining an extraordinary capacity for longer, sustained aerobic endurance exercise, the scientists said. "By changing the way skeletal muscles respond to low-oxygen levels, we've developed muscles that appear to be superiorly adapted or trained for long bouts of . . . aerobic exercise," Johnson said. The gains in the UCSD study were temporary and came at a price, however. After four days of exercise tests, the genetically engineered mice endured much more muscle damage than normal mice, and they could no longer run or swim as much as their unaltered counterparts. "It's a double-edged sword," said Johnson, a professor of biology who headed the study. "But these muscles also become damaged more easily than normal muscles during exercise, and we don't know why." In Evans' study, no adverse side effects were found in the engineered mice. Instead of improving times by fractions of a second that athletes strive for, the genetically enhanced marathon mice ran twice as far and nearly twice as long as naturally bred rodents. The engineered mice ran 1,800 meters before quitting and stayed on the treadmill an hour longer than the natural mice, which were able to stay running for 90 minutes and travel 900 meters. When it is activated, the "fat switch" gene begins the process of creating "fatigue-resistant" muscles while helping the heart and nervous system create endurance. UCSD's study didn't start out to examine the athletic prowess of modified mice. Johnson is a cancer biologist. Two years ago, he and a graduate student in his lab, Steven Mason, found that mice without HIF-1 in their white blood cells had a reduced tendency of those cells rushing to areas of infection &endash; a primary cause of inflammation. But they also found that the same mice appeared to have increased endurance on a treadmill. In the study profiled today, Johnson and Mason set out to confirm what they saw anecdotally in the cancer study. They subjected two groups of 4-month-old mice to exercise routines. One group was engineered without the HIF-1 gene in their skeletal muscle, and other group was normal. In a swimming test, normal mice typically swam for 150 minutes before they were exhausted. Mice without the HIF-1 gene swam an average 45 minutes longer. Then the two groups ran on treadmills tilted up 5 degrees. The treadmills were started at a speed of 10 meters per minute and increased in speed every five minutes until the mice could no longer run. Normal mice averaged about 50 minutes on the uphill test, while mice without the HIF-1 gene ran an average 10 minutes longer. The additional 10 minutes also included two increases in speed. Most daily activities are performed aerobically, in which muscles make full use of oxygen. When the demands on muscles exceed the oxygen available to them, during sprinting or lifting a heavy object, for example, the HIF-1 gene is activated. The protein it produces enables muscle to switch to the more explosive anaerobic process, which does not use oxygen and generates lactic acid as a byproduct. The lactic acid may help warn the body it has become overexerted. Scientists suspect the HIF-1 gene plays a significant role in the ability of muscle to sense oxygen deprivation, switch muscle metabolism from aerobic to anaerobic and then trigger a sense of exhaustion that prevents muscle damage. "By not having the sensation of exhaustion . . . they start getting more and more muscle damage," Johnson said of the genetically engineered mice in his study. The findings could someday help scientists learn more about what happens to the body during exercise, and that may lead to more optimal training for athletes, Johnson said. But for how, many important questions about exercise and training remain unanswered. High-performance athletes may have a greater physiological tolerance than others for lactic acid. Others argue that superior athletes have the ability to clear lactic acid from their bodies more quickly than normal. In either case, these people can endure long periods of athletic performance, but there is a long-term risk of muscle damage. A separate debate focuses on training and how the level of oxygen in muscle affects performance. One of the things we want to try to do in future studies is understand exactly how the process of training is impacted by the loss of the ability to adjust to low oxygen within the muscle," Johnson said. "Hopefully, by doing that, we can try to understand the overall process of training better." Besides Johnson and Mason, other UCSD scientists who contributed to the study included Richard Howlett, Matthew Kim, Mark Olfert, Michael Hogan, Wayne McNulty and Peter Wagner. Reed Hickey and Fran Giordano from the Yale University medical school and C. Ronald Kahn from the Harvard University medical school were also coauthors of the study, which was paid for by the National Institutes of Health. Staff writer Bruce Lieberman contributed to this report.

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33. Gene therapy for rapid weight loss

PNAS online 9th February 2004 (Internet),
http://www.healthandage.com/Home/gm=1!gid1=5493

When animals receive the hormone leptin via gene therapy, their fat cells are transformed into fat burning cells. Leptin is a hormone which plays a role in regulating satiety. Its functioning is complex but there has been hope that it may be used as a drug to induce weight loss by helping control the appetite. In a new study, researchers at the University of Texas show how leptin administered to rats by gene therapy led to some dramatic changes. The animals’ weight dropped from 280 grams to 207 grams within 14 days while their food intake went down by a third. Leptin levels went up steeply as a result of the gene therapy. And analysis showed that the animals’ fat cells had reduced in size, acting now like fat burning cells according to their pattern of gene activity. The researchers say the fat loss found here is different from that which occurs by calorie restriction. Further research along these lines may help realize the dream of using leptin as a drug which can help to control obesity.

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34. Gene therapy that melts away the fat

BBC (Internet), 10.2.2004
http://www.medicalnewstoday.com/index.php?newsid=5801

American and Swiss scientists have used gene therapy to transform fat-storing cells into fat-burning cells. This could lead to a revolutionary new treatment for obesity. The scientists added leptin (a protein) to cell. When they experimented on rats, the animals lost enormous amounts of weight (fat) without any side effects (apparently). Some extremely obese people have had leptin injected into them. This work is still in its initial stages. Scientists hope that leptin treatment will become more widespread. They worked on rats that had been genetically predisposed to develop diabetes. When they injected the rats with leptin (with a virus), they lost on average, 280 grams to 207 grams in 14 days. The rats also ate less and stayed healthy and active. Energy centres Microscopic evaluation of individual fat cells found that the cells shrank in size, and developed more energy centres called mitochondria. In addition, levels of enzymes known to promote fat metabolism increased while those that impede fat metabolism decreased. The rats showed no signs of the side effects associated with fat loss induced by starvation or insulin deficiency. These can include loss of lean body mass, hunger and the build up toxic substances called ketones in the blood. The researchers also found that when they force-fed rats, those given the leptin gene put weight on at a slower rate. The researchers stress that more work is needed to reveal the precise mechanism behind the changes. However, they suggest that the results may have important implications for the treatment of obesity in humans. Writing in the journal, they say: 'The fat loss induced here was far more rapid and profound than can be induced by caloric restriction.' Lead researcher Dr Roger Unger, director of the Touchstone Center for Diabetes Research at the University of Texas Southwestern Medical Center in Dallas, said: 'The structure of the cells changed from the normal appearance of a fat cell to a very novel cell that's really never been seen before. 'There's no precedent for a cell that appears like this.' Leptin is normally produced by fat cells, or adipocytes, but is somehow prevented from interfering with the accumulation of surplus fat. Scientists believe this is to ensure fat cells maintain their vital function of storing fuel during times of food shortage. Dr Andrew Hill, chairman of the Association for the Study of Obesity, told BBC News Online a quick and effective treatment for obesity would be extremely welcome. But he said: 'There is an enormous distance between what these researchers have done and a GP using it to treat 400-500 patients on their list for obesity.' Dr Hill said many scientific advances had promised the prospect of a quick fix for obesity, but for most people the only solution was to work hard at maintaining a low weight throughout their life.

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35. Enrolment in Clinical Studies for Angiogenic Gene Therapy Candidate Stopped

biz yahoo (Internet), 30.1.2004
http://biz.yahoo.com/prnews/040130/ukf008_1.html

BERLIN, January 30 /PRNewswire-FirstCall/ -- Schering AG, Germany (FSE: SCH, NYSE: SHR) announced today that it has stopped enrolment in its Phase IIB/III clinical studies for Ad5FGF-4, its investigational, non-surgical angiogenic gene therapy product being developed for the treatment of patients with stable exertional angina due to coronary artery disease. The company's analysis of the interim data of one of the studies has led it to conclude that the studies, as currently designed, will not provide sufficient evidence of efficacy to warrant continued enrolment. The company is instructing investigators to stop patient enrolment. All patients who have already been treated will continue to be evaluated. Each study is being monitored by an independent Data Safety Monitoring Board (DSMB). No evidence of important safety concerns was found. Schering AG continues to evaluate and explore the potential of this highly innovative technology. Schering AG is a research-based pharmaceutical company. Its activities are focused on four business areas: Gynecology & Andrology, Diagnostics & Radiopharmaceuticals, Dermatology as well as Specialised Therapeutics for disabling diseases in the fields of the central nervous system, oncology and cardiovascular system. As a global player with innovative products Schering AG aims for leading positions in specialised markets worldwide. With in-house R&D and supported by an excellent global network of external partners, Schering AG is securing a promising product pipeline. Using new ideas, Schering AG aims to make a recognised contribution to medical progress and strives to improve the quality of life: making medicine work This press release has been published by Corporate Communication of Schering AG, Berlin, Germany.

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36. Designer Virus Delivers Long-lasting Gene Therapy

Betterhumans Staff (Internet),
http://www.betterhumans.com/News/news.aspx?articleID=2004-01-19-5

Special delivery: A modified virus could advance gene therapy by sidestepping the liver's tendency to keep foreign objects out of the bloodstream A designer virus has been developed for delivering long-lasting gene therapy anywhere in the body from a single injection. The delivery system, developed by researcher Andrew Baker and colleagues from the University of Glasgow in Scotland, could overcome a key problem of gene therapy. "It may be possible to design and construct genetically engineered 'designer' gene therapy for selectively delivering genes to any part of the body," says Baker. Liver cleansing Gene therapy involves the introduction of genes into a person's DNA in order to treat disease. Currently, most techniques use viruses to carry the genes. The viruses are either harmless to humans or have their disease-causing components removed. The viruses are injected or inserted into the body where they "infect" a specific area with therapeutic genes. Because the liver cleanses the blood of foreign material, the viruses must be delivered directly into areas to be treated. The liver sequesters anything injected into the bloodstream. This has made viral delivery systems less practical for clinical settings and many genetically based diseases. Infectious treatment Baker and colleagues have now redesigned an adeno-associated virus that's harmless to humans so that it is not quarantined by the liver. The virus remains in the bloodstream long enough to infect cells in the body. It targets vascular endothelial cells, which are cells that line the inside of blood vessels. "Vascular endothelial cells, which are in continuous contact with the bloodstream and integrally involved in cardiovascular abnormalities, are appropriate targets for gene therapy," says Baker. The researchers modified the virus with two small peptides that bind specifically to endothelial cells. In laboratory studies, they found that the unmodified virus was 100 times more infectious in liver cells than in endothelial cells, while the modified virus was far more inclined to infect endothelial cells. In mouse studies, the modified viruses accumulated at lower levels in the major organs, predominantly the liver. It also remained in blood circulation longer, likely because of reduced liver sequestration. Single dose According to Baker, the virus has the potential for long-lasting gene expression from a single dose. In laboratory studies on hemophilia, a single dose provided at least five years of therapy&emdash;possibly longer because the studies are ongoing. "The concept of developing systematically injectable gene transfer vehicles is important for a number of potential cardiovascular gene therapies, particularly in those conditions where access to the target site can only realistically be achieved via the bloodstream," says Baker. "This work shows for the first time that this is possible using cell-specific peptides to modify AAV vehicles for systemic gene delivery." The study is published in the journal Circulation

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37. Gene Therapy in Salivary Glands Could Lead to Promising Applications in Oral Diseases

NIH.gov (Internet), 22.1.2004
http://www.nih.gov/news/pr/jan2004/nidcr-22.htm

Although gene therapy has shown much promise over the past decade, one of its major challenges continues to be controlling the expression of a transplanted gene once it has been delivered into a cell. As many scientists already have reported, transplanted genes may switch off prematurely, or, in some cases, they might not turn off fast enough, causing an undesirable overproduction of its replacement protein. One way around this problem is to control the expression of the transplanted gene with a system controlled by a small molecule, for example rapamycin, a well-characterized immunosuppressive drug. As scientists have envisioned the strategy, they stitch a chemical switch next to the gene that only rapamycin (or derivative) molecules can control. Upon administration of the drug, the gene will turn on leading to protein production. Already, researchers have demonstrated the effectiveness of this approach in the liver, muscle, and eye. Now, a team of scientists report in an article published online today in the journal Gene Therapy they succeeded in getting the so-called "rapamycin gene-activation" system to work in the salivary glands. As the scientists noted, their finding could one day have important implications in treating a variety of oral and possibly systemic conditions with gene therapy. "Our data mark an important first step toward applying the technique in the salivary glands," said Dr. Bruce Baum, the senior author on the paper and a scientist at the NIH's National Institute of Dental and Craniofacial Research. "Our next goal will be to refine the technique further and apply it to treat disease." For decades, most biologists have subscribed to the dogma that glands in the body either secrete proteins into the bloodstream, called endocrine secretion , or channel them outward through a duct, for example, to the mouth or intestine, called exocrine secretion . But, according to the dogma, they can't do both. Salivary glands are an exception to the dogma. They not only secrete proteins into saliva then into the mouth in an exocrine manner, but also partly into the bloodstream in an endocrine fashion. This dual secretory feature has made the salivary glands an intriguing, though often overlooked, target for gene therapy experiments. "What's fascinating is, in theory, one could use gene therapy in the salivary glands to treat either oral and systemic conditions or single-gene disorders, such as diabetes and growth hormone deficiency," said Baum, who has studied the salivary glands for nearly three decades. Baum's group and others already have confirmed this theory in animal studies. They found that, depending on the signal for the protein, the salivary glands can secrete in either direction. The group also established in a series of gene therapy experiments that the salivary glands will secrete the transferred gene's protein products in a normal, physiological way, showing that their potential as a site for gene therapy will extend well beyond the mouth. In the current Gene Therapy paper, Wang et al. hypothesized that the rapamycin system also might work in the salivary glands. In a series of experiments, the scientists showed that the production of human growth hormone (hGH) and its secretion into the saliva of rats could be regulated with rapamycin for at least three times in 16 days. Rats that were not given rapamycin had no hGH in their saliva. hGH was used as a surrogate exocrine protein in this study. Normally, however, hGH is secreted via a "regulated pathway" in the anterior pituitary gland, which leads to its secretion in blood. However, in salivary glands, secretion from a regulated pathway secretion leads into the saliva. The scientists found only a small amount of hGH in the blood compared to saliva, showing the salivary gland released this protein in the same manner as the pituitary gland normally does. To deliver the therapeutic gene and the rapamycin-based system, two adenoviruses were injected through the oral duct of the rat's salivary glands. "The system has several important components," Dr. Jianghua Wang, a scientist in Dr. Baum's lab and lead author on the paper, explained. "One virus contains the hGH gene and the second virus supplies genes encoding a DNA-binding domain protein and an activation domain protein." Wang added that the DNA-binding domain protein can attach to a site next to the hGH gene in the first virus. Both the activation and DNA-binding domain proteins are fused to a rapamycin binding site. Upon administration, rapamycin will link both domains leading to the activation of the hGH gene. Without rapamycin, no linkage and therefore no activation can occur. Rapamycin is generally used to prevent rejection in patients receiving organ transplantation. It suppresses the immune system and currently, other scientists are investigating whether it also works to treat cancer. The application of rapamycin in a gene-regulating system could be complicated by its immunosuppressive actions. When used in patients, it could lead to infections the body is unable to respond to. Several scientists in the field have tried to find a way around this problem and examined analogs of rapamycin. Some of the analogs turned out to be as effective as rapamycin itself, but are without the immunosuppressive effects. Wang noted that adenoviruses are not ideal vectors for gene transfer in people because they evoke a large immune response. However, they are helpful to study potential gene therapy in animals. Also, they may be useful for short term applications in humans. To help suppress the immune reaction in the rats initiated by the adenovirus, the scientists also injected dexamethasone, a drug commonly used to reduce immune reactions in clinical practice and animal studies. "Given these results," said Wang, "it may be possible to transfer, and control, genes into the salivary glands to treat a variety of oral conditions." For example, oral ulcers or infections are common, but difficult to treat. With the rapamycin approach, the duration of the treatment could be controlled." Fungal and bacterial infections, for instance, usually require treatment for about 10-14 days, a period the scientists showed could be achieved with their system. Oral ulcers, which can result for example from irradiation of the mouth or chemotherapy, could benefit from therapy with a single gene. Certain growth factors or cytokines, which are proteins involved in regulating immune responses, have been tested for ulcers. These protein drugs need to be given with frequent injections or require local application with creams. "The large advantage of gene therapy is, that only one gene delivery, instead of multiple protein injections, is needed, making it less expensive and easier to tolerate for patients," explained Wang. Additionally, therapeutic genes injected into the salivary gland could provide a higher local concentration of medication than is possible with general injections into blood or muscle. "The next step will be the use of rapamycin in the delivery of genes for long-term expression with an adeno-associated virus (AAV)," Baum said. AAV has the advantage over adenovirus in that only a minimal immune response is seen after delivery, meaning this virus has the potential for broader use in humans. Also, the expression of the protein is much longer than can be achieved with adenovirus. "Our group has shown protein expression with an AAV up to one year in mice." Collaborating with were Drs. Baum and Wang were Drs. Antonis Voutetakis and Changyu Zheng. The study is titled, "Rapamycin control of exocrine protein levels in saliva after adenoviral vector-mediated gene transfer" and it was published online in the journal Gene Therapy on Thursday, January 22, 2004.

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38. Tracing the Cause of Leukemia in Gene Therapy Trial

Genome News Network (Internet), 23.1.2004

Last year, researchers in France halted a gene therapy trial because two young patients developed leukemia. The patients suffered from a rare genetic disorder caused by a missing immune system gene. In October, researchers determined that the delivery of the missing gene was somehow activating a known cancer-causing gene in the patients’ genomes. Now a new study in mice offers an explanation of what went wrong. Researchers from the National Cancer Institute in Frederick, Maryland, report in Science that the gene delivered to the patients is itself a possible cancer-causing gene. The high incidence of leukemia perplexed researchers. Ninety-nine percent of the genome doesn’t code for any genes at all. The likelihood of the missing gene getting inserted within or near any gene, much less a cancer-causing gene, was thought to be extremely rare. But out of 10 patients enrolled in the trial, two had the same insertion in the same gene and both developed leukemia. The patients were being treated for severe combined immune deficiency, also known as the bubble baby disease because patients have such compromised immune systems that they must live in isolated environments. The patients were missing a gene called IL2RG. Replacing the gene effectively cured the immune disease, but caused leukemia in the two patients. These patients have been treated with chemotherapy and are in now in remission. No other patients have developed leukemia. Most studies show that in order for any cancer to develop, several cancer-causing genes must be disrupted within the same cell. Inserting one cancer-causing gene into another cancer-causing gene increases the chances that leukemia could develop, according to Neal Copeland who led the recent study. "Cancer doesn’t result from one mutation," says Copeland. "But our data suggest that putting a cancer gene in a virus causes it to be overactive. By itself, it’s not enough to cause cancer. But if it happens to land near another cancer-causing gene then you get cancer." Copeland has developed a database that contains a long list of potential cancer-causing genes in mice. He has found two tumors in mice with insertions in IL2RG, the gene replaced in the gene therapy trial. He also found two tumors with insertions in the same gene that became activated in the leukemia patients. And he found one tumor with insertions in both genes. In the trial, the replacement gene was overactive and not regulated the same way it is in a normal cell. As a result, says Copeland, cells expressing the replacement gene could grow better than those without the gene. In a few rare cells, the replacement gene ended up near another cancer-causing gene, and cells could grow even faster. It’s likely that these fast-growing cells rapidly replaced healthy cells in the patients’ blood, causing leukemia. "Before conducting any further gene therapy trials it might be wise to first check our database and see if the gene of interest is a potential cancer-causing gene," says Copeland. "If the gene is listed in our database, it might not be a wise choice for gene therapy."

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39. Unsportsmanlike behavior

Washington Times (Internet) , 23.1.2004
http://washingtontimes.com/op-ed/20040122-082812-2585r.htm

When it was released in October, Beyond Therapy, a report from the President's Council on Bioethics, prophesied that professional athletes would have a difficult time not using genetic agents for enhancing muscle mass once they had been discovered and developed. The report perspicaciously called attention to insulin-like growth factor 1 (IGF-1), a protein that promotes the growth and repair of muscles. It was developed as a therapy for those with muscle-wasting diseases, such as individuals weakened by either muscular dystrophy or the process of aging. IGF-1 has shown a dramatic ability to build muscle mass in lab mice. Yet, even though IGF-1 has not been tested for safety, healthy individuals &emdash; namely athletes &emdash; have already expressed interest in using the compound. Professional athletes may already be using it, according to "In Pursuit of Doped Excellence," the cover story of last Sunday's New York Times Magazine by Michael Sokolove. The scientists he talked to suggested that dosing athletes with IGF-1 would be easy to do and almost undetectable. Amateur athletes also can dose themselves with IGF-1, since it can be easily procured on the Internet. Hucksters proclaim that IGF-1 will produce all sorts of remarkable effects, ranging from building muscle to repairing nerves. The abuse of such substances raises substantial safety issues, but even more significant ethical ones. After all, should the FDA deem IGF-1 to be dangerous, it could ban it, just as it did Ephedra. While sanctions would not stop all individuals willing to pay any price for high athletic performance, the worst abuses might be limited. Yet, as Beyond Therapy noted, doping not only corrupts the excellence of the achievement, but it also distorts powers not innate to the athlete. This, the council said, "deforms ... the character of human desire and aspiration." Athletes are already close to that point. Mr. Sokolove pointed out that "elite athletes in many different sports routinely consume cocktails of vitamins, extracts and supplements ... The cheaters and noncheaters alike are science projects." Professor John Hoberman told Mr. Sokolove, "The current doping agony is a kind of very confused referendum on the future of human enhancement." Considering its potential ramifications, the issue needs far greater clarity and far more open debate. After all, the use &emdash; and abuse &emdash; of IGF-1 is a likely prelude to the dangers and dilemmas of other artificial genetic muscle enhancers. Their availability seems certain, their desirability seems assured and their dehumanization seems likely. The president's council argued that it would be easy to turn "our would-be heroes into slaves, persons who exist only to entertain us." There is no sure way to avoid such dangers, although the old proverb, "Forewarned is forearmed" seems especially relevant. To better arm the public against such perils, the president's council recently made an anthology of literature on the dilemmas of bioethics freely available to the public (www.bioethics.gov). The site should be visited by those interested in forearming themselves.

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40. Drugs in sports a fact of life

Sun Times (Internet), 21.1.2004
http://www.suntimes.com/output/telander/cst-spt-rick21.html

The dirty doping results keep coming in, as they have for years, not in gushing numbers, but in spurts, trickles and drips. The pattern is probably most like that of a drainpipe carrying runoff from a field. The volume ebbs and flows from week to week and month to month, occasionally drying up entirely, but it is fairly constant over time. Recently, we have had female Olympic shot-put champion Yanina Korolchik of Belarus (the anabolic steroid clenbuterol), U.S. hammer-throw champion Melissa Price (designer steroid THG) and British sprint champion Dwain Chambers (THG) get nailed. Kelli Smith, the only U.S. woman to win the 100- and 200-meter sprints at the same outdoor world championships, failed her postrace drug test last fall in Paris (the stimulant modafinil), and other track stars such as shot putter Kevin Toth and middle-distance runner Regina Jacobs (both THG) recently have been busted. Of course, there are NFL players such as Bill Romanowski, Dana Stubblefield and Barret Robbins (all THG) who have been found dirty. But there are also less-expected dopers such as cyclist Adham Sbeih, the first U.S. athlete to test positive for the red-blood-cell-boosting hormone EPO; Termell Sledge, a minor-league baseball player in the Montreal Expos' system (the steroid nandrolone); and former U.S. Open tennis runner-up Greg Rusedski (nandrolone). One of the more interesting positive tests was produced by a 30-year-old rookie in the Italian pro soccer league, Saadi Gadhafi. Performance-enhancing steroids were the culprit, leading one to wonder whether the dope was sent via secret agent from the player's father, Libyan leader Moammar Gadhafi. The point here is not simply to announce the cheaters' names and say, "Tsk-tsk, bad boys and girls!'' The world more or less has been doing that for years, and the ethics of doping, cheating and performance-enhancing through dubious means has been debated and worried about ad nauseam. And more and stricter doping controls randomly have sprung into effect, only to lose impact as the general public grows tired of the whole cat-and-mouse game between cheaters and those who would like to believe they somehow can create the proverbial even playing field. Indeed, we have become so bored with doping tales and so unenthusiastic in our policies of prevention and education that it seems we might have reached a fork in the road. Last week, International Olympic Committee president Jacques Rogge and UNESCO director-general Koichiro Matsuura signed an agreement in Paris aimed at a more unified global fight against doping. And that was a nice thing. But short of bankrupting the economy of the sports worlds, there is only so much agencies can put into tracking down athletes and testing them in a foolproof fashion, while at the same time stopping the renegade chemists and/or trainers and coaches from developing and dispensing the latest dope to elude the latest tests. The fight should go on, but it more and more is one of ethics. Where and how to draw the cheating line is the real issue. And if athletes are not dissuaded by their consciences, the anti-doping folks are fighting a miserable battle of morality legislation. It is so hard now to prove that an athlete is cheating with drugs that, as any expert will tell you, it is only the brazen, the naive and the foolish who get caught. What are we to think of baseball sluggers who have toyed with the old power numbers? Sammy Sosa, like Barry Bonds and Mark McGwire, has changed physically as much as anyone in the sport. But he never has tested positive for performance-enhancing drugs. Then, too, until a few months ago, Major League Baseball didn't even have a drug-testing policy. Just the other day, Sosa told Cubs manager Dusty Baker how much he was looking forward to the coming season. "I'm so pumped, so hyped,'' Sosa said. "This is the first time I've started lifting weights in December.'' Maybe Sosa didn't know what he was implying with that statement, but I think many people assumed Sosa had put on his 40 or so pounds of muscle from lifting maniacally year-round. Moreover, designer drugs are always a step ahead of the testing regulations. And with the genetic engineering and laboratory experimentation going on, performance-enhancers made from animal testosterone and stimulants someday will seem as primitive as strychnine and leeches. In an article titled "In Pursuit of Doped Excellence'' in the New York Times Magazine last Sunday, writer Michael Sokolove checked out sports doping and also checked up on gene-therapy experiments at the University of Pennsylvania used on mice to make them big, strong and perpetually youthful. The key juice is IGF-1, or insulin growth factor-1, a protein that enhances muscle growth and health. "They were built like cattle,'' Sokolove writes of the doped lab mice, "with thick necks and big haunches. They belonged in some kind of mouse rodeo.'' Of course, the Penn scientists have good intentions for their research. The point is not to develop saddle-wearing mice but to aid elderly humans and those with muscle-wasting diseases. But the connection from the lab to sports and performance enhancement would be a short one. We have seen how quickly jocks learn their science. Not coincidentally, the cover story in Newsweek this week -- "Girl or Boy? Now You Can Choose. But Should You?'' -- reports how the once-unthinkable notion of picking your child's sex is as easy as going to Dr. Jeffrey Steinberg's Fertility Institute in Los Angeles and plunking down $18,480. Oh, and getting pregnant. But science is winning this battle. "There will come a day when they just have to give up,'' Penn researcher H. Lee Sweeney, the chairman of the school's department of physiology, tells Sokolove. Sweeney is speaking of the Olympic and world anti-doping federations, of which he approves but feels sorry for. "It may be 20 years away, but it's coming.'' If it isn't here.

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41. Gene therapy promising for muscular dystrophy Quebec scientists pioneer research Cells transplanted into young patients

Toronto Star (Internet), 19.2.2004
http://www.thestar.com/NASApp/cs/ContentServer?pagename=thestar/Layout/Article_Type1&c=Article&cid=1077145816497&call_pageid=968332188492&col=968793972154

In a world first, Quebec researchers have devised a gene therapy to treat a severe form of muscular dystrophy, using a method of cell transplantation that could also benefit heart attack survivors and hemophiliacs. "This research really is a very strong indicator of a direction that we will pursue aggressively and can lead to treatment," said George Henderson, spokesperson for Muscular Dystrophy Canada. "We don't want to overstate that or be too aggressive on that. It's a very important first step along a long path. It will be some time before we see it translated into bedside care or treatment for Duchenne muscular dystrophy," he added. Duchenne is an early onset form of the disease, typically striking between ages 2 and 6. Researchers found that cells transplanted into Duchenne patients could actually trigger the growth of a protein crucial to muscle development that's missing in people with muscular dystrophy. "I think it's a very good step forward," lead investigator Dr. Jacques Tremblay of the Centre Hospitality du Universidad Quebec, a research centre affiliated with Laval University, said in an interview. People with muscular dystrophy aren't able to produce a protein called dystrophin, which protects muscle fibres from breaking as they contract. Myoblast cells can repair broken muscle fibres, but because the fibres are so easily broken in muscular dystrophy patients, the cells eventually wear out and the fibres stay broken. That leads to the general muscle breakdown and weakness that puts muscular dystrophy patients in wheelchairs and leads to early death. Researchers in Tremblay's lab harvested myoblast cells from the fathers of three Duchenne patients. The cells were then grafted them into the boys' shin muscles, in a technique that was tested 10 years ago, but mostly abandoned when the results proved inconclusive. The theory is that the transplanted cells would compensate for the missing gene and begin producing the absent protein. This time around, Tremblay's team proved that a trio of patients, aged 8, 10 and 16, were producing dystrophin thanks to their fathers' donated cells. Each was first treated with immunosuppressants to keep their immune systems from attacking the cells, then received 25 injections of the cells. The results are published in the latest issue of Molecular Therapy, the journal of the American Society for Gene Therapy. The trial is small, but enormously promising, since the same kind of therapy could be applied to hemophiliacs or people with incontinence, experts say. Even patients whose heart walls are thinning, and whose only hope for survival is a heart transplant, could benefit.

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42. Gene Therapy Shows Promise for Cystic Fibrosis

Reuters (Internet), 19.2.2004
http://www.reuters.co.uk/newsArticle.jhtml?type=healthNews&storyID=4397956§ion=news

NEW YORK (Reuters Health) - Using an inhaler to deliver a specific gene to the lungs of cystic fibrosis (CF) patients is well tolerated and shows signs of effectiveness, new research indicates. Cystic fibrosis is a hereditary disease involving the lungs that is caused by mutations in a gene called CFTR, short for cystic fibrosis transmembrane regulator. As reported in the medical journal Chest, Dr. Richard B. Moss at Stanford University in California and colleagues, used inhalers to deliver either a normal copy of the CFTR gene or inactive "placebo" to the lungs of 37 patients with mild CF. With this delivery method, modified viruses were used to transport the gene to the cells that line the patients' airways. Repeat treatments were given on a monthly basis. Adverse events were similar in the patients given the gene and those who received placebo, the authors report, and no subject withdrew from the study because of side effects. No events were deemed definitely related to the active treatment. Patients treated with CFTR showed improvements in all measures of lung function at day 30. With longer follow-up, however, the differences between these patients and those treated with placebo disappeared. This gene delivery method "is safe in patients with mild-to-moderate CF lung disease," Moss told Reuters Health. Normal CFTR can be delivered this way to airway cells and it persists for at least one month. Early improvements in airflow and reduction of signs of inflammation in the sputum are evidence of effectiveness, Moss added. "However, these findings must be replicated in more patients. Moreover, crucial questions of long-term effect...need to be answered in further studies." Moss said that an ongoing trial of the current technique at 12 centers across the United States is estimated to yield results in mid-2005. "Eventually, the role of stem cells in this as well as many other diseases offers exciting potential for a true, single-dose cure," he added.

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43. Give gene therapy time to deliver on promise

Straight Times (Internet), 20.2.2004
http://straitstimes.asia1.com.sg/techscience/story/0,4386,236281,00.html

WHEN four-year-old Ashanthi DeSilva was injected with a new and improved dose of her own white blood cells in 1990, she was finally able to lead a normal life. Without this transfusion of cells, genetically altered by scientists to contain the gene the American was born without, her faulty immune system would have confined her to a sterile life in a bubble. That landmark case heralded a brave new world of medicine, one where faulty disease-causing genes could be swopped with healthy ones, changing the body's genetic make-up and potentially erasing the need for drugs. Switching bad genes for good seemed so simple that the media and public were hooked. As most diseases have a genetic component, gene therapy became a beacon of hope for a wide range of diseases where conventional therapy was useless or, at most, far from perfect - from inherited ones like cystic fibrosis to devastating illnesses such as cancer and Aids. But 13 years on, the medical miracles are still pending, and there seems little reason to celebrate. There have been few successes despite hundreds of clinical trials, and the plug has been pulled on many of them because of lacklustre results. There was also the death of 18-year-old American Jesse Gelsinger, who suffered massive organ failure after he was injected with a gene-carrying virus meant to cure a liver disorder in 1999. And in 2002, two young boys suffering from 'bubble boy' disease contracted leukaemia from a gene injection, the very treatment meant to save their lives. 'There have been a lot of difficulties, and a decade is not long enough for us to see the field take off,' said Professor Malcolm Brenner of the departments of molecular and human genetics, and medicine and paediatrics at Baylor College of Medicine in the United States. 'There's still a lot of stupidity. People, including some scientists, have no concept of just how long these developmental procedures will take,' he said, adding it could be decades before gene therapy bears fruit. Because of the vast number of different genes needing to be transported, and the wide variety of organs and tissues whose cells must be targeted for the treatment, a broad range of gene delivery technologies is necessary, said Dr Hui Kam Man, director of cellular and molecular research at the National Cancer Centre here. 'This is a very delicate system. We have to balance many, many things,' he said. The failures have largely been due to techniques for delivering enough healthy genes to the right place - scientists are still learning to sneak genetic material past the immune system's defences. While electrical pulses and fat molecules are also used, viruses are the most efficient form of carrying DNA to the cells, given their evolutionary need to infect cells. But viruses can be a double-edged sword, and studies have shown that they could turn on cancer-causing genes. 'Gene therapy is like a computer program where we're still trying to iron out the glitches,' said Prof Brenner, who is also director of the Centre for Cell and Gene Therapy in Texas. It is complex because there are many areas where something can go wrong - the gene, the material used to deliver the DNA into the cell, or the cell itself, when it reacts to the new genetic material. But, said Dr Hui, these issues are slowly being ironed out as researchers understand better the molecular workings of disease, and fine-tune the gene therapy process. To, say, kill a liver cancer cell, researchers would make sure the DNA used to kill the cell would home in specifically on abnormal liver cells. As added insurance, even the virus transporting this package would be engineered to latch onto cancer cells only. Look at the results so far in perspective, say scientists. The two boys, for example, have responded well to leukaemia treatment. Without a bone marrow transplant, their immune disease could have killed them within a year. And Jesse Gelsinger's death, though tragic, is a far cry from the thousands killed every day while undergoing surgery and conventional treatments. Strict regulations and high costs - it takes at least US$1 million (S$1.7 million) to conduct a trial on 10 people, said one researcher - have also hampered efforts in the field. Hounded by bad press, many scientists have chosen to do their gene therapy work away from the spotlight. But research continues, as shown by a major cancer gene therapy conference here this weekend, with 30 speakers who are leaders in the field and 150 international delegates. Said Dr Stephen Russell of the Mayo Clinic's Molecular Medicine Programme: 'The field has continued to grow and, for me, it's not a question of whether gene therapy is going to be successful but how long it's going to take.' So, can gene therapy deliver on its promise? Yes, given more time and caution. It is important not to write it off entirely for that would be throwing the baby out with the bath water

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44. Study May Improve Gene Therapy Safety GENE THERAPY, VECTOR, MODIFIED VIRUS, GENE EXPRESSION

UNC Chapel Hill (Internet), 23.2.2004
http://www.newswise.com/articles/view/503391/

New research may hold keys to improving the safety of human gene therapy. The study showed that the messenger can be as important as the message: Viruses genetically engineered for use as delivery vehicles for transferring therapeutic genes into the body may alone influence gene expression. Newswise &emdash; New research from the University of North Carolina at Chapel Hill may hold keys to improving the safety of human gene therapy. The study showed that the messenger can be as important as the message: Viruses genetically engineered for use as delivery vehicles for transferring therapeutic genes into the body may alone influence gene expression, or which genes are turned on. Moreover, depending on the type of virus, they may do so in potentially harmful ways. "This basically tells us that the messenger plays an important role in gene expression," said study co-author Dr. Richard J. Samulski, professor of pharmacology and director of UNC's Gene Therapy Center. "At the molecular level, a cascade of cell signaling events occurs irrespective of the therapeutic gene. This is something we didn't anticipate." The report will appear in the March issue of Molecular Therapy, the American Society of Gene Therapy's journal. The study focused on two viruses: adenovirus and adeno-associated virus, or AAV. These viruses, when genetically engineered, have shown particular promise in laboratory studies as gene transfer vectors and have been used in more than 170 clinical trials. Samulski and UNC co-author Dr. Jackie L. Stilwell, a postdoctoral researcher, reported there has been significant progress in understanding how viral gene therapy vectors behave in laboratory animals, in terms of acute toxicity effects. However, they wrote, "systematic comparison of their effects upon cells at the molecular level has not been established." In that regard, the new research offers important new and potentially useful information for predicting the safety of gene delivery in people. "The field has advanced so rapidly that we can now do toxicity profiles inside an individual cell," Samulski said. "And basically what we're doing here is asking what happens to the genome if a vector or virus comes into the cell." In their experiments on cultured lung cells, Samulski and Stilwell used DNA microarray technology to examine gene expression. This technology can monitor the whole genome on a single silicon chip, giving researchers a better picture of the interactions among thousands of genes simultaneously by displaying patterns of gene expression. Exposure to AAV either as an intact virus or as a recombinant vector shell affected gene expression minimally and in patterns not associated with potential harm to the host organism, the report said. In addition, exposure to the empty capsid of AAV - the protein coat of the virus devoid of DNA - also produced minimal response from the genes. For example, only 1.9 percent of genes showed changes in expression in cells infected by recombinant AAV. In contrast, gene expression after exposure to intact adenovirus and recombinant adenovirus vector was much broader and included the activation of immune and stress response genes. Lung cells exposed to empty adenovirus capsid showed a decrease in changes in cellular gene expression, although some were related to stress response genes. This study provides a systematic explanation for the relative safety profiles of two commonly used gene therapy vector classes, Samulski said. "The take-home message here is we can now monitor the genes that get turned on when you put a vector on the cell," he said. "We can then make changes to the vectors and observe how their safety profiles improve prior to their use clinically." "As we make architectural changes to the delivery system, we can see the cell's response to it," Stilwell added. "The study represents the start of a useful database of gene expression signatures for people involved in vector development." The next step for the UNC researchers is to continue exploring the vector-associated gene expression signatures in other cell types and to extend the work to whole animals. "I think everybody involved in designing vectors will continue to build on this database," Samulski said. "We're laying the foundation here for that to happen." Samulski, a pioneer in AAV research, said the new study further confirms his choice to study and develop altered AAV for gene therapy. Along with its potential for fewer toxic effects than that of many other viruses studied for use in gene therapy, a gene delivered via AAV remains active in cells for months or even years, he added. Last year, a form of gene therapy created and developed in Samulski's laboratory and based on AAV was approved by the U.S. Food and Drug Administration and given to children with Canavan disease, a rare, inherited neurological disorder. It marked the first FDA approval for clinical use of an AAV vector for gene therapy that was produced by a U.S. academic institution. The new research was supported by grants from the National Institutes of Health and the Cystic Fibrosis Foundation.

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45. World's first gene therapeutic medicine approved for market in China

people daily (Internet), 4.3.2004
http://english.peopledaily.com.cn/200403/02/eng20040302_136315.shtml

Chinese State Food and Drug Administration (SFDA) approved on Jan. 20 the recombined human p53 adenovirus injection for production, marking it China's and also the world's first gene therapeutic medicine approved for entering into the market. Chinese State Food and Drug Administration (SFDA) approved on Jan. 20 the recombined human p53 adenovirus injection for production, marking it China¡¯s and also the world¡¯s first gene therapeutic medicine approved for entering into the market. As learned at an exhibition on achievements by returned overseas students started on Feb.29 and according to requirements of departments concerned, scientists will next put the medicine into use in grade-3 A-level hospitals all over the country and probe further for its functions. ¡°Father of gene therapy¡± US scientist Dr. W. French Anderson sent his congratulations to domestic scholars for the achievement. Expert said this milestone of biological hi-tech development would help push the development of the research on gene therapy and the entire industry forward, thereby making contribution to the health of human beings. Clinical experiments on gene therapy started in 1990. It is to inject gene into a specific carrier and introduce in human cells to cure diseases. Up to now, there are nearly hundred companies dedicated to gene therapy and clinical programs of gene therapy have come to more than 700. However, there has been no safe and effective gene therapy medicine approved so far in the world as new medicine. The United States approved clinical experiments of products on recombination p53 adenovirus. Through tumor local injection, intravenous injection, interposing medicine supply and thoracic and stomach infusion etc. US, Japan ese, Canadian and European scientists started gene therapy on dozens of kinds of tumors such as head and neck squamous cell carcinoma, breast cancer, esophageal cancer through recombined adenovirus p53 products. China is one of the pioneers in the research on gene therapy. With the support of the National 863 Program, national significant scientific and technological project and local governments and approved by State Food and Drug Administration (SFDA), Professor Peng Zhaohui etc. with Shenzhen SiBiono GeneTech Co., Ltd, China's first specialized company of its kind started in 1998 clinical experiment on recombination of human p53 adenovirus injection for therapy and established in Shenzhen production lines and factory as certified by state GMP (Good Manufacturing Practice or Good Practice in the Manufacturing and Quality Control of Drugs). To date, over 300 people at home and abroad have received treatment with recombined human p53 adenovirus injection. The gene therapy medicine has shown generality in tumor-resistance, marked coordinated effect with traditional radiotherapy and great effect in curing head and neck squamous cell carcinoma such as nasopharyngeal cancer. Peng Zhaohui remains sober though the medicine has gone into the market, saying that gene therapy only creates an effective method. There is a long way to go for human beings to conquer the carcinoma. By People's Daily Online

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46. Good Gene Therapy Viruses Sorted from Bad Studying their impact on gene activity could improve genetic treatments

Molecular Therapy journal (Internet), 24.2.2004
http://www.betterhumans.com/News/news.aspx?articleID=2004-02-24-3

Some viruses used to deliver therapeutic genes may themselves influence gene activity in harmful ways, researchers have found, and identifying them could improve gene therapy. The study, by researchers from the University of North Carolina at Chapel Hill, shows that when it comes to gene therapy, the messenger can be as important as the message. "This basically tells us that the messenger plays an important role in gene expression," says researcher Richard Samulski . "At the molecular level, a cascade of cell signaling events occurs irrespective of the therapeutic gene. This is something we didn't anticipate." Imperfect science Gene therapy is a technique that involves inserting genes into a person's DNA, usually to replace existing abnormal or defective genes. A carrier molecule called a vector is used to deliver therapeutic genes to target cells. The most common types of vectors in gene therapy are genetically disabled viruses into which DNA is packaged. There are major shortcomings with virus-based vectors, however, including recognition by the immune system. When familiar viruses are detected, the body sends antibodies to kill them. Researchers have also found that viruses can inject their DNA package into inappropriate spots, with unanticipated consequences. Altered expression For their study, Samulski and colleagues focused on two viruses that have shown particular promise in laboratory studies as gene transfer vectors: Adenoviruses and adeno-associated viruses. The researchers used DNA microarrays to examine the impact of the viruses on gene expression in cultured lung cells. They found that complete adeno-associated viruses had no impact on gene expression. They also found that the empty shell of adeno-associated viruses&emdash;the protein coat but no DNA&emdash;produced a minimal genetic response. In contrast, intact adenovirus and recombinant adenovirus vectors activated immune and stress response genes. "The take-home message here is we can now monitor the genes that get turned on when you put a vector on the cell," says Samulski. "We can then make changes to the vectors and observe how their safety profiles improve prior to their use clinically." Laying the foundation The next step for Samulski and colleagues is to continue exploring the vector-associated gene expression signatures in other cell types and to extend the work to whole animals. "I think everybody involved in designing vectors will continue to build on this database," Samulski says. "We're laying the foundation here for that to happen." The research is reported in the journal Molecular Therapy.

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47. Swedish committee stirs debate Recommendations supporting cloning are not universally welcomed

biomed central (Internet), 25.3.2004
http://www.biomedcentral.com/news/20040325/04/

The Scientist com: Research directed at developing methods that could theoretically be used for germline gene therapy should be permitted in Sweden, a government-appointed committee has recommended. The Committee of Genetic Integrity, established in 2001, believes such inheritable gene therapy should still be forbidden in practice. "But the prohibition we have for research today is hard to interpret," its chairman, Johan Munck, told The Scientist. "If we do not allow the possibility of doing research or even thinking [about a subject such as germline therapy], we will never get anywhere." The recommendations have received a mixed response from the scientific community. Elisabeth Rynning , professor of medical law at Uppsala University, says the suggestion to change the law in this way was made explicitly to support therapeutic cloning. She is concerned that the committee's recommendation to leave decisions about regulating this area to regional research ethics committees may not provide sufficient means of controlling research. "If it is not possible to legally define the area of justifiable research, then the research should at least be controlled centrally," Rynning said. If the regional committees do not refer to the central ethics committee, difficult and principally important questions may be decided upon regionally, she said. The committee has already stirred up trouble with earlier recommendations that proposed legalizing nuclear transfer. The government is expected to make a decision on these earlier recommendations soon, but there are already hints it will allow the technique. For example, health minister Lars Engqvist and science minister Thomas Östros voiced their support for the proposal in an article in the daily paper Dagens Nyheter. Allowing therapeutic cloning would violate a convention set down by the Council of Europe on human rights and biomedicine, which forbids the creation of embryos for the purpose of research Sweden has already signed this convention, but gaining an exemption would be legally possible, Rynning said. "But to do this, we must present some good arguments for why this is needed. I do not personally believe that the committee has provided sufficient discussion on this issue." "The committee has not considered the philosophical tradition that lies behind the convention… focusing on the inherited dignity of human life," comments Per Landgren, a member of the Christian Democratic party in the Swedish Parliament and a member of the committee. Landgren did not approve of the committee's suggestion to allow research into methods that could be used to cause inheritable genetic effects. "I could approve of the principle in a context wholly conducive to life, but I don't want to sacrifice hundreds of thousands of human lives in the early phases before the researchers know how to do this." Jan Wahlström, professor of clinical genetics at Gothenburg University, is one who supports the recommendation. There is a long way to go before there will be any medical use for this kind of research, he admits. "But it is important for research that the prohibition is taken away. By doing so, it will increase the possibility to find out the advantages and risks with the method." Links for this article Genetics, Integrity and Ethics, Final Report from the Committee of Genetic Integrity, SOU 2004:20. http://www.social.regeringen.se/propositionermm/sou/ Elisabeth Rynning http://www-hotel.uu.se/juri/personal/english/rynning.html Summary of the Genetic Integrity Report concerning embryonic stem cell research http://social.regeringen.se/propositionermm/sou/pdf/sou2002/sou2002_119_en.pdf Lars Engqvist http://www.social.regeringen.se/inenglish/ministry/ministers/engqvist/index.htm Thomas Östros http://utbildning.regeringen.se/ostros/pdf/tocv_eng.pdf L. Engqvis, T. Östros, "Terapeutisk kloning tillåts," Dagens Nyheter, March 6, 2004. http://www.dn.se/DNet/jsp/polopoly.jsp?d=572&a=240946 Convention for the Protection of Human Rights and Dignity of the Human Being with regard to the Application of Biology and Medicine: Convention on Human Rights and Biomedicine, Council of Europe Explanatory Report, CETS no. 164. http://conventions.coe.int/treaty/en/treaties/html/164.htm Christian Democratic Party in Sweden http://int.kristdemokrat.se/

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48. Tenth Annual Report of the Gene Therapy Advisory Committee published

medical news today (Internet), 26.3.2004
http://www.medicalnewstoday.com/index.php?newsid=6827

Medical news today UK - The Tenth Annual Report of the Gene Therapy Advisory Committee (GTAC) for 2003, is published today. It includes details of all proposals for clinical gene therapy studies reviewed by GTAC last year, as well as looking at recent scientific developments and the regulation of gene therapy over the year. The Committee advises on the ethics of gene therapy proposals, taking into account the scientific merits and the potential risks and benefits of gene therapy to patients. GTAC also advises Ministers on developments in gene therapy research. Thirteen new studies to conduct gene therapy trials were approved in 2003 - this is twice the number approved in 2002. The majority of these focus on treating cancer. Lord Warner said: "I am delighted to welcome the publication of the GTAC Tenth Annual Report and congratulate the committee on its work. The government is committed to supporting the latest advances in gene therapy research, which have the potential to improve the lives of thousands of patients. That is why it is investing £10 million into to gene therapy research. The UK leads Europe in this field and the committee plays an essential role in setting and maintaining the high standards applied to clinical gene therapy". Related links Gene Therapy Advisory Committee http://www.advisorybodies.doh.gov.uk/genetics/gtac/ Notes to editor 1. GTAC’s membership is drawn from a wide range of medical, scientific, legal and lay expertise. GTAC Members o Professor Norman Nevin (Chairman), Emeritus Professor of Medical Genetics, Queen’s University, Belfast. o Dr Richard Ashcroft, Medical Ethicist, Imperial College London o Mrs Deborah Beirne, Senior Research Nurse, St.James Hospital, Leeds o Dr Caroline Benjamin, Macmillan Genetic Counsellor, Liverpool Women's Hospital NHS Trust o Professor Martin Gore, Consultant Clinical Oncologist, The Royal Marsden Hospital, London o Professor Terence Hamblin, Consultant Haematologist, University of Southampton and Royal Bournemouth Hospital o Dr Peter Harris, Technical Director, KuDOS Pharmaceuticals Ltd. o Professor David Harrison, Professor of Pathology and Medical Researcher, Department of Pathology, Edinburgh University o Mr Michael Harrison, Barrister, London o Professor Nicholas Lemoine, Professor of Molecular Pathology, Cancer Research UK Molecular Oncology Unit, Hammersmith Hospital, London o Dr Adrian Lepper, Chartered engineer, Hertfordshire o Professor Andrew Lever, Professor of Infectious Diseases, University of Cambridge o Professor Alex Markham, Professor of Molecular Medicine, University of Leeds o Professor James Neil, Professor of Virology and Molecular Oncology, University of Glasgow o Reverend Dr Lee Rayfield, Vicar and former Immunologist, Berkshire o Mrs Fiona Sandford, Patient Advocate, Hertfordshire o Dr Michael Waterhouse, Television Producer and Author, Southborough 2. Since approving the first gene therapy trial in the UK in 1993, GTAC has approved 90 gene therapy clinical trials, involving over 700 patients. These gene therapy studies target inherited disorders such as Cystic Fibrosis and Hurler’s Syndrome, infectious diseases such as HIV infection, and vascular disease. The majority (72%) of trials are for the treatment of cancer, including breast, ovarian, cervical, pancreatic, prostate, bladder, head & neck, colorectal and liver cancer as well as skin cancer. Media enquiries ONLY to: Contact Press Office Phone Vicky Wyatt 020 7210 5656

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49. NIH and FDA Launch New Human Gene Transfer Research Data System

NIH, FDA (Internet), 26.3.2004
http://www.nih.gov/news/pr/mar2004/od-26.htm

GeMCRIS will facilitate faster reporting of adverse events in human gene transfer trials The National Institutes of Health (NIH) and the Food and Drug Administration (FDA) announced today that they have launched a new Genetic Modification Clinical Research Information System (GeMCRIS) &emdash; a Web-accessible database on human gene transfer. GeMCRIS, developed collaboratively by the two agencies, is a unique public information resource as well as an important new electronic tool to facilitate the reporting and analysis of adverse events on these trials. The new system will provide information to the public directly and will improve the government's ability to monitor adverse events in gene transfer research, also known as gene therapy. NIH Director Elias A. Zerhouni, M.D., said, "GeMCRIS is an important achievement and a unique resource for scientists, patients, and the public. GeMCRIS will help advance gene therapy, while allowing NIH, FDA, and the research community to maintain appropriate oversight." Acting FDA Commissioner Lester M. Crawford, D.V.M, Ph.D., emphasized that "the development of GeMCRIS illustrates the government's commitment to addressing public and patient concerns about safety while advancing gene therapy. Providing accurate and complete information about ongoing gene therapy studies is the best way to achieve this goal." GeMCRIS will enable patients, research participants, scientists, sponsors, and the public at large to become better informed about human gene transfer research. Through drop-down menus and preformatted reports, individuals can easily navigate the GeMCRIS site to view information on particular characteristics of clinical gene transfer trials. For example, GeMCRIS users can learn where trials are taking place, which diseases or health conditions are being studied, and what investigational approaches are being taken. While offering a rich array of information of value to many types of users, GeMCRIS also includes special security features to protect patient privacy and confidential commercial information. Investigators and sponsors conducting human gene transfer trials will now be able to report adverse events using a secure electronic interface on the GeMCRIS system. With this tool, reports can be submitted instantaneously to the NIH. Investigators and sponsors can save their NIH submission on their own computer and send a copy to the FDA in accordance with 21 CFR 312.32 together with a FDA Form 1571. This can be done either by mailing or faxing a signed hard copy or by making an electronic submission in accordance with 21 CFR 11. FDA Form 1571 can be found at http://forms.psc.gov/forms/FDA/fda.html. Those submitting electronic documents should also refer to the Draft Guidance for Industry: Providing Regulatory Submissions in Electronic Format-General Consideration at http://www.fda.gov/cber/gdlns/elecgenrev1.htm and related guidance at http://www.fda.gov/cber/esub/esubguid.htm. Additional copies can be routed to Institutional Review Boards, Institutional Biosafety Committees, Data Safety and Monitoring Boards, and other local officials and review bodies as appropriate. The electronic reporting tool is key to efforts by both agencies to improve safety oversight and reporting in human gene transfer trials through the harmonization of NIH and FDA reporting requirements. The public GeMCRIS site is available at: http://www.gemcris.od.nih.gov/. Investigators and sponsors who wish to use the system to report adverse events occurring on human gene transfer trials should send a written request on institutional letterhead by U.S. mail or fax to: GeMCRIS Systems Administrator, NIH Office of Biotechnology Activities, 6705 Rockledge Drive, Suite 750, Bethesda, Maryland 20892; Fax: 301-496-9839.

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50. Focus: What was cancer, great grandad?

The Sunday Times (Internet), 28.3.2004
http://www.timesonline.co.uk/article/0,,8123-1055612,00.html

Fatal heart attacks could soon be a thing of the past. The battle against many cancers is also being won. So are we on the brink of a new medical revolution? At 7am on February 6, John McCutcheon, 55, woke up in his farmhouse with a burning sensation deep in his chest. His heart was seizing up, but like many heart attack victims, he had no idea of the danger he was in. He got up, made a cup of coffee and hoped the burning would go away. It got worse. By 8am, the sensation was spreading upwards and outwards, along his arms and up into his face. A blood clot had lodged in McCutcheon’s chest and his heart muscle was slowly being starved of blood and destroyed. Without help, either his heart would be irreparably damaged or he would have died. Thirty minutes after the burning sensation started, the father of two was being attended to by two paramedics and his local GP in his kitchen, a cardiac monitor strapped to his chest. "Look, don’t be too alarmed," his doctor told him. "But you’re having a heart attack." McCutcheon, who lives near Durham, said: "I got to hospital and it was ‘action stations’. One of the nurses had this nasty-looking syringe and said they wanted my permission to give me this new clot-busting drug." The shot dissolved the clot and restored the flow of blood to his heart. Within a week, he was back home, vowing to cut back on fatty foods and begin a new exercise regime. McCutcheon is a lucky man. Had he suffered his heart attack just two years earlier he might not have survived. The clot-busting drug which saved him has only recently become widely available. Costing nearly £600 a shot, it is part of a powerful new arsenal of designer drugs and treatments that doctors are now successfully using to protect against some of Britain’s biggest killers. Experts predict that the new drugs’ potency in treating degenerative diseases such as heart disease and cancer will eventually be as marked as the impact penicillin and its derivatives had against infectious disease in the 20th century. The new medical revolution was dramatically highlighted last week when the government produced an expert report, saying that virtually nobody under the age of 65 should die from a heart attack in Britain in 10 years’ time if improvements in treatment continue at their current rate. The report showed that the number of deaths from heart disease &emdash; Britain’s biggest killer &emdash; have already fallen dramatically. The number of men and women dying from coronary heart disease has almost halved since 1990. Death rates, which the government described as being "scandalously high" when it took office in 1997, have come down steadily since then and could fall to zero by 2013. "Seven years ago, cardiac services were in a terrible state," crowed John Reid, the health secretary. "Patients could wait for years for diagnosis and more than two years for surgery." Although successes in the fight against cancer are less dramatic, here too big advances in both science and the treatments available are being made. Total deaths from cancer in Britain among the under-75s, for example, have fallen by 10% since 1995. The future looks brighter still. The mapping of the human genome, stem cell research and the creation of advanced vaccines are finally producing results in the battle against degenerative disease. Scientists say that a whole array of new treatments is only a matter of years away and that when it arrives, life spans could be extended dramatically. Stephen Helfand, associate professor of genetics at the University of Connecticut in America, said: "Almost everyone of these notions that we hold about ageing aren’t true at all. It’s not necessarily the case you can live for ever, but there are no physical laws which say you can’t replace things in your body as they break." NOT long ago, when people contracted cancer, doctors shook their heads and told their patients to prepare for the worst. Not any more. Although some cancers, particularly lung and pancreatic, are still hard to overcome, others such as prostate, testicular and breast cancer, are increasingly being successfully tackled. The death rate for breast cancer has fallen by 21% in the past decade and 95% of men with testicular cancer are successfully treated. A complete cure for the disease may still be decades away, but scientists are more optimistic now than they have ever been. Dr Lesley Walker, Cancer Research UK’s director of information, said: "In the decades to come we will be able to cure many more cancers, and if we can’t cure them, we will be able to control them. We expect the number of people who survive to rise dramatically." Cancer vaccines are expected to play a significant role in the treatment of a disease that comes in more than 100 different forms. Unlike the vaccines of the 20th century, which prevented disease, these are designed to treat it. In a study published last month, researchers in Dallas, Texas, revealed that a small number of patients in the advanced stages of lung cancer were cured after being given an experimental vaccine. Similar medicines are being tested for a range of other cancers. Scientists trying to find a cure for prostate cancer &emdash; the commonest form among men in Britain &emdash; are looking at taking prostate cancer cells from the patient, growing them in the laboratory and modifying them so that when they are injected back into the patient, they trigger the immune system to kill both them and cancerous cells. In heart disease too there is more progress to be made. In the future it may even be possible to reverse the damage caused by a heart attack. Researchers are studying whether stem cells, the building blocks of the body, could be injected into a patient to repair damaged muscle. Doctors in Brazil last year revealed that they had injected heart patients with their own stem cells. Within weeks the heart wall was able to contract better, improving the flow of blood around the body. More ambitiously tissue engineers believe that one day they may be able to grow an entire heart from a few cells taken from a patient. The organ would then be transplanted into the patient. IT MAY seem the stuff of science fiction, but we have beaten such mass killers before. A century ago it was a revolution in public health that led to huge cuts in death rates from diseases such as typhoid and cholera. A second health revolution of the 20th century was spearheaded by the arrival of immunisation and antibiotics. Vaccination led to the worldwide eradication of smallpox, the elimination of polio in many countries, and control over diseases such as measles, rubella, tetanus, whooping cough, and diphtheria that once killed and disabled millions. Doctors believe that medicine is now tackling a third revolution. This time the enemy is the range of chronic diseases and ailments that have come with greater life span, from cardiovascular disorders to Alzheimer’s disease. Genetic therapies, stem cell technology and designer drugs are among the weapons to be used. The sequencing of the human genome &emdash; the mapping of our DNA &emdash; is expected to result in a huge increase in new drugs, perhaps 3,000 of them, by 2020. The future will also see increasing numbers of genetic disorders being eliminated by the use of IVF and pre- implantation genetic diagnosis to identify and reject foetuses that carry the diseased gene. Genetic therapy could also be used to replace defective genes that predispose people to particular diseases. Although there is no cure for Alzheimer’s and other forms of dementia, which afflict one in five people over the age of 80, researchers hope that stem cells injected into the brain may help rebuild damaged tissue. Doctors in the future may use bespoke drugs devised specifically to match an individual’s DNA. The treatment will be matched to the patient’s genetic profile, increasing the chance of a cure and reducing the risk of side effects. SO IS this all too good to be true? In the early 1990s many scientists declared that gene therapy cures for untreatable diseases were almost in our grasp, but more than a decade of trials have produced no significant breakthroughs. Stem cell research has been more encouraging. Although British biotechnology companies involved in this much-hyped area have foundered, there have been enough positive results to suggest stem cell research could revolutionise medical treatment. Whether or not these breakthroughs are made, there is no dispute that incremental medical advances are already saving more lives. However, some warn the government’s good news on cutting deaths from heart disease should carry a health warning. They point out that the initial steep reduction in heart deaths trumpeted by the government will start to plateau. Many people who suffer heart attacks die before receiving medical attention and some will die anyway, whatever drugs or treatment they get. There is also no evidence of the fundamental changes in our eating and exercise habits required to attack the root cause of heart disease and other diseases. Indeed, we just seem to keep getting fatter. Dr Tim Bowker, associate medical director at the British Heart Foundation, said: "The bulk of heart disease is caused by our western lifestyle, sitting in front of the television and driving everywhere. "People are surviving their heart attacks for longer and staying alive because of better treatment. But they still have heart disease and if you want to get rid of that then you have to change people’s lifestyles." Experts say lifestyle changes are also needed to combat cancer. A third of all cases of the disease are believed to be linked to diet. Although more people are surviving cancer too, its prevalence in the general population is increasing. The government insists it is not neglecting people’s lifestyles. It supports a range of programmes aimed at encouraging a healthier way of life, including anti-smoking campaigns, free fruit given to children at school and initiatives encouraging more people to walk and cycle. Earlier this month, it published the consultation document Choosing Health?, which asks the public for suggestions for tackling the country’s health problems, such as obesity. It is a battle that needs to be won. Until we change the way we live, warn the experts, we won’t ever truly beat the biggest killers &emdash; no matter what the advance of science brings. At best, we will just delay them. NEW WEAPONS IN THE WAR AGAINST DISEASE Genetic testing As emphasis switches from cure to prevention, genetic testing will become standard. DNA profiling of babies could identify what illnesses threaten them, allowing lifestyles and medication to be tailored. Gene therapy This is a technique for correcting defective genes, which allow disease to develop. An infectious virus is genetically modified to carry the desired gene into the target cells. The first major successes are now emerging from research programmes. Advanced radiotherapies Radiation-based cancer treatment kills nearby healthy cells, making it especially risky in the spinal cord and brain. Intensively Modulated Radio Therapy avoids this by sculpting radiation to the shape of the tumour. The first generation of machines is arriving in Britain and the therapy could replace some surgery. Cell therapies These involve replacing diseased or dysfunctional cells with healthy, functioning ones. Cancers, neurological diseases such as Parkinson’s and Lou Gehrig’s disease, spinal cord injuries and diabetes are already being treated in this way. Replacing dead cells with new ones in the retina may some day cure eye diseases such as glaucoma and macular degeneration. It may also be possible to replace diseased heart cells. It is similar to organ transplantation, but with cells. Stem cells, cells that have not become specialised for particular functions and which can keep replicating, hold the most promise. Therapeutic cloning Used with cell therapies, this would allow doctors to clone a patient and use stem cells taken from the resulting embryo to grow replacement tissues. Such tissues would have almost identical DNA to the patient, so the risk of an immune reaction would be overcome. Cancer vaccines The idea is to stimulate the body’s defences to attack cancer cells. In animal experiments, researchers have eliminated 40% of lung cancers. Aventis, the drug company, is conducting trials on patients with colorectal cancers and is working on vaccines for breast and skin cancer. Patients are infected with a virus that is taken up by the cancer cells. The virus provokes the cells to produce a protein that is recognised as dangerous by the immune system. White blood cells then destroy the cancer cells. Anti-ageing therapies Drugs that reverse osteoporosis are being tested. Scientists at Edinburgh University will this week announce that they have reversed loss of mental abilities in elderly patients. Several of the genes that cause Alzheimer’s have also been discovered.

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51. Signs of progress in gene therapies

Drugresearcher (Internet), 19.4.2004
http://www.drugresearcher.com/news/news-NG.asp?id=51479

Growing knowledge about how to manage the risks of gene therapy could improve the chances of developing successful treatments, said experts meeting last week to weigh up the progress made on the technology to date. Trials on gene therapy, which involves replacing, removing or introducing genes, for example, to produce a specific missing protein, were brought to a halt last year after French researchers reported a higher than expected number of cases of leukaemia among children being treated for the fatal ‘baby-in-a-bubble’ syndrome, severe combined immunodeficiency (X-SCID). After three years of successful gene therapy for X-SCID, leukaemia occurred in the two youngest patients undergoing treatment. This news followed the death of a US patient after an inhaled adenoviral vector-delivered gene therapy led to an inflammatory response in the lungs. But despite these setbacks, the first successful treatments have also been reported from clinical gene therapy studies. Speaking at the third conference of Euregenethy, a Europe-funded network of scientists aiming to develop standardised gene therapy regulations and encourage discussions on related ethical issues, Alessandro Auiti from the San Raffaele Telethon Institute for Gene Therapy in Milano, Italy, reported that in patients suffering from the congenital immunodeficiency disease ADA-SCID (adenosine deaminase (ADA)-deficient severe combined immunodeficiency disease (SCID)), a largely normal immune system has been restored by using genetically modified blood stem cells. In some cases restoration lasted over a period of several years. Using retroviral vectors, a functional gene is transferred into stem cells from the patient’s bone marrow as a replacement for the defective gene. The modified blood stem cells differentiate within the patient’s body into normal blood cells thus improving immune functions. "It is foreseeable that gene therapy will offer improved or novel therapies," noted Klaus Cichutek, vice president of the Paul-Ehrlich-Institut, near Frankfurt, which hosted the conference. "However, with increasing efficacy, we are also facing side effects. We have to learn how to balance these risks against the benefits for the patients and how to manage such risks." To minimise risks as much as possible, a benefit/risk analysis is required in all cases, based on the exact knowledge of the individual gene therapy approach, Cichutek said. He added that in the trial on SCID-X1, two out of ten patients had developed leukaemia as an adverse reaction but after appropriate treatment they are alive. And according to current knowledge, the overall risk of this gene therapy is still lower than that of the available conventional therapy. The leukaemia cases triggered by the SCID-X1 gene therapy were observed only in this special type of gene therapy. They were previously known only as a theoretical adverse reaction of retroviral vector use. The conference heard that Christof von Kalle at the University of Freiburg, Germany, is testing a special method for the early diagnosis of this adverse reaction. "Adverse reactions must be communicated rapidly at an international level and must be evaluated by the scientific community by experts familiar with gene therapy," urged Euregenethy coordinator Odile Cohen-Haguenauer. According to the group, the principal investigator of the SCID-X1 gene therapy study in Paris, Alain Fischer, has shown excellent conduct communicating internationally the clinical findings of his study and the subsequent investigations of the causes of the leukaemias observed. Other highlights of the conference included reports on the clinical development of the gene therapy of rheumatoid arthritis by Barrie Carter, Targeted Genetics Corporation, Seattle, and reports by other scientists on gene therapy of cardiovascular disease and cancer as well as first trials of preventive vectored vaccines. Another subject of discussion was the experience gained from the use of HIV-derived replication-incompetent gene transfer vectors. Inder Verma from The Salk Institute in La Jolla, California, reported on further development of lentiviral vectors. These vectors are replication-incompetent particles derived from the Human Immunodeficiency Virus or other related lentiviruses. A study by Boro Dropulic, Virexsys, is currently underway in the USA using lentiviral vectors transferring HIV-inhibitory genes for the treatment of HIV-infected patients. The first three patients were treated without showing any adverse reactions. Lentiviral vectors are expected to improve gene transfer efficiency, thus improving ways of treating other severe diseases, such as cancer. Researchers believe that so-called tumour suppressor genes could be used to suppress the replication of malignant cells. Inder Verma claimed that such vectors would bring about tremendous progress since they could also be used to modify resting cells which occur frequently in the body.

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52. MELANOMA,TERAPIA GENICA A MILANO: UN «VACCINO» DA PROVARE SU 22 MALATI (Corsera)

Luca Coscioni (Internet), 24.5.2003
http://www.lucacoscioni.it/cms/article.php?sid=217

San Raffaele-Istituto dei tumori: si attende il sì del ministero. Il 31 maggio dovrebbe arrivare il via libera anche per la cura di una rara dermopatia congenita 24 Maggio 2003 - Italia prima in terapia genica, Italia unico Paese al mondo in cui le nuove sperimentazioni sono rimaste congelate dopo i due casi francesi di leucemia indotta da terapia genica. Entro fine maggio dovrebbe arrivare il parere di una commissione dell’Istituto superiore di Sanità: sbloccare le sperimentazioni come hanno già fatto le autorità competenti negli Stati Uniti, in Germania, in Gran Bretagna e in Francia o continuare con la moratoria che prevede l’approvazione caso per caso e solo di protocolli già approvati. Sarebbe un paradosso in un Paese dove i gruppi di ricerca in questo campo sono punti di riferimento. L’ultimo annuncio di Telethon riguarda una possibile terapia per almeno 8 rare malattie ereditarie. Restando al presente, due nuove cure giacciono da mesi nel cassetto del ministero della Salute in attesa del parere dell’Istituto superiore della Sanità: una riguarda il melanoma, il cosiddetto cancro della pelle, il cui protocollo è stato presentato dal San Raffaele-Istituto dei tumori di Milano; l’altra una dermopatia congenita (sempre di pelle si tratta), che può essere anche mortale, la cui cura tramite cellule staminali modificate geneticamente è stata messa a punto da Michele De Luca, direttore scientifico della The Veneto Eye Bank Foundation di Venezia. Ma è il «vaccino» per il melanoma (35 casi ogni 100 mila abitanti in Italia) ad attirare l’attenzione internazionale. Perché l’ipotesi di lavoro potrebbe un domani essere applicata anche per altri tipi di tumori solidi. «E’ noto che le cellule esprimono particolari molecole dette antigeni, che possono essere riconosciute dal sistema immunitario - spiega Marco Bregni che con Marco Russo è responsabile della sperimentazione per il San Raffaele -. Nel caso del melanoma uno degli antigeni identificati è quello espresso dal gene Mage-3: è presente nel 70% delle cellule del melanoma e solo in esse». Una delle caratteristiche del tumore, però, è quella di «spegnere» il sistema immunitario. La novità è quella osservata dai ricercatori italiani: prendendo i linfociti del paziente e trasferendo nel loro Dna il gene Mage-3 tramite un retrovirus i linfociti vengono «riattivati» ad attaccare e distruggere le cellule del melanoma. I linfociti manipolati geneticamente, una volta «reiniettati» nel paziente, trasmettono la nuova «memoria» alle altre cellule difensive e parte l’attacco al tumore. «Questo è quanto accade in vitro - spiega Giorgio Parmiani, responsabile del progetto per l’Istituto nazionale per la cura dei tumori di Milano, che da anni studia questa strada contro il melanoma -. Ora dobbiamo vedere se è lo stesso nell’uomo. Il protocollo clinico, prevede l’arruolamento di un totale di 22 pazienti affetti da melanoma metastatico, nei quali le cure sono ormai palliative. Vedremo... Se funziona, non è escluso che questo diventi un vero e proprio "vaccino" per i soggetti a rischio melanoma». Il protocollo, sottoposto all’approvazione il 18 ottobre 2002, è ancora in stand by a causa della sospensiva ministeriale di tutte le terapie geniche fino al 31 maggio 2003. Ma perché vi è stato questo blocco, nonostante i successi italiani nella cura della rara Ada-Scid (una grave immunodeficienza genetica) e dell’uso dei «geni suicidi» nel guarire dalle ricadute delle leucemie? Claudio Bordignon, direttore scientifico del San Raffaele, e pioniere a livello internazionale della terapia genica spiega: «Il problema è nell’uso dei retrovirus come "taxi" del gene da inserire nel Dna della cellula da curare o modificare. In una sperimentazione francese il retrovirus, in due casi, è andato a collocarsi dove non doveva, inducendo poi una forma di leucemia. Allora tutti i protocolli con vettori retrovirali sono stati sospesi. Poi, visto che il problema riguardava solo la tecnica francese e che i due bambini colpiti stanno ora bene, in tutto il mondo la situazione è ripartita. Solo in Italia siamo ancora in stallo». di Mario Pappagallo

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53. Italia e Francia a confronto

UIDM (Internet), 1.9.1999
http://www.uildm.org/dossier/genetica/francesi.htm

Malattie neuromuscolari verso le terapie? Italia e Francia a confronto: questo il tema del Convegno internazionale medico-scientifico organizzato dalla UILDM in occasione delle XXXVI Manifestazioni Nazionali dell’Associazione, svoltesi in maggio a Genova. Italia e Francia, infatti, giocano oggi un ruolo di primo piano nella ricerca neuromuscolare europea e in questa occasione autorevoli esperti italiani e d’oltralpe sono intervenuti in merito alle strategie terapeutiche presenti nei rispettivi paesi. E’ stato Corrado Angelini, presidente della Commissione medico-scientifica UILDM, a ricordare in esordio come nel nostro Paese si stia dedicando particolare attenzione all’approccio farmacologico alle patologie neuromuscolari, nel tentativo di migliorare la situazione dei pazienti, che non possono attendere la messa a punto di una terapia genica risolutiva la quale, per una sorta di divisione strategica dei compiti, costituisce invece l’ambito di studio privilegiato dai ricercatori francesi. Tra i vari trial farmacologici condotti in Italia, particolarmente interessanti risultano quelli con il deflazacort, uno steroide, condotti nell’ambito della distrofia di Duchenne: una serie di studi in doppio cieco ha infatti evidenziato un rallentamento della perdita di forza che ha ritardato fino a un anno e quattro mesi la perdita della deambulazione. Rimangono da chiarire il momento più opportuno per iniziare il trattamento e i dosaggi più indicati per minimizzare gli effetti collaterali (il più importante di essi è l’aumento di peso), contro i quali, ad esempio, sembra utile una terapia a giorni alterni. Per quanto riguarda altri trial sulle distrofinopatie in corso nel mondo, è da segnalare la sperimentazione, negli Stati Uniti, dell’anabolizzante oxandrolone, che sembra dare un certo beneficio. Per altre malattie neuromuscolari si sta valutando anche l’impiego di altri fattori che promuovono la massa muscolare, in particolare ibeta-agonisti adrenergici, capaci di indurre in alcuni modelli animali una redistribuzione del grasso e dei substrati energetici, influendo perlopiù sulla mobilizzazione degli acidi grassi e dell’insulina: essi sono oggi studiati in fase preliminare nella distrofia facio-scapolo-omerale, nella sclerosi laterale amiotrofica e nella glicogenosi II da difetto dell’enzima maltasi acida. Per quest’ultima patologia, interessanti novità provengono dall’Olanda, dove una ditta farmaceutica ha sviluppato la glucosidasi acida, che si intende impiegare nei bambini affetti. Nella malattia di McArdle, invece, è in corso in Inghilterra un trial con piridossina e vitamina B6 e altrove si è tentato anche il trapianto dei mioblasti. Riguardo poi la miastenia grave, per cui esistono già numerosi trattamenti, si è appurato recentemente che l’uso dell’azatioprina, un immunosoppressivo, consente di diminuire le dosi di prednisone, limitandone in questo modo gli effetti collaterali, così come laciclosporina permette di ridurre l’uso di altri steroidi, in particolare nelle forme severe. Successivamente Jon Andoni Urtizberea, delegato generale dell’Istituto di Miologia di Parigi e direttore dell’European NeuroMuscular Centre (recentemente succeduto ad Alan Emery), ha ricordato come, in campo neuromuscolare, dopo la descrizione della distrofia di Duchenne nella seconda metà del secolo scorso, non si siano avuti progressi fino al 1987, momento di svolta, grazie alla scoperta della distrofina negli Stati Uniti. Ma si è trattato, in quell’occasione, di un anno significativo anche per l’avvio del Telethon francese, voluto dall’AFM, l’Associazione francese contro le miopatie, una realtà nata nel 1958 per la volontà di pazienti e familiari che ha sùbito avvertito la necessità di una strategia ben precisa in ambito di ricerca. Innanzitutto, per poter studiare una cura di queste malattie, era necessario identificarne le cause genetiche: a questo scopo è stato creato Genethon - laboratorio specializzato che ha sviluppato questo settore e che a tutt’oggi dispone della banca di DNA più grande d’Europa - avviando al contempo collaborazioni internazionali. I progressi degli ultimi anni nell’identificazione dei geni alterati nelle miopatie hanno permesso di ipotizzare varie tecniche di terapia genica, per introdurre il gene sano nelle cellule muscolari malate, tra cui l’utilizzo di virus o di altri vettori sintetici. L’Istituto di Miologia di Parigi - struttura voluta dall’AFM tre anni fa, in cui collaborano proficuamente medici, ricercatori e pazienti - sta preparando un primo esperimento di terapia genica su pazienti Duchenne, che dovrebbe consistere in un’iniezione del gene distrofina nel bicipite, per testare, prima ancora dell’efficacia stessa del trapianto genico, una sua eventuale tossicità, giacché è ancora prematuro pensare di poter curare la malattia in questa fase. La sperimentazione dovrebbe partire in autunno e coinvolgere non più di cinque pazienti; le modalità non sono ancora ben definite, anche perché si attende il parere dell’Agence de Medicament, l’organismo governativo che in Francia decide sulle sperimentazioni. Va detto comunque che, anche se l’ambito specifico delle malattie neuromuscolari poco interessa i grandi gruppi farmaceutici, di contro, quello della terapia genica in generale gode di grandi investimenti da parte di tali aziende. Per studiare la terapia genica, l’AFM ha creato una specifica struttura, Genethon III, mentre l’impegno per coinvolgere maggiormente le realtà farmaceutiche si è concretato nel progetto chiamato Genopoli, grazie al quale decine di piccole ditte di biotecnologie sono confluite a Evry, nella stessa sede di Genethon, con la "benedizione" del potere politico. Ma oltre che per queste e per altre interessanti prospettive di terapia (tra cui l’utilizzo dell’utrofina e la terapia cellulare), l’AFM mantiene costante l’attenzione anche per tutti gli aspetti della presa in carico quotidiana di un paziente neuromuscolare, come la gestione dei problemi respiratori, cardiaci e ortopedici e l’utilizzo di adeguati supporti tecnologici: i progressi in questi settori hanno positivamente inciso sulla speranza e la qualità della vita di questi soggetti. Marc Y. Fiszman, membro del Comitato scientifico dell’AFM, ha spiegato dal canto suo come gli scopi di questo organismo siano essenzialmente di promuovere presso le istituzioni e le realtà accademiche lo studio del muscolo e della terapia delle miopatie e di fornire al contempo consulenza all’Associazione in merito alle strategie di ricerca. La terapia genica delle miopatie richiede ancora tempo e la risoluzione di importanti problemi tecnici, tra cui quello di riuscire a somministrare diffusamente i vettori dei geni terapeutici in tutto il corpo in una grande varietà di muscoli. Nei laboratori francesi qualche dato incoraggiante è emerso per quanto riguarda il cuore, che è un "organo isolato": si sta infatti mettendo a punto un metodo di somministrazione capace di far esprimere un gene (per il momento un "marcatore") nel 50% della massa cardiaca. Bisognerà poi verificare se l’espressione di un gene terapeutico in questa percentuale comporterà effettivamente dei benefici significativi per tale organo. Un altro significativo problema è quello di far durare per tutta la vita l’espressione del gene terapeutico. Nei ratti si è riusciti a prolungarla fino a quasi due anni, ma si tratta di modelli animali molto differenti dall’uomo e dalla vita breve, e non è ancora chiaro quanto ciò incida su questi tempi, i quali potrebbero magari allungarsi in altri organismi. Occorrono dunque ulteriori verifiche, fermo restando anche il problema della risposta immunologica, che si sta comunque affrontando con risultati promettenti. Fiszman ha ricordato infine anche i progressi in ambito di terapia cellulare, tecnica che consiste nel reintrodurre nell’organismo vere e proprie cellule sane, con un principio che ricorda quello dei trapianti. Per essa si intravedono nuove prospettive, tra cui quella aperta dai ricercatori italiani Cossu e Mavilio, che hanno scoperto precursori di cellule muscolari in derivati del midollo osseo, cioè in elementi capaci di viaggiare nella circolazione sanguigna: ciò fa pensare alla possibilità - per il momento solo teorica - di prelevare tali precursori, ottenerne in laboratorio un numero adeguato, inserirvi il gene mancante e reintrodurli attraverso il sangue nei muscoli per ricostruirli, senza dover ricorrere a iniezioni nei tessuti. Francesca Pasinelli, coordinatrice scientifica del Telethon italiano, ha concluso ricordando che, grazie alla maratona televisiva, l’Italia da due anni supera la media europea negli investimenti per la ricerca genetica. La ricerca su una malattia genetica può dividersi in quattro momenti: l’individuazione del difetto genetico; lo studio della fisiopatologia, ovvero del funzionamento del gene e delle conseguenze delle sue alterazioni; la costruzione di modelli animali; la ricerca clinica. In termini pratici, le prime fasi hanno ripercussioni positive soprattutto sulle possibilità di diagnosi e di individuazione dei portatori. Per quanto riguarda la parte clinica, le uniche possibilità di intervento risolutivo sono rappresentate dalla terapia genica e dagli interventi in utero. Ma ricerca clinica significa anche studiare la storia naturale della malattia ed eventuali trattamenti farmacologici non risolutivi, ma che possano portare un beneficio. Gran parte dei finanziamenti di Telethon vengono destinati alla ricerca di base e notevoli risultati sono stati finora ottenuti nell’identificazione dei geni e dei meccanismi di molte malattie. Tra i risultati clinici, è da ricordare il trattamento con terapia genica dell’immunodeficienza combinata grave e il trapianto di midollo in utero. Come accade anche negli Stati Uniti, si sente ora la necessità di premere sulla ricerca clinica e sulla collaborazione con le industrie farmaceutiche. Telethon sta lavorando per attivare questa sinergia tra le aziende e i vari centri specializzati e per favorire la reciproca collaborazione di questi ultimi, in modo che - insieme all’approfondimento degli aspetti clinici delle varie malattie - si possano distinguere i trattamenti in qualche modo utili da quelli che non lo sono e proporre nuovi protocolli con farmaci debitamente testati.

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54.

pedriatria uni pd (Internet),
http://www.pediatria.unipd.it/repser/dbase2generale.asp?attivita=121

La terapia genica si basa sul trasferimento di materiale genetico allo scopo di prevenire o curare una malattia. Le malattie genetiche sono causate, infatti, da un'alterazione del patrimonio genetico di un individuo (DNA) che si traduce in difetti fisici più o meno gravi. Nel caso delle malattie genetiche in cui un gene è difettoso o assente, la terapia genica consiste essenzialmente nel trasferire la versione funzionante del gene in modo da rimediare al difetto. Nello specifico, l'attività del Servizio attivo presso il Dipartimento di Pediatria si è concentrata sullo sviluppo di metodologie di trasferimento genico mediante vettori virali e sintetici per la terapia genica di malattie genetiche e metaboliche rare e tumori pediatrici. Il gruppo ha sviluppato metodologie per la terapia genica di: o malattie da accumulo lisosomiale (Mucopolisaccaridosi tipo II MPSII) o fibrosi cistica o tumori cerebrali pediatrici Tali metodologie implicano la generazione di vettori virali (adenovirus, retrovirus) e sintetici (vettori poliaminici) allo scopo di produrre alti livelli di enzimi circolanti (iduronato-solfatasi per laterpai genica di MPSII), sovraespressione di trascritti per la correzione di difetti di canali trasporanti Cloro (terapia genica della Fibrosi Cistica), geni killer per le masse tumorali cerebrali (sistema timidino-kinasi per la terapia genica dei medulloblastomi. Recentemente, sono stati attivati progetti per la terapia genica di leucemie e nuovi vettori virali in grado di integrare la sequenza terapeutica nelle cellule di midollo umano (vettori chimerici adeno-retrvirali). Inoltre, sono allo studio dei bioreattori contenenti cellule esprimenti ad alti livelli enzimi circolanti. Attualmente, il gruppo sta saggiando i vari sistemi su modelli animali per le singole patologie allo scopo di disegnare protocolli terapeutici per trials di fase I da attivare su pazienti affetti.

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55. Cos'è la terapia genica?

www.ica-net (Internet),
http://www.ica-net.it/pascal/biotecnologie/la7.htm

La terapia genica è una scienza giovane: il primo tentativo fu effettuato negli Stati Uniti da Michael Blaese nel 1990 su una bambina affetta da una grave immunodeficienza ereditaria; da allora, nonostante gli indubbi progressi raggiunti, sono ancora pochissimi i tentativi di terapia genica per i quali si possa parlare di un successo da un punto di vista clinico. Ad oggi, le numerose sperimentazioni in corso in tutto il mondo hanno soprattutto lo scopo di migliorare le conoscenze biologiche di base e le metodiche di terapia genica perché possa finalmente diventare uno strumento efficace nelle mani dei medici Per terapia genica si intende l'introduzione di un gene che ha come effetto quello di prevenire o correggere una condizione patologica. In teoria esistono due tipi di terapia genica: una che prevede l'inserimento di un gene in cellule della linea germinale, e un'altra che tende ad eliminare i difetti genetici in cellule somatiche con effetti limitati all'individuo. Il primo tipo di terapia è per il momento improponibile per motivi etici e pratici; il secondo invece permette la regressione di alcune malattie ereditarie, determinate da un'alterazione del genoma, attraverso l'introduzione nelle cellule del paziente di una copia del gene corretto. Tale terapia, per avere efficacia, deve essere applicata nelle patologie dovute ad una mutazione puntiforme e all'assenza di una parte o dell'intera sequenza nucleotidica del di un gene a una sua modificazione. La terapia genica è un procedimento terapeutico che designa una serie di tecnologie biomedicali volte ad introdurre in una cellula bersaglio un gene medicamento per correggere una disfunzione del suo genoma. Oltre alle malattie neuromuscolari, le terapie geniche si applicano all'insieme delle malattie genetiche ma anche ad altre come il cancro, e di tipo virale (AIDS), insomma il loro campo di applicazione può essere potenzialmente immenso, anche se le difficoltà sono moltissime: per trasportare un gene medicamento all'interno di una cellula bisogna avere un mezzo di trasporto. Il veicolo, che si chiama vettore, è generalmente un virus reso innocuo, o più raramente un composto artificiale. La prima difficoltà è di mettere a punto dei vettori adatti per trasportare i geni nelle cellule bersaglio. L'insieme vettore gene medicamento può essere riconosciuto come estraneo dall'organismo e suscitare una reazione immunitaria; la seconda difficoltà è rivolta a risolvere il problema dato da questa difesa naturale del corpo umano. Un terzo problema è riscontrabile nel momento in cui il vettore deve giungere alle cellule bersaglio nel giusto distretto e per la durata desiderata; le conoscenze attuali non consentono ancora un buon trasporto anche se è già possibile prelevare delle cellule del paziente, modificarle in laboratorio ed introdurle in gran numero nell'organismo (in questo caso si parla di terapia genica ex vivo). Esistono altri metodi di terapia genica che consistono nel coltivare in certe condizioni delle cellule in laboratorio senza dover ricorrere obbligatoriamente al transfert del gene, ed in seguito introdurre queste cellule con la speranza che la modificazione biologica porti agli effetti sperati. Le malattie genetiche umane mostrano diversi schemi di ereditarietà a seconda del tipo di mutazione che la causano; ad esempio abbiamo visto come la distrofia muscolare Duchenne sia causata da una mutazione recessiva del cromosoma X e mostra un tipico schema di segregazione legato al sesso. La fibrosi cistica, invece, dipende da una mutazione recessiva in un cromosoma; questo tipo di mutazione ha uno schema di segregazione molto diverso, maschi e femmine possono essere colpiti dalla malattia in uguale misura (si ha il 25% di probabilità di ereditare dai genitori entrambi i geni mutati ed essere affetti dalla malattia, 50% di ricevere un allele normale e uno mutato ed essere portatore, il 25% di essere normale). Mappare un gene autosomiale è più complicato che mappare le mutazioni legate al sesso; una volta che il cromosoma è stato individuato con l'analisi di linkage (vicinanza di altri geni o marcatori) per identificare altri marcatori noti sullo stesso cromosoma per disegnare la mappa. Attraverso l'analisi delle frequenze di ricombinazione tra due geni o marcatori specifici, è possibile stabilire la distanza dei marcatori stessi. Dal punto di vista della ricerca medica degli scienziati americani hanno individuato la presenza di larghe quantità di una particolare proteina nel cervello dei malati del morbo di Alzheimer e sospettano che proprio l'attività di questa proteina giochi un ruolo chiave nello sviluppo della patologia; inoltre la proteina potrebbe risultare un fattore significativo anche per altri disturbi neurologici. La causa di tutto ciò sembra essere la proteina p25 che da il via ai primi mutamenti molecolari che portano all'accumulo di fibre e placche nel cervello dei malati conducendoli alla demenza. In realtà questa proteina è in realtà un frammento di una più grande e già nota proteina p35 che attiva l'enzima chiave per lo sviluppo del sistema nervoso.

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56. TERAPIA GENICA UMANA

utenti lycos (Internet),
http://utenti.lycos.it/biotecnologie_4as/settori/terapia.htm

E' una tecnica che, utilizzando un particolare insieme di metodi di laboratorio, agisce sulle cellule e quindi sugli organismi per modificarne il corredo genetico. Ha avuto inizio grazie alla migliore conoscenza dei meccanismi di regolazione dell'espressione genica, di una più esatta localizzazione dei geni nelle catene di DNA (progetto genoma), dell’identificazione per alcuni di essi della loro localizzazione specifica. L’obiettivo è quello di far assumere ad un organismo funzioni che non sono proprie del suo corredo genico nel tentativo di eliminare malattie ereditarie. I candidati più promettenti per la terapia applicata alle cellule somatiche sono le malattie provocate dalla presenza di un singolo gene mutato che, essendo ormai stato individuato e clonato, può essere sostituito mediante il trapianto di un gene sano. Oggi la vera e propria sostituzione dei geni difettivi con quelli sani non è ancora possibile, mentre è relativamente semplice affiancare a geni mutati, di solito recessivi, geni sani dominanti.Queste disfunzioni dovrebbero essere più facilmente correggibili di quelle provocate da geni multipli. Il trapianto genico non deve limitarsi a riparare un cattivo funzionamento dei geni. Dovrebbe anche servire a potenziare la capacità delle cellule, soprattutto quelle difensive contro le malattie. Steven A. Rosenborg e altri collaboratori del National Cancer Institute hanno dimostrato che i linfociti prelevati dal tumore di un paziente e coltivati in vitro con interleuchina 2 (un attivatore dei linfociti T) possono ridurre di dimensioni alcuni tipi di tumori. La terapia genica si avvale di alcune tecniche del DNA ricombinante, in particolare della possibilità: - ottenere costrutti di DNA contenenti il gene che si vuole introdurre nel paziente; - disporre di vettori adeguati all'inserimento del gene; - disporre del promotore opportuno a far sì che il gene inserito venga espresso nelle cellule bersaglio. In questo campo vi sono tre metodi: ex vivo, in situ, in vivo. I vettori più utilizzati sono i retrovirus ( modificati per evitare che causino malattie). Il rischio che questi provochino il cancro, è estremamente ridotto, ma aumenta se si fanno moltiplicare i virus all’interno di un organismo e li si fanno propagare di cellula in cellula. Un’importante sfida è stata quella di progettare metodi in grado di impedire ai vettori di riprodursi. Gli sforzi compiuti hanno dato vita ad almeno una tecnica: i prodotti ottenuti hanno un involucro esterno normale e contengono tutte le proteine del virus, ciò permette la penetrazione di tali virus nelle cellule e la liberazione del loro contenuto nel citoplasma. Gli enzimi virali trasformano attraverso la trascrittasi inversa l’RNA in DNA e contribuiscono ad inserire questo DNA nel genoma della cellula ospite. Questo passaggio è però l’ultimo per il virus che poi muore. SUCCESSI: -TALASSEMIA -SCID -CELLULE TOTIPOTENTI -TERAPIA GENICA per l'AIDS -inoltre FIBROSI CISTICA, DISTROFIA MUSCOLARE di DUCHENNE, EMOFILIA A e B LIMITI: la terapia genica delle cellule somatiche può essere considerata dal punto di vista etico simile ai trapianti di organi, una pratica accettata ed ormai consolidata. Questo tipo di terapia deve essere riservata al trattamento di gravi disturbi clinici, evitando qualsiasi applicazione a scopo non terapeutico. Oggi si potrebbe anche applicare sulle cellule germinali, ma questa possibilità solleva problemi etici. Ci si chiede se debba essere applicata solo per impedire l'avvento di una malattia ereditaria o anche per migliorare la discendenza di un individuo; se la società vuole davvero rischiare di introdurre nel patrimonio genico cambiamenti che potrebbero rivelarsi dannosi per la specie; se abbiamo diritto di interferire nell'evoluzione umana. I temi connessi con la ricerca sul genoma umano, ricerca utilizzata per individuare malattie ereditarie e la conseguente applicazione della terapia genica preoccupano l'opinione pubblica. Ci si chiede: - come comportarsi circa la risevatezza dei dati personali relativi ai test - se l'introduzione di un costrutto biotecnologico nell'uomo a scopo terapeutico è efficace o può comportare rischi causando il sorgere di nuovi problemi (potrebbe essere interrotta una sequenza di geni associati) - se l'introduzione come vettore di un virus, anche se privo di DNA, può causare delle malattie (come nel caso della "mucca pazza" o BSE-encefalopatia spongiforme bovina, un prione che potrebbe aver indotto nell'uomo l'HSE-encefalopatia spongiforme umana) Le norme elaborate, la cui prima versione è stata approntata in un solo anno e modificata successivamente in modo meno restrittivo, prevedono che i ricercatori ottengano autorizzazioni ad eseguire certe ricerche da parte di Enti adibiti al controllo. Questa normativa, pur non avendo un vero valore legale, costituisce un punto di riferimento importante per i ricercatori. Anche il Consiglio d'Europa nel 1982 ha stilato raccomandazioni di cautela, soprattutto su potenziali interventi sull'uomo; di fatto però non esistono vincoli di alcuna natura e ogni Stato ha finito con il deliberare autonomamente o, come l'Italia, con l'ignorare il problema. Senza una continua sorveglianza da parte di tutti, dunque, queste misure potrebbero non essere sufficienti a prevenire deviazioni di uno scienziato "folle" o di una industria troppo avida (gli interessi economici in gioco sono enormi, la terapia genica sta rivoluzionando l'industria farmaceutica). D'altra parte non si può rinunciare a perseguire la conoscenza in un campo che promette di essere di grande aiuto nel risolvere molti problemi dell'umanità, a condizione di usare giudizio. 1° INSUCCESSO: il 25 dicembre 1999, a due mesi dalla sua morte, i medici hanno ammesso la causa del decesso di Jesse Gelsinger, affetto da una rara malattia ereditaria al fegato: una reazione immunitaria scatenata dalla terapia genica cui il ragazzo americano si sottoponeva.

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57. Terapia genica con cellule somatiche nei bambini

bionet online (Internet),
http://www.bionetonline.org/Italiano/Content/db_cont5.htm

2004 Fino ad ora, la terapia genica della linea germinale in ovuli, spermatozoi o embrioni è stata effettuata su embrioni animali, essendo attualmente illegale sugli esseri umani. Tuttavia, è possibile modificare i geni malformati presenti nelle cellule di un bambino già cresciuto o di un adulto per curare malattie come la fibrosi cistica. Queste metodo si chiama terapia genica con cellule somatiche. Recentemente, i terapeuti genici sono riusciti a curare un bambino da unostato genetico che metteva la sua vita in pericolo, impedendogli di sviluppare il sistema immunitario aiutandolo a sviluppare il suo sistema immunitario. Il bambino soffriva di una malattia denominata immunodeficienza combinata grave (SCID), provocata da un unico gene malformato. Cellule sviluppate in laboratorio Fino alla cura con terapia genica con cellule corporali effettuata nel marzo del 2001, il bambino di 18 mesi visse in una bolla sterilizzata, per evitare che contraesse infezioni. I medici hanno estratto parte del suo midollo osseo e hanno utilizzato un virus non infettivo per trasportare una versione sana del gene alle cellule immuni del suo midollo osseo. Il midollo osseo migliorato fu allora reimpiantato nel bambino, dove produsse gradualmente nuove cellule immuni. Queste cellule immuni entrarono nella circolazione sanguigna proteggendolo così dalle infezioni. Adesso, grazie alla terapia genica, può giocare senza limitazioni e il suo sistema immunitario è simile a quello degli altri bambini della sua età.

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58. La terapia genica delle patologie ereditarie

torino scienza (Internet),
http://www.torinoscienza.it/dossier/apri?obj_id=106

Le patologie ereditarie su cui si può ipotizzare un intervento di terapia genica sono essenzialmente quelle legate al difetto di un singolo gene dette monogeniche, tra cui l'ipercolesterolemia familiare che è dovuta ad un difetto nell'espressione del recettore delle LDL nel fegato. Il trial clinico di questa malattia è stato tra i primi avviati ed è ancora in atto. Dopo una parziale epatectomia, gli epatociti estratti sono stati coltivati in vitro e quindi trasfettati con il recettore per le LDL e un gene che veicolasse la resistenza ad un antibiotico, l'igromicina. Le cellule del fegato che durante la trasfezione avevano incorporato il vettore sono state poi selezionate con l'igromicina e quindi reimmesse nel fegato del paziente mediante iniezione intravena. Nel fegato si è potuta così indurre una certa espressione di recettore per le LDL che è in grado di supplire almeno in parte il deficit della malattia. Un'altra patologia che si presta bene all'utilizzo della terapia genica è il deficit di adenosin deaminasi (ADA). La mancanza di questo enzima porta ad una devastante immunosoppressione e basta anche pochissimo enzima a ridurre drasticamente gli effetti dannosi del deficit. Proprio per questo motivo un malattia di per se stessa terribile come questa è diventata il modello per la terapia genica. Del resto il trattamento non porta comunque alla completa indipendenza del paziente che deve comunque ricorrere alla terapia sostitutiva. Diverse malattie genetiche sono suscettibili in grado differente a terapia genica. Le comuni malattie genetiche non mendeliane possono implicare una complessa correlazione tra loci genetici e/o fattori ambientali differenti per cui le varie strategie di terapia genica possono essere non facili. Le patologie determinate da un unico gene sono invece i candidati più ovvii per la terapia genica. Grazie al clonaggio posizionale ed alle altre stategie di identificazione dei geni si stanno isolando e caratterizzando un numero sempre maggiore di geni responsabili di patologie di questo tipo. Tuttavia, le diverse modalità di patogenesi fanno si che alcune di queste malattie siano più adatte di altre ad essere trattate con tecniche di terapia genica. Tra le malattie ereditarie quelle ad ereditarietà recessiva concettualmente sono le più facili da trattare con la terapia genica. Le affezioni in cui la malattia è il risultato di una deficienza di uno specifico prodotto genico generalmente sono le più idonee a questo tipo di terapia: l'introduzione di un allele normale con un'elevata espressione dovrebbe essere sufficiente a far superare il deficit. Le malattie ad eredità recessiva costituiscono candidati particolarmente interessanti per la terapia genica perché le mutazioni determinano quasi sempre perdita di funzione. I soggetti ammalati presentano un'espressione deficitaria di entrambi gli alleli per cui il fenotipo patologico è dovuto all'assenza completa o quasi della normale espressione genica. Gli eterozigoti, invece, hanno circa il 50% del prodotto genico normale e solitamente sono asintomatici. Inoltre, si riscontra in alcuni casi un'ampia variazione dei normali livelli di espressione genica, per cui una percentuale relativamente bassa dei normali livelli di espressione può essere sufficiente a restaurare il fenotipo normale. Spesso si osserva anche che in queste malattie la gravità è inversamente proporzionale alla quantità del prodotto che viene espresso. Di conseguenza, anche se l'efficienza del trasferimento genico è bassa, anche modesti livelli di espressione del gene introdotto possono determinare un drastico miglioramento a livello del fenotipo. Benché le malattie ad eredità recessiva siano, almeno in teoria, adatte a una terapia genica basata sull'aggiunta di copie geniche, certe malattie lo sono meno di altre. Oltre al problema dell'accessibilità ai tessuti interessati dalla patologia, vi sono anche altre ragioni in grado di ostacolare la terapia genica. Un esempio è fornito dalla b-talassemia che deriva dalla mutazione del gene della globina detto HBB. Questa è una grave malattia che colpisce centinaia di migliaia di persone in tutto il mondo ed è piuttosto diffusa in Italia. Ad una valutazione superficiale sembrerebbe un ottimo candidato per la terapia genica infatti il gene dell'HBB è molto piccolo ed è stato ampiamente caratterizzato, la malattia è ad ereditarietà recessiva ed interessa le cellule del sangue. Un iniziale tentativo di terapia genica per questa malattia fu iniziato nel 1980 e fallì soprattutto per l'inefficienza del trasferimento genico e della scarsa espressione dei geni della ?-globina introdotti. Anche se ora si sa molto di più su come questi geni siano prodotti non si sono più fatti esperimenti di terapia genica. Permane infatti il problema del controllo dell'espressione genica dopo l'inserimento del gene normale della b-globina nelle cellule ospiti: la quantità di b-globina deve infatti essere uguale alla quantità di ?-globina. Se si produce una delle due forme più dell'altra si crea uno squilibrio tra le due catene di globina che determina il fenotipo dell'b-talassemia.

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59. L’ADA

torino scienza (Internet),
http://www.torinoscienza.it/dossier/apri?obj_id=107

L'ADA o adenosina deaminasi è un enzima coinvolto nel salvataggio delle purine nel percorso di degradazione degli acidi nucleici ed è un enzima indispensabile in molti tipi cellulari. La sua carenza causa una patologia recessiva molto rara che ha effetti molto seri sui linfociti T, una delle principali classi di cellule del sistema immunitario. I pazienti con questo deficit detti ADA- soffrono infatti di una grave e complessa immunodeficienza. Questa patologia è adatta per la terapia genica per varie ragioni: il gene ADA è piccolo ed era già stato clonato prima del 1990, le cellule bersaglio sono i linfociti T che sono facilmente ottenibili dal paziente (basta un prelievo di sangue) e facilmente coltivabili in modo da permettere una terapia genica ex-vivo. Inoltre l'espressione normale di questo gene non è rigidamente controllata ma può variare negli individui sani da un livello del 10 ad uno del 5000% dei valore medio. Il primo successo si ebbe con un trial clinico iniziato il 14 settembre 1990 su una paziente, Ashanthi DeSilva, che aveva solo 4 anni. In questo caso si osservò che il trasferimento di geni ADA normali nei linfociti T di un paziente ADA- determina la restaurazione del fenotipo normale. Per questa patologia esistono anche trattamenti alternativi: quello di elezione è il trapianto allogenico di midollo osseo che porta alla guarigione nell'80% dei casi, ma occorre un donatore di midollo HLA compatibile. Dove il trapianto non è attuabile si può anche ricorrere alla sostituzione enzimatica, che consiste in iniezioni intramuscolari di ADA coniugata a polietilenglicole (PEG) da effettuarsi con scadenza settimanale. Il PEG serve a stabilizzare l'enzima permettendogli di rimanere attivo nel corpo umano per diversi giorni. Del resto la terapia enzimatica non fornisce una completa ricostruzione della capacità immunitaria e quindi l'attesa di vita del paziente è comunque minore infatti i linfociti T sono necessari per la realizzazione di risposte immuni efficaci contro i microrganismi invasori e la prevenzione dei tumori. La terapia dell'ADA implica essenzialmente quattro passaggi: - Il clonaggio del gene sano dell'ADA in un vettore retrovirale - Il trasferimento del ricombinante ADA nei linfociti T ADA- del paziente - L'identificazione dei linfociti diventati ADA+ e la loro espansione numerica in coltura - Il reimpianto di queste cellule nel paziente Poiché i linfociti T hanno un periodo di vita limitato nel tempo è necessario ripetere la terapia ogni uno o due mesi per questo motivo le ricerche sono ora orientate a trasferire il gene dell'ADA nelle cellule del midollo osseo. La terapia sulle cellule del sangue è infatti un trattamento e non una cura che invece richiederebbe proprio il trasferimento del gene dell'ADA nelle cellule staminali del midollo osseo del paziente. Il problema di quest'approccio è che le cellule staminali del midollo osseo sono molto difficili da isolare benché ormai siano possibili degli arricchimenti utilizzando anticorpi monoclonali come quello che riconosce il CD34 (un recettore selettivamente presente nella popolazione cellulare totipotente del midollo osseo). La frequenza delle infezione dei pazienti trattati con terapia genica è sicuramente diminuita, ma l'efficacia della terapia genica da solo è ancora difficile da dimostrare perché Ashanthi e gli altri pazienti sinora trattati con terapia genica hanno anche ricevuto iniezioni intramuscolari di enzima PEG-ADA

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60. I problemi tecnici della terapia genica

torino scienza (Internet),
http://www.torinoscienza.it/dossier/apri?obj_id=122

Idealmente il trasferimento di geni esogeni dev'essere sicuro per il paziente e per gli operatori, efficiente sia in termini quantitativi sia qualitativi e selettivo per un determinato bersaglio cellulare. La sicurezza si basa sulla necessità di non causare danni diretti al paziente e soprattutto di non modificare stabilmente il patrimonio genetico umano. A questo proposito per ora la maggior parte degli sforzi della terapia genica sono diretti verso quelle patologie che non lasciano prevedere la possibilità che il paziente abbia dei figli (ad esempio su malati terminali di neoplasie), ma in futuro la necessità di non modificare stabilmente la linea germinale potrebbe divenire un problema reale e concreto. Comunque la terapia genica attuale è esclusivamente somatica sia a causa delle perplessità etiche che solleverebbe la manipolazione di cellule germinali sia a causa dei limiti tecnologici esistenti. L'efficienza della terapia invece dipende dal numero di geni che si riesce a veicolare in un paziente e dalla facilità con cui questi possono venir inseriti stabilmente nelle cellule. Ogni sistema cellulare presenta sotto quest'aspetto caratteristiche differenti, ma è per ora ancora l'efficienza che si riesce ad ottenere variando la scelta delle strategie note è ancora troppo bassa rispetto alle ambizioni terapeutiche. Un altro problema è quello della selettività del bersaglio cellulare da trasfettare, questa è maggiore se si procede ex-vivo, mentre è più difficile da garantire in-vivo, soprattutto se si utilizzano vettori virali che almeno in teoria non hanno limiti d'infezione. Al momento sono in studio differenti strategie per permettere l'ingresso del vettore solo in cellule specifiche anche in-vivo, ma nonostante il grande impegno i risultati sono stati per ora modesti. Un altro aspetto non trascurabile è legato all'immunogenicità che alcuni vettori o che l'inserimento di determinati segmenti genici in determinate cellule può creare. L'immunogenicità infatti può causare fenomeni acuti come ad esempio le reazioni simil-influenzali o renitiche conseguenti all'uso di vettori adenovirali o fenomeni subacuti e cronici in particolare causati dagli anticorpi prodotti dall'organismo ospite che riescono a riconoscere le cellule bersaglio trasfettate e quindi a distruggerle annullando tutti gli effetti della terapia genica.

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61. Gentherapie bei Mukoviszidose . Suche nach dem Genvektor

thieme verlag (Internet),
http://www.thieme.de/viamedici/medizin/wissenschaft/mukoviszidose.html

Einleitung Die Mukoviszidose ist nach wie vor eine tödliche Krankheit, obwohl die Lebenserwartung der Patienten in den letzten Jahren enorm gestiegen ist. Seit kurzem jedoch besteht die Hoffnung auf eine endgültige Heilung mit Hilfe der Gentherapie: Forscher sind auf der spannenden Suche nach geeigneten Vektoren, um die fehlende Erbinformation in die menschliche Zelle einzubringen ... Die Euphorie war groß, als eine amerikanische Forschergruppe 1989 den für die Mukoviszidose verantwortlichen Gendefekt lokalisieren konnte. Die Heilung der Stoffwechselkrankheit schien in greifbare Nähe gerückt und die Forschungsereignisse überstürzten sich: Bereits ein Jahr später konnte gezeigt werden, daß der Defekt in menschlichen Mukoviszidose-Zellen in vitro durch den Transfer des gesunden Gens korrigierbar ist. Nur ein weiteres Jahr verging, bis die Genübertragung in vivo an Bronchialepithelien von Tieren gelang. Heute liegen die ersten Ergebnisse klinischer Studien vor. Wird die Gentherapie in kurzer Zeit die Standardtherapie bei Mukoviszidose sein? Sekretstau in allen Richtungen Die Veränderung im Erbgut der Mukoviszidose-Kranken erscheint minimal: Ein für die zelluläre Chloridpermeabilität verantwortliches Protein wird nicht oder nur funktionsuntüchtig gebildet. Folge ist eine Störung der Wasser- und Elektolytströmung durch die Zellwand. Dies wirkt sich funktionell auf die Zellen exkretorischer Organe aus: Sie sezernieren ein wasserarmes hochvisköses Sekret, das die Ausführungsgänge der Drüsen verstopft, zu reaktiven Entzündungen und anschließend zum Funktionsverlust der Organe führt. Betroffen sind vor allem Lunge, Pankreas und Leber, wobei das Ausmaß der pulmonalen Beteiligung der lebenslimitierende Faktor ist. Über 700 Genmutationen bekannt Der Gendefekt der Mukoviszidose, die auch zystische Fibrose (CF) genannt wird, ist auf dem langen Arm des Chromosoms 7 lokalisiert. Hier fehlt die Aminosäure Phenylalanin in Position 508 des Proteins, weshalb die Mutation den Namen Delta-F-508 erhielt. Gleichzeitig wurde auch das Genprodukt bekannt: Der "Cystic Fibrosis Transmembrane Conductance Regulator" (CFTR) ist ein Protein, das die Chloridpermeabilität an der Zellmembranoberfläche herabsetzt. Bei der Mutation Delta-F-508 wird ein räumlich inkomplett strukturiertes CFTR-Protein gebildet, das seine eigentliche Lokalisation in der Zellmembran nicht erreicht. Ca. 70% der Patienten weisen diese Mutation auf. Inzwischen sind über weitere 700 Genveränderungen bekannt, die mit dem Krankheitsbild der Mukoviszidose einhergehen. Hoffnung Gentherapie Moderne medizinische Verfahren auf dem Gebiet der Früherkennung und der Therapie konnten die Lebenserwartung der Mukoviszidose-Patienten in den letzten Jahren enorm verbessern. Während die Betroffenen früher meist nicht älter als fünf Jahre wurden, erreichen sie heute in der Regel das Erwachsenenalter. Ein in den 90er Jahren geborenes Kind mit Mukoviszidose hat eine Chance von über 90%, das Erwachsenenalter zu erreichen &endash; entsprechend nimmt auch die Prävalenz der Erkrankung zu. Eine endgültige Heilung gibt es bisher noch nicht. Es bestehen aber berechtigte Hoffnungen, daß die Gentherapie in absehbarer Zeit eine Behandlungsmöglichkeit für die Mukoviszidose wird: "Die fehlende Erbinformation in die menschliche Zelle zu bringen ist das Ziel neuer gentherapeutischer Ansätze. Man sucht zur Zeit nach geeigneten Vektoren, um das Gen optimal zu übertragen", berichtet Dr. Heike Diekmann, Medienreferentin des Deutschen Mukoviszidose e.V., von der 21. European Cystic Fibrosis Conference, die im Juni diesen Jahres in Davos stattfand. Intrauterine Therapie: erfolgreich &endash; aber umstritten Neuester Traum der Forscher ist eine intrauterine Therapie, bei der das fehlende Gen direkt in die Keimbahn eingebracht wird. Zumindest im Tierversuch ist dies keine Zukunftsmusik mehr: Im März 1997 berichtete der Lancet (The Lancet 1997; 349: 619&endash;620) über die erste erfolgreiche intrauterine Gentherapie bei Mukoviszidose. Die Wissenschaftler aus New Orleans behandelten hierzu Feten von sog. S489X-Knock-out-Mäusen. Diese Mäuse weisen einen CFTR-Gendefekt auf, der zur gastrointestinalen Symptomatik der Mukoviszidose führt. Den Mäusefeten wurde mittels Amniozentese ein Gen injiziert, das die korrekte Aminosäurensequenz zur Ausbildung von CFTR enthielt. Es konnte so in das Erbgut der Mäuse eingeschleust werden. Während in der Kontrollgruppe alle Tiere noch vor dem 50. Lebenstag verstarben, waren die gentherapierten Mäuse 250 Tage nach der Geburt gesund und zeigten keine Anzeichen einer Pankreas-Symptomatik. In Deutschland sind diese Eingriffe in die Keimbahn aus ethischen Gründen sehr umstritten: "Die intrauterine Gentherapie ist sicher keine Therapie-Option für die Mukoviszidose. Ich sehe einen besseren Ansatz in der somatischen Gentherapie, bei der nur einzelne Körperzellen verändert werden. Da das Erbgut unangeastet bleibt, kann man sie praktisch mit einer Organtransplantation vergleichen", meint Dr. med. Joachim Bargon von der Universitätsklinik in Frankfurt. Die Vektor-Frage Auch auf dem Gebiet der somatischen Gentherapie ist in den letzten Jahren viel geforscht worden. Eine Frage, die die Wissenschaftler zur Zeit beschäftigte, ist die Wahl des geeigneten Vektors: Um eine In-vivo-Gentherapie der Lunge durchzuführen, benötigt man ein Medium, das als Überträger und Protektor der korrekten DNA-Version dient und die Zielzelle mit dem Ort der CFTR-Exprimierung erreicht. Ist dies gelungen, muß das intrazellulär abgelesene, funktionsfähige Membranprotein (CFTR) immunhistochemisch und elektrophysiologisch nachzuweisen sein. Adenoviren mit Expressionskassetten Als möglicher Genüberträger steht das Adenovirus mit seiner Fähigkeit, spezifisch Atemwegsepithelien zu infizieren, zur Debatte. Das Virus wird durch das Entfernen einer bestimmten Region seiner DNA replikationsunfähig gemacht. An dieser Stelle wird eine Expressionskassette eingebaut, in der ein Promotor die eingesetzte CFTR-DNA antreibt. Das Virus bindet zunächst an spezifische Rezeptoren der Epithelzellen, die sog. Coated pits, über die es endozytotisch in die Zelle gelangt. Intrazellulär wird es als fremd erkannt und zum Endosom verpackt. Als solches kommt es in den Zellkern hinein und streift dort seine Proteinhülle ab. Auf diese Weise konnte sowohl in vitro als auch in vivo an Ratten die fehlende CFTR-DNA in Bronchialepithelien übertragen werden. Die virale DNA mit dem CFTR-Promotor liegt im Zellkern neben den Chromosomen und wird dort exprimiert.In klinischen Studien an CF-Patienten stellte sich der Erfolg leider noch nicht ein. Für ein effizientes Transferergebnis mußte man bisher das Virus mehrmals applizieren (a Abb. 6) und brauchte zu hohe Dosen mit der Folge, daß diese schlecht vertragen wurden: Entzündungsreaktionen, Bildung neuer Genkombinationen aus Virus- und Wirtszellgenom (Rekombination) und zelluläre sowie humorale Immunantworten stellten sich ein. Da manche Adenoviren in vitro Tumoren induzieren können, bleibt auch dies ein Unsicherheitsfaktor für die Anwendung am Menschen. Nun arbeitet man an der Herstellung einer neuen Generation von Adenoviren, die weniger adenovirales Genom aufweisen und so eine geringere Immunantwort auslösen. Des weiteren versucht man die initiale Immunantwort des Patienten medikamentös zu blockieren. Adenoassoziierte Viren &endash; zuwenig erforscht Ein weiterer Kandidat für die Gentherapie ist der nicht humanpathogene adenoassoziierte Virus (AAV). Seine Vorteile bestehen einerseits in seiner geringen Toxizität und andererseits darin, daß die DNA gut ins Zellgenom integriert wird. Mit diesem Vektor konnte eine Korrektur des Chloriddefektes und eine Transgenexpression über mehrere Monate nachgewiesen werden. Allerdings wagte man sich noch nicht daran, den Vektor breit anzuwenden: Die Auswirkungen der Integration ins Genom des Wirtes sind noch zu wenig erforscht, und es gibt Probleme bei seiner Herstellung. Mit Liposomen zum Erfolg? Einige Wissenschaftler setzen derzeit auf Liposomen als geeignete Vektoren. Im Gegensatz zu den viralen Kandidaten haben diese weniger antigene Wirkungen auf den Organismus. Zudem besteht bei der virusfreien Übertragung keine Gefahr einer Rekombination. Die mizellenartigen Fettemulsionen sind positiv geladen und verbinden sich mit der negativ geladenen DNA zu einem zellmembrangängigen Komplex. Helferlipide, z.B. Dioleoylphosphatidylethanolamine (DOPE), erleichtern das Eindringen der DNA in die Zielzelle durch Membranfusion oder Endozytose. Intrazellulär verfügen die Liposomen nicht wie die Viren über einen Mechanismus, der sie in den Zellkern bringt: Ein großer Teil der DNA wird im Zytoplasma der Zelle noch vor dem Erreichen des Zellkernes durch lysosomale Enzyme zerstört. Es müssen also große Mengen von Liposomen-DNA-Komplexen eingesetzt werden, um eine effiziente Korrektur in vivo zu erreichen. Eine klinische Liposomen-Studie in England zeigte bereits erste Erfolge: Bei 15 Mukoviszidose-Patienten wurden Komplexe aus Liposomen und CFTR-DNA in die Nasenhöhle appliziert. Die Ergebnisse deuten auf eine partielle Normalisierung der Chloridsekretion in der Nasenschleimhaut hin. Der Beweis, mit den Liposomen eine CFTR-Expression in der Lunge zu erreichen, steht derzeit noch aus. Da aber auch bei Liposomen die Behandlung regelmäßig wiederholt werden muß, bleibt bislang offen, wie die Patienten auf eine häufige Applikation der hohen Menge DNA und Liposomen reagieren. Einen Schritt weiter &endash; aber noch immer ungelöste Fragen Die Zukunft wird zeigen, welcher Vektor für die Therapie der Mukoviszidose am besten geeignet ist. Das Problem der kurzen Wirkdauer der Gentherapie ist ebenfalls noch nicht gelöst. Schließlich sind mit der somatischen wie mit der intauterinen Gentherapie zahlreiche Risiken verbunden. Erst wenn alle Fragen beantwortet sind und die Risiken der Gentherapie minimiert wurden, kann der große Durchbruch in der Mukoviszidose-Therapie gelingen. Optimistische Forscher sind davon überzeugt, daß die entscheidenden Schritte noch vor der Jahrtausendwende erfolgen werden.

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62. Gentherapie - Taxi für Gene

life science (De) ((Internet),
http://www.lifescience.de/bioschool/sheets/30.html

Gentherapie - wozu? Mit Pillen, Spritzen oder Krankengymnastik lassen sich meist nicht die eigentlichen Krankheitsursachen, sondern eigentlich nur deren Symptome bekämpfen. Besonders, wenn ein Gendefekt die Ursache der Krankheit ist, bleibt mit konventionellen Therapieformen eine wirkliche Heilung unmöglich. Das heißt für viele Patienten, daß sie ein Leben lang Medikamente zu sich nehmen müssen. Seit den 70er Jahren wird nun an der direkten Therapie der Gene, das heißt den Ersatz des defekten Genes durch ein gesundes, geforscht. Der erste Einsatz von Gentherapie Ashanti DaSilva hat ein krankes Gen. Wäre es gesund, würde dieses Gen das Enzym Adenosindesaminase (ADA) herstellen, das eine wichtige Rolle im Stoffwechsel des Immunsystems spielt. Aufgrund ihres ADA-Mangels ist Ashanti überempfindlich für Infektionen aller Art. Nur wenn sie das Enzym ADA künstlich aufnimmt, kann sie sich in öffentlichen Räumen aufhalten, in die Schule gehen. Damit sie nicht permanent ADA von außen braucht, sondern ihr Körper den Stoff selbst produziert, wurde 1986 an Ashanti erstmals ein gentherapeutischer Ansatz getestet. Entschärfte Viren halfen dabei, in Zellen von ihr ein gesundes ADA-Gen einzuschleusen. Mit anscheinendem Erfolg: Durch die Gentherapie verbesserte sich Ashantis Krankheitsbild. Bei genauerem Hinsehen stellt sich allerdings heraus, daß die Ärzte außer Gentherapie noch andere Medikamente einsetzten. Niemand vermag nun genau zu sagen, ob die Verbesserung von Ashantis Zustand wirklich der Gentherapie zuzuschreiben ist - ein gutes Beispiel für die heutzutage vorherrschende Skepsis in Bezug auf Nutzen und Wirkung der Gentherapie, die sich momentan noch in der Testphase befindet und nicht als offizielle Therapieform zugelassen ist. Potentielle Anwendungen der Gentherapie ADA-Mangel, Mukoviszidose oder Hypercholesterinämie, eine Fettstoffwechselstörung, sind Beispiele für Erbkrankheiten, die von einem einzigen defekten Gen verursacht werden. Diese Krankheiten haben, aufgrund des genau definierten genetischen Defektes, die besten Chancen auf Heilung durch Gentherapie. Ein anderer wichtiger Anwendungsbereich sind erworbene Erkrankungen wie Tumoren, Herzinfarkt oder Viruserkrankungen, bei denen man die genetische Steuerung des Krankheitsverlaufes kennt. Gentherapie-Arten In der Testphase befindet sich derzeit nur die somatische Gentherapie. Dabei werden therapeutische Gene in betroffene Organe oder Körperzellen eingeschleust. Im Gegensatz dazu würde bei derKeimbahn-Gentherapie das Gen in die Eizelle oder Spermien eingebracht werden und sich damit auf die Nachkommen übertragen. Obwohl der Transfer von Genen in Forpflanzungszellen bei Tieren regelmäßig angewandt wird (zum Beispiel beim Pharming oder bei der Herstellung transgener Mäuse), ist er beim Menschen verboten und mit Haftstrafen belegt. Gen-Taxi in die Zelle Zwei Methoden kennt man, mit denen das gesunde Gen eingeschleust werden kann. Die ex-vivo Strategie bei der somatischen Gentherapie Therapeutische Gene werden im Labor mittels Vektoren in zuvor entnommene Körperzellen des Patienten eingeschleust. Die so behandelten Zellen werden dem Patienten wieder zugeführt. Mit freundlicher Genehmigung des VCI. Bei der ersten Methode entnimmt der Arzt dem Patienten körpereigene Zellen (Autosomen), schleust das therapeutische Gen mit Hilfe von Vektoren ein, vermehrt die Zellen daraufhin im Labor und führt sie dem Patienten wieder zu. Diese sogenannte ex-vivo Strategie ist zum Beipiel zur Bekämpfung von Blut- oder Knochenmarkskrebs oder AIDS denkbar. In-vivo Gentherapie für Mukoviszidose-Patienten Bei der Inhalationstherapie der Mukoviszidose soll mit Hilfe von viralen Gentaxis das rettende Gen in die Lunge der Patienten eingeschleust werden, um dort den für die Krankheit typischen zähflüssigen Schleim abzubauen. Bei der in-vivo-Strategie wird das Gen durch sogenannte Gentaxis direkt zum Zielort im Körper transportiert. Diese Gentaxis oder Genfähren können, wie bei Ashanti DaSilva, entschärfte Viren sein, die Zellen zwar infizieren können und das therapeutische Gen dabei einschleusen, sich selber aber nicht mehr vermehren oder ausbreiten können. Auch Liposomen, eine Art Fettkügelchen, die leicht mit Zellmembranen verschmelzen können, dienen als Gentaxis. Zur Anwendung könnte das in-vivo-Prinzip unter anderem bei der Bekämpfung von Tumoren wie dem Gehirntumor Glioblastom kommen. Allerdings sind die meisten der heute bekannten Vektorsysteme noch nicht ausgereift genug, um einen zuverlässigen Gentransfer zu gewährleisten. Sowohl die Pharmaindustrie als auch die Biomedizin versucht hier, bessere Grundlagen zu schaffen

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63. Doppelter Erfolg mit neuartiger Diabetestherapie

Ärzte Zeitung (Internet), 8.9.2004
http://www.aerztezeitung.de/docs/2004/09/08/160a1102.asp?cat=/medizin/gentechnik/gentherapie

INDIANA (ple). Bei Patienten mit Typ-2-Diabetes ist der Glukagon-Spiegel im Blut erhöht. Zumindest im Tierversuch lassen sich offenbar Zeichen eines Typ-2-Diabetes durch Blockade des Gens für den Glukagon-Rezeptor abschalten. Wie US-Forscher in Zusammenarbeit mit deutschen Wissenschaftlern herausgefunden haben, gelingt es bei Mäusen mit kurzen Nukleinsäure-Ketten - Antisense-Moleküle - den Blutzucker zu normalisieren, die Glukosetoleranz zu verbessern und die Insulinsekretion zu erhalten (J Clin Investigation 113/11, 2004, 1571). Durch die Gen-Hemmung wird die Glukagon-vermittelte Glukose-Synthese in der Leber unterdrückt. Zudem steigt die Serumkonzentration von Glukagon-ähnlichem Peptid sowie die Insulinspiegel in den Beta-Zellen des Pankreas.

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64. Gentherapeuten haben die Hoffnung noch nicht aufgegeben

Ärzte Zeitung (Internet), 7.4.2004
http://www.aerztezeitung.de/docs/2004/04/07/065a1501.asp?cat=/medizin/gentechnik/gentherapie

In 15 Jahren ist der Großteil der Studien in der Phase I steckengeblieben / Methoden werden verfeinert / Nicht-virale Verfahren favorisiert MÜNCHEN (rom). Der erste klinische Versuch mit der Gentherapie startete 1990: An den National Institutes of Health in Bethesda im US-Staat Maryland erhielt eine vierjährige Patientin weltweit erstmals eine Gentherapie. Knapp 15 Jahre später hat das Behandlungsverfahren einen schlechteren Stand als zum Zeitpunkt dieser Premiere. Alles deutet darauf hin, daß die Gentherapie viel zu früh initiiert wurde, um erfolgreich sein zu können. "In den USA haben die National Institutes of Health ab 1990 viel Geld in völlig neue Ansätze gepumpt", erinnerte Professor Bernd Gänsbacher auf einem Workshop der BioM AG in Martinsried bei München. Die Koordinationsstelle der Biotech-Region München hatte den Direktor des Instituts für Experimentelle Onkologie und Therapieforschung der TU München eingeladen, um eine Rückschau auf fast 15 Jahre Gentherapie zu halten. In den USA wurde viel in die Therapieforschung investiert Gänsbacher provozierte bewußt: "Warum wurde die Gentherapie viel zu früh in die Klinik eingeführt? Weil die Schulmedizin riesige Probleme hat, Krankheiten zu heilen!" Beispiel Krebs: Immense Summen wurden in den USA und auch in Deutschland in die therapeutische Forschung gesteckt, doch die Mortalitätsraten blieben nahezu stabil. So wurden mehrere Forschungsrichtungen eingeschlagen, mit dem Ziel, die Krebstherapie erfolgreicher zu machen. Die Therapie mit Antikörpern ist ein innovativer Sektor, der ein positives Beispiel abgibt: Antikörper haben sich zumindest in der Onkologie weitgehend etabliert. "Für die Gentherapie gilt das noch lange nicht", so Gänsbacher. Im Gegenteil: Nicht zuletzt durch die Hiobsbotschaften Ende 2002 aus Paris, wonach zwei von zehn Kindern mit angeborener, schwerer Immunschwäche (SCID) nach der Gentherapie - wie berichtet - an Leukämie erkrankten, hat sich die Stimmung noch weiter ins Negative gekehrt. Nach einem Stop hat die Arbeitsgruppe um Professor Marina Cavazzana-Calvo am Hôpital Necker des Enfants Malades das Projekt wieder aufgenommen. Das Negativ-Image besteht aber zu Unrecht, so Gänsbacher. Denn anhand dieser einzelnen, gleichwohl tragischen Ereignisse dürfe man nicht den Wert einer gesamten Behandlungsstrategie beurteilen. "Wenn man sich die Geschichte der Chemotherapie oder der Knochenmarktransplantation anschaut, sieht das noch viel weniger rosig aus", erinnerte Gänsbacher. Noch weit weniger relevant für eine globale Verurteilung der Gentherapie sei der Tod des US-Amerikaners Jessie Gelsinger im Jahr 1999 in Philadelphia. Wie der Onkologe sagte, waren damals vielmehr die drei schweren und 25 leichten Therapie-Protokoll-Verstöße auf Seiten der behandelnden Ärzte ausschlaggebend. In Deutschland sind die Studien ausgereifter als in den USA So nimmt es nicht Wunder, daß über die etwa in der Onkologie insgesamt 113 derzeit offenen klinischen Gentherapie-Studien mit Argusaugen gewacht wird. Auch in Deutschland. Zuständig ist hier die Kommission für Somatische Gentherapie (KSGT), welche die Lage längst nicht so negativ sieht. Eine "Phase der Konsolidierung" konstatierte KSGT-Vorsitzender Professor Klaus Cichutek vom Paul-Ehrlich-Institut in Langen bei Frankfurt am Main Ende Februar auf dem Deutschen Krebskongreß in Berlin. Gänsbacher beurteilt die Studiensituation in Deutschland generell als etwas besser, verglichen mit den USA. "In Deutschland handelt es sich überwiegend um größere multizentrische Untersuchungen, die ausgereifter sind." Im Zuge einer Art Goldgräberstimmung hätten sich in den 90er Jahren des vergangenen Jahrhundert transatlantisch viele Biotech-Firmen in klinische Studien gestürzt, obwohl die Ideen nicht ausgereift waren. Bester Beleg, so Gänsbacher: Ein Großteil der Studien blieb in Phase I stecken. Weltweit wurden bislang etwa 6000 Patienten gentherapeutisch behandelt, davon etwa 300 in Deutschland. Am Anfang stand der Gentransfer mit viralen Vektoren im Vordergrund. Jetzt läßt sich eine Verschiebung hin zu nicht-viralen Methoden ausmachen. Dadurch lassen sich nach Angaben von Gänsbacher zwar viele Probleme vermeiden, aber dafür verlaufen die Transferraten längst nicht so effizient. Analoges gilt für das methodische Vorgehen in vivo oder ex vivo: Problematischer ist die Anwendung bei Patienten. Doch die Alternative, Patienten etwa Leukämiezellen zu entnehmen, gentechnisch zu verändern und dann zu reimplantieren, wartet bislang nur mit dürftigen Erfolgen auf. Gänsbacher, der auch Präsident der European Society of Gene Therapy ist, hält generell ein intensives Methodenstudium für notwendig, bevor es ein Stückchen weiter gehen kann mit der Gentherapie. "Wir müssen die Techniken verfeinern, um die beobachteten Gefahren unwahrscheinlich zu machen - ganz ausschließen wird man sie allemal nicht können." Doch eine rundweg optimistische Stimmung läßt sich nicht heraufbeschwören: Im Zuge des Wirbels um die Mißerfolge ist es schwieriger geworden, gutes Geld und gute Leute für gentherapeutische Forschung zu gewinnen.

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65. Gentherapeutisch veränderte Haut zieht Cholesterin aus dem Blut

Ärzte Zeitung (Internet), 18.4.2000
http://www.aerztezeitung.de/docs/2000/04/18/072a0301.asp?cat=/medizin/gentechnik/gentherapie

Hintergrund Die Gentherapeuten entdecken die Haut, nicht nur für die Behandlung von Patienten mit Hautkrebs - einem der ersten Ziele gentherapeutischer Intervention überhaupt -, sondern auch zur Immunisierung gegen Viruserkrankungen wie HIV oder zur Behandlung bei genetisch verursachten Stoffwechselerkrankungen wie Hypercholesterinämie. Auch bei Wundheilungsstörungen oder Viruserkrankungen, die sich auf der Haut manifestieren, Feigwarzen etwa, könnten künftig gentherapeutische Ansätze verwirklicht werden. Das ist auf einem Internationalen Kongreß über Gentechnologie und Gentherapie der Haut an der Universitäts-Hautklinik Essen deutlich geworden. Gründe dafür, warum die Forschung zu Gentherapien über die Haut derzeit boomt, nannte der Essener Dermatologe Privatdozent Ulrich Hengge: die einfache Zugänglichkeit für therapeutische Gene, aber auch für Substanzen, die die Therapie unter Kontrolle halten, die Fähigkeit von Zellen in der Haut, fremde Erbsubstanz aufzunehmen und nach ihrer Bauanleitung Eiweißmoleküle herzustellen sowie die ausgeprägte Fähigkeit von Zellen der Haut, Immunreaktionen anzukurbeln. Keratinozyten können "nackte" DNA aufnehmen Keratinozyten können unverpackte, zu Ringen geschlossene Erbsubstanz ("nackte" Plasmid-DNA ) aufnehmen, wie Hengge in Tierversuchen und an menschlicher Haut belegt hat. Diese Fähigkeit ist sonst nur von Skelettmuskelzellen bekannt. Die nackten DNA-Plasmide bleiben außerhalb der Chromosomen und können damit nicht die Funktion von Genen verändern. Werden sie mit Hilfe einer feinen Nadel zwischen Dermis und Epidermis injiziert, beginnen die epidermalen Keratinozyten schon wenige Stunden nach der Injektion, die von den Genen kodierten Eiweißmoleküle zu bilden. Die Wirkung hält etwa drei Tage an. Daß die Keratinozyten auch therapeutisch wirksame Konzentrationen fremder Proteine herstellen können, belegten Hengge und seine Kollegen am Hund. Bei Hunden bilden sich ebenso wie beim Menschen nach Infektion mit Papillomviren Feigwarzen, die in der Anal- und Zervikalregion bekanntlich maligne entarten können. Die Essener Forscher fanden in einer doppelblinden Untersuchung mit 17 Beagles heraus, daß lokal in Papillome der Mukosa des Oberkiefers injizierte DNA-Plasmide die Papillome abheilen lassen können. Die therapeutischen Plasmide enthielten das Gen für Interferon alpha, welches antiviral wirkt. Jetzt will Hengge testen, ob die Methode bei Menschen sicher ist und sich für die klinische Anwendung nutzen lassen könnte. Epidermal growth factor schließt Wunden Auch bei Wundheilungsstörungen könnte die Gentherapie genutzt werden. Professor Elof Eriksson aus Boston im US-Staat Massachusetts hat am Schwein belegt, daß sich Wunden rascher schließen, wenn gentherapeutisch veränderte Zellen in ihre Nähe gebracht werden. Die Zellen werden dem zu behandelnden Individuum entnommen, ex vivo vermehrt, mit den Genen transfiziert und beim chirurgischen Eingriff zurückverpflanzt. In Erikssons Experimenten produzierten die Zellen den epidermal growth factor (EGF) in höheren Konzentrationen als unbehandelte Zellen. Die Synthese des Wachstumsfaktors stand unter der Kontrolle eines Tetrazyklin-regulierten Genes, das dem EGF-Gen benachbart war. Über die Gabe des Antibiotikums kann die Synthese der gewünschten Proteine an- und abgeschaltet werden. Auch für chronische Wundheilungsstörungen, etwa durch venöse Gefäßerkrankungen, sehen die Wissenschaftler in der Gentherapie Perspektiven. Die Arbeitsgruppe um Ulrich Hengge hat herausgefunden, daß Keratinozyten von Menschen, die Krampfadern in Kombination mit schlecht heilenden Wunden haben, im Bereich der offenen Stellen vermindert das Eiweiß TIMP bilden. TIMP hemmt eine Matrix-Metalloproteinase, die Kollagen zerstört. So wichtig die Aktivität dieser Metalloproteinase ist, um bei einer frischen Wunde Zellreste zu entfernen, so hinderlich ist sie, wenn die Läsion im zweiten Schritt verschlossen werden soll. "Wenn wir wissen, daß eine Wundheilungsstörung mit einer verminderten oder erhöhten Expression eines bestimmten Proteins einhergeht, könnte die Gentherapie mit transfizierten Keratinozyten eine Perspektive sein, zumal die Synthese der therapeutischen Proteine in unserem System nur wenige Tage anhält", so Hengge zur "Ärzte Zeitung". Es gäbe also keine Gefahr, daß die Behandlung zum "Selbstläufer" wird. Professor Thomas Jensen aus Aarhus in Dänemark hat eine Idee, mit genetisch veränderten Keratinozyten zu therapieren: Die Haut als größtes menschliches Organe könnte als metabolische Stütze bei Stoffwechselstörungen fungieren. Als Beispiel nannte er die Hypercholesterinämie. Keratinozyten lassen sich mit Genen für den LDL-Rezeptor transfizieren, so daß das LDL-Bindeprotein in den Hautzellen überexprimiert wird. Die veränderten Zellen nehmen bis zu zehn Mal mehr Cholesterol auf, als es unveränderte menschliche Körperzellen tun, wie Jensen bereits im Mausmodell belegt hat. Jetzt will er messen, wie stark sich die Cholesterolkonzentrationen im Blut der Mäuse längerfristig senken lassen. Jensen: "Sollte unser Ansatz bei Menschen funktionieren, bräuchte man unseren Berechnungen nach ein Hautstück von etwa 18 mal 18 Zentimetern Größe, um therapeutisch relevante Effekte zu erzielen."

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66. Somatische Gentherapie bei Patienten mit Fanconi-Anämie (FA)

uni klinik duesseldorf (Internet),
http://www.uniklinik-duesseldorf.de/deutsch/Unternehmen/Kliniken/Klinik%20f%fcr%20Kinder-Onkologie,%20-H%e4matologie%20und%20-Immunologie/Forschung/Somatische%20Gentherapie%20bei%20Fanconi%20An%e4mie/index_1.html

Fanconi-Anämie (FA) ist eine seltene angeborene, autosomal rezessive Erkrankung des Kindes- und Jugendalters, die klinisch durch die Trias multiple angeborenene Fehlbildungen, ein chronisch-progredienten Knochenmarkversagen und eine Neigung zu bösartigen Erkrankungen charakterisiert ist. Pathogenetisch gehört die FA zu den DNA-Reparaturerkrankungen, wobei die Patienten relativ spezifisch kreuzvernetzte doppelsträngige DNA nur sehr unzureichend reparieren können. FA wird durch Defekte in mindestens acht verschiedenen Genen (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG) verursacht, wobei sieben (alle bis auf FANCB) kloniert bzw. identifiziert wurden. Nach gegenwärtigen Vorstellungen interagieren die ersten fünf identifizierten Gene (ACEFG) in einem Proteinkomplex miteinander und aktivieren das sechste Gen (D2), dass dann mit den beiden Brustkrebsgenen BRCA1 und BRCA2 interagiert. So werden im Rahmen der homologen Rekombinationsreparatur Schäden der DNA beseitigt. Überraschenderweise wurde BRCA2 im Sommer 2002 ebenfalls als ein FA-Gen identifiziert, d. h. Patienten mit Defekten in beiden Allelen des BRCA2-Gens leiden klinisch unter einer schweren FA; dagegen sind Patienten mit nur einem defekten BRCA2-Allel klinisch gesund, entwickeln aber im Laufe ihres Lebens zu bis zu 80% ein Mamma-, Pankreas- oder Ovarialkarzinom. Die Hämatopoese ist ein streng hierachisch strukturiertes, dynamisches System, das aufgrund einer Vielzahl von geregelten Proliferations- und Differenzierungsvorgängen eine kontinuierliche Produktion von reifen hämatopoetischen Zellen gewährleistet. Alle Zellen des hämatopoetischen Systems stammen von den sogenannten hämatopoetischen Stammzellen ab, die dadurch charakterisiert sind, daß sie sich ständig selbst erneuern und in jede Zelle des hämatopoetischen Systems differenzieren können. Aufgrund der unzureichenden Reparatur von DNA-Schäden kommt es bei FA-Patienten zu einer deutlich gesteigerte Apoptose (Suizid der Zelle) auf allen Ebenen des hämatopoetischen Systems. Die Apoptose insbesondere in Stammzellen resultiert über Jahre hinweg in einem kontinuierlichen Verlust an Stammzellen im Knochenmark, so dass die Patienten aufgrund des fehlenden Nachschubs reifer, funktionsfähiger Blutzellen klinisch durch die vermehrte Infektionsneigung (Granulozytopenie), durch eine besondere Blutungsneigung (Thrombozytopenie) und/oder durch die ausgeprägte Anämie charakterisiert sind. Trotz aller supportiven Massnahmen versterben die Patienten im Rahmen ihrer Panzytopenie meistens in der ersten Dekade nach Manifestation der Panzytopenie. Die einzige kurrative Therapie für die hämatologischen Manifestationen der Erkrankung ist der Austausch der defekten Hämatopoese im Rahmen einer allogenen Stammzelltransplantation. Bei dieser aufgrund des zugrundeliegenden DNA-Reparaturdefektes sehr belastenden Therapie wird allerdings der Defekt nur im blutbildenden System, nicht aber in den anderen Körperzellen der Patienten korrigiert. Somit summieren sich zu den endogenen DNA-Schäden (durch die FA) noch die durch die Konditonierung im Rahmen der Transplantation gesetzten DNA-Schäden hinzu. Diese Schäden werden für die deutlich gesteigerte Inzidenz an Malignomen bei den FA-Patienten nach Transplantation verantwortlich gemacht. Diese erhöhte Inzidenz kann wahrscheinlich auch durch eine mildere Konditionierung mit Verzicht auf DNA-kreuzvernetzende Substanzen nur teilweise gesenkt werden. Mit der somatischen Gentherapie von hämatopoetischen Stammzellen als Zielzellen für den Gentransfer soll eine Heilung der Ausfälle im hämatopoetischen System erzielt werden. Analog zu einer Transplantation mit normalen hämatopoetischen Stammzellen sollte das Einbringen der "gesunden" Kopie eines defekten Gens in die Stammzellen eines Patienten zu einer Heilung der Krankheit führen, da die korrigierten Stammzellen das hämatopoetische System des Patienten repopulieren können. Aufgrund des großen Selektionsvorteils in vivo und der stark gesteigerten Apoptose der kranken Zellen sollte bei der FA auch eine geringe Anzahl gesunder, korrigierter Stammzellen ausreichend sein, um das gesamte hämatopoetische System mit gesunden Zellen zu repopulieren - sofern ausreichend Zeit vorhanden ist. Auch wenn das Prinzip einfach erscheint, gibt es - neben dem primären technischen Problem, ein gewünschtes Gen stabil in bestimmte Zielzellen einbringen zu wollen - verschiedene Hindernisse, die für eine erfolgreiche Therapie überwunden werden müssen. Auch wurde 2002 bei zwei Patienten mit x-chromosomalem schweren kombinierten Immundefekt eine durch die Gentherapie durch Insertion des onkoretroviralen Vektors in das Genom der Stammzellen entstandene T-Zell-Leukämie als zusätzliches Problem beschrieben. Obwohl diese beiden Vorfälle von Insertionsmutagenese noch nicht vollständig verstanden sind, hat diese Problematik die Gentherapie als Therapieoption bei diesen Patienten - bis dahin 9 von 11 Patienten ohne Nebenwirkungen geheilt - relativiert. Fragestellungen 1.) Lässt sich ein funktioneller Test entwickeln, mit dessen Hilfe die defekten Gene schon bei Diagnose erkannt werden können? 2.) Wie ist die Aufteilung der Gendefekte bei den FA Patienten in Deutschland/Europa bzw. in den Amerika? 3.) Welche Ergebnisse lassen sich mit klassisches retroviralen Vektoren in Mäusen erzielen? 4.) Wie lässt sich ein klinisches Protokol für eine somatische Gentherapie umsetzen? Ergebnisse Zuerst wurden replikationsdefekte rekombinante Retroviren kloniert, die die Gene FANCA, FANCC, FANCD2, FANCE, FANCF und FANCG enthalten und damit stabile retrovirale Produzentenzellen hergestellt. In Zusammenarbeit mit PD Dr. Schindler, Institut für Humangenetik, Universität Würzburg, und Frau Dr. Arleen Auerbach, Internationales FA Register (IFAR) wurden Zellen von mehr als 500 FA-Patienten aus Deutschland/Europa und Nord/Südamerika mit diesen Retroviren transduziert und bei 90% der Patienten das defekte Gen identifiziert. Nach unseren Untersuchungen ist die Verteilung der Patienten in Deutschland/Europa bei 216 Patienten folgendermaßen: 59% FANCA, 7% FANCC, 1% FANCD1, 3% FANCD2, 2% FANCE, 2% FANCF, 13% FANCG, 11% FANC-nonACEFG und 2% FANC-nonACD1D2EFG. Bei 298 FA-Patienten aus Nord/Südamerika gab es folgende Verteilung: 55% FANCA, 10% FANCC, 5% FANCD1, 2% FANCD2, 2% FANCE, 4% FANCF, 12% FANCG, 3% FANC-nonACEFG und 7% FANC-nonACD1D2EFG. Material von 72 Patienten konnte nicht analysiert werden, da die EBV-immortalisierten Zellinien gegenüber DNA-kreuzvernetzende Substanzen resistent geworden waren und somit den Phänotyp der FA verloren hatten. Im Rahmen dieser Untersuchungen konnten wir in Europa 14 Patienten identifizieren, bei denen es zu einem Mosaik im peripheren Blut gekommen ist, bei denen sich also im Blut neben FA-Zellen auch "normale" Zellen nachweisen lassen. Bei bisher zwei Patienten konnten wir zeigen, dass diese normalen Zellen sich auf Ebene der genomischen DNA in allen Zellreihen des Blutes nachwiesen ließen und es somit in einer hämatopoetischen Stammzelle dieser Patienten zu einer spontanen Reversion in dem defekten Gen zu einem normalen Allel gekommen sein muss. Diese einzelne normale Stammzellen hat den über einen Zeitraum von Jahren hinweg kontinuierlich Nachkommenschaft hervorgebracht, d. h. normale Blut- und Immunzellen gebildet. Gleichzeitig sterben die defekten FA-Zellen kontinuierlich ab, so dass die Patienten hämatologisch normal und klinisch geheilt sind. Wie unsere Untersuchungen zeigen, scheint eine wichtige Voraussetzung für diese "natürliche" Gentherapie zu sein, dass die Patienten eine spezielle Art von Mutationen haben, bei denen eine Reversion überhaupt möglich ist. Vor diesem Hintergrund könnte eine Gentherapie von hämatopoetischen Stammzellen den genetisch korrigierten Zellen in vivo einen Wachstumsvorteil bringen, so daß auch bei niedriger Gentransfereffizienz allmählich eine Repopulation des gesamten hämatopoetischen Systems mit korrigierten Zellen erfolgen würde. Deshalb wurde in einem MultiCenter-Ansatz (Cincinnati, Düsseldorf, Indianapolis, Memphis, Seattle) ein Gentherapieprotokoll verfasst, das bereits von der Food & Drug-Administration der U.S.A. zur Begutachtung prinzipiell genehmigt wurde. Durch die zwei Fälle von Insertionsmutagenese sind allerdings von der FDA weitreichende zusätzliche Untersuchungen gefordert worden, die vor einer Aktivierung des Protokolls noch erbracht werden müssen. Die zwei Vektoren mit dem korrekten FANCA- und FANCG-Gen werden zur Zeit in Indianapolis bzw. Cincinnati für einen klinischen Einsatz unter GMP-Bedingungen hergestellt und sicherheitsgetestet. Beide Vektoren wurden in Düsseldorf konstruiert. In Kooperation mit Dr. Juan Bueren, CIEMAT, Madrid, Spanien konnten wir zeigen, dass sich mit unseren onkoretroviralen Vektoren auch in Zellen von fanca-/- (knock-out) Mäusen der zelluläre Phänotyp der Zellen korrigieren ließ. Zukünftige Untersuchungen Auch weiterhin werden wir bei den neu diagnostizierten FA-Patienten mittels retroviralem Gentransfer in vitro das betroffene Gen identifizieren und somit für die kooperierenden Humangenetiker, Frau Dr. Auerbach, und Herrn PD Dr. Schindler, die Voraussetzungen zu schaffen, um in dem jeweiligen Gen die beiden Mutationen zu finden. Zur Zeit ist es noch unklar, welchen Stellwert die somatische Gentherapie als Therapieoption bei monogenetischen Erkrankungen in der Zukunft haben wird. Insertionsmutagenese als Komplikation der Stammzellgentherapie mit onkoretroviralen Vektoren ist nur bei den beiden SCID-Patienten aufgetreten, nicht aber bei den anderen 230 Patienten, die weltweit z. T. mit gleicher Erkrankung und gleichem Vektor behamdelt wurden. Leider gibt es bisher auch kein geeignetes Tiermodell, in dem man systematisch den Mechanismus der Leukämogenese untersuchen und entsprechende Vermeidungstrategien entwickeln könnte.

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67. Gene Therapy Reduces Drinking in Rats with Genetic Predisposition to "Alcoholism" Finding confirms earlier result using better model for human alcohol abuse

bnl.gov (Internet), 5.5.2004
http://www.bnl.gov/bnlweb/pubaf/pr/2004/bnlpr050504a.htm

UPTON, NY &emdash; As a follow up to previous work showing that gene therapy can reduce drinking in rats trained to prefer alcohol, scientists at the U.S. Department of Energy’s Brookhaven National Laboratory have used the same technique to cut drinking in rats with a genetic predisposition for heavy alcohol consumption. The findings, along with additional results on the effects of long-term ethanol consumption on certain aspects of brain chemistry, are published in the May 2004 issue of Alcoholism Clinical and Experimental Research. "Though we are still early in the process, these results improve our understanding of the mechanism or mechanisms of alcohol addiction and strengthen our hope that this treatment approach might one day help people addicted to alcohol," said Panayotis (Peter) Thanos, who lead the study in Brookhaven Lab’s medical department. Genetically predisposed alcohol-preferring rats are a much better model for human alcoholism than the rats used previously, which the scientists had to train to prefer alcohol. Without any training, the genetic alcohol-preferring rats drink, on average, more than five grams of ethanol per kilogram of body weight per day when given a free choice between alcohol and plain water. Genetically non-preferring rats, in contrast, typically consume less than one gram of ethanol per kilogram of body weight per day. In this study, both groups were treated with gene transfer to increase the level of a brain receptor for dopamine, a chemical important for transmitting feelings of pleasure and reward and known to play a role in addiction. After the gene treatment, the alcohol-preferring rats exhibited a 37 percent reduction in their preference for alcohol and cut their total alcohol consumption in half &emdash; from 2.7 grams per kilogram of body weight before treatment to 1.3g/kg after. Non-preferring rats also reduced their drinking preference and intake after gene treatment, but not in nearly as dramatic a fashion. The greatest reductions in alcohol preference and consumption were observed within the first few days after gene treatment, and both preference and consumption returned to pre-treatment levels by day 20. The gene administered was for the dopamine D2 receptor, a protein shown in various studies to be relevant to alcohol and drug abuse. For example, low levels of dopamine D2 receptors in the brain have been postulated to lead to a reward deficiency syndrome that predisposes certain people to addictive behaviors, including drug and alcohol abuse. The alcohol-preferring rats used in this study have about 20-25 percent lower levels of dopamine D2 receptors when compared to the non-preferring rats, which may, in part, explain their tendency toward heavy drinking. The scientists delivered the gene by first inserting it into a virus that had been rendered harmless. They then injected the virus directly into the rats’ nucleus accumbens, the brain’s pleasure center. The idea behind this type of gene therapy is to use the virus as a vector to carry the gene to the brain cells, which can then use the genetic instructions to make the D2 receptor protein themselves. As an additional measure in this study, the scientists used micro-positron emission tomography (microPET) imaging to non-invasively assess the effects of chronic alcohol consumption on D2 receptor levels in alcohol-preferring and non-preferring rats. They measured D2 levels seven weeks after the gene therapy treatment (well after the effects of gene therapy had worn off). D2 receptor levels in alcohol-preferring rats were significantly lower (about 16 percent) compared to that in non-preferring rats. These levels were similar to previous data in naïve preferring and non-preferring rats. In future studies, the D2 connection to alcoholism will be examined in transgenic mice that are totally depleted of D2. In addition, the scientists plan to develop a second generation D2 vector approach that will provide a longer period of treatment. "These findings further support our hypothesis that high levels of D2 are causally associated with a reduction in alcohol drinking and may serve as a protective factor against alcoholism," Thanos said. This study was funded by the Office of Biological and Environmental Research within the Department of Energy’s Office of Science and by the National Institute of Alcohol Abuse and Alcoholism within the National Institutes of Health

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68. Parkinson: Gentherapie, Wachstumsfaktoren (GDNF, NGF)

med knowledge (Internet), 26.6.2004
http://www.medknowledge.de/abstract/med/med2004/06-2004-26-parkinson-gentherapie-da.htm

Schlüsselwörter: Krankheit, Gene, Parkinson, Gentherapie, Wachstumsfaktoren, GDNF, NGF, Nerve Growth Factor, Therapie, Behandlung, glial cell line-derived neurotrophic factor, Gehirn, Implantation und Operation. Mit Genen und Wachstumsfaktoren den Parkinson stoppen "Während die meisten Experten die Versuche, den Morbus Parkinson durch konventionelle Zelltransplantationen zu heilen, als gescheitert betrachten, gibt es neue Versuche, die Erkrankung direkt im Gehirn zu behandeln. Zwei interessante Ansätze wurden auf der Jahrestagung der American Academy of Neurology in San Francisco vorgestellt. Am Hammersmith Hospital in London wurden fünf Patienten mit fortgeschrittener Parkinson-Erkrankung mit einem Wachstumsfaktor namens "glial cell line-derived neurotrophic factor" (GDNF) behandelt. GDNF soll die Fähigkeit der Nervenzellen zur Produktion von Dopamin wiederherstellen ("neurorestorative Effekt"). Das Molekül wird zu diesem Zweck über einen Katheter direkt in die posteriore dorsale Region des Putamens infundiert. Bei einigen Patienten erfolgte die Behandlung beidseitig, bei den anderen einseitig. Wie Gary Hotton auf dem Kongress berichtet, werden die Patienten seit zwei Jahren behandelt. Die Symptome der Patienten sollen sich deutlich verbessert haben...Die Studiengruppe um Mark Tuszynski von der Universität von Kalifornien in San Diego verfolgt einen ähnlichen Ansatz. Die Wachstumsfaktoren, in diesem Fall der "Nerve Growth Factor" (NGF), werden aber nicht in das Gehirn infundiert, sondern von genetisch veränderten Hautzellen "vor Ort" abgegeben. Die Zellen wurden in das Gehirn implantiert, genauer gesagt in fünf Regionen des Nucleus basalis. Das ist eine Region im Vorderhirn, in der sich cholinerge Nervenzellen befinden..."

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69. Gentherapie bei Parkinson Die Therapie mit menschlichen Genen zur Behandlung von Parkinson macht Hoffnung

aerztliche praxis (Internet), 6.9.2004
http://www.aerztlichepraxis.de/aktuell/artikel/1094457071/neurologie/aktuell

Zum ersten Mal ist ein Patient zur Behandlung der Parkinson-Krankheit einer Gen-Therapie unterzogen worden. . 06.09.04 - Berichten der Society of Chemical Industry zufolge hat der Patient die erste Phase ohne Schwierigkeiten überstanden, was Aussagen der Forscher bekräftigt, dass die Therapie sicher und effektiv ist. "Bis jetzt sind keine unerwünschten Nebenwirkungen in Zusammenhang mit der Gentherapie aufgetreten", sagte der Leiter der Studie Matthew During von der Universität von Auckland. "Unsere Therapie ist extrem sicher und wir hoffen, dass sie zu symptomatischen Verbesserungen führt." Vor einem Jahr war dem ersten Patienten, Nathan Klein, ein Virus injiziert worden, das ein Gen in einen Teil seines Gehirns beförderte. Er berichtet von einer Verbesserung um 40 bis 60 Prozent hinsichtlich seiner Symptome, wenn er zusätzlich Medikamente nimmt, und einer Verbesserung um zehn bis 20 Prozent, wenn er diese nicht nimmt. Vor dem Eingriff hatte er unter starkem Zittern der rechten Körperhälfte gelitten. Roger Barker, ein Parkinson-Experte von der Universität Cambridge, der dem Gen-Versuch kritisch gegenüberstand, sagte, die Tatsache, dass es keine unerwünschten Nebenwirkungen gäbe, sei eine gute Nachricht. Jedoch bekomme der Patient nur niedrige Dosen dieser Medikation, daher sei es nicht möglich zu bestimmen, ob die Gen-Therapie wirkungsvoller ist als eine aggressivere medikamentöse Behandlung oder eine subthalamische Tiefenhirnstimulation. Die Behandlung verwendet einen harmlosen Virus, um einem Teil des Gehirns, der bei Parkinson-Patienten überaktiv ist und ruckartige Bewegungen verursacht, ein Gen zuzuführen. Dieses Gen löst die Erzeugung natürlicher Chemikalien aus, die die überaktiven Gehirnzellen blockieren. Durings Gruppe ist weltweit die einzige, die eine Therapie mit menschlichen Genen zur Behandlung von Parkinson anwendet. Experten gehen davon aus, dass die Gentherapie der Stammzellentherapie bei der Behandlung von Parkinson um 20 Jahre voraus ist.

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70. Gentherapie lässt Fettpolster schwinden

netz zeitung (Internet), 10.2.2004
http://www.netzeitung.de/spezial/gentechnik/272676.html

Im Tierversuch ist gelungen, was viele Übergewichtige vergeblich versuchen: Fettspeichernde Zellen wurden zu Fett verbrennenden Zellen «umprogrammiert». Eine Gentherapie könnte sich als wirksame Behandlung für extrem Übergewichtige erweisen. Forscher konnten mithilfe der genetischen Manipulierung von Fettzellen bei Ratten einen Kalorienverbrauch auslösen, der die Fettreserven weit schneller und nachhaltiger aufzehrte als jegliche Beschränkungen der Kalorienaufnahme. Reduzierung um ein Viertel Die Wissenschaftler um Roger Unger von der US-amerikanischen University of Texas benutzten ein Virus, um das Gen für das Protein «Leptin» in das Erbgut der Fettspeicher-Zellen einzufügen. Leptin ist ein Hormon, das in Fettzellen produziert wird. Es wirkt auf das Gehirn und vermittelt dort das Gefühl von Sättigung. Durch die Gentherapie wurden die Zellen der Versuchstiere zu einer Überproduktion dieses Hormons gebracht. Bei den behandelten Ratten, die genetisch zur Entwicklung von Diabetes veranlagt waren, führte die Gentherapie innerhalb von 14 Tagen zu einer deutlichen Gewichtsreduktion. Von durchschnittlich 280 Gramm Gewicht nahmen sie auf 207 Gramm ab, reduzierten ihr Körpergewicht um etwa ein Viertel. Die Tiere verringerten zudem ihre Nahrungsaufnahme um etwa dreißig Prozent, berichten die Forscher in der aktuellen Ausgabe des Magazins «Proceedings of the National Academy of Sciences». Mechanismus unklar Die mikroskopische Untersuchung der Fettzellen zeigte, dass die Fettspeicher deutlich an Größe abnahmen. «Die Struktur der Zellen veränderte sich von der normalen Erscheinung einer Fettzelle zu einer neuartigen Zelle, die so noch nicht gesehen wurde», sagte Unger dem Online-Dienst des britischen Senders BBC. Auch das Zellinnere veränderte sich: Die Zahl der Mitochondrien, der Energie-produzierenden Organellen, nahm zu. Die Leptin-Gentherapie ließ zudem die Konzentration der den Fettstoffwechsel anregenden Enzyme ansteigen und die der hemmenden sinken. Schädliche Nebenwirkungen wie sie etwa bei einer Nulldiät auftreten können, blieben aus, sagen die Forscher. Der Abbau betraf ausschließlich Fettzellen und nicht Muskelmasse oder andere Gewebe. Es gab auch keine Anreicherung von Fett-Abbauprodukten im Blut. Zwar sei bislang ungewiss, wie das im Übermaß produzierte Leptin in den Stoffwechsel eingreift, die Ergebnisse ihrer Studie könnten sich jedoch auf die Behandlung von Fettleibigkeit bei Menschen auswirken, sagen die Forscher. Dazu bedarf es aber weiterer Studien. So hat die Leptin-Gentherapie bei Ratten, die genetisch nicht zur Entwicklung des Diabetes veranlagt waren, innerhalb von sieben Tagen zum Verlust des gesamten sichtbaren Körperfetts geführt. Bis aus dem Gentherapie-Ansatz eine sichere Behandlung Übergewichtiger entwickelt wird, bleiben Bewegung und die Kontrolle der Kalorienaufnahme die wichtigsten Mittel zur Körperfett-Reduzierung. Für das Web ediert von Patrick Eickemeie

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71.

home t-online (Internet),
http://home.t-online.de/home/skruip/html/gentherapie.html

Die CF-Gentherapie scheiterte bislang u.a. daran, dass den Viren der Zugang zu menschlichen Lungenepithelzellen verwehrt wurde. Forscher von der University of North Carolina in Chapel Hill haben jetzt ein kleines Signalmolekül (UTP) an die Viren gekoppelt, das wie ein Türöffner an der Zellmembran wirkt. Das UTP interagiert mit einem Rezeptor der Zellen und läßt die Viren als trojanische Pferde so aussehen, als ob sie in die Zellen gelangen sollen. Spectrum der Wissenschaft am 31.05.00 schrieb außerdem, dass die Methode demnächst am CF-Mausmodell getestet werden soll. Die Gentherapie der Immunschwäche SCID, bei der die Kinder in abgeschirmten Plastikhüllen leben müssen, gelang erstmals nach 10 Jahren Forschung am Pariser Necker Krankenhaus. Die zwei Kinder haben mit Antikörpern auf diverse Impfungen reagiert. Damit scheint die schwere Krise der Gentherapie nach einem Todesfall in den USA überwunden, wie ABC-News am 27.04.2000 meldete. Das Londoner Forscherteam um Prof. Bob Williamson erzielte mit einer Gentherapie mit Liposomen in der Nasenschleimhaut von Mukoviszidose-Patienten einen Teilerfolg. Die genbedingten Störungen wurden meßbar reduziert, allerdings nur zu etwa 20 % und lediglich zwei bis drei Tage lang. Liposomen sind winzige ölige Fett-Tröpfchen, die sich natürlicherweise mit der Zellmembran vereinigen und durch ein Aerosolspray in die Nase gelangten. Die im Liposom verpackten Gene werden auf diese Weise in die Zellen aufgenommen. Diese "Lipofektionsmethode" soll nun durch den Einsatz von Promotern ("Leistungssteigerern") hinsichtlich Wirkung und Wirkungsdauer verbessert werden. Die Forscher hoffen angesichts ihrer eigenen Erfolge, in zwei oder drei Jahren einen deutlichen Therapieerfolg am Patienten erzielen zu können, wie die Therapiewoche (Nr. 24/95, S. 1406) berichtete. Obwohl zuvor in Labor- und Tierversuchen vielversprechend verlaufen, ist die Gentherapie mit Adenoviren an zwölf Mukoviszidose-Patienten fehlgeschlagen, wie das Team unter Richard C. Boucher von der Universität von North Carolina im New England Journal of Medicine (Bd. 333, Nr. 13, S. 823) im September 95 berichtete. In Kommentaren wurde aber davor gewarnt, diesen Mißerfolg gleich als Todesstoß für die Gentherapie zu verstehen. Er sei lediglich ein Anstoß für weitere Forschungsarbeiten. Diese dpa-Meldung vom 29.09.95 wurde unter anderem von Neues Deutschland und die tageszeitung gedruckt. Zur Zeit sind sich alle gesellschaftlichen Gruppen in Deutschland einig, daß keine Keimbahntherapie am Menschen durchgeführt werden darf, da die veränderten Gene über die Veränderung der Keimzellen an nächste Generationen weitergegeben würde ("Menschenzüchtung"). Bei der somatischen Gentherapie sollen nur Zellen behandelt werden, die nicht die Fortpflanzung betreffen. Da sich die Forscher aber nicht so sicher waren, daß die Erbanlage nicht doch in die Keimbahn gelangt, hatten bislang nur Männer mit CF an der Gentherapie mit Liposomen teilgenommen. Männer mit CF sind unfruchtbar, sodaß kein Risiko besteht, daß das Gen weitergegeben wird. Inzwischen haben englische Forscher durch Versuche an Mäusen geklärt, daß die zusätzliche Erbanlage nicht in die Eizellen gelangt, sodaß erstmals auch Frauen mit CF an der nächsten Studie teilnehmen werden, wie die Frankfurter Rundschau am 04.10.95 berichtete.

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72. Ex-vivo-Gentherapie der Epilepsie

uni zuerich (Internet),
http://www.unicom.unizh.ch/journal/archiv/1-99/gentherapie.html

Der Ausdruck Gentherapie löst Neugier und Faszination, aber auch Vorbehalte aus. Am Beispiel der Epilepsie soll erläutert werden, unter welchen Bedingungen eine Gentherapie Anwendung finden kann. VON HANS M…HLER Die Epilepsie ist eine häufige Störung der Gehirnfunktion, die ein Prozent der Bevölkerung betrifft und auch zahlreiche Persönlichkeiten der Geschichte nicht verschonte, unter anderem Alexander der Grosse, Julius Cäsar, Napoleon, Moli_re, Van Gogh, Tschaikowsky und Alfred Nobel. Die Symptome reichen von kurzen Bewusstseinsabsenzen, zum Beispiel Episoden eines starren Blicks bei Kindern, bis zu generalisierten Krämpfen des gesamten Körpers. Die Anfälle beruhen auf episodischen ÇEntladungsgewitternÈ der erregenden Nervenzellen im Gehirn und sind bei chronischem Verlauf mit einer zunehmenden Zelldegeneration verbunden. Trotz einer Fülle von antiepileptischen Medikamenten, die zudem oft nicht frei von Nebenwirkungen sind, gelingt es nur bei siebzig bis achtzig Prozent der Patienten, die Symptome medikamentös zu beherrschen. Ein riskanter neurochirurgischer Eingriff bleibt oft die einzige Alternative. Um in Zukunft auch für die Therapie-resistenten Patienten eine medikamentöse Behandlungsmöglichkeit zu eröffnen, ist eine neue Therapiestrategie notwendig. Voraussetzung für ein effektives Medikament ist ein neues Wirkungsprinzip, welches keinem der heute verwendeten Antiepileptika zugrunde liegt. Körpereigenes Adenosin Ein solches Therapieprinzip bietet die körpereigene Substanz Adenosin. Experimentell unterdrückt Adenosin sehr effizient die erregende Neurotransmission im Gehirn und bietet den Nervenzellen Schutz vor Degeneration. Adenosin kann jedoch nicht konventionell als Medikament eingenommen oder injiziert werden, da die Substanz selbst und ihre synthetischen Analoge starke negative Auswirkungen auf den Kreislauf ausüben. Um die krampfblockierende und neuroprotektive Wirkung von Adenosin auszunutzen, ist eine direkte Applikation ins Gehirn notwendig. Dies kann erreicht werden durch eine Transplantation von Zellen, welche Adenosin freisetzen. In einem Forschungsansatz werden Zellen tierischen Ursprungs experimentell durch¤nderung ihrer Genstruktur so manipuliert, dass sie Adenosin kontinuierlich abgeben. Zur Vermeidung von Abstossungsreaktionen im Organismus werden die Zellen zunächst in dünne Plastikkanülen verkapselt, die nur für niedermolekulare Substanzen durchlässig sind. Zur Prüfung der antikonvulsiven Wirksamkeit der Adenosin-freisetzenden Zellen werden die Zellkapseln unter Anästhesie in den Hirnventrikel von Ratten transplantiert. Nach der Transplantation können bei den Tieren keine epileptischen Anfälle mehr ausgelöst werden. Dieser antikonvulsive Effekt hält bisherüber mehrere Wochen an. Bemerkenswert ist dabei, dass die Tiere keinerlei erkennbare Nebenwirkungen zeigen, insbesondere keine Schläfrigkeit, Bewegungsarmut oder Einschränkung des sozialen Kontakts. In Kontrollversuchen blieben Kapseln mit gentechnisch nicht veränderten Zellen ohne Wirkung. Ausblick Die Strategie einer Ex-vivo-Gentherapie zur Unterdrükkung von epileptischen Anfällen erscheint prinzipiell realisierbar. Jetzt müssen weitere wichtige Bedingungen erfüllt werden: anhaltende Freisetzung von Adenosinüber Monate beziehungsweise Jahre, Einbau von zellulären Sicherungssystemen und Steuerbarkeit der Adenosinfreisetzung. Falls diese Kriterien erfüllt werden, kann eine Anwendung bei Therapie-resistenten Patienten in Betracht gezogen werden. Der jetzt gelungene erste Schritt macht Mut, diesen Weg weiterzuverfolgen in Zusammenarbeit mit experimentellen Forschern und Klinikern. Die Gentechnik wird hierbei eingesetzt, um Zellen im Reagenzglas (ex vivo) gentechnisch so zu verändern, dass sie als ÇMedikament-SpenderÈ eingesetzt werden können. Nach der Transplantationüben die genetisch veränderten Zellen ihre therapeutische Wirkung aus.

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74. Thérapie génique de la drépanocytose - Guérison de la souris

Science, n°5550, vol. 294 (Internet), 1.1.2002
http://www.forumlabo.com/2002/actus/actus/INSERM/0102therapie.htm

Janvier 2002 - Les résultats publiés en décembre dans Science obtenus grâce à une collaboration franco-américaine coordonnée par Philippe Leboulch, directeur de l'Equipe Inserm 111, responsable d'un laboratoire au MIT et professeur à Harvard Medical School, marquent une étape importante vers la thérapie génique de la drépanocytose, une des maladies génétiques les plus fréquentes au monde due à une mutation d'un gène de l'hémoglobine. En collaboration avec Yves Beuzard (Equipe Inserm 111, Institut universitaire d'Hématologie, Hôpital Saint-Louis, Paris), Philippe Leboulch vient de guérir des souris drépanocytaires en introduisant chez ces animaux un gène produisant une hémoglobine " anti-drépanocytaire " en quantité élevée. Ces recherches ont reçu le soutien de l'Association française contre les myopathies (AFM), organisatrice du Téléthon. La drépanocytose, également appelée anémie falciforme, est causée par la mutation d'un gène de l'hémoglobine : le gène de la globine. L'hémoglobine produite, l'Hémoglobine S, est alors anormale. Elle forme des fibres dans les globules rouges quand elle libère l'oxygène qu'elle transporte des poumons aux tissus. Ces fibres déforment et rigidifient les globules rouges, qui bloquent les petits vaisseaux sanguins. Il en résulte des douleurs très vives et des complications qui peuvent être mortelles ou responsables de séquelles invalidantes. Il s'agit d'une maladie héréditaire qui affecte aussi bien les filles que les garçons et qui se manifeste seulement lorsqu'on hérite de deux exemplaires du gène muté. Les parents n'en possédant qu'un seul sont en bonne santé. La drépanocytose touche chaque année 300 000 nouveaux enfants dans le monde. Ils deviennent malades quelques mois après leur naissance lorsque l'hémoglobine fœtale est remplacée par l'Hémoglobine S. Les régions les plus touchées sont l'Afrique subsaharienne (un nouveau né sur 100 y est atteint), l'Inde, l'Amérique, les Antilles et la Guyane, certaines régions méditerranéennes et le Moyen-orient. En France métropolitaine, quelque 200 futurs malades sont dépistés à la naissance chaque année. Le nombre de patients y est de l'ordre de 5000. Le travail de l'équipe de Philippe Leboulch a consisté à transférer le gène thérapeutique ( globine) dans les cellules souches hématopoïetiques ( = qui donnent naissance à toutes les cellules du sang) de souris drépanocytaires et à l'intégrer dans l'ADN cellulaire. Ce transfert a été fait grâce à un vecteur dérivé d'un lentivirus, dont les gènes codant pour les protéines virales ont été éliminés. A la place, les chercheurs ont inséré un gène de globine " antidrépanocytaire " et les éléments nécessaires à son expression à un niveau élevé et uniquement dans les cellules précurseurs des globules rouges, les érythroblastes. L'hémoglobine produite a, non seulement, une fonction normale, mais empêche également la formation des fibres d'Hémoglobine S. Résultat : les souris drépanocytaires traitées ont été guéries à long terme, en toute sécurité. Avant d'envisager un premier essai clinique de cette thérapie chez l'homme, les scientifiques devront vérifier que l'efficacité thérapeutique et l'innocuité des lots cliniques sont confirmées sur les cellules humaines. Les chercheurs espèrent également, dans l'avenir, parvenir à augmenter encore le nombre de cellules souches corrigées. Ces travaux marquent une avancée importante sur la voie de la thérapie génique de la drépanocytose chez l'homme. A l'heure actuelle, la greffe de moelle osseuse ou de sang de cordon constitue le seul traitement radical possible pour les malades atteints de drépanocytose, mais seule une minorité peut en bénéficier faute de donneur compatible ou encore du risque thérapeutique jugé trop élevé. La thérapie génique permettra de guérir les malades avant même que les manifestations de la maladie n'apparaissent.

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76. THERAPIE GENIQUE POUR TRAITER LE DIABETE

Nature Medecine Vol 9(5):596-603, mai 2003., 1.9.2003
http://www.gircor.net/actu/act8.htm

Le diabète est touche entre 3 et 4 % de la population française. Il concerne les enfants et les adultes avec une incidence plus importante chez les personnes âgées. Actuellement, plusieurs voies de thérapie génique sont envisagées pour traiter cette maladie. Le diabète Stockage de l'énergie après un repas Le diabète est une maladie due à un défaut d’absorption du sucre apporté par l’alimentation. Lors des repas, le sucre produit par la digestion des aliments est transporté par le sang vers le foie. Dans le foie, une hormone produite par le pancréas, l’insuline, permet la mise en réserve du sucre sous forme de glycogène. En dehors des repas, le glycogène est utilisé pour redonner du sucre, et ainsi de l’énergie, à l’organisme. Chez les personnes diabétiques, ce processus de stockage du sucre ne se fait pas correctement : chez certains malades la quantité d’insuline produite n’est pas suffisante (diabète de type I ou diabète insulinodépendant), chez d’autres, l’insuline produite n’est pas capable d’agir pour faire entrer le sucre dans les cellules (diabète de type II ou diabète non-insulinodépendant). Dans tous les cas, ces anomalies se traduisent par une augmentation du taux de sucre dans le sang (= augmentation de la glycémie) généralement associée à de la fatigue, un amaigrissement et des effets néfastes pour l’organisme à long terme. Les traitements Les traitements actuels sont essentiellement basés sur l’injection d’insulinepour faire baisser la glycémie. Ces traitements sont très contraignants car ils nécessitent des contrôles fréquents de la glycémie du patient et plusieurs injections d’insuline par jour. Pour essayer de diminuer ces contraintes, l’utilisation des greffes de cellules du pancréas est une voie de recherche en cours de développement. Mais comme pour toute greffe, ce traitement implique la compatibilité entre le donneur et le receveur. De plus, il faut généralement plusieurs donneurs pour greffer un seul patient ce qui rend ce type de traitement très difficile à mettre en pratique. La thérapie génique Les progrès de la thérapie génique ont amené les chercheurs à tester de nouvelles approches pour traiter le diabète. Ces approches ont pour but essentiel de maintenir la glycémie à un niveau normal. Cette thérapie aurait donc le même effet que l’injection d’insuline mais le traitement serait beaucoup moins contraignant. o La voie de thérapie génique la plus souvent explorée pour traiter le diabète consiste à introduire dans les cellules du foie un gène permettant la production d’insuline. Cependant, l’insuline, normalement synthétisée dans le pancréas, doit être activée par des enzymes que le foie ne possède pas. Pour que cette approche soit efficace, il faudrait trouver le moyen de faire fabriquer ces enzymes par le foie. o D’autres chercheurs ont exploré une autre voie de thérapie génique. L'objectif est de favoriser dans le foie le développement de cellules semblables à celles du pancréas. Ces cellules produiraient non seulement l’insuline mais également les enzymes nécessaires à son activation. C’est cette approche est actuellement étudiée en collaboration par deux équipes de chercheurs japonais et américains. Ces chercheurs ont publié en mai dans la revue Nature Medecine les résultats d’une étude faite chez des souris qui développent un diabète aigu sous l’action de la streptozotocine. Ils ont introduit par thérapie génique une combinaison de deux gènes (Neurod et Btc) dans ces souris. Le vecteur de transfert est un adénovirus modifié afin ne pas induire d’effets secondaires toxiques chez l’animal. L’introduction de ces deux gènes se traduit par l’amélioration de l’état de santé des souris qui retrouvent une glycémie normale. De plus, il se forme, dans le foie de ces souris, des îlots de cellules capables de produire de l’insuline et les enzymes nécessaires à son activation. Ces expériences ont donné des résultats très encourageants qui confirment qu’il est envisageable d’utiliser la thérapie génique pour le traitement du diabète.

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78. MODELE EXPERIMENTAL DE THERAPIE GENIQUE DES METASTASES HEPATIQUES DU CANCER PANCREATIQUE PAR TRANSFERT IN VIVO DU GENE SST2

SNF GE .org (Internet), 26.3.2002
http://www.snfge.org/01-Bibliotheque/0A-Resumes-JFPD/2002/Mardi/seance_pleniere/1.htm

(1) INSERM U531, Institut Louis Bugnard, CHU Rangueil, 31403 Toulouse (2) Institut Louis Bugnard, CHU Rangueil, 31403 Toulouse Introduction Nous avons récemment proposé une approche pré-clinique de thérapie génique du cancer pancréatique exocrine, basée sur l'effet anti-oncogénique du récepteur sst2 à la somatostatine. En effet le rétablissement ex vivo de l'expression du récepteur sst2 est capable d'induire dans des modèles expérimentaux de cancers pancréatiques une inhibition de la croissance tumorale, une inhibition de la progression métastatique et un effet anti-tumoral bystander in vivo . Le but de notre travail était de créer un modèle de métastases hépatiques pancréatiques chez le hamster et d'y réaliser un transfert in vivo du gène du récepteur sst2 et d'en évaluer l'effet anti-oncogénique. Matériel et méthodes Les métastases hépatiques étaient obtenues par injection intra portale (J0) de cellules cancéreuses pancréatiques de hamster PC-1.0. A J15, après laparotomie, un transfert intra-tumoral du gène sst2 était réalisé sur une métastase cible dont le volume était préalablement déterminé (groupe sst2, n = 10 hamsters). Le transfert de gène était effectué à l'aide d'un transporteur synthétique, le polyéthylenimine (PEI 22 kDa). Le groupe témoin recevait le gène lacZ (groupe lacZ, n = 9). A J20 (5 jours après transfert), les animaux étaient sacrifiés avec évaluation : du volume tumoral, de l'expression du transgène, de l'index PCNA et de la TUNEL reaction. Résultats Notre modèle était reproductible avec un taux de métastases hépatiques à J15 de 89,3 % avec un volume moyen de 71mm3 ± 26 et de 100 % à J20. Cinq jours après transfert in vivo, l'expression du récepteur sst2 était retrouvée par RT-PCR dans toutes les métastases cibles. Les métastases exprimant le récepteur sst2 présentaient une inhibition significative de la progression tumorale par rapport au groupe témoin lacZ (groupe sst2 progression de 202 ± 47 % versus 622 ± 143 pour le groupe LacZ &emdash; p < 0,02). Par immunohistochimie PCNA, une diminution significative (p < 0,001) de l'index de prolifération tumorale était mesurée dans le groupe sst2 par rapport au groupe lacZ (groupe sst2 : 43 % versus 69 % pour le groupe LacZ). En parallèle une activité pro apoptotique significative (p < 0,004) était mesurée par TUNEL reaction dans le groupe sst2 par rapport au groupe lacZ. Conclusion Un modèle original et reproductible de métastases hépatiques de cancer pancréatique a été créé. Par ailleurs nous avons montré que le transfert du gène sst2 in vivo à l'aide du PEI 22 KDa exerce un effet anti-tumoral en diminuant la prolifération cellulaire et en augmentant les phénomènes d'apoptose dans les métastases hépatiques. Ces derniers peuvent être à l'origine d'un effet anti-tumoral « bystander » local. Il peut s'agir d'une approche thérapeutique novatrice dans la prise en charge palliative du cancer pancréatique.

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79. De nouvelles perspectives sur les rétinopathies pigmentaires.

Le Rétino n° 42, juin 2002. (Internet), 1.6.2002
http://www.retina-france.asso.fr/ret42-rp.html

José Sahel est Professeur d'ophtalmologie, Directeur de l'équipe Inserm physiologie cellulaire et moléculaire de la rétine, récemment nommé Chef de service au Centre Hospitalier National d'Ophtalmologie des Quinze-Vingt à PARIS. "Longtemps, la spécificité de la maladie a conduit à ne pas la faire figurer parmi les priorités de l'industrie pharmaceutique et à rejoindre le cortège des affections dites orphelines" explique le Professeur José Sahel. Grâce notamment aux efforts financiers d'associations de malades, d'autres voies de recherche ont pu être explorées parmi lesquelles la thérapie génique et les transplantations cellulaires. 1. Les bâtonnets sous l'oeil de la thérapie génique. Depuis une dizaine d'années, la découverte de mutations à l'origine de la rétinopathie pigmentaire a évolué à grande vitesse, 120 locus (segments de chromosome) responsables ont été repérés, et la moitié caractérisés. "Le premier gène identifié a été celui de la rhodopsine, rappelle le Professeur Sahel. Il s'agit d'un pigment initiant la transduction visuelle au niveau du segment externe des bâtonnets. L'apoptose (mécanisme de mort cellulaire programmée) a été mise en évidence au niveau des bâtonnets en 1993. Plus de 60 gènes ont été caractérisés depuis." Les travaux récents d'une équipe américaine en thérapie génique ont consisté dans le cadre d'expérimentations animales, à réintroduire un gène (RPE65) exprimé dans l'épithélium pigmentaire nécessaire au métabolisme de la vitamine A (pigments visuels). Des chiens porteurs d'une forme grave de rétinopathie pigmentaire ont pu retrouver certaines capacités visuelles. La thérapie génique ouvre donc des espoirs devant la restauration fonctionnelle de la rétine, même si de nombreuses étapes demeures à valider. 2. Limiter la disparition des cônes par implants de bâtonnets. Les défauts génétiques mis en évidence ont parfaitement expliqué la mort des bâtonnets et la perte de vision nocturne mais ne pouvaient, par la suite, expliquer la dégénérescence des cônes et la perte de vision centrale en résultant. "Mon équipe de recherche, explique José Sahel, a privilégié deux hypothèses. La première était celle d'une neurotoxicité initiale ayant entraîné la mort des bâtonnets. La seconde était l'idée d'une perte de viabilité cellulaire des cônes en l'absence de bâtonnets." Cette dernière hypothèse semble aujourd'hui validée par les effets de transplantations de bâtonnets en couches sur des animaux porteurs de rétinopathie pigmentaire. Réalisées à un moment où l'ensemble des bâtonnets a dégénéré, et où les cônes ont seulement entrepris leur processus de dégénérescence, elles ont permis de ralentir de moitié la vitesse d'élimination des cônes. Il est prouvé aujourd'hui que la survie des cônes est liée à la libération d'une protéine par les bâtonnets. La transplantation de ces photorécepteurs a été proposée sur l'humain par l'équipe du Professeur Sahel. Elle a été freinée par un manque d'infrastructures, mais aussi par la nécessité de trouver des donneurs à coeur battant, dans le cadre de prélèvements multiples ; enfin par la législation sur le risque de transmission de prions, l'oeil étant en effet un organe cible pour la maladie de Creutzfeld Jacob. 3. Des thérapies à valider, d'autres pistes à explorer. En matière de thérapie génique, il a donc été démontré dans des modèles animaux que la restauration fonctionnelle était possible. Mais la multiplicité des mutations, les problèmes posés par les mutations dominantes, la nécessité de valider la thérapie en toute sécurité laissent encore beaucoup de travail aux chercheurs. En matière de transplantations, l'avancement des travaux est plus avancé puisque l'on travaille déjà sur la pathologie humaine. Mais la validation reste à faire pour mesurer dans le temps l'évolution du champ visuel central et de l'acuité visuelle centrale de l'oeil opéré par rapport à l'oeil non greffé. "Un recul de trois à cinq ans est nécessaire, estime José Sahel, pour avoir de véritables résultats." "D'autres pistes sont aujourd'hui explorées, consistant notamment à tenter de caractériser la ou les molécules impliquées dans la signalisation assurant la viabilité des cônes. Leur identification définitive sur la chaîne ADN et des administrations locales permettraient d'éviter la greffe des photorécepteurs. Des approches pharmacologiques peuvent elles aussi être envisagées, qu'il s'agisse d'inhibiteurs de l'apoptose, de métabolites de la vitamine A ou de bloquants des canaux calciques. Quelles que soient les solutions thérapeutiques envisagées, les chercheurs de l'équipe du Professeur Sahel savent que toutes protections permettant de protéger les photorécepteurs de la dégénérescence constituent un enjeu essentiel. Une population résiduelle de 25 % de bâtonnets, même non fonctionnels, suffirait à assurer la survie des cônes. Et une population de 10 % des cônes pourrait permettre une vision conservée et donc une vie normale. "Il devient aujourd'hui possible d'envisager, explique José Sahel, pour le patient atteint de RP, de prolonger l'usage de sa vision centrale. Et il n'est pas interdit d'envisager que nos connaissances sur la préservation des cônes puissent aboutir à moyen terme, sur des résultats applicables à la DMLA, qui demeure la principale cause de cécité de l'adulte".

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80. Treatment hope for nerve disease

bbc news (Internet), 27.5.2004
http://news.bbc.co.uk/1/hi/health/3750125.stm

Thursday, 27 May, 2004, 00:52 GMT 01:52 UK Cells are lost in the brain Scientists may have developed a gene therapy treatment for the most common form of motor neurone disease (MND). In lab tests on mice the therapy slowed onset and progression of Amyotrophic Lateral Sclerosis (ALS). It also extended life expectancy by 30%. Writing in the journal Nature, the research team at biopharmaceutical firm Oxford BioMedica stressed the work is at an early stage. MND affects about 5,000 people in the UK and there are 1,000 new cases a year.The disease is caused by the death of cells - called motor neurones - that control movement in the brain and spinal cord. There is currently no known cure.ALS is a form of the disease which affects adults, leading to paralysis and death within five years for most patients. Key gene The new treatment - called MoNuDin - essentially consists of a gene which triggers production of a chemical called a vascular endothelial growth factor (VEGF).The gene is injected into the muscles, but stimulates VEGF production in the nerve cells of the spine.ALS has been linked to reduced levels of VEGF in both mice and humans. It is thought that the chemical plays a key role in protecting nerve cells from damage.Tests on mice showed that a single shot of the new therapy was enough to produce a significant beneficial effect.Professor Alan Kingsman, Oxford BioMedica chief executive, said: "Although these results published in Nature are still at a preclinical stage, the data suggests that VEGF gene therapy could provide an effective treatment for ALS."Dr Brian Dickie, of the MND Association, welcomed the findings.He said: "These findings reflect current optimism amongst researchers that gene therapy represents a viable strategy for the treatment of ALS and other neurodegenerative diseases, overcoming problems of access of drugs to the central nervous system, which can occur with more conventional approaches to treatment." The Oxford BioMedica team worked on the treatment in collaboration with the Center for Transgene Technology and Gene Therapy in Belgium.

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81. Genetic "smart bomb" knocks out hepatitis

Nature Biotechnology (Internet),
http://www.newscientist.com/hottopics/tech/article.jsp?id=99993642&sub=Biotechnology

Human liver cells harbouring the hepatitis C virus can be selectively targeted and destroyed by a new gene therapy approach, according to new research.The key is a genetically-engineered "suicide" gene, delivered aboard a harmless virus, which is triggered only when it enters a hepatitis-infected cell.The two current treatments for the debilitating liver disease - alpha interferon and ribavarin - can reduce the level of infection, say researchers, but the virus usually comes back. The new gene therapy approach could one day "offer the potential of a total cure" for many people, says virologist Christopher Richardson, at the Ontario Cancer Institute in Toronto, Canada, and one of the research team. It might also help tackle other viruses, such as HIV. About 200 million people worldwide are affected by hepatitis C and infections are increasing. In advanced cases, the virus causes the liver to fail completely or become cancerous. Achilles heel The research began when Richardson and colleague Eric Hsu identified an "Achilles heel" in hepatitis C - a unique protease enzyme produced by the virus.Some proteases in human cells trigger proteins to kick-start the process by which the cell commits suicide. So the team removed the genetic code that allows the protein to recognise the human protease and replaced it with code specific to the hepatitis C protease. The DNA for the modified protein was then smuggled into cells using a harmless adenovirus. If a cell is infected, then the viral protease causes it to order its own death. "It's like a suicide vector, a smart bomb," Richardson told New Scientist. No rebound The therapy successfully cleared low and medium level hepatitis C infections in mice with implanted infected human liver cells. In mice suffering high levels of infection, the gene therapy slashed levels of the virus by a factor of 1000. Importantly, the virus did not "rebound" after the gene therapy, as it can do with existing treatments. This is true for at least 28 days after gene therapy and the team is now doing further work to see if this effect lasts longer."It's an incredibly novel approach," said Nigel Hughes, chief executive of the British Liver Trust and an adviser on the UK government's strategy to tackle hepatitis C. "But I have some reservations. If you had this massive cascade of cells dying in the human liver, what would the body's response be? Would you create more harm?"The approach is "futuristic", admits Richardson: "It is very drastic and we would say it should not be used immediately for human trials." An intermediate approach could be to apply the therapy outside the body, he says.In an ex vivo therapy, relatively healthy liver cells could be extracted from patients with an advanced infection, cultured and then exposed to the gene therapy. This would kill any infected cells, meaning healthy cells could be transplanted back and restore some of the liver's function.

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82. New cancer case halts US gene therapy trials

NewScientist.com news service (Internet), 15.1.2003
http://www.newscientist.com/news/news.jsp?id=ns99993271

Nearly 30 US gene therapy trials were halted on Tuesday following the announcement that a second child in a pioneering French gene therapy trial has developed leukaemia following the treatment.The French trial is testing a treatment for "bubble boy" disease, or X-SCID (X-chromosome-linked Severe Combined Immunodeficiency). The initial results of the trial were hailed one of the first great successes for gene therapy.But the trial was halted in October 2002 following the first diagnosis of leukaemia in one of the boys. Three similar US gene therapy trials were suspended at the same time. A similar trial in the UK was not halted, as British doctors argued that without the treatment many of patients would certainly die. Cure rates for childhood leukaemia can be 90 per cent, but boys with SCID almost always die within a year without a bone marrow transplant.However, the second leukaemia case has prompted the US Food and Drug Administration to suspend other trials that use the same type of virus to shuttle therapeutic genes into blood cells. "Precautionary measure" The FDA has no evidence of leukaemia caused by gene therapy in US studies, but says the suspension of trials using retroviruses is a "precautionary measure". The agency will consider specific requests to allow new patients into gene therapy trials tackling life-threatening disorders for which there are no other treatments.The UK's Gene Therapy Advisory Committee says they will maintain their position and not suspend the British X-SCID trial. However, no new patients will be treated until the evidence from the French trial has been looked at, says Stephen Cox, of Great Ormond Street Children's Hospital where the British patients are being treated. The patients have all been assessed in the last month and none shows any sign of leukaemia.Norman Nevin, chairperson of GTAC, speculates that the adverse French results may be due to minor differences in the techniques used. "The design of the vector is not quite the same in the UK as in the French study," he toldNew Scientist.Philip Noguchi, head of gene therapy issues at the FDA, remains optimistic about the overall prospects of gene therapy. "We continue to see gene therapy as a promising therapy for all those who have not benefited from current technologies," he says. No resistance Boys with SCID have no resistance to infection due to a faulty copy of an X-chromosome gene that makes an immune protein called interleukin-2. The gene therapy corrects the genetic defect by shuttling a correct copy of the gene into the patient's cells using a virus. The treatment appears to have cured a number of boys.The two boys with leukaemia are now being treated with chemotherapy and are clinically stable. The leukaemia may have been caused by the fact that the injected DNA cannot be targeted to insert into a specific part of a chromosome.Scientists suspect the first child developed the cancer because the gene inserted next to an oncogene, called Lmo2, in a single white blood cell. This could have triggered the cell to proliferate uncontrollably, causing the disease.According to Reuters, gene therapy experts called to an emergency meeting on Friday at the US National Institutes of Health have said that the development of leukaemia could be unique to the SCID gene therapy trial.

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83. Undercover genes slip into the brain

New Scientist Online News (Internet), 20.3.2003
http://www.newscientist.com/hottopics/tech/article.jsp?id=99993520&sub=Biotechnology

A molecular Trojan horse that can slip past the brain's defences has proved to be very effective at delivering genes to the brains of primates. It could be used to treat a host of brain disorders, from Parkinson's to epilepsy. Treating the brain is very difficult because of the "blood-brain barrier" created by the tight junctions between the cells lining the capillaries. Only molecules recognised by the cell receptors can get in, unless they are very small. The viruses most gene therapists use to deliver genes are too big, and have to be injected directly instead. Even then, the genes are not expressed widely and evenly throughout the brain."Quite frankly, the existing delivery systems have been woeful failures," says William Pardridge of the University of California, Los Angeles. Instead, his team has been perfecting a way to get genes into the brain hidden inside fatty spheres called liposomes.First the team coats the liposomes with a polymer called polyethylene glycol (PEG), without which they would be purged from the blood within minutes. Next, antibodies that latch on to some of the brain-capillary receptors are tethered to a few of the PEG strands. The antibodies trick the receptors into letting the liposomes pass, where they can deliver their cargo to brain cells. Bright light Pardridge's team has already shown that the technique works in rats, by delivering the gene for the luminescent protein luciferase (New Scientistprint edition, 10 June 2000). Now the team has tested the liposomes in rhesus monkeys, using antibodies specific to primate brain receptors. Not only did it work, but the amount of luciferase produced was 50 times greater than in rats (Molecular Therapy, vol 7, p 11)."I haven't seen anything like this for viral or non-viral vectors," says Savio Woo, director of gene therapy at the Mount Sinai School of Medicine in New York. "To reach the central nervous system through the blood-brain barrier in a non-human primate with this kind of efficiency - that's absolutely fantastic."The liposomes do not appear to have any toxic side effects, though they do deliver genes to other organs besides the brain. But the team has shown that by choosing the right switch to turn on the gene, the gene will be active only in the desired tissues.Because the genes are not integrated into the genome, weekly or monthly injections would be needed for long-term treatment. But Pardridge sees this as an advantage, because there's no risk of genes lodging permanently in the wrong place and triggering cancer - a worry with some gene therapy viruses. Abnormal movements The method shows promise for treating Parkinson's. The team gave rats a neurotoxin that causes Parkinson's-like symptoms by cutting production of the key enzyme tyrosine hydroxylase.Four weeks later, the team injected the rats with liposomes containing a gene that boosts production of the enzyme. Three days after that, the rats' abnormal movements were reduced by 70 per cent.The liposomes can also deliver cargoes other than genes, including drugs and "antisense" RNA. The lifespans of mice with brain tumours doubled when the liposomes were used to deliver antisense RNA to block production of a growth factor.And in studies yet to be published, the team has exploited a mechanism called RNA interference, delivering fragments of double-stranded RNA that "silence" cancer genes. From 14:48 20 March 03

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84. "Human Gene Therapy: Present and Future"

The Institute for Human Gene Therapy (Internet), 1.2.1999
http://www.ornl.gov/sci/techresources/Human_Genome/publicat/hgn/v10n1/15wilson.shtml

Institute for Human Gene Therapy, University of Pennsylvania In his presentation at the 1998 Cambridge meeting, James Wilson characterized gene therapy as a novel approach in its very early stages. Its purpose, he said, is to change the expression of some genes in an attempt to treat, cure, or ultimately prevent disease. Current gene therapy is primarily experiment based, with a few early human clinical trials under way. Theoretically, he continued, gene therapy can be targeted to somatic (body) or germ (egg and sperm) cells. In somatic gene therapy the recipient's genome is changed, but the change is not passed along to the next generation. This form of gene therapy is contrasted with germline gene therapy, in which a goal is to pass the change on to offspring. Germline gene therapy is not being actively investigated, at least in larger animals and humans, although a lot of discussion is being conducted about its value and desirability. Gene therapy should not be confused with cloning, which has been in the news so much in the past year, Wilson continued. Cloning, which is creating another individual with essentially the same genetic makeup, is very different from gene therapy.Listing three scientific hurdles in gene therapy, Wilson emphasized the concept of vehicles called vectors (gene carriers) to deliver therapeutic genes to the patients' cells. Once the gene is in the cell, it needs to operate correctly. Patients' bodies may reject treatments, and, finally, there is the need to regulate gene expression. Wilson expressed optimism that many groups are making headway and cooperating to overcome all these obstacles.Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to take advantage of the virus's biology and manipulate its genome to remove the disease-causing genes and insert therapeutic genes. These gene-delivery vehicles will make this field a reality, he said. In the mid-1980s, the focus of gene therapy was entirely on treating diseases caused by such single-gene defects as hemophilia, Duchenne's muscular dystrophy, and sickle cell anemia. In the late 1980s and early 1990s, the concept of gene therapy expanded into a number of acquired diseases. When human testing of first-generation vectors began in 1990, scientists learned that the vectors didn't transfer genes efficiently and that they were not sufficiently weakened. Expression and use of the therapeutic genes did not last very long. In 1995, Wilson continued, a public debate led to the consensus that gene therapy has value although many unanswered questions require continued basic research. As the field has matured over the last decade, it has caught the attention of the biopharmaceutical industry, which has begun to sort out its own role in gene therapy. This is critical because ultimately this industry will bring gene therapies to large patient populations. Wilson reviewed several specific gene-therapy cases involving high cholesterol, hemophilia, and cystic fibrosis. He emphasized that the response to any therapy in a heterogeneous patient population will be quite variable.He asked the audience to think about gene therapy, not necessarily to treat genetic disease but as an alternative way to deliver proteins. Protein therapeutics currently are manufactured by placing genes in laboratory-cultured organisms that produce the proteins coded by those genes. Examples of such manufactured proteins include insulin, growth hormone, and erythropoietin, all of which must be injected frequently into the patient. Recent gene therapy approaches promise to avoid these repeated injections, which can be painful, impractical, and extremely expensive. One method uses a new vector called adeno-associated virus, an organism that causes no known disease and doesn't trigger patient immune response. The vector takes up residence in the cells, which then express the corrected gene to manufacture the protein. In hemophilia treatments, for example, a gene-carrying vector could be injected into a muscle, prompting the muscle cells to produce Factor IX and thus prevent bleeding. This method would end the need for injections of Factor IX --a derivative of pooled blood products and a potential source of HIV and hepatitis infection. In studies by Wilson and Kathy High (University of Pennsylvania), patients have not needed Factor IX injections for more than a year. In gene therapies such as those described above, the introduced gene is always "on" so the protein is always being expressed, possibly even in instances when it isn't needed. Wilson described a newer permutation in which the vector contains both the protein-producing gene and a type of molecular rheostat that would react to a pill to regulate gene expression. This may prove to be one of gene therapy's most useful applications as scientists begin to consider it in many other contexts, he said. Wilson's group is conducting experiments with ARIAD Pharmaceuticals to study the modulation of gene expression. Wilson stated that only so much can be done in academia and that the biopharmaceutical industry has to embrace gene therapy and handle issues of patents, regulatory affairs, and the optimum business model. An example of a dilemma that society may be facing can be seen in the treatment of hemophilia. Infusing a patient with the replacement protein, which stops bleeding episodes but doesn't prevent them, currently costs about $80,000 a year. Why would a vector to prevent bleeding for 5 to 10 years be commercialized when it would displace such a lucrative treatment, and how would this gene therapy be delivered to the public? Wilson concluded his presentation by outlining future milestones in the field: proof of concept in the next few years in model inherited diseases, followed by cancer and cardiovascular diseases; continued explosive activity in technological development; development of regulatory policy (with the Food and Drug Administration); and commercial development. [

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85. Gene Therapy for Thyroid Cancer: Current Status and Future Prospects

medscape.com (Internet), 26.7.2004
http://www.medscape.com/viewarticle/483773?src=search

Abstract Despite multimodality treatment for thyroid cancer, including surgical resection, radioiodine therapy, thyrotropin (TSH)-suppressive thyroxine treatment, and chemotherapy/radiotherapy, survival rates have not improved over the last decades. Therefore, development and evaluation of novel treatment strategies, including gene therapy, are urgently needed. A variety of gene therapy approaches have been evaluated for the treatment of follicular cell-derived and medullary thyroid cancer, including corrective gene therapy (p53 restoration, expression of a dominant negative RET mutant), cytoreductive gene therapy (suicide gene/prodrug strategy herpes simplex virus-thymidine kinase [HSV-tk]/ganciclovir, antiangiogenic therapy with endostatin) and immunomodulatory gene therapy (expression of interleukin (IL)-2 and IL-12). Furthermore, cloning of the sodium iodide symporter (NIS) gene has paved the way for the development of a novel cytoreductive gene therapy strategy based on NIS gene transfer followed by the application of radioiodine therapy (131I). NIS gene delivery into medullary and follicular cell-derived thyroid cancer cells has been shown to be capable of establishing or restoring radioiodine accumulation and might therefore represent an effective therapy for medullary and dedifferentiated thyroid tumors that lack iodide accumulating activity. The data summarized in this review article clearly demonstrate that the currently available strategies represent potentially curative novel therapeutic approaches for future gene therapy of thyroid cancer. The combination of different therapeutic genes has been demonstrated to be very useful to enhance therapeutic efficacy and seems to have a promising role at least as part of a multimodality approach for advanced thyroid cancer. Introduction Thyroid carcinoma represents the most common endocrine malignancy, accounting for the majority of deaths from endocrine cancers. The vast majority of thyroid carcinomas (94%) are papillary and follicular thyroid carcinomas, differentiated tumors derived from follicular epithelial cells. Five percent of thyroid carcinomas are medullary thyroid carcinomas, which arise from C cells, and the remaining 1% consists of anaplastic and poorly differentiated thyroid carcinomas. While the overall survival rates for papillary (98%) and follicular (92%) thyroid cancer are high, the survival rate for medullary thyroid cancer is only 80% and for anaplastic cancer it is as low as 13%.[1] Despite multimodality treatment for thyroid cancer, including surgical resection, radioiodine (131I) therapy, thyrotropin (TSH)-suppressive thyroxine treatment, and chemotherapy/radiotherapy, survival rates have not improved over the last decades. Therefore, development and evaluation of novel treatment strategies, including gene therapy approaches, are urgently needed. Gene therapy for thyroid cancer represents a new technology that, more than any currently available therapy, takes direct advantage of our new understanding of thyroid carcinogenesis at the molecular level. Gene therapy is particularly attractive for the treatment of thyroid cancer because of the possibility of selective targeting of therapeutic genes to tumor cells by application of tissue-specific promoters, such as the thyroglobulin and calcitonin promoter, thereby reducing extratumoral toxicity. In addition, with the possibility of complete thyroid hormone replacement therapy the thyroid gland represents a "dispensable" organ, which allows the clinician to pursue therapeutic strategies, including gene therapy, that might ablate normal as well as malignant thyroid cells. The term gene therapy encompasses a range of approaches (Fig. 1), such as: o Corrective gene therapy: to restore the normal function of a deleted or mutated gene (usually a tumor suppressor gene) or negate the effect of a tumor-promoting gene (oncogene). o Cytoreductive gene therapy: to deliver an exogenous gene that causes cell death or allows the application of toxic agents. o Immunomodulatory gene therapy: to induce gene expression that enhances immune responses against tumor tissues. Corrective Gene Therapy Most poorly differentiated thyroid tumors have lost expression of the normal p53 tumor suppressor gene through inactivating mutations. p53 is a transcription factor mediating critical cellular responses, including cell cycle arrest and apoptosis, after exposure to DNA-damaging stimuli.[2] Mutations in the p53 gene seem to be late genetic events associated with loss of differentiation and are, at least in part, responsible for the aggressive behaviour of advanced and/or dedifferentiated tumors. Restoration of wild-type (wt) p53 expression has recently been used in a variety of experimental cancer models including thyroid cancer and has been tested in human clinical trials.[3-9] p53 restoration has been shown to be associated with a bystander effect, which means that not only p53-transduced cells are killed, but also that surrounding nontransduced cells are killed by the transduction of their neighbors. A bystander effect is highly desirable for a therapeutic gene, because it reduces the level of transduction efficiency required for successful gene therapy, which represents one of the crucial technical hurdles for in vivo gene therapy applications. The bystander effect of p53 gene therapy results from its antiangiogenic effect, which is the result of downregulation of vascular endothelial growth factor (VEGF) and upregulation of thrombospondin, a potent inhibitor of angiogenesis.[2] Several studies of p53 gene therapy of thyroid cancer have been reported. Expression of wild-type tumor suppressor gene p53 (wt-p53) in a p53-null thyroid carcinoma cell line (FRO) resulted in decreased cell growth in vitro and inhibition of tumorigenesis in vivo in nude mice. 40% of mice inoculated with p53-transfected FRO were tumor-free and 60% developed small hypovascular tumors indicating suppression of neovascularization.[4] In another study, retroviral p53 gene transfer into p53 mutant papillary thyroid cancer cells (NPA) resulted in a dose-dependent inhibition of tumor cell growth and enhanced chemosensitivity to adriamycin in vitro and in vivo.[5]Using a replication-deficient adenovirus expressing wild-type tumor suppressor gene p53 (wt-p53), Nagayama et al.[4] evaluated the therapeutic efficacy of p53 restoration in four human anaplastic thyroid cancer cell lines harboring p53 mutations (ARO, FRO, NPA, WRO) and normal human thyroid follicular cells in vitro and in vivo. Adenovirus-mediated p53 expression resulted in dose-dependent cell killing in thyroid cancer cell lines, whereas normal thyroid cells were relatively resistant to p53-mediated cell death despite their highest adenovirus infectivity. The mechanism of cell killing was shown to be apoptosis. In addition, wt-p53 expression sensitized some of the cell lines to the chemotherapeutic effect of doxorubicin (FRO and NPA cells) and 5-fluorouracil (FRO cells). In vivo experiments using FRO and NPA cell xenografts in nude mice showed inhibition of tumor growth following direct injection of the adenovirus expressing wt-p53. This effect was augmented by combination with doxorubicin, resulting in tumor regression.[6] To develop an adenoviral gene transfer system that replicates exclusively in wt-p53-deficient cancer cells thereby limiting the cytotoxic effect of a replication-competent adenovirus lacking E1B55K, Nagayama et al. used the geneinactivation strategy using a p53-regulated Cre/loxP system consisting of two recombinant adenoviruses. One contains an expression unit of the synthetic p53-responsive promoter and the Cre recombinase gene, and the other adenovirus contains two expression units: the first consists of the E1A gene flanked by a pair of loxP sites downstream of the constitutive CAG promoter, and the second consists of the E1B19K gene under the control of the cytomegalovirus (CMV) promoter. Coinfection of these two adenoviruses into p53 expressing cells leads to expression of Cre recombinase, which then excises the E1A gene that is flanked by a pair of loxP sites, thereby stopping virus replication. In cells without p53 expression, however, Cre recombinase is not expressed, the E1A gene not excised, and virus replication takes place thereby causing cell lysis.[7] Another, more recent study by Imanishi et al.,[8] indicated that the histone deacetylase inhibitor depsipeptide enhances apoptotic killing by p53 gene transfer in anaplastic thyroid cancer cell lines (FRO and WRO cells), suggesting that this combination treatment strategy might also be useful in the treatment of undifferentiated thyroid carcinomas. Inhibition of oncogenic RET signaling by expression of a dominant-negative RET mutant, a new corrective gene therapeutic approach in medullary thyroid cancer, has been investigated by Drosten et al.[10,11] More than 95% of medullary thyroid carcinomas harbor dominant activating mutations in the RET proto-oncogene, which play a central role in the development of medullary thyroid cancer. The human RET proto-oncogene encodes for a transmembrane receptor (a common receptor for the glial cell derived neurotrophic factor family ligands) that consists of three functional domains: the extracellular ligand binding domain, the transmembrane segment and the intracellular domain formed by a tyrosine kinase. Mutations of the RET gene result in constitutive activation of RET tyrosine kinase with aberrant downstream signaling and initiation of tumor formation. Adenoviral vectors expressing dominant-negative RET mutants were used to investigate the effects of RET inhibition in TT cells (human medullary thyroid carcinoma cells). Because of amino acid changes in the extracellular domains of these dominant-negative RETmutants, which naturally occur associated with Hirschsprung's disease, the glycosylation process is disturbed resulting in hampered protein transport to the cell surface. In addition, the dominant-negative mutants dimerize with oncogenic RET protein in the endoplasmic reticulum (ER), thereby preventing expression of both dominant-negative and oncogenic RET protein on the cell surface. Using an adenoviral vector expressing dominant-negative RET under the control of a C-cell-specific synthetic calcitonin/calcitonin gene-related peptide promoter, a pronounced shift of endogenous oncogenic RETprotein localization from the cell surface to the ER was demonstrated, resulting in strong inhibition of cell viability caused by induction of apoptosis in vitro. Adenoviral mediated transfer of dominant-negative RET mutant in TT cell tumors in vivoin nude mice resulted in prolonged survival, while inoculation of ex vivo transduced TT cells in nude mice led to complete suppression of tumor growth in vivo.[10,11] In contrast to p53 restoration, inhibition of oncogenic RET expression by dominant-negative RET mutant delivery is not associated with a bystander effect requiring high levels of in vivo transduction efficiency, thereby limiting its therapeutic efficacy in vivo. Recently, downregulated expression of tyrosine phosphatase _ (PTP_), which encodes a receptor-type tyrosine phosphatase protein with tumor-suppressor activity, was demonstrated in human thyroid carcinomas. Adenovirus mediated transfer of rat PTP_ resulted in inhibition of cell growth in four thyroid carcinoma cell lines (ARO, FB-1, NIM, TPC-1) in vitro and significant reduction of growth of anaplastic thyroid cancer cell (ARO) xenografts in nude mice. These data suggest that gene therapy based on restoration of PTP_ function has potential in the treatment of human thyroid malignancies.[12] Gadd45 family (growth arrest and DNA damage-inducible gene family) proteins have been implicated in a variety of growth-regulatory mechanisms, including DNA replication and repair, G2/M checkpoint control, and apoptosis. Chung et al.[13] demonstrated significantly lower levels of Gadd45_ RNA levels in anaplastic thyroid cancer cells compared with normal primary cultured thyrocytes. In addition, adenovirus-mediated reexpression of Gadd45_ significantly inhibited the proliferation of anaplastic thyroid carcinoma cells (ARO, FRO, NPA) resulting from apoptosis.[13] Moreover, high mobility group I (HMGI) proteins are overexpressed in several human malignant tumors, and it has been demonstrated that inhibition of HMGI synthesis is capable of preventing thyroid cell transformation. Using an adenovirus carrying the HMGI(Y) gene in an antisense orientation (Ad-Yas), Scala et al.[14] showed induction of programmed cell death in two human anaplastic thyroid carcinoma cell lines (ARO, FB-1) but not normal thyroid cells in vitro. ARO cell xenografts in nude mice revealed a drastic reduction in tumor size following intratumoral application of Ad-Yas. Therefore, suppression of HMGI(Y) protein synthesis by an HMGI(Y) antisense adenoviral vector may represent a useful treatment strategy for thyroid malignancies, in which HMGI(Y) gene overexpression is a general event.[14] Cytoreductive Gene Therapy A common strategy for cytoreductive gene therapy is the suicide gene/prodrug strategy herpes simplex virus thymidine kinase/ganciclovir. Expression of herpes simplex virus thymidine kinase (HSV-tk) in tumor cells followed by administration of ganciclovir (GCV), which is phosphorylated by HSV-tk and competes with deoxyguanosine triphosphate in DNA polymerization, results in arrest of DNA synthesis and cell death.[15] To minimize extratumoral toxicity, thyroid-specific promoters, such as the thyroglobulin (Tg) promoter and the calcitonin promoter, have been used to target the suicide gene to thyroidal cells. Nishihara et al.[16] demonstrated a therapeutic effect of ganciclovir in thyroid carcinoma cell lines FRO and WRO after retrovirus-mediated HSV-tk gene transfer, which was associated with a significant bystander and radiosensitizing effect in vitro andin vivo in xenografted tumors in nude mice. In order to improve effectiveness and safety ofHSV-tk/GCV gene therapy, Braiden et al.[17] applied the Tg promoter to target the HSV-tk gene to Tg-expressing thyroid carcinoma cells using a retrovirus. They showed an in vitro cytotoxic effect selectively in Tg expressing thyroid carcinoma cells (FRTC) in contrast to anaplastic thyroid carcinoma cells without Tg expression (FRO) with a significant growth inhibition in vivo in transduced FRTC tumors in nude mice.[17] This study demonstrates the ability of the Tg promoter to transcriptionally target therapeutic genes to thyroid carcinoma cells, albeit with lower efficacy than constitutive viral promoters such as the CMV early promoter. Nagayama et al.[18] therefore successfully applied the Cre/loxP system to enhance the therapeutic efficacy of the HSV-tk/GCVsystem driven by the Tg promoter. Zhang et al.[19] performed adenovirusmediated HSV-tk gene transfer under the control of the Tg promoter, and demonstrated significant and selective suppression of cell growth in Tg-expressing cells in vitro with low in vivo toxicity after systemic administration of the adenovirus (no significant changes of serum transaminase levels and histologic liver abnormalities). Takeda et al.[20] used the telomerase reverse transcriptase promoter to achieve tumor-specificHSV-tk/GCV gene therapy in undifferentiated thyroid carcinoma cells and demonstrated a tumor-specific therapeutic effect in vitro and in vivo. The HSV-tk/GCV system has also been applied to therapy of medullary thyroid cancer. In rat medullary thyroid cancer cells (rMTC) the HSV-tk/GCV system was limited by a low bystander effect in vitro, which correlated well with the limited antitumor efficacy n vivo. Intratumoral application of an adenovirus carrying the HSV-tk gene under the control of the CMV promoter followed by GCV treatment resulted in destruction or stabilization of smaller tumors without a therapeutic effect in larger tumors.[21] These data confirm that a significant bystander effect is an important advantage for effective suicide gene therapy. Minemura et al.[22] performed C cell-specific adenoviral HSV-tk gene transfer by introduction of the HSV-tk cDNA into exon 4 of the calcitonin mini gene (exon 3-5) coupled to the calcitonin promoter taking advantage of C-cell-specific alternative RNA splicing. After alternative splicing, which only occurs in thyroid C cells and medullary thyroid cancer cells, exon 4 is joined to exon 3 resulting in C-cell-selectiveHSV-tk expression. In contrast, in neural cells, which also express the calcitonin gene, exons 1 to 3 are spliced to exons 5 and 6 to form calcitonin gene-related peptide (CGRP), therefore noHSV-tk expression will be induced using the adenovirus. Using this C-cell-specific strategy, significant suppression of cell growth was shown in vitro in human and rat medullary thyroid cancer cells, accompanied by reduced expression of HSV-tk in other cancer cell lines.[22] In a more recent study, Zhang et al.[23] evaluated the effectiveness of adenovirusmediatedHSV-tk/GCV therapy driven by the Tg promoter (AdrTgtk/GCV) in a human Hurthle cancer cell line (XTC-1). A significant therapeutic effect was shown in vitro as well as in vivo in XTC-1 cell xenografts in BALB/c-SCID mice after intratumoral injection of AdrTgtk/GCV with low in vivotoxicity of AdrTgtk/GCV compared to an adenovirus carrying the noncell-specific CMV promoter (AdCMVtk/CGV).[23] Tissue- or tumor-specific promoters, such as the Tg- and calcitonin-promoter, tend to be weaker than constitutively activated viral promoters, which is often a concern regarding transduction efficiency which has to be sufficiently high to yield a therapeutic effect. Strategies to enhance Tg promoter activity by treatment with histone deacetylase inhibitors and 8-bromo cyclic adenosine monophosphate (cAMP), by use of a tandemly repeated Tg core promoter, and to extend its applicability to poorly differentiated and anaplastic thyroid cancer with lost Tg expression by cotransfection with TTF-1 and PAX-8 have been evaluated with promising results.[24-28] Activity and tissue-specificity of the calcitonin promoter was enhanced by combination of a minimal human calcitonin promoter with multiple copies of tissue-specific enhancer elements, and by taking advantage of C-cell-specific RNA splicing as described above.[22,29-31] Taken together, in vitro as well as in vivo experiments in several follicular cell-derived and medullary thyroid cancer cell lines have clearly demonstrated a therapeutic effect of the HSV-tk/GCV strategy, which therefore seems to be a promising therapeutic approach for future therapy of advanced thyroid cancer. For a tumor to grow it must recruit blood supply through the process of angiogenesis, which is regulated by proangiogenic factors, such as basic fibroblast growth factor and VEGF, and antiangiogenic factors, such as angiostatin and endostatin. Endostatin is one of the most potent antiangiogenic factors and has been shown to effectively inhibit angiogenesis and tumor growth in a variety of in vivo models. In follicular thyroid carcinoma cells (FTC-133), recombinant endostatin significantly inhibited the growth of FTC-133 xenografts in nude mice. Xenografts derived from FTC-133 cells stably expressing endostatin following retrovirusmediated gene transfer revealed significantly lower tumor growth in vivo than parental FTC-133 cells. This effect was associated with reduced microvessel density in the tumors and decreased systemic levels of vascular epithelial growth factor. This study demonstrates the therapeutic efficacy of antiangiogenic strategies, such as application of recombinant endostatin protein and endostatin gene transfer in follicular cell-derived thyroid cancer.[32] The cytopathic effect of an E1B gene-defective adenovirus (ONYX-015), which replicates only in tumor cells lacking functional p53 and causes cell death, was evaluated in human thyroid carcinoma cell lines (ARO, FRO, KAT-4). ONXY-015 induced cell death in these three anaplastic thyroid cancer cell lines, but not in a normal rat thyroid cell line. Moreover, growth of ARO xenograft tumors in nude mice was significantly reduced by local injection of ONYX-015. The ONYX-015 virus acted synergistically with the antineoplastic drugs doxorubicin and paclitaxel in inducing ARO and KAT-4 cell death.[33] Furthermore, ONYX-015 treatment enhanced radiation induced cell death in human anaplastic thyroid carcinoma cells in vitro and in vivo.[34] These data strongly suggest that ONYX015 may be a valid tool in the treatment of anaplastic thyroid cancer, in particular in combination with chemotherapy and/or radiotherapy. Immunomodulatory Gene Therapy Many cancers express tumor-associated antigens that can be recognized by the immune system. Tumor-associated antigens are released from tumor cells physiologically or after cytotoxic therapy. The antigens are then taken up through phagocytosis by antigen presenting cells, which process and present the tumor-associated antigens to CD8+ cytotoxic T cells and CD4+ helper T cells in the context of major histocompatibility complex (MHC) class I and II and B7.1/B7.2 costimulatory molecules. Tumors often demonstrate downregulated expression of MHC class I or costimulatory molecules, resulting in poor T-cell responses, and thereby evade the immune system. Mobilization of the immune system by delivery of genes that enhance immunogenicity of tumors and responsiveness of the immune system, is associated with a number of advantages, including inherent specificity of the immune system (decreasing normal tissue toxicity), a systemic immunogenic effect, signal amplification, and permanent antitumor immunity because of inherent memory of the immune system. Local expression of certain cytokines is able to elicit an immune response against the tumor by stimulating surrounding immunocompetent cells, targeting cytotoxic T cells and natural killer cells, thereby inducing rejection of tumor cells. Cytokines with antitumor activity include interferon-_, tumor necrosis factor-_, interleukin-2 (IL-2), and interleukin-12 (IL-12). IL-2 has been examined in various studies for genetic immunotherapy of thyroid cancer. Zhang et al.[35,36] used a replication-defective adenovirus harboring the IL-2 gene for treatment of medullary thyroid tumors in mice and rats. Intratumoral injection of the adenovirus resulted in tumor regression in smaller tumors and tumor stabilization in larger tumors with low in vivo toxicity after systemic application of the adenovirus. The antitumor effect was shown to be dependent on cytotoxic T lymphocyte activity against the tumor, which also prevented tumor growth after reinjection of tumor cells, indicating development of long-term antitumor immunity. To enhance therapeutic efficacy in thyroid cancer further, the combination of suicide and immunomodulatory gene therapy has been evaluated by several groups.[37-40] Zhang et al.[38]developed an adenovirus expressing both HSV-tk and human IL-2 (AdCMVTKhIL2), which was shown to have an antitumor effect in rat medullary thyroid tumors after intratumoral injection superior to that of each single vector. In addition, systemic and long-term antitumor immunity was established in most rats after intratumoral injection of AdCMVTKhIL2.[38] Barzon et al.[40] used a retroviral vector for combined transfer of the human IL-2 and the HSV-tk gene in differentiated and anaplastic thyroid carcinoma cells and showed an enhanced therapeutic effect compared to IL2 alone. In vivo studies in nude mice showed complete eradication of xenografts derived from retrovirally transduced anaplastic thyroid tumors, and more than 80% reduction of tumor size of differentiated thyroid carcinoma xenografts. The therapeutic effect of the combination of IL-2 andHSV-tk gene therapy was associated with a significant bystander effect in vitro and in vivo.[40] To further optimize this therapeutic approach, a transcriptionally targeted retroviral vector was generated, replacing the viral enhancer with the enhancer sequence of the human Tg gene, which allowed selective transgene expression and cell killing in differentiated thyroid tumor cells, but not in anaplastic thyroid carcinoma cells or nonthyroid cells.[39] Another cytokine with antitumor activity, IL-12, causes proliferation of natural killer cells and CD8+T cells, and activation of macrophages. Zhang et al.[41] generated an adenovirus carrying two subunits of the murine IL-12 gene and showed efficient antitumor activity and development of long-term antitumor immunity after intratumoral injection of the adenovirus into rat medullary thyroid tumors.[41] Using the same adenovirus, a significant therapeutic effect with long-term antitumor immunity was also demonstrated in a rat thyroid follicular cancer cell line (RTC-R2) in vivo after intratumoral injection of the virus. In vivo toxicity was low after intratumoral or systemic application of the adenovirus, and in rats with two tumors, injection of the adenovirus in one tumor resulted in antitumor activity in the injected as well as noninjected tumor, indicating systemic antitumor immunity.[42] With the aim of tissue-specific antitumor activity in medullary thyroid cancer, another adenovirus was generated in which the two subunits of the murine IL-12 gene were linked to a modified calcitonin promoter. IL-12 was selectively expressed in rat medullary thyroid carcinoma cells, resulting in a significant therapeutic effect in medullary thyroid tumors in rats after intratumoral injection of the adenovirus. Tissue-specific IL-12 gene transfer was also associated with development of long-term antitumor immunity and low in vivo toxicity after local and systemic adenovirus application. Moreover, intratumoral injection of the adenovirus induced antitumor activity in injected as well noninjected tumors in the same rat.[43] Taken together, immunomodulatory gene therapy, in particular in combination with suicide gene therapy, seems to be a promising therapeutic approach for treatment of follicular cell-derived and medullary thyroid cancer. NIS Gene Therapy Cloning of the NIS gene and its extensive characterization has not only revolutionized our understanding of the physiology and pathophysiology of thyroidal iodide accumulation, but also provided us with a powerful new diagnostic and therapeutic gene.[44,45] As an intrinsic plasma membrane glycoprotein, NIS mediates the active transport of iodide at the basolateral membrane of thyroid follicular cells. NIS cotransports one iodide ion against its electrochemical gradient together with two sodium ions along their electrochemical gradient (Fig. 2).[46] Functional NIS expression in the thyroid gland is responsible for thyroidal accumulation of iodide, an essential constituent of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). The unique property of thyroid follicular cells to trap and concentrate iodide because of expression of NIS allows imaging as well as highly effective therapy of differentiated thyroid carcinomas and their metastases by administration of radioiodine, thereby improving the prognosis of thyroid cancer patients significantly.[47] Differentiated thyroid carcinomas are usually treated by total or near-total thyroidectomy, followed by 131I ablation of the thyroid remnant and occult microscopic carcinomas. Subsequent postablative 131I total body scanning can diagnose local and metastatic residual and recurrent disease. Therapy with 131I has been successfully used for more than 40 years in the treatment of differentiated thyroid cancer. Recurrence rates are significantly higher in patients treated with surgery and thyrotropin suppression by thyroxine alone compared to those who also receive radioiodine treatment.[47] The efficacy of radioiodine therapy is reflected in the low mortality of patients suffering from metastatic thyroid cancer who are treated with 131I (3%) as compared to those who are not (12%). Even young patients with diffuse pulmonary metastases at initial presentation can be successfully treated by 131I, achieving a 10-year survival of more than 80%.[47] Thyroidal NIS expression therefore opens the door to effective cancer therapy with remarkably low incidence of serious adverse affects. Cloning of the NIS gene[44,45] has paved the way for the development of a novel cytoreductive gene therapy strategy for the treatment of thyroidal and extrathyroidal malignancies based on NIS gene transfer followed by radioiodine therapy. Targeted expression of functional NIS in cancer cells would enable these cells to concentrate iodide from plasma, and would, therefore, offer the possibility of radioiodine therapy. Application of the NIS gene as novel therapeutic gene therefore extends the diagnostic and therapeutic use of 131I and the extensive experience with radioiodine in the management of differentiated thyroid cancer to the treatment of nonthyroidal cancer and dedifferentiated, anaplastic, and medullary thyroid cancer. NIS further represents a therapeutic gene that is associated with a bystander effect, because not only NIS-transduced cancer cells, but also surrounding non-transduced cells are destroyed by the crossfire effect of the _-emitter131I, that is characterized by a path length of 0.2-2.4 mm.[48] Several investigators have explored the efficacy of NIS gene transfer into nonthyroidal tumor cells for induction of radioiodine accumulation allowing radioiodine therapy of extrathyroidal malignancies. Using various gene delivery techniques, including electroporation, liposomes, adenoviral and retroviral vectors, radioiodine accumulation was induced in vitro and in vivo in a variety of cancer cell lines (glioma and neuroblastoma cells, melanoma, cervix, breast, lung, liver, colon and ovarian carcinoma cells, myeloma cells, pancreatic neuroendocrine tumor cells) by NIS gene delivery.[49-57] In addition, prostate cancer (LNCaP) cells were shown to be selectively killed by accumulated 131I after induction of tissue-specific iodide uptake activity by prostate-specific antigen (PSA) promoter-directed NIS expression in vitro.[58,59] Iodide accumulation was confirmedin vivo in LNCaP cell xenografts in athymic nude mice and was high enough to allow a therapeutic effect of 131I in vivo. A single therapeutic 131I dose of 3 mCi was administered and shown to elicit a dramatic therapeutic response in NIS-transfected LNCaP cell xenografts with an average volume reduction of more than 90%.[59] As a next crucial step toward therapeutic application of NIS gene delivery followed by radioiodine therapy in patients with prostate cancer in a clinical setting, a replication-deficient human adenovirus carrying the human NIS gene linked to the CMV promoter (Ad-5CMV-NIS) was used to perform in vivo NIS gene transfer into LNCaP cell tumors. After intraperitoneal injection of a single therapeutic dose of 3 mCi 131I 4 days after adenovirus-mediated intratumoral NIS gene delivery, LNCaP xenografts showed a clear therapeutic response with an average volume reduction of more than 80%.[60] These studies clearly showed for the first time that NIS gene delivery into nonthyroidal nonorganifying tumor cells is capable of inducing accumulation of therapeutically effective radioiodine doses, and might therefore represent an effective and potentially curative therapy for extrathyroidal tumors. While in differentiated thyroid cancer functional NIS expression allows effective therapy with radioiodine, patients with poorly differentiated thyroid cancer with low TSH-stimulated NIS expression levels or patients with medullary thyroid cancer do not benefit from 131I therapy because of insufficient or absent 131I accumulating activity. In these patients NIS gene transfer could be used to restore or induce 131I accumulation, thereby establishing effective 131I therapy. Early studies in malignantly transformed rat thyroid cells (FRTL-Tc) without iodide transport activity showed that transfection with rat NIS cDNA using electroporation is able to restore radioiodine accumulation in vitro and in vivo. However, the effective half life of 131I in NIS-transfected FRTL-Tc xenografts in rats was only 6 hours and did not allow a therapeutic effect of 131I (1 mCi).[61] More recently, stable transfection of a NIS-defective follicular thyroid carcinoma cell line (FTC-133) with the NIS gene was able to reestablish iodide accumulation activity in vitro and in vivo.[62] In the same NIS-transfected follicular thyroid carcinoma cell line, thyroid ablation and low-iodide diet were able to increase the biologic half-life of accumulated radioiodine in vivo from 3.8 hours to 26.3 hours leading to postponed xenotransplant development in nude mice after administration of a therapeutic dose of 2 mCi 131I.[63] Petrich et al.[64]transiently transfected a variety of papillary, follicular, and anaplastic thyroid carcinoma cell lines (B-CPAP, K1, WRO, 8505C, FTC-133) with the NIS gene, thereby inducing perchlorate-sensitive accumulation of 125I. These studies show that NIS gene delivery into thyroid cancer cells is capable of restoring 131I accumulation, and might therefore represent an effective therapy for dedifferentiated and anaplastic thyroid tumors that lack iodide-accumulating capacity. Lee et al.[65] used a recombinant adenovirus to transduce a panel of thyroid carcinoma cell lines (ARO, FRO, NPA) with the NIS gene. They demonstrated significant iodide accumulation associated with rapid iodide efflux in vitro, which the authors explained by low expression levels of Tg, TSH-receptor, and thyroid peroxidase (TPO) genes involved in iodide organification.[65] The radiation dose responsible for a possible therapeutic effect of trapped 131I is determined by the rate of uptake of iodide and its retention time in the tumor cell, which is affected by rate of iodide efflux, iodide recirculation, and iodide binding to cellular proteins or lipids, a process termed iodide organification. In the thyroid gland trapped iodide is organified by TPO-catalyzed oxidation and incorporation into tyrosyl residues along the Tg backbone. Enhancing the retention time of131I in the thyroid gland, iodide organification is generally believed to be a crucial prerequisite for131I therapy, although no clear data exist to prove this hypothesis. In fact, our recent studies in LNCaP cells, that do not organify iodide after PSA promoter-mediated NIS gene transfer, clearly demonstrate that iodide organification is not required for a therapeutic effect of 131I in tumor cells.[58-60] Several investigators have asked if coupling of NIS gene transfer with the delivery of the TPO gene in nonorganifying tissues is capable of inducing iodide organification thereby enhancing the iodide retention time and achieved radiation dose in the target tissue. In a recent study, Boland et al.[66] showed that coinfection of human cervix carcinoma cells with two different adenoviral vectors encoding the rNIS gene and the hTPO gene, respectively, does not increase iodide retention time in the tumor cells, although enzymatically active TPO was produced and a significant increase in iodide organification could be observed. In contrast to these findings, Huang et al.[67] demonstrated that cotransfection of the NIS and TPO genes in non-small-cell lung cancer cell lines was capable of decreasing iodide efflux suggesting that the degree of organification-mediated iodide retention might be cell-type-specific. Whether TPO-mediated organification can indeed be used as a therapeutic strategy to enhance the efficacy of NIS-based radioiodide concentrator gene therapy remains to be confirmed in further studies. Recently, the high energy _-emitter 211astatine (211At) and the potent _-emitter 188rhenium (188Re), have been introduced as alternative radionuclides to 131I for treatment of thyroidal and nonthyroidal tumors after NIS gene transfer.[68-71] It has been known from cell culture and animal experiments that 211At and 188Re accumulate in the thyroid similar to iodide and pertechnetate, suggesting that they are transported by NIS. Petrich et al.[70] demonstrated sodium-dependent and perchloratesensitive accumulation of 211At in papillary and anaplastic thyroid carcinoma cell lines (B-CPAP, K1, 8505C) after stable transfection with the NIS gene, resulting in a significant increase of the tumor absorbed dose from 3.5 Gy/MBqtumor for 131I to 50.3 Gy/MBqtumor for211At. These data suggest that application of 211At may enhance the differentiated follicular cell-derived thyroid carcinomas and therapeutic efficacy of NIS-based gene therapy, in particular medullary thyroid cancer after NIS gene transfer (Fig. 3). in thyroid tumors with low iodide retention time.[70] Finally, recently, in medullary thyroid cancer, a therapeutic effect of radioiodine was demonstrated following induction of tissue-specific iodide uptake activity by calcitonin promoter-directed NIS gene transfer in vitro.[72] Taken together, these studies demonstrate the potential of NIS as a novel therapeutic gene allowing 131I therapy of dedifferentiated follicular cell-derived thyroid carcinomas and medullary thyroid cancer after NIS gene transfer (Fig. 3)

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