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Gene therapy in the world and in Switzerland
S. Rusconi, Swiss Medical Weekly 1999, 129. 1769-1778
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Until the mid-seventies, biology used to be taught as an interesting, yet rather "useless" discipline in our high schools. The advent of molecular biology has drastically changed this image. Now, applied molecular genetics has been shown to have the potential to revolutionise many aspects of our life, including the paradigms of medicine. In a first phase, gene knowledge has allowed medical diagnosis with previously unimaginable precision. In a second wave, gene transfer in micro-organisms has produced a plethora of biopharmaceuticals. This decade has seen the third era of molecular medicine, in which direct gene transfer into humans is being developed. This article comments on the most recent developments and concepts in the field of human gene transfer (also called "gene therapy"). Some essential methods are briefly presented and a great deal of attention is devoted to the technical hurdles still to be overcome in achieving efficient and safe gene therapy protocols. The final paragraph attempts to clear up some myths and misunderstandings that are commonly propagated when people talk or think about gene therapy. The purpose of this article will be fulfilled if at the end the reader is convinced that gene therapy is not necessarily dedicated exclusively to hereditary disorders, that Switzerland undertaken an intensive and competitive experimental effort in this direction, that gene therapy has already proven its efficacy and has great potential, but that it will take a couple of decades before some applications are routinely used in the clinics.
Keywords: gerle transfer; molecular biology; somatic gene therapy; gene repair; oligoucleotides; recombinant viruses; infection; transfection; hereditary and acquired disorders; clinical trials
Bis Mitte der 70er Jahre wurde Biologie an den Gymnasien im allgemeinen als zwar interessantes, aber letztlich nutzloses Fach dargestellt und vermittelt. Die Molekularbiologie hat dieses Bild dramatisch verandert. Die angewandte Molekulargenetik hat das Potential, viele Aspekte unseres Lebens zu revolutionieren, inklusive der Paradigmen der Medizin. In einer ersten Phase ermoglichte die Kenntnis der Gene eine medizinische Diagnostik von zuvor unvorstellbarer Genauigkeit. In einer zweiten Welle hat der Gentransfer in Mikroorganismen zu einer Vielzahl von biopharmazeutischen Produkten gefuhrt. In diesem Jahrzehnt erleben wir die dritte Phase der Molekularmedizin, in welcher der direkte Gentransfer in den Menschen vorangetrieben wird. Dieser Artikel fasst die jungsten Entwicklungen und Konzepte im Bereich des Gentransfers in den Menschen (auch als <<Gentherapie>> bezeichnet) zusammen; einige grundlegende Methoden werden kurz vorgestellt. Ausfuhrlich wird auf technische Probleme eingegangen, die noch gelost werden mussen, damn' sich die Gentherapie als sicher und wirksam erweist. Der letzte Abschnitt geht auf gangige Mythen und Missverstandnisse ein, die in der Diskussion um Gentherapie haufig anzutreffen sind. Dieser Artikel hat sein Ziel erreicht, wenn der I eser/ die Leserin am Schluss weiss, class die Gentherapie nicht ausschliesslich bei Erbkrankheiten zum Einsatz kommt, class die Schweiz in diesem Bereich grosse Forschungsanstrengungen unternimmt, class die Gentherapie ihre Wirksamkeit bereits bewiesen hat und ein grosses Potential in sich tragt, class es aber immer noch Jahrzehnte dauern wird, bevor diese Verfahren in der Klinik routinemassig zur Anwendung kommen.
Keywords: Gentransfer; Molekularbiologie, somatische Gentherapie; Genreparatur; Oligonukleotide; rekombinante Viren; Infektion, Transfektion; erbliche und erworbene Krankheiten; klinische Versuche
Ageing of the population
The western population has doubled life expectancy at birth since the beginning of this century (see graph in fig. 1A). Linked with ageing there is a number of degenerative disorders with severe consequences such as cancer (fig. 1B) or Alzheimer's disease (fig. 1C). Thus, many disorders that had a minor clinical impact in the early 1900, have now become major trouble makers in the public health scenario. From the "good news" of prolonging life through better hygiene, better nutrition and better conventional medicine we have reached the realm of "bad news" of the unsolved problem of "lower life quality after the age of 50". Solving this problem will be one of the major challenges for our immediate future medicine and it will require the combined effort of many medical disciplines and the discovery of novel paradigms for the treatment or the healing of late-age-onset disorders. Gene technology promises to be one of the fields that will reveal some of these new solutions.
Impact of molecular bioloy techniques in various medical fields
Gene technology is essentially a "cut-andpaste-select-grow-and-transfer" ceremonial which allows isolation, characterisation, experimental manipulation and transfer of individual genes. A gene is an elementary hereditary module (similar to what a cell is for an organism, or a person for a society). Physically, a gene is a segment of DNA which upon two phases of expression (transcription and translation) converts a string code of nucleic acids bases into a string code of amino acids which forms a specific protein. Proteins are therefore the "final" products of genes. In general, a gene produces a single type of protein (e.g. insulin, collagen, alpha globin, interferon alpha, etc.) and each protein represents a specific function (enzyme, structure, signal, transport, etc.). The entire gene "package" (the genome) is precisely duplicated upon each cell duplication and selected genes are expressed during the development. For the basic functions of the cell metabolism about 10 000 genes are necessary (the so-called house keeping genes), whereas another set of 70 to 90 000 is destined to be expressed only in some specific tissues, conferring to them their differential activity. All vital processes depend on the coherent expression of genes, thus all pathologic processes are characterised by some incapacity of restoring this equilibrium. Many disorders (at least 4000) are directly inherited in form of a gene malfunction, whereas the occurrence of other disorders can be enhanced or reduced according to complex gene configurations (genetic susceptibility or predisposition, see example in figure IC for some alleles that change the susceptibility to develop Alzheimer's disease). An increasing number of genes whose malfunction is directly responsible for causing disorders or susceptibilities has been characterised. This has profoundly altered our vision of diagnostics by allowing analyses of almost mathematical precision. These molecular diagnostics have been applied for several years in prenatal genetic counselling and are also getting into the more demanding field of predictive diagnostics as exemplified by some cancer susceptibility alleles, Alzheimer's predisposition and so on. The ethical and social implications of these predictive diagnostics have not yet been worked out completely and everybody in the field is "learning by doing".
Definition of gene therapy: the DNA as a first-hand pharmaceutical
Of course the power of gene technology is not limited to the descriptive level. The genes for several pharmaceutically interesting products (insulin, interferons, erythropoietin, coagulation factors, anti- coagulation factors, etc.) have been isolated and engineered to be abundantly expressed from foreign organisms: bacteria, yeast, cell cultures, transgenic animals or transgenic plants. The expressed products are easily purified and are commercialised as pharmaceuticals for many indications (vaccinations, treatment of immune disorders, treatment of hormonal imbalances, etc. ). As in many other cases this still juvenile market has shown the weak side of our society, by favouring misuse (see the doping of bike riders with Epo). However, this technology has proven superior to conventional preparations of pharmacologically active macromolecules by allowing pathogen-free production of vaccines, growth factors, coagulation factors and so on. In our country there were more than 30 registered biotechnologically prepared pharmaceuticals by the end of 1998, with an increase of one per month. Thus, our pharmaceutical industry has learned how to use the power of genes to produce healing substances. However, this is not yet "gene therapy". This term is reserved to the situation in which genes (or fragments thereof) are directly transferred to the organism to restore a malfunction. The malfunction does not need to be inherited. For instance, there is increasing optimism for so-called DNA-based vaccinations, in which genes encoding the antigens to be immunised are temporally transfected intramuscularly instead of injecting the antigens in form of inactivated viruses, bacteria or protein extracts. It seems that the organism produces a better immune response when those antigens are directly expressed. Furthermore, a DNA-based vaccination offers the advantage of a very inexpensive preparation which has a very long shelf life (up to several years without refrigeration). This would enormously reduce the costs of mass vaccinations and facilitate programmes especially in underdeveloped countries. DNA-based vaccination is thus a bona fide "gene therapy" and will probably be the first form of broad scale application of this concept of human gene transfer. In conclusion, the formal definition of gene therapy is: a human gene transfer aimed at correcting or preventing acquired or inherited physiological disorders. The gene transfer can be conducted on the intact organism or on explanted tissues or organs that are reimplanted after transfer. There are some approaches such as the utilisation of encapsulated gene-engineered heterologous cells to reconstitute expression of diffusible factors such as neuro-regenerative hormones, blood clotting and so on. These "artificial xeno-glands" do hardly belong "under the roof" of gene therapy although operationally very close to the use of ex vivo engineered autologous cells.
The three major technical problems in human gene transfer
In the preceding paragraphs we have defined the problem: transfer genes into a sufficient number of cells of a tissue to produce a therapeutic effect. The percentage of transfected cells that are necessary to reequilibrate the almost normal functions varies according to the type of disorder. If willing to eradicate a tumour with a pro-drug activating gene, we may need to transfer the gene in a very high percentage, possibly in all the several tens of billions of tumour cells, while for an efficacious vaccination it may be sufficient to transfect few millions of cells. There are many artificial ways to transfer genes into mammalian cells: they range from physical approaches and micropojectile bombardement to electroporation and chemically mediated protocols utilising various aggregating compounds. Many of these methods allow a significant percentage of cells (from 5 to more than 50%) to be transiently transfected in vitro (in monolayer cell cultures). A small fraction of the transient transfectants (less than 1 in 100) will stably incorporate the gene into the own genome at an undetermined position. Thus, with the best nonviral transfections we can expect to stably transform at most 1% of the cultured cells. Now, one has to further reduce this fraction to 0.001-0.1% when attempting to stably transform cells in a complex, three-dimensional tissue.
This is the reason why many of the current gene transfer protocols foresee a biological method in which the desired gene is first engineered into the genome of a virus and the recombinant virus is used for "ferrying" the gene into cells by natural infection. Those recombinant viruses (fig. 2) require the establishment of dedicated packaging cell lines in which the essential genes of the original virus (fig. 2A) are stably anchored into the host cell genome, making way for the therapeutic gene (fig. 2B). Recombinant viruses allow the incorporation of a defined maximal size (from 3 to 30 kb, depending on the virus type).
Independently of the transfer vector there are two classes of transfer methods: in vivo and exvivo (fig. 3). In vivo means that the gene formulation is applied directly on the intact organism either in the blood circulation (systemic delivery, fig. 3A) or by local application (intramuscular, intratumoral, by local permeation, etc., fig. 3B). Ex vivo means that the tissue or organ is first surgically removed, briefly cultured in vitro and mixed with the transfection formulation. The transfected tissue or organ is then reimplanted in the hope that it will reconstitute the normal function. A variation of the ex vivo approach is the reconstitution of artificial glands with autologous cells (fig. 3 C). Besides efficiency two other keywords summarise the technical hurdles in gene therapy: specificity and persistence.
Specificity of transfer is important when the expression of the therapeutic gene must be restricted to a particular subset of cells to avoid side effects (e.g. when trying to provoke selective suicide of tumour cells). The easiest way to ensure specificity is to transfer the gene locally or on explanted tissues (see below). It is the most difficult when willing to deliver the gene formulation systemically. A major effort in the field is currently devoted to improving the targeting capacity of systemically applied gene formulations.
Persistence of expression of the "transgene" is important in the treatment of metabolic or chronic disorders. It is not easy to guarantee persistence especially in protocols where the transgene integrates at random positions of the host genome. The natural consequence is that in the majority of integration sites the gene is more or less rapidly switched off (the phenomenon is also called "transgene silencing"), as it does not have aufficient additional information to be reorganised as a permanent "locus". The use of viral vectors strongly limits the size of the insert and this problem becomes even more acute. Finally, even when the transgene maintains its activity, one has to cope with the natural turnover of the targeted cells. If those are shed and replaced by newly differentiating untreated cells, then the therapeutic effect will have the same half-life as the target cells. Thus, there is also a big effort in trying tc target the pool of cells that regenerate each tissue or organ, the so-called "stem cells". However, studies on somatic stem cells are still toa crude to permit a precise and efficacious targeting of those. If the chosen protocol does not allow to aim at the stem cells, then the gene transfer must be repeated at regular intervals. This may be interesting for pharmaceutical industries but is certainly not a suitable scenario for maintaining a cost-effective public health programme.
Furthermore, there are other inherent obstacles that especially apply to the use of viral vectors. Many of the viral capsid proteins have some intrinsic toxicity for the cells or have at least the potential to raise neutralising antibodies. Thus, a virally mediated gene transfer is generally poorly suitable for regularly repeated gene transfer (see above).
Finally, all the current working protocols for permanent gene transfer are based on the notion of random integration into the host genome. This random gene addition ("shotgun approach" ) raises one additional important problem besides "transgene silencing" (see above): it is in itself a highly mutagenic process. Random insertion can disturb or enhance the activity of resident genes and among tumour suppressor or tumour-promoting genes. To circumvent this, several protocols foresee the cotransfer of accompanying conditional suicide genes, whose effect can be activated by the addition of some pro-drugs that are converted to toxic compounds. This should allow the destruction of hyper-proliferating engineered cells. None of this suicide-control approaches is guaranteed to work and therefore the shotgun gene addition is destined to be limited to severe disorders, where the risks associated with the therapy are less than the risk of a nonintervention. The shotgun approach also renders any type of voluntary or involuntary germ-line intervention potentially dangerous, this momentarily excludes this type of therapy. We shall see towards the end of this article that some novel approaches of targeted gene correction promise to offer genomic alterations with "surgical" precision. If those protocols can be implemented, they will solve many of the problems linked to the above mentioned keywords " specificity " and "persistence " .
Paradigmatic examples: working and non-working protocols
The first clinical trial was registered in 1990 by F. W. Anderson and colleagues [1]. Since then more than 200 clinical trials dealing with some kind of gene transfer have been registered. The majority (>95%) of those was at clinical phase I, which aims principally at assessing the degree of side effects at a single dose and not focused on measuring the therapeutical effect. I therefore would say that the first ten years of gene therapy were principally aimed at proving the concept rather than the efficacy of this novel class of therapy. Thus we can better understand why there have been very few reports of therapeutical success.
The first gene transfer [1] was designed to cure the adenosine deaminase deficiency upon transfer of the healthy ADA gene into explanted bone marrow cells of a young patient. The vector chosen for this ex vivo gene transfer was a recombinant murine retrovirus, which is notoriously incapable to infect nonproliferating cells. After reinfusion of the treated bone marrow cells the authors were able to demonstrate that cells harbouring the additional gene were still present several months after the treatment, however, the expression of the transgene was generally switched off after a few weeks. Several other attempts by other research groups led to the same conclusion: the transduction with recombinant retroviruses leads to progressive silencing of the transgene.
Another classical example was the reported treatment of an LDL deficiency which caused severe hyperlipidaemia [2]. In this case a liver biopsy of several grams was disaggregated and the cells were put in culture for a short time, then exposed to a transducing recombinant retrovirus that was supposed to bring in an additional copy of the healthy LDL gene. The transfer was reportedly successful although there has been no published follow-up of that experiment. Lxamples of attempts to treat monogenic diseases can be cited also for cystic fibrosis where either recombinant adenoviral vectors L31 or liposome aerosols were adopted. The results of those early attempts were very disappointing although a large number of yet unpublished clinical trials (partly presented at the January Keystone meeting in Salt Lake City 1999) seems to indicate that some hurdles may have been passed. The major barrier in this case seems to be the refractoriness of the lung epithelial cells to many kinds (virally-mediated or not) of gene transfer. Further details will be found in the parallel contribution to this series by Dr Rochat.
Now, some good news dedicated to all those scepticals who like to believe that gene therapy has not yet demonstrated its effectiveness: the treatment of critical limb ischaemia (see [4]). Surprisingly, this comes from a protocol of deceiving simplicity: by injecting a DNA solution into the muscles. Not surprisingly, this comes from a case in which the gene transfer does not have to obey any of the strict rules of efficiency, specificity and persistence. In fact, the success of this gene transfer depends largely on its reduced efficiency and its short-lived persistence. For the treatment of critical limb ischaemia, the researchers have injected intramuscularly and in proximity of the necrotic tissue a certain amount of DNA solution bearing a vector that can express the vascularising factor VEGF. When expression occurs, VEGF is secreted and diffuses attracting thereby the migration of endothelial cells and the neovascularisation of the tissue. A high load of VEGF would cause angiomas and this is exactly one of the problems when applying VEGF directly. Thus, the gene transfer by intramuscular macroinjection produces just about the proper amount during just about the proper time necessary to restore a revascularisation of the affected limb. With this type of treatment the team of Dr Isner has successfully healed dozens of patients who were condemned to amputation and this already in a clinical phase I trial. Needless to say, this technique is now entering clinical phase II (dosage testing and larger number of patients) and is being extended into cardiac revascularisation [5].
Finally, we could not conclude the listing of clinical trials without mentioning antitumour therapies. The space in this short review is insufficient to comment on the numerous attempts to tackle tumours. Some details will certainly emerge from parallel contributions to this series by Dr Fey and others. Therefore, I will limit myself to mention the most relevant strategies. Several protocols foresee the possibility of raising the resident immune system against the metastasising tumour. This strategy has been very promising in animal models of melanoma and is currently tested in several clinical trials. Another type of strategy aims at specifically toxifying tumour cells by the selective expression of enzyme genes whose product can catalyse the conversion of a pro-drug into a toxic compound. A paradigmatic example is the use of the herpes virus thymidine kinase gene that converts the relatively untoxic gancyclovir (GC) into its toxic derivative GC-monophosphate. These protocols require a high percentage of efficacy, a high specificity of gene transfer and a substantial level of carryover effect on nontransformed cells (the socalled "bystander" effect). Other protocols aim at reinforcing the endogenous immune system and making it resistant to chemotherapy, while others tend to make tumour cells more sensitive to conventional cytotoxic therapies (radio or chemical therapy). Finally, in a promising class of experimental designs special viruses are constructed that abundantly replicate in tumour cells while sluggishly carrying on in normal cells. The virus is of a Iytic type and is destined to selectively Iyse the tumour cells. Recent unpublished results by ONYX (data from the talk of D. Kirn, Salt Lake City, January 1999 and [6]) showed that this type of selectively killing viruses is very efficacious in patients with head-and-neck cancer.
Inherited and acquired disorders
The wrong image that the term "gene therapy" evokes in people is linked with the treatment of inherited disorders. A simple representation of the registered clinical trials and the corresponding number of enrolled patients (fig. 4A), is sufficient to demolish this image. The most impressive growth of protocols has been reserved for anticancer gene-assisted therapies and less than 20% is currently dedicated to inherited disorders. The growth of trials aimed at the treatment of cardiovascular malfunctions has just started and common knowledge predicts this type of protocols to match the anticancer protocols within a few years. This is easy to understand if we remember that 45% of deaths in the western population are due to cardiovascular failure of some kind. This aspect has some ethical implications because the frequency of cardiovascular failures could be substantially reduced if simple life-style rules (diet and exercise) could be favoured. The ethical question can simply be formulated as follows: do we want to invest millions in research and treatment of the devastating effects of relaxed diet and low exercise or rather invest in much less expensive prevention programme?
The situation with cancer treatment has some analogous implications since it has been demonstrated that the incidence of several cancer types is related to life-style although the links are more difficult to establish than for cardiovascular disorders. Considering that many types of cancer also have a strong inherited susceptibility component, it may indeed be justified to invest much in the molecular therapy approaches. Switzerland has a very high reputation and certainly a solid tradition in the life sciences research field and it is therefore less surprising to note that our country counts a substantial percentage of gene therapy-enrolled patients (Fig. 4B). About 10°\, of the worldwide clinical efforts are conducted in Switzer land. In 1996 the National Research Programme number 37 (NFP37) "Somatic Gene Therapy" entered its active phase. The programme has a budget of 15 million Sfr to be invested over five years (expires in 2001). Currently the NFP37 is financing about 20 research teams whose activity classes are summarised in Fig. 4B. In the two years of the scientific direction of this programme I could witness how the initial efforts have brought the first fruits in terms of increased intercampus and international collaborations. The second phase of the NFP37 has been launched in 1998 and it was very pleasing to note that the fraction of clinical trials was substantially increased (see www.unifr.ch/NFP37). One must bear in mind that the NFP37 represents only a small fraction of the general effort in the direction of gene therapy (see Fig. 4B). Thus, wc can proudly say that gene therapy gets out of its adolescence also in our country. Our commitment for the coming years must be to maintain a high standard of academic and industrial research in this field to avoid having to purchase this technology from foreign countries at exorbitant prices in the future.
Myths versus facts in human gene transfer and some ethical considerations
Besides the common belief that gene therapy is mainly dedicated to inherited disorders (see above) there are several other "myths" that may deserve a comment. Many critics predict that gene therapy will be invariably expensive. The example of DNA-based vaccination should be sufficient to illustrate that genebased interventions can become much less expensive than conventional therapies if properly designed. Another current belief is that gene therapy is withdrawing substantial public funds from conventional research. A simple calculation shows that less than 1% of the public research funds are currently put into this field in our country and less than 2 % in the US. The reason is simple: most of the financial effort in the field of gene therapy is done by private investment. Another common statement is that gene therapy has not yet proven its effectiveness. The example of the treatment of critical limb ischaemia is currently our best exam ple to counteract this statement. The reason that people had high expectations in gene therapy is probably due to the same reason expressed above, i.e. that most of the research is funded by venture capital. This situation forces many small companies to overstate their results to guarantee their mid- term financing. The mass media with their inherent need to spectacularise have of course not helped in setting the proper equilibrium between hopes and reality. There are also two "technical" myths that can easily be disputed: (a) the recombinant viruses are exclusively of the nonautonomously replicating type and (b) current gene therapy protocols exclusively work with "random" gene addition. Point (a) has been discussed already when we mentioned the use of selectively replicating viruses for the killing of tumour cells (see above). For point (b) we shall mention again that there are emerging protocols that utilise oligonucleotide-mediated gene repair [7] and have raised the possibility of curing disorders by specifically correcting the resident "sick" genes. The technique employing chimeric oligonucleotides (also called "chimeraplasty" ) has so far been reproduced only by few research teams, and in particular there is a heavy debate on its real effectiveness. However, the chimeraplasts repair strategy holds a titanic potential of opening the way to a "real" gene surgery. All specialists are currently very much flirting with the idea to first consolidate then improve the effectiveness of those gene-repair protocols. If proven to be adoptable for in vivo protocols, chimeraplasty would solve the problem of specificity (since only one particular gene is targeted by the repair-inducing oligonucleotide) as well as the problem of persistence of gene expression (since the targeted gene is in its own natural locus, which should not be silenced as it is the case with the random gene addition protocols). Of course, only a subset of disorders can be treated in this manner but this technique remains one of the only hopes we have to perform specific interventions in the patient's genome. If precise interventions can be performed, this would reopen the possibility of proposing germ-line (i.e. inheritable) interventions.
With that concept we have come to the only specific problem raised by gene therapy: the possibility of actively interfering with hereditary potential. The consequences of such interventions can only be measured over many generations and therefore escape any rational prediction. Currently, interventions on the germline are rightly forbidden in many countries, including our own, and this is fully just)fied by the current inability of our gene transfer protocols to drive the transgene to a precise chromosomal position. If one day the germ-line interventions become biologically justified due to the increased precision of gene transfer, then we must be prepared to discuss the rules that we want to apply to this new paradigm. It is not sufficient to radically refuse germ-line therapy while accepting somatic therapy. In fact, accepting the idea of somatically curing severe inherited disorders and permitting those patients to propagate the sick alleles in future generations has a clear consequence of increasing both the genetic burden of future generations and their health costs. Thus, the concept of germ-line therapy deserves to be rehabilitated and thoroughly discussed before the advent of the corresponding technical capacity. In my opinion there are no other inherent ethical implications for gene therapy. All the other discussions such as social availability, dignity of the patient, safety considerations, exaggerated costs of high-tech protocols and so on are not specific because they apply to all forms of advanced medicine. Gene therapy therefore must be seen as a crystallisation centre or a catalyst of the revival of preexisting ethical problems. In fact, I believe that the importance of a technology is directly proportional to the amount of revived preexisting structural problems. Paradoxically, the number of raised problems is good news for all those who believe that gene therapy is an important technique.
Quo vadis gene therapy?
Gene therapy has started making headlines long before having concretised the most elementary requirements. This is not necessarily a bad situation because it may permit, for the first time (!), to thoroughly discuss the legal, social and ethical implications of a novel technique long before it can be implemented. This was not the case with many revolutionary technical advances inside and outside the medical world (from the anti-baby pill, the cellular phone to Prozac). We can say the nastiest things about gene therapy but we will never be able to deny that it has had enough time to mature its ecological niche in our social and cultural context. When caught by our endemic optimism, we like to think this should help in preventing the occurrence of some misuses and abuses.
But when will gene therapy become a clinical reality? I will use a metaphoric comparison with the history of aviation to illustrate my view on the progress of gene therapy. Gene therapy before 1990 can be compared to Icarus's flight: many dreams and many ending in a sad way. Gene therapy anno 1990 reminds me of Leonardo's attempts to sketch flying objects: the first rational approaches. Gene therapy anno 1995 has the smell of the Mongolfier's achievements: "yes, it can fly!" Gene therapy today (anno 2000) can be compared to the awkward engine-aeroplanes by the Wright brothers: we are bumping on an grass-andmud takeoff track, we take off but it is difficult to control flight. I see gene therapy anno 2005 as the first World War nostalgic aeroplanes: noisy and fragile but somewhat effective. The year 2010 may bring with the first real achievements of mid- to long-term therapy the enthusiasm of the first and heroic transatlantic flights. Gene therapy protocols with a precision and reliability similar to the bombers of the World War II may be achieved around 2015. Finally, I shall probably have been retired for a long time when the gene therapy equivalent of real routine "commercial flights" or with the poignant properties of the "stealth bombers" will be available. This will not be achieved without many frustrations and regrettable accidents but this perspective should not discourage us from attempting to overcome the hurdles. Last year, I started writing my welcome address to the annual meeting of our NFP37 with the words: "Gene therapy is not a field for pessimists!" In this spirit, I confess that in our crew we are all proud to belong to those who prepare this promising new therapeutical field for future generations.
Acknowledgements: I thank A. Genilloud for providing the graphic material for figure 4, and Drs. J. Isner and D. Losordo for communication of their unpublished data.
References
1. Blaese RM, Culver KW, Chang L, Anderson WF; Mullen C, Nienhuis A, et al. Treatment of severe combined immunc~deficiency disease (SCID) due to adenosine deaminase deficiency with CD34+ selected autologous peripheral blood cells transduced with a human ADA gene. Hum Gene Ther 1993;4: 521- 7.
2. Grossman M, Raper SE, Kozarsky K, Stein EA, Engelhardt JF, Muller D, et al. Successful ex vivo gene therapy directed to liver in a patient with familial hypercholesterolaemia. Nat Genet 1994j6:335-41.
3. Crystal RG. Transfer of genes to humans: early lessons and obstacles to success. Science 1995;270:404-10.
4. Baumgartner I, Pieczek A, Manor O, Blair R, Kear~ey M, Walsh K, et al. Constitutive expression of phVI GF165 .~fte~intramuscular gene transfer promotes collateral vessel devel opment in patients with critical limb ische~nia. Circ~latic' 1998j97:1114-23.
5. Isner J, Losordo D. Personal communication.
6. Pennisi E. Will a twist of viral fate lead to a new cancer treatment? Science 1996;274:342-3.
7. Kmiec E. Targeted gene repair. Gene Ther 1999;6:1-3.
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Figures
Figure 1 Ageing of the population and disease
frequency.
A) Plot representing life expectancy at birth (y axis) during this
century (x axis), source Swiss office of statistics; B) plot of the
incidence of cancer (y axis) as function of age (x axis), source NIH;
C) plot representing Alzheimer's free fraction (y axis), as a
function of age (x axis). Symbols: M: average of the population;
E2/E2, E3/E4, E4/E4: represent curves for allelic configurations of
the apoliproprotein E gene. (notes from a lecture of A. Roses, USGEB
1998).
Figure 2, Therapeutic viruses.
Natural viruses (2A) are composed of one or more protective layers that are made by proteins expressed from viral genes (envelope or capsid proteins) and that protect the nucleic acids that compose the viral genome (RNA or DNA). Natural viruses contain in their genome all the genes that are necessary for producing the coat and envelope and for ensuring viral genome replication after infection. The genes responsible for viral replication are changing the cell's genetic programme and are therefore the basis of the pathogenic potential of viruses. In recombinant viruses (2B) parts of the genome are substituted by the gene(s) of interest (therapeutic or marker gene). This occurs at the costs of essential viral genes. Thus, such recombinant viruses are incapable to grow autonomously. They can be grown in especially engineered cells (the so-called "packaging cell lines" which contain in their chromosomal genome the complementing functions). Efficient titers of recombinant viruses can be obtained with adenoviruses, retroviruses (including HIV), Herpes viruses and even paramyxoviruses. Those recombinant viral particles are fully capable of infecting cells but incapable of propagation and are currently the most efficient vehicles to deliver genes into primary cells. One of the disadvantages of recombinant viruses is their limited size capacity for foreign genes (from 3000 to 10 000 base pairs, with exceptional cases exceeding 30 000 bp). Other disadvantages are discussed in the text.
Figure 3, Principal methods for human gene transfer.
Explanations are in the text.
Figure 4, Trends in gene therapy.
A) Gene therapy world-wide. The graph shows the evolution of
approved clinical trials (y axis left) or the number of patients (y
axis right) as function of time (x axis, from 1990 to 1998). Solid
line: clinical trials aimed at cancer treatment; dotted line:
clinical trials aimed at treatment of hereditary disorders. Other
abbreviations: vase = vascular disorders; infect = infectious
disorders.
B) Gene therapy in Switzerland. The table reports the number of
approved clinical trials (left column) and the corresponding number
of patients (rightmost column) as of February 1998 (source SKBS), and
the number of preclinical experiments aimed at gene therapeutic
applications (~deduced from NF database). The lines under the title
"type of disease" or "type of gene transfer" indicate the
distribution into the different categories. The last two lines
indicate the number of clinical and preclinical trials performed in
the first phase (1996-1998) of the National Research Programme 37
"Somatic Gene Therapy" (NFP37, for details please consult our WEB
site http://www.unifr.ch/nfp37). For the second phase (1999-2001) the
NFP37 has received three additional grant applications dealing with
clinical trials.
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