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Lists with links to individual abstracts
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WEDNESDAY 3.10
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Time |
Name |
Team |
Abs Nr |
min |
Title |
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900 |
Graham, I. |
Univ. London, Egham, UK |
45 | ||
|
950 |
Arsenijevic, Y. |
GUEST |
5 | ||
|
955 |
Lobianco, C. |
AEBISCHER |
5 | ||
|
1000 |
Regulier, E. |
AEBISCHER |
5 | ||
|
1005 |
Odermatt, A. |
FREY/Rusconi |
5 | ||
|
1010 |
Ashrafuzzaman, M. |
GUEST |
5 | ||
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1015 |
Esslinger, C. |
GUEST |
5 | ||
|
1020 |
Schafer, B. |
GUEST |
5 | ||
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1025 |
Afanasieva, T. |
KLEMENZ |
5 | ||
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1030 |
BREAK |
COFFEE |
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|
1050 |
Schultz, J. |
MOELLING |
5 | ||
|
1055 |
Schuemperli, D. |
SCHUEMPERLI |
20 | ||
|
1115 |
Brun, C. |
SCHUEMPERLI |
5 | ||
|
1120 |
Jilek, S. |
WALTER |
5? | ||
|
1125 |
Kralisch, S. |
WALTER |
5 | ||
|
1130 |
Li, J. |
VASSALLI |
5 | ||
|
1135 |
Blaese, M. |
VALIGEN Inc, |
45 | ||
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1220 |
BREAK |
POSTERS & LUNCH |
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1230 |
Bensadoun, J. |
AEBISCHER |
- | ||
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1230 |
Guillot, S. |
AEBISCHER |
- | ||
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1230 |
Hussain-khan, H. |
AEBISCHER |
- | ||
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1230 |
Ridet, J. |
AEBISCHER |
- | ||
|
1230 |
Schneider, A. |
SCHNEIDER/ Rusconi |
- | ||
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1230 |
Schwenter, F. |
AEBISCHER |
- | ||
|
1230 |
Stolarczyk, D. |
GUEST |
- |
| |
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1300 |
BREAK |
POSTERS & LUNCH |
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1400 |
Jones, G. |
MERLO |
20 | ||
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1420 |
Kolakofsky, D. |
KOLAKOFSKY |
20 | ||
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1440 |
Scully, J. |
REHMANN-SUT. |
20 | ||
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1500 |
Moelling, K. |
MOELLING |
20 | ||
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1520 |
BREAK |
REFRESHMENTS |
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1540 |
Walter, E. |
WALTER |
20 | ||
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1600 |
Bisoffi, M. |
STADLER/ Cecchini |
20 | ||
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1620 |
Mermod, N. |
MERMOD |
20 | ||
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1640 |
High, K. |
Children's Hosp., Phila. |
45 | ||
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1730 |
End-day2 |
REFRESHMENTS |
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1900 |
Dinner |
Invitees |
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Time |
Name |
Team |
Abs Nr |
min |
Title |
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900 |
Mertelsmann, R. |
Uni-Klinikum, |
45 | ||
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955 |
Kok, M. |
DREHER |
5 |
Live Salmonella Vectors For Antigen Transfer Into Human Dendritic Cells. | |
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1000 |
Obregon, C. |
DREHER |
5 |
Salmonella Typhimurium Sipb Protein Modulates Activation And Release Of Il-18 ... | |
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1005 |
Dreher, D. |
DREHER |
20 |
From Genomics To Vaccination: Treatment Of Latent Tuberculosis By Recombinant ... | |
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1025 |
BREAK |
COFFEE |
. |
. |
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1045 |
Galeazzi, R. |
INVITED PANELIST symp |
none |
30 |
NF and clinical research, report |
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1115 |
Diggelmann, H. |
INVITED PANELIST symp |
none |
10 |
NF policy |
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1125 |
Cavalli, F. |
INVITED PANELIST symp |
none |
10 |
The view of a Dr med/politician |
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1135 |
Dorsch, K. |
INVITED PANELIST symp |
none |
10 |
Biosafety rules update |
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1145 |
All |
EVENT/ |
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panel and general discussion |
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1220 |
BREAK |
POSTERS & LUNCH |
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1400 |
All |
EVENT/ |
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presentation of steering committee GT |
|
1415 |
Schneider, H. |
Children's Hosp. Erlangen, De |
45 | ||
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1500 |
Seemayer, C. |
GAY |
20 | ||
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1520 |
BREAK |
REFRESHMENTS |
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1540 |
Wiznerowicz, M. |
TRONO |
20 |
Hematopoietic Stem Cells-directed Gene Therapy: Chronic Granulomatous Disease | |
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1600 |
Colman, A. |
PPL Therapeutics Midlothian, UK |
45 |
Potential Of Animal Cloning Technologies In Future Therapies | |
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1700 |
Closure |
Safe return! |
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Name |
Abstract Nr |
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Ackermann M | |
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Aebischer P | |
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Afanasieva T | |
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Allemann N | |
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Arsenijefic Y | |
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Ashrafuzzaman | |
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Audige A | |
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Azzouz M | |
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Ball AK | |
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Baumgartner I | |
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Beck-Sickinger | |
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Bensadoun J-C | |
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Bernasconi L | |
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Bisoffi M | |
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Blaese M | |
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Bouche N | |
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Brun C | |
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Bueeler H | |
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Buehlmann E | |
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Bueler H | |
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Burg G | |
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Burgunder J-M | |
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Chiodini F | |
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Cochand L | |
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Colman A | |
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Danos O | |
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Deglon N | |
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Doebbeling U | |
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Dollenmaier G | |
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Dong Z | |
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Dreano M | |
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Dreher D | |
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Driscoll R | |
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Dudler J | |
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Dummer R | |
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Dunant P | |
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Esslsinger C | |
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Feldon J | |
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Ferger B | |
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Fleury S | |
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Fontana A |
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Name |
Abstract Nr |
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Fraefel C | |
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Frey FJ | |
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Frick C | |
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Fuhrman-B, E. | |
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Geertsen R | |
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Genilloud A | |
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Gonin J | |
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Graham I | |
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Greber U | |
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Guillot S | |
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Heinzerling | |
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Heister T | |
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Hellwig R | |
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Hemmi S | |
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Herrera P | |
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High K | |
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Hornfeld D | |
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Hossle JP | |
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Hussain-Kahn H | |
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Iggo | |
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Jilek S | |
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Johnson L | |
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Jones G | |
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Keyser J | |
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Klemenz R | |
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Kok M | |
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Kolakofsky D | |
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Kostic C | |
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Kralisch S | |
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Li J | |
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Lobianco C | |
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Lochmuller H | |
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Losordo D | |
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MacDonald HR | |
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Malipiero U | |
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Margue CM | |
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Matheux F | |
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Merkle H-P | |
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Mermod N | |
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Mertelsmann R | |
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Moelling K | |
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Mueller M | |
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Nicod LP | |
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Nurnier F |
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Name |
Abstract Nr |
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Obregon C | |
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Pach R | |
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Pajusola K | |
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Pauli C | |
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Pavlovic J | |
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Pereira de A., L. | |
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Peter I | |
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Pralong WF | |
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Regulier E | |
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Reith W | |
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Ridet JL | |
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Rusconi S | |
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Salmon P | |
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Schaefer BW | |
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Schaffner W | |
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Schneider A | |
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Schneider H | |
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Scholl FA | |
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Schuemperli D | |
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Schultz J | |
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Schwenter F | |
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Scully J | |
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Seemayer C | |
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Seger RA | |
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Sommer B | |
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Spicher A | |
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Suter D | |
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Tan T | |
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Tekaya M | |
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Textor M | |
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van Adel BA | |
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Vassalli G | |
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Vassalli J-D | |
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Villard J | |
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Vitaliti A | |
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Voros J | |
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Walter E | |
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Weis J | |
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Willers J | |
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Wittmer M | |
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Wiznerowicz M | |
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Wodnar-F., A. | |
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Wyder L | |
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Zala D | |
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Zurn AD |
L01// ONCOLYTIC VIRUSES
Leisa Johnson, Onxy Pharmaceuticals, Richmond, USA
Keywords: Adenovirus, Cancer, Retinoblastoma, P53, E2F
L02// BIOLOGICAL AND CLINICAL FEATURES OF AAV
VECTORS
Olivier Danos, Genethon, Paris, France
Keywords: Adeno-associated virus, viral vectors, immune response,
skeletal muscle, neuromuscular diseases
L03// EARLY RESULTS FROM CLINICAL TRIALS OF GENE
THERAPY FOR MYOCARDIAL ANGIOGENESIS
Douglas Losordo, Tufts University School of Medicine, Boston, USA
Keywords: Angiogenesis, VEGF, randomized clinical trial, endothelium,
coronary arthery disease
L04// OLIGONUCLEOTIDE-BASED GENE CORRECTION
STRATEGIES: APPLICATIONS TO NEUROMUSCULAR AND CARDIVASCULAR
DISEASES
Ian Graham, School of Biological Sciences, University of London,
Egham, UK
Keywords: apolipoproteins, dystrophin, gene targeting,
hyperlipidemia, muscular dystrophies, point mutation, antisense, RNA
splicing
L05// THERAPY BY GENE REPAIR
Michael Blaese, Valigen Inc., Newtown, USA
Keywords: Genoplasty, DNA repair, monogenic diseases, DNA sequence
conversion, MutS, chimeraplast
L06// GENE-ASSISTED TREATMENT OF HEMOPHILIA
Katherine High, Children's Hospital, Philadelphia, USA
Keywords: Adeno-associated vectors, blood clotting factor, clinical
trial
L07// GENE THERAPY AND GENE MARKING CONCEPTS IN
CANCER TREATMENT
Roland Mertelsmann, Universitätsklinikum Freiburg i.Br.,
Germany
Keywords: gene marking, stem cells, cancer therapy, gene therapy
L08// IN UTERO GENE THERAPY
Holm Schneider, Children's Hospital, Erlangen, Germany
Keywords: aims, candidate diseases, choice of vectors, routes of
administratiuon, animal models, developmental windows, ethical
considerations
L09// POTENTIAL OF ANIMAL CLONING TECHNOLOGIES
IN FUTURE THERAPIES
Alan Colman, PPL Therapeutics, Midlothian, UK
Keywords: nuclear transfer, reprogramming, imprinting, stem cells,
cloning
M01// DIAGNOSTIC TEST FOR MHC CLASS II
DEFICIENCY PATIENTS BY DIRECT GENETIC CORRECTION
NFP37 Nr: 055159; Franck Matheux1, Aydan Ikinciogullari3, Madeleine
Zufferey1, Emanuèle Barras1, Walter Reith1 and Jean
Villard1,2; 1Department of Genetics and Microbiology, University
of Geneva Medical School, Centre Medical Universitaire (CMU),
2Immunology and Transplant Unit, University Geneva Hospital, 3Ankara
University, Department of Pediatric Immunology
MHC class II (MHC-II) deficiency is a genetically heterogeneous
immunodeficiency syndrome resulting from defects in four trans-acting
regulatory factors (CIITA, RFX5, RFXAP and RFXANK) required for
transcriptional activation of MHC-II promoters. In contrats to the
genetic heterogeneity of the disease, the clinical characteristics
are usually homogenous. By cell fusion analysis, patients have been
first classified in four different groups corresponding to the four
mutated genes. In some cases the diagnostic are clear with typical
clinical and immunological features or a family story. In other cases
it's not so evident and even the classical and biological diagnostic
is clear, it's not permitted to determine the genetic defect of the
patient. The isolation of the factors mutated in the different group
has permitted to envisage a rapid and easy diagnostic test. We have
transduce cell derived from a new Turkisch patient (PBL) with a
supernatant of lentivirus vector containing the gene of interest
(CIITA, RFX5, RFXAP or RFXANK). We have observed by FACS analysis,
the reexpression of MHC class II molecules at the cell surface of the
PBL stimulated with IL-2 and PHA with the lentivirus containing the
RFXAP gene only. Therefore, the patient is mutated in the RFXAP gene
and belongs to the complementation group D. The molecular analysis
confirm the defect in the RFXAP gene. The RFXAP cDNA of this turkish
patient was ampliffied and subcloned and we find a point mutation (C
to T) leading to a premature sto codon. The truncated RFXAP protein
derived from the mutated gene is unable to activated MHC class II
expression at the cell surfaces. By direct genetic correction this
new tool permits to classifiy easily new MHC-II deficient patient and
to envisage prenatal diagnostic analysis.
M02// ADENOVIRUSES WITH TCF BINDING SITES IN
MULTIPLE EARLY PROMOTERS SHOW ENHANCED SELECTIVITY FOR TUMOUR CELLS
WITH CONSTITUTIVE ACTIVATION OF THE WNT SIGNALLING PATHWAY
NFP37 Nr: 055140; Christophe Fuerer and Richard Iggo; Oncogene
Group; Swiss Institute for Experimental Cancer Research; Chemin des
Boveresses 155; CH-1066 Epalinges; Switzerland
Mutations in the adenomatous polyposis coli and b-catenin genes in
colon cancer lead to constitutive activation of transcription from
promoters containing binding sites for Tcf/LEF transcription factors.
We have constructed adenoviruses with Tcf binding sites in multiple
early promoters, in order to target viral replication to colon
tumours. Tcf regulation of the E1A or E2 promoters leads to selective
replication in cells where the wnt signalling pathway is active. We
show that p300 is a coactivator for b-catenin and E1A inhibits
Tcf-dependent transcription through sequestration of p300. Mutation
of the p300 binding site in E1A reduces E1A-mediated repression of
transcription from the Tcf-E1A and Tcf-E2 promoters in transient
transfection assays but does not increase promoter activity in the
context of the virus. On the contrary, mutation of the p300 binding
site in E1A leads to a 10-fold reduction in cytopathic effect of all
of the Tcf-regulated viruses. When Tcf sites are inserted in the E1A,
E1B, E2 and E4 promoters, the viruses are highly selective for cells
with activated wnt signalling but 10 to 1,000-fold attenuated
relative to wt Ad5. The virus with Tcf changes only in the E1A and E4
promoters (vCF11) shows 100 to 10,000-fold selectivity for cells with
activated wnt signalling in viral burst and cytopathic effect assays
and is active in a broad panel of colon cancer cell lines.
M03// LENTIVIRAL VECTORS FOR THE TREATMENT OF
NEURODEGENERATIVE DISORDER
NFP37 Nr: ; Patrick Aebischer
see abstracts Lobianco et al (P09) and Ridet et al (P11)
M04// LENTIVIRUS-MEDIATED GENE TRANSFER INTO
HEMATOPOIETIC PRECURSOR CELLS FROM UMBILICAL CORD BLOOD
NFP37 Nr: 055157; Luther-Wyrsch A (1), Kalberer CP (1), Siegler U
(1), Costello E (2), Buetti E (2), Thali M (2), Surbek D (3),
Holzgreve W (3), Nissen C (1) and Wodnar-Filipowicz A (1). (1)
Research Department and (2) Obstetrics Department, University
Hospital Basel; (3) Institute of Microbiology, University Hospital
Lausanne
Hematopoietic stem cells are important targets for gene therapy
of inborn and acquired diseases. This project examined the efficiency
of genetic modification and the maintenance of transgene expression
during long-term expansion and lineage-specific differentiation of
human CD34+ hematopoietic progenitor cells from cord blood (CB)
transduced with lentiviral vectors. Transfer vectors contained the
green fluorescent protein (GFP) gene under the control of PGK
promoter (Costello et al, Gene Ther 7, 596-604, 2000) or EF1a
promoter (kind gift of P. Salmon and D. Trono, University of Geneva).
The transduction rate was 25-35% when cells were exposed to growth
factors (IL-3, IL-6, flt3 ligand and SCF) or 5-10% without
stimulation. Analysis of transduction efficiency relative to cell
cycle phases demonstrated GFP expression distributed evenly in
dormant Go and cycling G1 and S/G2/M cells stained with pyronin Y and
Hoechst 33342. The expansion capacity of transduced hematopoietic
progenitors was equal to untransduced cells. In liquid cultures
supplemented with flt3 ligand and thrombopoietin, a 1000-fold
amplification of CD34+ cells was achieved and GFP expression was
maintained at the 25-35% level for 10 weeks after transduction, but
declined after 2 more months in culture. In the absence of GFP
expression, integrated GFP-specific sequences could be detected,
consistent with transgene silencing as a function of time. Next, we
determined whether genetically modified progenitors can be driven to
lineage differentiation without loss of transgene expression. Using
IL-15 and flt3 ligand, we obtained effective differentiation of CD34+
cells to natural killer (NK) cells. After 5 weeks in culture, the
content of CD56+CD3- NK cells was 40-70% including 10-20% of GFP+
population. In summary, these results demonstrated an efficient
lentivirus-mediated GFP gene transfer to hematopoietic progenitors
with high potential for self-renewal and lineage-specific
differentiation. Since CB cells are considered as targets for somatic
gene therapy of inborn defects before birth of an affected child, we
analysed the feasibility of transduction of CD34+ cells from CB of
early gestational ages. We found that fetal CB is rich in
hematopoietic progenitors (Wyrsch et al, Exp Hematol 27:1338-1345,
1999) and that fetal precursors can be transduced using lentiviral
vectors and expanded ex vivo with the efficiency comparable to
neonatal CB (Luther-Wyrsch et al. Human Gene Ther. 12, 377-389,
2001). These results suggest that CB progenitors obtained in the
second and early third trimester can be targeted for gene transfer
and may prove useful for autologous gene therapy of genetic diseases
amenable to prenatal stem cell transplantation.
M05// SELECTIVE ENHANCEMENT OF GENE TRANSFER BY
STEROID-MEDIATED GENE DELIVERY (SMGD)
NFP37 Nr: 044802; Alexandre Rebuffat (1), Alessio Bernasconi (1),
Maurizio Ceppi (2), Hans Wehrli (1), Stefano Brenz Verca (2), Merdol
Ibrahim (3), Brigitte M. Frey (1), Felix J. Frey (1), and Sandro
Rusconi (2) . 1) Division of Nephrology, Inselspital, Bern; 2)
Institute of Biochemistry, UNIFR, Fribourg; 3) Institute of
Histology, UNIFR, Fribourg.
The strategy that we name steroid-mediated gene delivery (SMGD) is
designed to facilitate the nuclear uptake of transfected DNA with the
help of steroid receptors, which are natural cytoplasm-nuclear
shuttles. In this work the SMGD principle was modelled with the
Glucocorticoid Receptor (GR) system. The challenge was to chemically
derivatize steroids to allow their covalent linkage to plasmid DNA
while maintaining their affinity and accessibility for the GR. We
synthesized and tested several bifunctional steroid derivatives to
finally focus on the compound named DR9NP that consists of a
dexamethasone backbone linked to a psoralen moiety with a 9-atom
chemical spacer. DR9NP is shown to be able bind to GR either in its
free or DNA crosslinked form and to induce nuclear translocation of
GR. The expression of DR9NP-decorated reporter plasmids transiently
transfected into dividing cells is reproducibly enhanced (up to 4
fold). Most importantly, the enhanced expression occurs only in cells
expressing the GR, is independent of the transactivation potential of
GR and correlates with the enhanced nuclear accumulation of
hormone-decorated plasmids in GR-positive cells. The SMGD effect can
be also reproduced in cells that naturally express the GR and gives a
markedly increased transfection advantage (up to 40 fold) to
hormone-decorated transgenes in non-dividing cell cultures.
Therefore, we propose the SMGD approach as a platform strategy for an
additional selective targeting in non-viral somatic gene transfer.
M06// PROGRESS IN SOMATIC GENE THERAPY OF
X-LINKED CHRONIC GRANULOMATOUS DISEASE
NFP37 Nr: 055165; Hossle JP (1), Saulnier SO (1), Steinhoff D (1),
Hellwig R (1), Al-Azzeh E (1), Grez M (2), Trono D (3), Ott MG (4),
Hoelzer D (4), Merget-Millitzer H (5), Dinauer MC (6), and Seger RA
(1); (1) Div. of Immunology/Hematology, Univ. Children's Hospital,
Zuerich, Switzerland; (2) Georg-Speyer-Haus, Frankfurt a.M., Germany,
(3) Dept. of Genetics and Microbiology, University of Geneva Medical
School, Geneva, Switzerland, (4) Hematology/Oncology, Univ. of
Frankfurt Medical School, Frankfurt a.M., Germany, (5) MainGen GmbH,
Frankfurt a.M. Germany, (6) Herman B Wells Center for Pediatric
Research, Indiana University School of Medicine, Indianapolis, IN,
USA.
The objectives of our project were the development and
implementation of clinically applicable protocols for somatic gene
therapy of X-linked chronic granulomatous disease (X-CGD). We
developed a bicistronic retroviral vector (SPsLdS) carrying the
therapeutic gp91phox gene and a truncated form of the low affinity
nerve growth factor receptor (deltaLNGFR) as a selectable marker.
After successful preclinical testing of this vector, including
long-term expression in a murine X-CGD model, PG13-SPsLdS
virus-containing supernatant was produced according to
good-manufacturing-practise (GMP) for clinical use of the produced
material. A collaborative first clinical phase I study has been
initiated earlier this year with adult X-CGD patients enrolled in
Frankfurt. This is the first gene therapy study worldwide to combine
the gene transfer with a mild myelosuppressive chemotherapy with
autologous reconstitution (cyclophosphamide conditioning). It is
planned to use the same retroviral vector in a phase I study to start
later in Zuerich for the treatment of X-CGD children. Most recent
further progress includes i) the generation of improved lentiviral
vector constructs for gp91phox gene transfer into not only resting
pluripotent hematopoietic stem cells but also featuring stronger and
potentially myeloid tissue-specific promoters and ii) construction of
human G-CSFR/estrogen chimeric receptor variants responsive to
4-hydroxy-tamoxifen, for to provide transduced progenitor cells with
a selection advantage. Finally, preliminary experiments in
EBV-transformed B-cells from an X-CGD patient suggest the feasibility
of direct oligonucleotide-mediated targeted gene repair in selected
X-CGD cases with single nucleotide point mutations.
M07//
Lessons learned from VEGF gene therapy; abstract not received.
M08// THE FOCAL ADHESION KINASE (FAK) IS HIGHLY
EXPRESSED IN HUMAN BRAIN TUMORS AND IS REQUIRED FOR TUMOR INVASION
AND SURVIVAL: NEW DIRECTIONS FOR ANTI-INVASIVE THERAPIES?
NFP37 Nr: 055167; JONES G, MERLO A; Molecular Neuro-Oncology,
Department of Biological and Clinical Sciences, Kantonsspital Basel,
Basel, Switzerland.
To improve the dismal outlook of patients suffering from glial brain
tumors, a new therapy must target both the diffuse infiltration of
healthy brain tissue and the rapid proliferation of tumor cells. We
have investigated the role of the focal adhesion kinase (FAK), which
modulates cellular migration and survival, in human glial tumors.
Examination of tumor samples consistently showed increased expression
of FAK in low- and high- grade astrocytomas, but not in the more
benign oligodendroglioma. Increased FAK immunoreactivity was also
observed during glioblastoma progression following radiotherapy.
Expression of the C-terminal focal adhesion targeting domain (FAT) of
FAK, which targets FAK to the focal adhesions, inhibited FAK in
cultured glioblastoma cell lines in a dominant-negative manner,
resulting in loss of invasion and enhanced sensitivity to apoptotic
stimuli. Significantly, the inhibition of FAK resulted in loss of
integrin and growth factor-dependent signalling. Investigation of how
FAK might integrate these pathways in glioblastoma cells revealed
that FAK was required for sustained EGFR activity at the cell
membrane and cell migration. Unexpectedly, FAK was not only present
at the cell membrane, but also in the nucleus, predominantly as a
truncated N-terminal variant. Although FAK was present in the nucleus
of normally growing cells, exposure to apoptotic stimuli induced the
aggregation of FAK in the nucleus, which was enhanced by expression
of the FAT domain. In summary, our results identify FAK expression as
a marker of brain tumor progression and as a signalling platform
integrating growth and invasive pathways. Finally, the novel
observation that FAK is present at the membrane and in the nucleus
provides clues as to how such integration might occur. Publications:
Cancer Res 59: 5479-5482, 1999. Cancer Res. 61: 5688-5691, 2001.
Cancer Res. 61:4978-4981, 2001.
M09// PROLONGING THE LIFE SPAN OF MATURE
DENDRITIC CELLS
NFP37 Nr: 044711; Dominique Garcin and Daniel Kolakofsky,
University of Geneva
Linear paramyxovirus RNA genomes contain promoters at each end which
control genome and antigenome synthesis (the leader and trailer
regions). One of the first rSeV we prepared, called GP42, contains
the first 42 nt of its genomic promoter replaced with 42 nt of the
antigenomic promoter. Remarkably, this virus infects most cell lines
and grows almost normally, but GP42 is non-cytopathic and leads to a
persistent infection of cell lines. SeV-GP42 infection appears to
interfere with the normal cellular apoptotic program, because these
cells also become resistant to the relative absence of serum factors
(GP42-persistently-infected Hela cells can be split 1 : 1000 !). Part
of this work has been published ; Garcin, D., G. Taylor, T.
Tanebayashi, R. Compans, and D. Kolakofsky. The short Sendai virus
leader region controls induction of programmed cell death. Virology,
243: 340-353, 1998. We have since determined that of the 42 nt that
have been exchanged in GP42, only 11 nt (the palindrome UUUUAAAUUUU,
positions 31-41) are important for preventing programmed cell death.
This palindrome represents a 40 kD host cell protein binding site,
and the non-cytopathic virus phenotype apparently results from the
presence of this binding site on the short RNAs now expressed from
both viral promoters (manuscript in preparation). A large effort is
underway to use ´ programmed ª dendritic cells for
immuno-therapy, e.g., DCs loaded with tumor antigen peptides ex vivo
as anti-tumor agents. If the DCs also express an anti-apoptotic gene
like bxl (in a mouse model), their anti-tumor activity is much
improved. We have therefore examined the effects of SeV-GP42
infection on human DCs generated from CD14+ monocyte precursors, to
see whether SeV-GP42 can increase the life span of such cells which
normally have short life spans. In particular, dendritic cells which
have been induced to mature, ie, to present antigens via HLA class II
at their surface, normally all die well within 10 days following
maturation, by apoptosis. SeV, like many other viruses, is relatively
efficient in inducing human dendritic cell maturation, and dendritic
cells infected with wild-type SeV also all die within 10 days.
Dendritic cells infected with SeV-GP42, on the other hand, remain in
excellent health for at least 15 days, as determined by staining with
vital dyes. Moreover, analysis by FACS sorting determined that these
living cells were indeed mature dendritic cells, and immunoblotting
showed that these cells continue to produce quite respectable amounts
of viral antigens.
Given these encouraging first results, we have prepared two new
´ types ª of GP42 viruses for further studies.
a) Wild-type and GP42 viruses containing either an aditional red-FP
or GFP transgene. These viruses will allow us to follow the on-going
virus infection simply by following red or green fluoresence, using
the other color to follow some aspect of dendritic cell maturation.
This will alow us to study the effect of the GP42 virus on human
dendritic cells in detail, in real time, and more accurately, by
FACS. b) The same wild-type and GP42 viruses as above, except that
their F (fusion) genes carry the TR5 mutation so that their fusion
proteins are no longer activated (cleaved) by trypsin-like proteases
(but which grow in eggs supplemented with chymotrypsin). These
viruses are safe for mouse infections (and are, in fact, used as SeV
mouse vaccines) because they undergo only a single round of infection
in the animal (the virus progeny are not infectious).
We have begun a collaboration with Claire-Anne Siegrist in Geneva
(who can legally infect mice with TR5 SeV), to evaluate these viruses
as antigen presenters in mice. We know that control TR-5 immunization
produces robusts antibody responses in adult mice. We can then
evaluate the levels of IgG1 and IgG2a antibodies to Sendai following
intramuscular injection, as these antibodies are acceptable surrogate
markers for APC-Th1/Th2 interactions and for APC- B cell activation.
M10// PERCEPTIONS OF HEALING NEEDS: SOMATIC GENE
THERAPY, DISABILITY AND IDENTITY
NFP37 Nr: 053073; Scully JL, Rippberger C, Loew C, Rehmann-Sutter C.;
Arbeitsstelle fuer Ethik in den Biowissenschaften, Institut fuer
Geschichte und Epistemologie der Medizin, Universitaet Basel,
Schoenbeinstrasse 20, CH-4056 Basel
This project has analysed differences between interest groups in the
perception of the ethical issues associated with somatic gene therapy
(SGT). We use well established qualitative research methodologies to
analyse questionnaire and interview data from potential consumers and
from the medical profession. Our results demonstrate that there are
significant differences between these two groups in (a) the ethical
issues they find significant, and (b) the approaches they bring to
decision making, in the area of SGT. The key areas of difference
identified include: a) perception of disability and illness as part
of the person's identity; b) belief in the alteration of fundamental
identity by gene therapy; c) issues of power differentials and trust
in/mistrust of the medical profession and biotechnology; d) the
time-scale over which ethical evaluation is made, and use of factors
other than the medical, e.g. emotional, financial factors. Currently
we are analysing in more detail how these differences may reflect the
underlying perception of the moral meaning of a medical condition and
its connection with personal and group identity. We have also made
preliminary identification of differences between affected groups.
People with the late-onset condition of multiple sclerosis (MS)
generally were much more positive towards SGT than people whose
conditions were stable and/or of early onset (achondroplasia,
deafness). Some achondroplasiacs, however, distinguished between
separate manifestations of the condition. Interviewees with cystic
fibrosis (CF) were more ambivalent than the other groups. We relate
these differences to different constructions of the relationship
between identity and illness in these groups.
M11// DISRUPTION OF INTEGRIN FUNCTION BY
EXPRESSING PARTIAL REGIONS OF THE CYTOPLASMIC DOMAIN OF THE BETA3
SUBUNIT
NFP37 Nr: 055150; A.Foletti, D. Oguey, C. Paroz, and C. Ruegg;
Centre Pluridisciplinaire d'Oncologie, School of Medicine,
University of Lausanne CH-1011 Lausanne c/o ISREC CH-1066
Epalinges
Administration of tumor necrosis factor (TNF), interferon gamma
(IFNg) and chemotherapy through the isolated limb perfusion
technique, has emerged as a potent anticancer therapy in which
disruption of the tumor vasculature is an essential component. Since
systemic administration of TNF induce severe side effects before
therapeutic level, we are investigating alternative treatments, which
acting in synergy would allow reduction of TNF dose and so systemic
administration. Endothelial cells adhere to the basement membrane
mainly via the integrins beta1 (quiescent vessels) and beta3
(angiogenic vessels). Since TNF/IFNg treatment suppress alphavbeta3
mediated endothelial cell adhesion we are aiming at further
destabilise angiogenic endothelial cells interfering specifically on
beta3 subunit. Dominant negative constructs encoding for membrane
bound cytoplasmic domain of beta1 or beta3 induce detachment of
confluent monolayers of HUVEC, block re-adhesion to different matrix
proteins but have transdominant effects. To improve the specificity
of the dominant negative effect to beta3, FLAG peptidic tag or GFP
protein were fused to parts of the beta3 cytoplasmic domain.
Introduced in confluent HUVEC monolayer by electroporation, the
constructs were expressed at the same level and with the same kinetic
within 24h. beta3 fragments fused to the FLAG had no effects on
adhesion of HUVEC. We think to an intracellular degradation. Among
GFP fused proteins only the construct expressing the full length
beta3 cytoplasmic domain induced detachment of confluent HUVEC
monolayers and blocked re-adhesion to different matrix proteins but
without any specificity towards beta1 or beta3 integrin mediated
adhesion. These data demonstrate that, membrane proximal sequence is
required for dominant negative activity of isolated beta3 cytoplasmic
domain and that full length beta3 tail has trans dominant negative
activity as both membrane anchored or cytoplasmic soluble chimeric
protein.
M12// CYTOKINE-ENCODING DNA FOR CANCER GENE
THERAPY
NFP37 Nr: 048767; Moelling, K; Institute of Medical Virology,
University of Zuerich, Gloriastrasse 30, CH- 8028 Zuerich
We have recently demonstrated that the cytokine Interleukin-12
(IL-12) induces a long-lasting anti-metastatic effect in a mouse
model using the melanoma cell line B16-F10 and plasmid DNA injected
intramuscularly. DNA encoding IL-12 is superior to DNA encoding
GM-CSF, CpG oligonukleotides or DNA coding for the tumor-associated
antigen gp100.The anti-metastatic effect involves CD8 positive T
cells as shown by genetically modified CD8 or perforin deficient
mice. Moreover, depletion of natural killer cells abrogates the
anti-metastatic effect. Interferon-gamma or TNF-alpha are not the
cause. DNA injected up to 30 days before tumor cell challenge
prevented formation of metastases. IL-12 expression is long-lasting,
up to 60 days due to an autocrine loop involving various cytokines. A
second animal model was employed as alternative to mice, grey horses,
which naturally develop malignant melanoma at a high incidence. IL-12
encoding plasmid injected intratumorally lead to tumor reduction in 7
horses with a total of 12 lesions. On the basis of these two animal
models and a 3 year follow up of a DNA vaccine trial with HIV
infected patients, who did not show any side effects, a clinical
trail was initiated at the University hospital in Zuerich.The study
includes a dose escalation of IL-12 DNA applied intratumorally in
patients with late stage malignant melanoma. The trial is ongoing
with no serious side effects in the patients treated so far. Schultz,
J., Pavlovic, J., Strack, B., Nawrath, M. and Moelling, K.:
Long-lasting anti-metastatic efficiency of IL-12-encoding plasmid
DNA. Human Gene Therapy 10, 407-417 (1999). Schultz, J., Heinzerling,
L., Pavlovic, J. and Moelling, K.: Induction of long-lasting cytokine
effect by injection of IL-12 encoding plasmid DNA. Cancer Gene
Therapy, 7, 1557-1565 (2000). Heinzerling, L., Feige, K., Rieder, S.,
Akens, M., Dummer, R., Stranzinger, G., Moelling, K.: Tumor
regression induced by intratumoral injection of DNA coding for human
interleukin 12 into melanoma metastases in gray horses. Journal of
Molecular Medicine, 78, 692-702 (2001).
M13/ MICROPARTICLE-MEDIATED TRANSFECTION OF
NON-PHAGOCYTIC CELLS IN VITRO
NFP37 Nr: 055144; Walter E (1), Dreher D (2), Merkle HP (1);
(1)Department of Applied BioSciences, ETH Zuerich, (2)Division of
Pneumology, University Hospital of Geneva
A novel approach involves the preparation of cationic
microparticles to adsorb DNA onto the cationic surface instead of
incorporation into the microparticle matrix (1). This eliminates the
difficulties of DNA degradation during the encapsulation process (2),
and optimises the release properties towards a faster release before
the actual degradation of the polymer. In this study, we prepared
cationic microparticles for loading plasmid DNA on the microparticle
surface. Two cationic polymers were chosen regarding their gene
transfer capacity and their ability to delivery DNA from the
endosomal compartments to the cytosol of the cells. Microparticles
exhibiting a positively charged surface were prepared by the
incorporation of the two selected cationic polymers into a
poly(D,L-lactide-co-glycolide) polymer (PLGA) core. The toxicity of
the different formulations was checked in two cell lines and was
found to be comparable to plain PLGA particles. Increased toxicity of
some formulations was observed in primary macrophages (MF) with high
phagocytosis activity. Plasmid DNA was efficiently adsorbed to the
microparticle surfaces, and the different formulations were checked
for their transfection efficiency in phagocytic and non-phagocytic
cells. Interestingly, the most pronounced gene transfer efficiency
was observed in a non-phagocytic 293 cell line when compared to a
macrophage cell line and primary MF. Possible mechanisms include the
dissociation of DNA-polymer complex and subsequent transfection of
the cells. Microscopic observation of fluorescent-labeled DNA in
primary MF revealed large amounts of DNA entering the cells, but no
detectable DNA inside the nuclei. We conclude that phagocytic
professional APC represent a group of cells which is especially
difficult to transfect when compared to other cell types. Although
DNA-loaded cationic microparticles resulted in significant
transfection of cells, toxicity and transfection efficiency was not
superior to that of DNA complexed with soluble cationic polymer. (1)
Hedley ML (1998) Nature Med. 4:365; (2) Walter E (1999) J. Control.
Rel. 61:361
M14// SCAVENGER RECEPTOR BLOCK AS IMPROVED
STRATEGY FOR THE IDENTIFICATION OF BONE MARROW HOMING PHAGES BY
PANNING IN VIVO RANDOM PEPTIDE PHAGE DISPLAYED LIBRARIES
NFP37 Nr: 044804; Finger AN, Bisoffi M, Wetterwald A, Gautschi E,
Hohenfeld U, Klima I, Thalmann GN, Stadler BM, Mazzucchelli L,
Cecchini MG; Gene Therapy Laboratory, Department of Clinical
Research and Department of Urology, and Institute of Immunology, and
Institute of Pathology, University of Bern, Inselspital, 3010 Bern,
Switzerland
Surface molecules exclusively expressed by cells of the bone
marrow (BM) are candidate targets for delivery of therapeutic agents
to this tissue. To identify ligands specific for the BM we screened a
series of random peptide phage displayed libraries in vivo. Some
phage clones, apparently BM-specific, could be identified after 5
consecutive panning cycles. However, a consensus motif did not emerge
and synthetic peptides were unable to inhibit the BM homing of the
corresponding phages, indicating that their binding to bone marrow
endothelium (BME) was not mediated by the peptide insert.
Furthermore, parental control phages, bearing no peptide insert,
showed a strong "natural" affinity to the BME that was abrogated by
polyanions. These results indicate that the BME is part of the
reticulo-endothelial system (RES). They also suggest that phage
trapping by this endothelium is mediated by scavenger receptors (SR).
To circumvent interference by SR, polyinosinic acid was administered
prior to phage panning in vivo. This led to the identification of
phage clones bearing a consensus motif that confers binding
specificity for a subpopulation of hemopoietic marrow cells. Thus, SR
inhibition, by avoiding phage trapping by the endothelium, seems to
allow phage particles to extravasate and reach parenchymal cells.
Accordingly, this panning strategy in vivo may be useful for the
identification of targeting motifs specific for cells located in the
extravascular space of various tissues.
M15// A REGULATORY NETWORK FOR THE EFFICIENT
CONTROL OF TRANSGENE EXPRESSION
NFP37 Nr: 044744; Mickail Bettan, Damien Saugy and Nicolas Mermod;
Laboratory of Molecular Biotechnology, University of Lausanne,
CH-1015 Lausanne
We previously constructed a doxycycline-regulated gene switch
system for gene therapy using a network of transcriptional repressor
and activator proteins. This allowed long term and tight regulation
the erythropoietin gene in stably transfected myoblastic cell lines.
We next turned to an in vivo gene transfer approach of mice muscles
in situ. In vivo gene transfer were performed using transcutaneous
injection of DNA into the tibialis cranialis muscles of mice, and
enhanced using the electrotransfer method. The settings determined in
vitro for the plasmid type and relative abundance were found to be
equally optimal in vivo. In the absence of doxycycline, the level of
transgene expression was close to background values, indicating
extremely tight transgene control, despite the high sensitivity of
the luciferase reporter system. When doxycycline was added to the
animals' drinking water, the transgene expression level was increased
by 3 to 4 orders of magnitude within several days. The network
compared favourably with the original tTA-based gene switch in terms
of fold activation and for its non leakiness in absence of
doxycycline. Transgene expression with the network was sustained for
weeks in nude mice, with no indication of expression decline or
toxicity. However, this system triggered an immune response in
immunocompetent mice, and transgene expression was extinguished
within 3 weeks. Studies are underway to identify the system
component(s) responsible for the immune response. In conclusion, the
system shows promising properties in terms of transgene regulation in
vivo, but it requires further work before human gene therapy
applications can be envisaged.
M16/ STABLE TRANSDUCTION AND EXPRESSION OF
ANTISENSE SMALL NUCLEAR RNAS FROM HIV- AND MURINE STEM CELL
VIRUS-DERIVED VECTORS
NFP37 Nr: 044704;; Reber U.1, Bouliong, E.1, Krähenbühl,
P.1, Liu S.-K.1, Zufferey R.2, Trono D.2, Kole, R.3, Schümperli
D.1. 1) Institut für Zellbiologie, Universität Bern,
Switzerland; 2) Département de génétique et de
microbiologie, Université de Genève, Switzerland; and
3) Lineberger Comprehensive Cancer Center, University of North
Carolina, Chapel Hill, NC, USA
We are exploring the potential of U7 small nuclear RNA (snRNA), a
factor involved in 3' end processing of animal histone mRNAs, as a
delivery system for antisense RNA sequences. The 5' end of U7 RNA
which is complementary to part of the processing signal in histone
pre-mRNAs can be replaced by antisense sequences to other targets.
The specific nuclear location is advantageous if early steps in the
metabolism of the target RNAs, such as specific splicing events, are
to be affected. For efficient in vivo application, the modified U7
genes must be introduced and expressed in primary cells of
experimental animals or patients. We have analysed gene delivery
using two types of retroviral vectors. With murine stem cell virus
(MSCV)-derived vectors4) that should be particularly suited for
hematopoietic cells, several murine and human cell lines could be
transduced albeit with low transduction efficiencies. However,
expression of the U7 snRNA gene was very inefficient and was only
observed when the vector contained a self-inactivating (SIN) deletion
in the 3' LTR. We have more successfully used an HIV-derived vector
system5) that allows for transduction of nondividing cells. With this
system, the transduction efficiencies were generally higher for human
than for murine cells and for hematopoietic as opposed to
fibroblastic cells. Murine erythroleukemia (MEL) cells were much more
efficiently transduced by a vector containing full-length LTRs than
by the corresponding SIN-deleted vector. With the HIV vector system,
the expression of the U7 snRNA gene was generally efficient, reaching
~30% of endogenous U7 snRNA levels. Moreover, at least in MEL cells,
the U7 snRNA levels were high irrespective of whether the vector had
full-length or SIN-deleted LTRs, indicating that transcriptional
interference by the HIV LTR promoter was not a problem. Thus the HIV
vector system may allow for efficient U7 gene transduction and
expression in a variety of cell types, including erythropoietic,
lymphopoietic and myogenic cells. We are currently testing this
vector in primary blood stem cells of murine and human origin. 4)
Cheng, L. et al., 1997. Gene Ther. 4, 1013. 5) Zufferey, R. et al.,
1998. J Virol. 72, 9873.
M17// FROM GENOMICS TO VACCINATION: TREATMENT OF
LATENT TUBERCULOSIS BY RECOMBINANT SALMONELLA
NFP37 Nr: 055164; Donatus Dreher (1), Menno Kok (2), Paul Imboden
(3), Stephen G. Kiama (4), David W. Muhindi (5), Costa Georgopoulos
(2), and Laurent P. Nicod (1); 1) Division of Pneumology,
University Hospital of Geneva; 2) Department of Medical Biochemistry,
University of Geneva; 3) Dr. PI Bioconsulting, Zollikofen,
Switzerland; 4) Department of Veterinary Anatomy, University of
Nairobi; and 5) Aga Khan Hospital, Nairobi, Kenya
BACKGROUND: One third of the world's population is infected with
Mycobacterium tuberculosis (Mtb). The majority of the infected
develop latent tuberculosis (TB), but remain at risk of switching to
active disease during lifetime. The conversion to active TB is
dramatically accelerated by AIDS. OBJECTIVE: To develop a bacterial
live vector that delivers Mtb genes for the immunotherapy of TB and
that would be safe, even for HIV-infected individuals. SUMMARY: We
have demonstrated that the facultative intracellular bacteria
Salmonella typhimurium (ST) can efficiently invade human dendritic
cells (DC) without killing the infected cells. The genetic background
of ST has profound impact on the cytokine pattern that is induced in
the DC. Moreover, DC infected with the attenuated strain PhoPc
secreted a T helper type 1 cytokine pattern, which is known to be
effective against TB, and became very potent inducers of IFN-gamma in
autologous T cells. Due to these unique properties of the ST vector,
our vaccine system was selected as one of the first nine approaches
worldwide to receive the Sequella Global Tuberculosis Foundation
grant for the development of TB vaccines. We have combined our live
vector with a new particular set of Mtb genes that are expressed when
the low oxygen conditions that Mtb encounters during latency are
reproduced in vitro. We have identified and cloned some of these
genes in the ST vector. The vector is currently being tested in vivo
at the University of Geneva. The proof of principle will be carried
out at the Kenya Medical Research Institute (KEMRI) in Nairobi, using
the Cornell mouse model, where a state of latent TB is obtained and
the conversion into disease is triggered by inducing
immunodeficiency. CONCLUSIONS: ST are not only a very efficient tool
for the transfer of plasmid-encoded antigens into human dendritic
cells, but they also stimulate the DC to induce a potent Th1 type
immune response. By combining this vector system with a new set of
Mtb genes expressed in the latent stage, we hope to provide a safe
and effective means for the immunotherapy of latent TB.
M18// GENE TRANSFER FOR TREATMENT OF RHEUMATOID
ARTHRITIS
NFP37 Nr: 055152; CA Seemayer1, S Kuchen1, P Kuenzler1, M Neidhart1,
WK Aicher2, BA Michel1, RE Gay1, S Gay1
1. SV40 transformation leads to proliferation, but not to invasion of
synovial fibroblasts into cartilage in the SCID mouse model. To
determine, whether SV40 transformation could increase the
invasiveness of synovial fibroblasts in the SCID mouse coimplantation
model of rheumatoid arthritis (RA). RA synovial fibroblasts (SF) and
normal (N)SF were transformed with an plasmid encoding for the SV40
T-antigen. Colony forming unit assays on fibroblast layers were
undertaken with the SV40 transformed cells and controls. Cell cycle
analysis was performed by flow cytometry. RASF, NSF and SV40
transformed RASF (ST-1) and NSF (ST-2) were coimplanted with normal
human cartilage under the renal capsule of SCID mice (n=22). After 60
days the invasion of the synoviocytes was assessed by a
semi-quantitative score, and the cellular density of synoviocytes
adjacent to the cartilage was analyzed by counting on HE stained
sections. The proliferative rate of RASF (n=5) was low (0.8 -5.5%
S-phase) compared to the SV40 transformed ST-1 and ST-2 (28% and
24%). In the SCID mice coimplantation model RA-SF revealed a
significantly stronger invasion into the cartilage than NSF and ST-2
(p=0.004 for both), and stronger than ST-1 (p=0.052). Of note, both
ST-1 and ST-2 cells formed tumor-like tissues adjacent to kidney and
cartilage, but without infiltration into neighbouring organs. In
comparison to RASF and NSF the cellular density adjacent to the
cartilage was significantly higher in the ST-1 (p=0.004 and p=0.017)
and higher in the ST-2 (p<0.1). In the colony forming unit assay
RASF and NSF did not form colonies, but the SV40 transformed ST-1 and
ST-2 lost contact inhibition. Although SV40 transformation of
synovial fibroblasts leads to an enhanced proliferation, it does not
result in a higher invasiveness. Therefore, the data suggest a
dissociation of proliferation and invasive behaviour. 2.
Cytokine-independent up-regulation of matrix-metalloproteinase 1 mRNA
expression by stress activated protein kinase 4. S Kuchen1, CA
Seemayer1, P Kuenzler1, T Pap1, RE Gay1, BA Michel1, M Neidhart1, S
Gay; 3. Targeting cathepsin L by specific ribozymes decreases protein
synthesis and cartilage destruction in rheumatoid arthritis. J
Schedel1, CA Seemayer1, W Zacharias3, T Pap1, M Neidhart1, S Kuchen1,
BA Michel1, RE Gay1, S Gay1; 4. Gene transfer of TIMP-1 and TIMP-3
inhibits proliferation of RA synovial fibroblasts and reduces
cartilage invasion in the SCID mouse model of RA. WH van der Laan4,5,
CA Seemayer1, PHA Quax4, JH Verheijen4, TWJ Huizinga4, BA Michel1, RE
Gay1, S Gay1, T Pap1 1Ctr Exp Rheum, Dept Rheum, Univ Hosp, Zuerich,
Switzerland 2Bas Sci Lab, Orth Surg, Tubingen, Germany; 3Dept Biochem
Mol Genetics, Univ Alabama, Birmingham, AL 35294, USA; 4TNO Prev
Health, Leiden, NL, 5Dept Rheum, Univ Med Ctr, Leiden, NL; All the
data under No. 2-4, will be presented at the ACR meeting in November
2001 in San Francisco.
M19// HEMATOPOIETIC STEM CELLS-DIRECTED GENE
THERAPY: CHRONIC GRANULOMATOUS DISEASE
NFP37 Nr: 046196; Maciej Wiznerowicz, Didier Trono. University of
Geneva, Faculty of Medicine, Department of Genetics and Microbiology,
Geneva, Switzerland. wizner@cmu.unige.ch
An optimal stem cell gene therapy approach should result in the
efficient transduction of HSCs, and considering the stem cells
plasticity, in the restricted expression of therapeutic genes into
specific mature blood cells. Third generation lentiviral vectors are
currently the most optimized tools for gene delivery into non-cycling
human HSC. Moreover, a self-inactivating design allows for the use of
tissue specific promoters without interference from the upstream LTR.
In a first series of experiments, a high percentage of UCB HSCs
transduction - up to 80% - was achieved by a lentivector in which a
central poly-purine tract was inserted upstream of an EF1a-GFP
expression cassette. High numbers of GFP positive cells were retained
after in vitro (cytokine cocktail) or in vivo (SCID/NOD mice
transplantation) differentiation of transduced HSCs. In order to
obtain specific transgene expression into differentiated neutrophils,
the ubiquitous EF1a promoter was replaced with transcriptional
elements (-1.5kb) of the sequence encoding human gp91phox subunit of
phagocyte NADPH oxidase. Transduction of HSCs with lentivector
carrying the gp91-GFP cassette followed by their in vitro or in vivo
differentiation resulted in GFP expression restricted exclusively to
mature monocytes and granulocytes. GFP expression from the gp91
promoter in mature neutrophils was enhanced by insertion of WPRE
without loss of specificity. Moreover, the gp91 promoter delivered
into HSCs by a lentivector, with or without WPRE, exhibited its
physiological responsiveness to INF-? in mature neutrophils. These
results show that the neutrophils-specific expression of a marker
gene can be efficiently achieved by third-generation lentivectors.
This represents an important step towards gene therapy of chronic
granulomatous disease.
M51// ADENOVIRUS-DISASSEMBLY REVEALS A FUNCTION
OF NUCLEAR HISTONE-H1 IN VIRAL DNA-IMPORT INTO THE NUCLEUS
NFP37 Nr: guest; Lloyd C. Trotman, Nicole Mosberger, Maarten
Fornerodð, Robert P. Stidwill & Urs F. Greber; University
of Zuerich, Institute of Zoology, Winterthurerstrasse 190 CH-8057
Zuerich, Switzerland; Netherlands Cancer Institute, Plesmanlaan 121,
1066 CX Amsterdam, The Netherlands
Adenovirus type 2 (Ad2) imports its DNA-genome through the
nuclear pore complex (NPC) of interphase cells for viral production.
Here we identify the NPC-filament protein CAN/Nup214 as a docking
site for incoming Ad2-capsids. Binding to CAN is independent of
cytosolic factors. Capsids disassemble at NPCs to free their DNA for
import. This process requires binding of nuclear histone-H1 to the
stably docked capsids and involves H1-import factors, restricting
this irreversible process to the proximity of the nucleus. Our
results provide a molecular mechanism for disassembly of Ad2 and
reveal an unexpected function of H1 in virus-mediated DNA import.
P00// A SINGLE-GENE APPROACH TO DEVISE INDUCIBLE
OR REMOVABLE THERAPEUTIC GENES: A GENERAL METHOD TO DRIVE CONDITIONAL
(TRANS-) GENE EXPRESSION
NFP37 Nr: 055105; Edya Fuhrman-Benzakein, Jean-Dominique Vassalli and
Pedro Herrera; Department of Morphology, University of Geneva
Medical School, 1 rue Michel-Servet, CH-1211 Geneva 4, Switzerland
1) Two-gene (binary) systems are no longer required. Inducible
recombinases can be used to temporally control the expression of a
given coding sequence. In our design, the gene of the tamoxifen
(4-OHT)-dependent Cre-ERT is flanked by its target sequences (loxP
sites). This cassette, (loxP-CreERT-loxP) or a derivative, may be
used to modulate the translation of a gene located on the same DNA
molecule. 2) Induction of expression. In a first configuration, the
loxP-CreERT-loxP cassette was placed upstream of an EGFP gene, within
the same DNA molecule, under the control of a potent promoter. The
transgene was introduced into BHK cells which, upon 4-OHT
administration, became fluorescent (NAR, 2000, Vol 28, e99). 3)
Repression of expression (transgene removal). Stopping the activity
of a gene is often a requirement in human gene therapy protocols.
With our approach, this can be achieved by introducing the gene of
interest, behind an internal ribosome entry site (ires), within the
Cre-ERT cassette, i.e. between the loxPs. We have thus shown that
EGFP expression can be abrogated in the presence of 4-OHT in BHK
cells bearing a loxP-CreERT ires EGFP-loxP gene. In another
experiment, a diphtheria toxin A (DT-A) coding sequence was placed
downstream of the loxP-CreERT-ires-EGFP-loxP cassette; 4-OHT
administration induced the death of fluorescent BHK cells transfected
with such a construct. In ongoing experiments, an additional
transgene encodes the angiogenic factor gene hVEGF instead of DT-A.
We are currently working on validating this conditional gene
expression system also in vivo: HeLa cells carrying the DT-A encoding
transgene will be injected into SCID mice. We seek to determine
whether tumor progression is inhibited when the animals are given
4-OHT. 4) Perspectives. We have shown that a segment of DNA capable
of inducible self-excision, the loxP-CreERT-loxP cassette, can
function as a rapid molecular switch to control the expression
(activation or silencing) of a physically associated transgene. The
simplicity and power of this approach makes it particularly suitable
for a variety of experimental and therapeutical designs involving
human gene therapy where conditional gene expression with biosafety
are required.
P01// SELECTION OF SPONTANEOUSLY TRANSLOCATING
NUCLEIC ACIDS
NFP37 Nr: 055154; Genilloud, A; Rusconi, S; Institute of
Biochemistry, University of Fribourg, Ch. du Musee 5, 1700 Fribourg,
Switzerland
We have designed a procedure to generate in vitro an
amplifiable/selectable library of randomized oligoDNA molecules.
Starting with a population of ssDNA molecules with a complexity of
1014, the deoxyoligonucleotides are selected for their capacity of
penetrating mammalian cells. Selected DNAs are PCR-amplified and
subjected to subsequent cycles of
selection-amplification/denaturation. Using the
limit-dilution-detection by PCR, we could show that the fifth-round
population of selected molecules has at least a one-log advantage
over the initial population. Sequencing of clones derived from this
resulting population will give informations about its components. If
redundant sequences are found, individual candidates could be tested
for their spontaneous translocation ability in mammalian cells.
P02// OLIGONUCLEOTIDE-MEDIATED TARGETED GENE
REPAIR OF X-LINKED CHRONIC GRANULOMATOUS DISEASES
NFP37 Nr: 055165; Hellwig R, Seger RA and Hossle JP; Div. of
Immunology/Hematology, Univ. Children's Hospital, Zuerich,
Switzerland
Chronic granulomatous disease (CGD) comprises a group of primary
immunodeficiencies characterized by failure of superoxide production
due to defects in one of the phagocyte NADPH oxidase (phox) genes.
Two thirds of the patients have mutations in their X-linked CGD gene
(X-CGD) encoding gp91phox, the largest subunit of the NADPH oxidase.
These mutations include several single nucleotide changes, which
represent possible targets for oligonucleotide-mediated targeted gene
repair. This technique is based on transfection of chimeric RNA/DNA
oligonucleotides carrying the correct sequence into target cells, in
which they provoke nucleotide conversion at the homologous genomic
site [1]. However, the actual mechanism is still under investigation
[2]. Recently, Gamper et al. demonstrated that single-stranded DNA
oligonucleotides with thioate backbone modifications are also capable
of gene repair [3]. We used an EBV-transformed B-cell line of a X-CGD
patient characterised by a C to T nonsense mutation at position 285
of gp91phox as a model system for gene repair. Transfection of
chimeric RNA/DNA oligonucleotides or DNA oligonucleotides with
terminal phosphothioate linkages was performed with DOTAP and
polyethylenimine (PEI), respectively. Determination of gene repair
frequencies was performed with SNUPE-ONCE (co-developed together with
Dr. G. Matyas, Div. of Metabolism and Molecular Pediatrics, in
house), based on single nucleotide primer extension products being
quantified by Gene scan analysis on an ABI 310 sequence analysis
system. Transfection of chimeric RNA/DNA oligonucleotides and DNA
oligonucleotides with terminal phosphothioate linkages using DOTAP
led to efficient gene conversion for both oligonucleotide types at
the molecular level. Using PEI as transfection reagent, gene
conversion was observed only with the phosphothioate oligonucleotide.
A test for functional reconstitution of superoxide production
indicated the presence of successfully converted cells at low
frequency as a result of specific gene repair. 1. Cole-Strauss, A.,
et al. Science, 1996. 273(5280): p. 1386-9 2. Gamper, H.B., et al.
Biochemistry, 2000. 39(19): p. 5808-16. 3. Gamper, H.B., et al.
Nucleic Acids Res, 2000. 28(21): p. 4332-9.
P03// GLIOBLASTOMA MULTIFORME GENE THERAPY
NFP37 Nr: 055155; Bernasconi L, Malipiero U, Fontana A; Institute
of clinical immunology, University Hospital Zuerich, Switzerland
Glioblastoma multiforme (GBM) is a tumour arising from glia cells
in the central nervous system (CNS). Its rapid growth and the lack of
an efficient treatment, confer a poor prognosis to this kind of
disease. The average survival of the patients ranges from six to
twelve months. We tested two different gene therapy approaches on a
murine and a rat model of the disease. Both approaches were performed
using recombinant adenovirus (rAd) modified to express genes that
interfere with the development of the tumour. In the first approach,
Fisher rats were stereotactically inoculated with F98 tumour cells
and then treated with rAd expressing Fas ligand (rAd-FasL) alone or
in combination with Topotecan. FasL operates via induction of tumour
cell apoptosis, whereas Topotecan (a topoisomerase I blocker) was
found to be additive to FasL induced glioma killing. The second
approach was performed on C57Bl/6 mice that were stereotactically
inoculated with GL261 tumour cells and subsequently treated with
rAd-IL-12 alone and or in combination with rAd-IP-10. IL-12 acts as
an immunostimulatory cytokine, whereas IP-10 is thought to be an
angiostatic factor. The combination of FasL/Topotecan and IL-12 alone
exert beneficial therapeutic effects in the two studied glioma
models, leading to an increase of the mean survival ranging from 50%
to 90%. To understand the effect of IL-12 as an immunoregulatory
cytokine affecting IFN gamma production by CD4 T cells, the GL261
tumour model was studied in terms of infiltration of dendritic cells
(DC). Both T cells and DC invade indeed GL261 tumours. Currently we
are investigating the degree of maturation and the functionality of
this antigen presenting cells after infection of GL261 with
rAd-IL-12.
P04// PARTIAL AND TRANSIENT RESTORATION OF
MHC-II MOLECULES IN RFX5-/- MICE BY LENTIVIRAL VECTOR
NFP37 Nr: 055159; Franck Matheux1,Walter Reith1 and Jean Villard1,2;
1Department of Genetics and Microbiology, University of Geneva
Medical School, Centre Medical Universitaire (CMU), 2Immunology and
Transplant Unit, University Geneva Hospital
The RFX5 mouse model reproduce all of the major features typical
of the diseases in term of functionnal immunodeficiency for B and T
cell compartiments (Clausen BE, Immunity, 1998 143-155). Therefore,
the mouse RFX5-/- represents a underestimate toll for the proof of
concept required to validate gene therapy in immunodeficiency
patients. In MHC-II deficiency, the key cellular compartments in
which MHC-II expression should be restored by gene therapy are bone
marrow derived antigen presenting cells including dendritic cells, B
cells and macrophages To achieve this, bone marrow cells are isolated
from RFX5-/- donor mouse. These cells are cultured during 24 hours
with IL-3, IL-6 and stem cell factor and then infected ex vivo with a
lentiviral vectors expressing the mouse RFX5 gene or the GFP reporter
gene (MOI 2 to 10). This is done with bulk bone marrow cells first
treated with 5-FU. After 6 to 10 hours, the cells are then injected
via the tail vein into irradited recipient six weeks old RFX5-/-
mice. Repopulation of the recipient mice by corrected (transduced)
bone marrow derived cells are then analyzed by examining MHC-II and
GFP expression by means of standard FACS analysis. Our results show
first that after one and two months, the GFP is expressed in 35% of
the peripheral blood and the spleen of the corrected transplantanted
mice. The analysis of the RFX5 transplanted mice demonstrated the
expression of MHC-II molecules in 15% of the B220 spleen cells
compared to the control after 30 days. In two other mice the
expression of MHC II is demonstrated in 15% of the B220 spleen cells
after 10 weeks. Two other mice sacrified after 4 month do not
expressed MHC-II molecules. We conclucled from these encouraging
preliminary results that expression of the of MHC-II proteins are
restored in B cells from RFX5-/- mice. These expression are present
in 10 to 20 % of the cells and seems to be transient, resulting
probably from the transduction of progenitors and not from stem
cells. Next experiments are underway with the transduction of an
enriched population of stem cells (SCA+ cell) at a higher MOI.
P05// EXPLORING THE LIMITS OF ORGANELLAR
TRANSLATION: A SINGLE GENE PRODUCT SPECIFIES CYTOSOLIC ELONGATOR AND
MITOCHONDRIAL INITIATOR TRNAMET IN TRYPANOSOMA BRUCEI
NFP37 Nr: 055154; T. Tan, N. Allemann, R. Pach and A. Schneider;
Department of Biology/Zoology, University of Fribourg,
Switzerland
The mitochondrion of T. brucei lacks tRNA genes. Its translation
system therefore depends on the import of cytosolic, nucleus-encoded
tRNAs. Thus, most trypanosomal tRNAs function in both the cytosol and
the mitochondrion and all are of the eukaryotic-type. This is also
the case for the elongator tRNAMet, whereas the only other
trypanosomal tRNAMet, the eukaryotic initiator, is found exclusively
in the cytosol. Unlike their cytosolic counterparts organellar
initiator tRNAsMet carry a formylated methionine. This raises the
question of how initiation of translation works in trypanosomal
mitochondria where only elongator tRNAMet is found. Using in
organello charging and formylation assays we have shown that
unexpectedly a fraction of elongator tRNAMet becomes formylated after
import into mitochondria. Furthermore, in vitro experiments with
mitochondrial extracts demonstrated that only the trypanosomal
elongator but not the initiator tRNAMet is recognized by the
formylation activity. Finally, RNA interference assays have
identified the gene encoding the trypanosomal formylase activity.
Whereas the predicted protein is homologous to prokaryotic and
mitochondrial methionyl-tRNAMet formyltransferases it has about twice
the mass of any of these proteins. Two aspect of the study of
mitochondrial tRNA import in T. brucei might be relevant for the
field of gene therapy. 1) Numerous protocols have been established to
target nucleic acids into cells where they can exert their effects,
however, despite these attempts an efficient system is still lacking.
A detailed knowledege of a natural occuring mitochondrial RNA import
systems may therefore help to develop more efficient protocols. 2)
Many mitochondrial cytopathies exist which are not amenable to
classic protocols of gene therapy, since they are caused by mutations
in mitochondrially encoded tRNAs. However, these diseases may be
treated if mitochondrial import of the corresponding cytosolic tRNAs
can be induced and if the imported tRNA can function in the
bacterial-type translation system of mitochondria.
P06// NEUROPROTECTIVE EFFECT OF INTERLEUKIN-6
AND IL6/IL6R CHIMERA IN THE QUINOLINIC ACID RAT MODEL OF HUNTINGTON'S
DISEASE
NFP37 Nr: 044718; Jean-Charles Bensadoun1, Luis Pereira de
Almeida1,3, Michel Dreano2, Patrick Aebischer1,4 and Nicole Deglon1;
1Division of Surgical Research and Gene Therapy Center, Lausanne
Medical School, Switzerland; 2Serono International SA, 1228
Plan-Les-Ouates/Geneva, Switzerland, 3Laboratory of Pharmaceutical
Technology; Faculty of Pharmacy and Center for Neuroscience;
University of Coimbra, Portugal; 4Swiss Federal Institute of
Technology, EPFL, Lausanne, Switzerland.
Ciliary neurotrophic factor prevents behavioral deficits and striatal
degeneration in rat and primate models of Huntington's disease (HD).
Another member of the cytokine family, the interleukin-6 (IL-6) and a
chimeric molecule (IL6/IL6R) consisting in a fusion of IL-6 and its
soluble receptor have been shown to exert trophic action on various
neuronal populations in the central nervous system. In the present
study, we have investigated the neuroprotective effect of these two
molecules in the quinolinic acid (QA) model of HD. LacZ-, IL-6-, and
IL6/IL6R-expressing lentiviral vectors were stereotaxically injected
into the striatum of rats. Three weeks later the animals were
lesioned through the intrastriatal injection of 180 nmol of QA. The
extent of the striatal damage was significantly diminished in the
IL-6- and IL6/IL6R-treated rats. The neuroprotective effect was
however more pronounced with the IL6/IL6R chimera than with IL-6 as
indicated by the lesion volume of 38.6 ± 10% and 63.3 ±
3.6% respectively, compared to 84.3 ± 2.9% in the control group.
Quantitative analysis of striatal interneurons further demonstrated
that the IL6/IL6R chimera has a more robust effect than IL-6 on ChAT
and NADPH-d-immunoreactive neurons. These results suggest that
IL6/IL6R chimera may hold potential for the treatment of HD.
P07// LONG-TERM GENE TRANSFER TO THE MOUSE
SPINAL CORD USING A LENTIVIRAL VECTOR
NFP37 Nr: 044718; 1S. Guillot, 1,3M. Azzouz, 1N. Deglon, 1,2P.
Aebischer, and 1A.D. Zurn; 1Division of Surgical Research and Gene
Therapy Center, Lausanne University Medical School, CHUV, Lausanne;
2Swiss Federal Institute of Technology Lausanne, EPFL, Switzerland;
3Oxford BioMedica, Oxford, United Kingdom.
The potential of a lentiviral vector to transfer the reporter
gene LacZ and the gene encoding the neurotrophic factor glial cell
line-derived neurotrophic factor (GDNF) to the spinal cord of
wild-type mice was investigated. An ad hoc developed system composed
of a stereotaxic frame and an automatic micropump allowed the
localized injection of 0.5 ml of viral stock solution in the anterior
horn of the spinal cord. Extensive rostrocaudal diffusion of the
virus within the spinal cord was observed at 5 weeks and 3 months,
with b-galactosidase-expressing cells and GDNF immunoreactivity
covering a distance of up to 3mm along the spinal cord, respectively.
Numerous motoneurons were strongly labeled with the GDNF antibody. At
the injection site, approximately 80% of the b-galactosidase-labeled
cells co-expressed the neuronal marker NeuN. To assess the proportion
of motoneurons infected with the viral vector, spinal motoneurons
were retrogradely labeled with fluorogold 1 week prior to sacrifice.
At the site of injection, 32.9 ± 4.5% of the fluorogold-labeled
motoneurons were found to be transduced with b-galactosidase. The
present study demonstrates that lentiviral vectors allow to express
transgenes in the spinal cord over long distances and for long
periods of time. This vector system may thus constitute an excellent
tool to evaluate potential protective therapies in mouse models of
motoneuron diseases such as amyotrophic lateral sclerosis and spinal
muscular atrophy.
P08// BIOARTIFICIAL PANCREAS: MATCHING DEVICE
AND CELL ENGINEERING
NFP37 Nr: 044718; 1H. Hussain-Khan, 1N. Bouche, 1,2P. Aebischer,
1,2WF. Pralong; 1Surgical Research Division and Gene Therapy
Center, CHUV, CH-1011Lausanne, 2Swiss Federal Institute of Technology
(EPFL/ETHL), Ecublens, CH-1014 Lausanne Switzerland
Cell-based therapies provide promising alternative to the
treatment of numerous diseases such as insulin-dependent diabetes
mellitus (IDDM) relying on the chronic administration of insulin. In
this context, cell encapsulation offer an attractive solution to
favor immunological acceptance of allogeneic transplanted cells and
to prevent acute reactivation of the underlying auto-immune disease
by insulating the implanted cells from host immune cells.
þTC-tet cells express a phenotype similar to primary pancreatic
cells. They represent therefore an ideal cell model to design and
test artificial devices capable of supporting b-cell immunoisolation
and in vivo cell function for the treatment of IDDM. Two
encapsulation systems were investigated in parallel with these cells.
Micro-encapsulation of b-cell clusters in polylysine-alginate beads
(PAB), as a non-limited diffusion model, was compared to b-cells
loading into thermoplastic polyether-sulfone flatsheet disks (PFD).
Following in vitro testing, which demonstrated similar glucose
responsiveness and cell survival, the two types of device were
implanted intraperitoneally (IP) in streptozotocin-induced diabetic
mice (blood glucose > 25 mM). PAB-encapsulated cells (2-3 x 106
cells) corrected glycemia (5 mM) and weight profiles within a few
days to control values. The effect was observed for more then 3
months with a good cell survival on histological analysis. In
contrast, PFDs (pore size < 1 mm) loaded with the same number of
cells were only transiently efficient on glycemia control and showed
poor cell survival. Genetic engineering of the cells with the
anti-apoptotic gene bcl-2, the gluco-incretin GLP-I together with the
use of more open PES membranes (pore size > 5mm) were necessary to
confer a good survival and functional properties in vivo to
PFD-encapsulated pure b-cells. It is concluded that non-genetically
modified pure b-cells can behave as a normal endocrine pancreas
substitute only in a non-limited diffusion microdevice system. In
contrast to previous observations made with primary islets
(constituted by different cell types also exerting paracrine effects,
i.e. a-cells secreting glucagon), pure b-cells may need, in addition
to optimized oxygen supply, specific engineering to provide them with
gluco-incretins in order to function properly in a macro-device
configuration.
P09// DOPAMINERGIC CELL LOSS INDUCED BY
LENTIVIRAL-MEDIATED OVEREXPRESSION OF WILD TYPE OR MUTANT HUMAN
A-SYNUCLEIN IN THE SUBSTANTIA NIGRA OF RATS
NFP37 Nr: 044718; C. Lobianco1, J.L. Ridet1, A. Zurn1, N. Deglon1, P.
Aebischer1,2. 1Division of Surgical Research and Gene Therapy
Center, Lausanne University Medical School, CHUV; 2Swiss Federal
Institute of Technology Lausanne, EPFL, Switzerland
Parkinson's disease (PD) is characterized by a progressive loss
of dopaminergic neurons and the appearance of Lewy bodies in neurons
of the substantia nigra pars compacta. Two missense mutations of the
a-synuclein (A30P and A53T) have been described in several families
with an autosomal dominant form of PD. Since then, it was reported
that a-synuclein constitutes one of the main component of Lewy bodies
in sporadic cases of PD. The lentiviral vector mediated
overexpression of both wild type and mutant forms of a-synuclein is
being evaluated both in in vitro and in vivo models. In vitro we
showed that the overexpression of both A53T and A30P mutant human
a-synuclein induces a substantial cell death in the SH-SY5Y human
neuroblastoma cell line. In order to develop genetic animal models of
PD, lentiviral vectors expressing either wild type or mutant human
forms of a-synuclein were injected into the substantia nigra of rats.
Five months post-injection, a significant loss of substantia nigra
dopaminergic cells was observed in animals injected with both wild
type and mutant forms of human a-synuclein as compared to rats
injected with viral vectors expressing b-galactosidase. Analysis for
the presence of inclusions, dopaminergic cell death specificity and
striatal dopaminergic nerve terminal loss is currently investigated.
This work is supported by the Swiss National Science Foundation
P10// LENTIVIRAL-MEDIATED DELIVERY OF MUTANT
HUNTINGTIN (HTT) IN THE STRIATUM OF RATS INDUCES A NEUROPATHOLOGY
MODULATED BY POLYGLUTAMINE REPEAT SIZE, HTT EXPRESSION LEVELS AND
PROTEIN LENGTH
NFP37 Nr: 044718; E.Regulier1, L.Pereira de Almeida1,2, D.Zala1,
P.Aebischer1,3, N.Deglon1; 1Univ. of Lausanne, Switzerland; 2Univ.
of Coimbra, Portugal, 3EPFL, Lausanne, Switzerland.
In the present work, we used lentiviral vectors as a gene
delivery system to produce models of Huntington's disease (HD) in
rats and assessed the importance of large polyglutamine CAG repeats,
Htt protein length and expression levels on the onset and specificity
of the pathology. The cDNAs encoding the first 171, 853 and 1520
amino acids of Htt protein with either 19 (wild type), 44, 66 and 82
CAG repeats (mutants) were cloned in SIN-W transfer vectors with
cytomegalovirus or mouse phosphoglycerate kinase 1 internal
promoters. The viruses expressing wild type or mutant Htt were
injected in the right and left striatum of adult Wistar rats and
lesion formation was followed over 6 months. A progressive pathology
was observed only in hemispheres injected with the mutant Htt
proteins and characterized by a cascade of successive
neuropathological events: appearance Htt of aggregates,
ubiquitination, loss of DARPP-32 stained neurones and
neurodegeneration. Earlier onset and faster kinetics occurred with
shorter Htt fragments, larger CAG repeats and higher expression
levels. Importantly, in animals injected with a lentiviral vector
expressing Htt171-82Q, a selective sparing of cholinergic and
GABAergic interneurons was observed at 12 weeks even though a drastic
loss of DARPP-32 staining and an atrophy of the striatum was observed
already at 1 month. Experiments are currently underway to explore the
relationship between mutant protein expression and progression of the
disease using tet-regulatable htt expressing lentiviruses.
P11// IN VIVO CHARACTERIZATION OF A
TETRACYCLINE-REGULATED LENTIVIRAL SYSTEM FOR A CONTROLLED DELIVERY OF
THERAPEUTIC MOLECULES
NFP37 Nr: 044718; Ridet JL1*, Spicher A1, Sommer B1, Deglon N1 and
Aebischer P1,2; 1Surgical Res. & Gene Therapy, CHUV, Lausanne,
2 Swiss Federal Institute of Technology Lausanne, EPFL,
Switzerland.
In vivo CNS gene therapy approaches require the development of
regulated gene expression systems especially for vectors such as
lentiviruses leading to the long-term expression of the transgene.
Lentiviral vectors carrying the tetracycline (tet)-regulatable system
for the controlled expression of green fluorescent protein (d2EGFP)
or glial cell line-derived neurotrophic factor (GDNF) were therefore
developed. In vitro, we showed that doxycycline (DOX), a tet
analogue, decreases d2EGFP expression by 50 to 100 fold in infected
293T cells. Lentiviral vectors encoding either for d2EGFP or GDNF
were then injected into the striatum of adult rats. DOX (200
microg/ml) was added in the drinking water of defined cohorts. One
week later, only scarce GFP-positive cells were detected in the
cohort that received DOX treatment whereas numerous neurons with
robust GFP expression were observed in the cohort that did not
received DOX. The ability to cycle several times the GFP expression
as a function of DOX administration was observed in subsequent
animals. GDNF expression was also regulated by DOX, although a
background revealing a certain level of leakage was observed. Based
on GDNF immunostaining the regulation reached 2-3 fold level. ELISA
assay revealed that DOX allowed a 5 fold decrease of protein level.
New vector constructs including silencers are currently being
developed to resolve the leakage problem. This work is supported by
the the Swiss National Science Foundation.
P12// OPTIMIZATION OF HUMAN ERYTHROPOIETIN
SECRETION FROM MLV-INFECTED HUMAN PRIMARY FIBROBLASTS FOR
ENCAPSULATED CELL THERAPY
NFP37 Nr: 044718; F. Schwenter1, N. Deglon1 and P. Aebischer1,2;
1Division of Surgical Research & Gene Therapy Center, Lausanne
University Medical School, CHUV Pavillon 4, 1011 Lausanne,
Switzerland 2Swiss Federal Institute of Technology, Lausanne, EPFL,
Switzerland
The transplantation of encapsulated cells genetically engineered
to secrete human erythropoietin represents an alternative to repeated
injections of the recombinant hormone for the treatment of
Epo-responsive anemia. In the present study, we have examined the
ability of primary human foreskin fibroblasts to produce
therapeutically relevant levels of Epo. First, the transduction
efficiency of these cells was evaluated using
b-galactosidase(LacZ)-encoding retroviral vectors derived from the
murine leukemia virus containing either an amphotropic envelope or
the G envelope glycoprotein of the vesicular stomatitis virus
(VSV-G). Human fibroblasts were then infected with hEpo-expressing
retroviral vectors. We demonstrated a 7.5-fold increase in Epo
production with the insertion in the vector of a regulatory element
from the woodchuck hepatits virus (WPRE) as well as of a Kozak
consensus sequence (KZ). Transgene expression was further optimized
by increasing multiplicity of infection of VSV-G-pseudotyped viruses
and selecting high producer cells with raising concentration of G418.
These modifications led to a 40-fold increase in hEpo secretion,
reaching approximately 200 IU hEpo/106 cells/day. The survival and
the transgene expression of modified fibroblasts were then evaluated
in vivo. The cells were encapsulated into microporous hollow fibers
and subcutaneously implanted in nude mice. An increase in the
hematocrit was observed during the first 2 weeks and elevated levels
exceeding 60% were maintained over a 6-week period. These results
indicate that genetically engineered primary human fibroblasts can
secrete therapeutically relevant amounts of hEpo and represent a
promising cell source for encapsulated cell therapy approach of
Epo-responsive anemia.
P13// SELECTION OF PEPTIDE LIGANDS BINDING TO
THE BASOLATERAL CELL SURFACE OF PROXIMAL CONVOLUTED TUBULES
NFP37 Nr: 044802; Annette Audige1, Christoph Frick1, Felix J. Frey1,
Luca Mazzucchelli2, and Alex Odermatt1; 1Division of Nephrology
and Hypertension, Department of Clinical Research, and 2Institute of
Pathology, University of Berne, 3010 Berne, Switzerland
Recently, we have developed a novel approach of screening
phage-display peptide libraries on microdissected intact renal
tubular segments for the identification of ligands that bind
specifically to the basolateral membrane. Here, we applied phage
libraries, that were depleted of ligands binding to ubiquitously
expressed receptors by preincubation with HEK-293 cells. Two distinct
ligands, that bind specifically to receptors expressed at the
basolateral membrane, were selected from screening libraries on
microdissected intact proximal convoluted tubules (PCT). Phage
expressing peptides with the consensus sequence GV(K/R)GX3(T/S) or
RDXR mediated 15-fold and 13-fold higher binding to PCT than control
phage, and binding to PCT was 13-fold and 21-fold higher than binding
to CCD, respectively. Both phage did not mediate significant binding
to various epithelial cell lines. Binding of both ligands was
abolished by the addition of the corresponding synthetic peptide.
Immunofluorescence experiments revealed a submembrane localization of
both ligands upon incubation with PCT.
Conclusions: Exploiting the versatility of phage-display and
biopanning allowed the identification of two distinct peptide ligands
that bind specifically to the basolateral membrane of PCT. Tubule
segment-specific ligands, such as the described PCT ligands, may be
useful for the analysis of cell-extracellular matrix interactions and
may contribute to the development of new therapeutic strategies for
renal diseases.
P14// IP-10 ENCODING PLASMID DNA IMMUNIZATION
EXHIBITS PROFOUND ANTITUMOR/ANTIMETASTATIC EFFICIENCY AND SYNERGISM
WITH IL-12
NFP37 Nr: 048767; Schultz J, Keyser J, Heinzerling L, Dollenmaier G,
Pavlovic J, Moelling K; Institute of Medical Virology, University
of Zuerich, Gloriastr. 30, CH-8028 Zuerich, Switzerland
Interferon-gamma inducible protein 10 (IP-10), a C-X-C chemokine,
has been shown earlier to display antitumoral properties through
immune system-mediated as well as angiostatic effects when applied by
genetically engineered tumor cells, recombinant defective
adenoviruses or as protein. We report here that IP-10 also elicits
strong antitumor and antimetastatic responses when administered by
DNA immunization. Intratumoral but not intramuscular DNA inoculation
profoundly reduced tumor formation in a B16F10/C57BL/6 malignant
melanoma- and a Lewis lung carcinoma model (LL/2/C57BL/6). Reduction
of tumor burden was partly mediated by immunological mechanisms,
since the antitumor effect was abrogated in NK cell-depleted mice.
Furthermore, IP-10 encoding plasmid DNA substantially abrogated the
establishment of metastases in the B16F10C57BL/6 model when injected
systemically by the intramuscular route. This effect was completely
mediated by NK cells, because it was abolished in NK cell-depleted
mice. When compared to IL-12 DNA therapy IP-10 DNA immunization
exhibited lower efficacy in the B16F10/C57BL/6 tumor and metastasis
model but higher efficacy in the LL2/C57BL/6 tumor model.
Coadministration of IP-10 with IL-12 DNA improved the induced
antitumor and antimetastatic effects in the B16F10/C57BL/6 model. Due
to this additive inhibition of tumor and metastasis formation the
amount of individual DNAs applied could be reduced, thereby lowering
the risk of potential side effects. IP-10 serum levels remained below
the detection limit and, correspondingly, no toxic side effects were
observed, suggesting that immunization with IP-10-, in particular in
combination with IL-12 encoding plasmid DNA, might have clinical
value for cancer therapy.
P15// EXON SKIPPING OF DYSTROPHIN mRNA BY
EXPRESSION OF ANTISENSE U7 snRNAs
NFP37 Nr: 044704; Brun C1, Pauli C1, Dunant P4, Schuemperli D2,
Lochmuller H4, Burgunder J-M3, Weis J1, Suter D1; 1Division of
Neuropathology, Institute of Pathology, 2Institute of Cell Biology,
3Neurological Clinic, Inselspital, University of Berne, Switzerland;
4Genzentrum, Ludwig-Maximilian-Universitaet, Munich, Germany
In most cases, Duchenne muscular dystrophy (DMD) is caused by
dystrophin gene mutations that disrupt the reading frame of the mRNA.
Artificial exclusion of additional exons (exon skipping) often would
restore the reading frame and might give rise to a truncated but
semi-functional dystrophin protein. Similarly, a natural alternative
splicing mechanism leads to rare dystrophin-positive, "revertant"
fibers in the mdx mouse and in DMD patients. Using antisense
sequences expressed on derivatives of U7 small nuclear RNA (snRNA),
we aim at blocking distinct splice sites and specifically inducing
artificial exon skipping. We first targeted the splice sites flanking
exon 23 and showed precise exon 23 skipping in the dystrophin mRNA of
"wild-type" C2C12 muscle cells. The same strategy was then
successfully adapted to immortalized mdx mouse muscle cells that
normally do not express functional dystrophin. Here, skipping of exon
23 carrying the nonsense mutation restores the reading frame. These
results demonstrate that dystrophin gene mutations can be corrected
on the level of pre-mRNA splicing by the use of custom-made U7
snRNAs.
P16// MONITORING THE MATURATION OF DENDRITIC
CELLS UPON ADDITION OF BIODEGRADABLE MICROPARTICLES
NFP37 Nr: 055144; Samantha Jilek, Hans P. Merkle and Elke Walter;
Department of Applied Biosciences, Institute of Pharmaceutical
Sciences, ETH Zuerich, 8057 Zuerich
Dendritic cells (DC) are the key cell type for the induction of
the immune response and represent the most important cell type for
antigen presentation. In their immature state, they are able to
phagocytose antigens or even microparticles. As mature cells, they
migrate into secondary lymphoid follicles to induce an immune
response by activating the T helper lymphocytes. Maturation is
induced by various factors, such as LPS or phagocytosis of bacteria
or viruses. Thus, the capacity to phagocytose antigenic material is
restricted to the immature state of DC. Targeting of immature DC
through antigen loaded microparticles could therefore represent an
efficient way to induce long lasting immune reponse. The focus of
this project was to study the capacity of microparticles to induce DC
maturation. For preparation of the microparticles we used two
biodegradable polymers, poly(D,L-lactide) (PLA) and
poly(D,L-lactide-co-glycolide) (PLGA), and the microparticles were
made by spray drying. Plasmid DNA or salmon DNA were either
encapsulated or coated on the microparticles' surface. After
isolation of monocytes from human blood, DC were differentiated over
7 days before incubation with the microparticles. On day 11, the
presence of different surface markers (CD11b, CD14, CD83, CD86) was
assessed by flow cytometry (FACS). Maturation of DC, as indicated by
upregulation of CD83 and CD86, was induced by both LPS and
microparticles, but was superior with LPS. Further experiments will
be carried out with CpG- or non CpG- oligonucleotides-coated
particles or with DNA directly added to the culture medium in order
to determine whether upregulation of CD83 and CD86, and thus
maturation of DC can be further enhanced.
P17// TARGETING OF DENDRITC CELLS BY SPECIFIC
PHAGOCYTOSIS OF RGD-SURFACE MODIFIED MICROSPHERES
NFP37 Nr: 055144; Kralisch S (2), Mueller M (3), Voros J (3), Thiele
L (1), Beck-Sickinger AG (2), Textor M (3), Merkle HP (1), Walter E
(1); (1)Department of Applied BioSciences, ETH Zuerich,
(2)Institute of Biochemistry, University of Leipzig, Germany, (3)
Laboratory for Surface Science and Technology, ETH Zuerich
Gene delivery to professional antigen presenting cells such as
dendritic cells (DCs) has great potential for the immunotherapy of
infectious diseases, cancer and for tolerance and is likely to
revolutionize future vaccine design. Thereby, synthetic gene delivery
approaches have emerged as a promising technology to circumvent
safety concerns involved with life vectors. Microspheres from
biodegradable poly(lactide-co-glycolide) (PLGA)-polymers have a
proven track record for the microencapsulation of therapeutics and
can be used to selectively target phagocytic cells such as DCs and
macrophages (Mo). A targeting approach to selectively address DCs by
avoiding Mo involves coating of PLGA particles with
polyethyleneglycol (PEG)-derivatives (1) as a first step. We
demonstrated resistance to protein adsorption to avoid opsonization
by serum proteins which generally initiates phagocytosis by Mo.
Subsequently, specific targeting to DCs is suggested by addressing
the alphavbeta3 integrin receptor which is selectively expressed on
the cell membrane of DCs (2). We demonstrated specific uptake of a
RGD-peptide ligand (3) in DCs. Furthermore, chemical conjugation of
this ligand to the surface of carboxylated polystyrene beads was
performed by linkage via a modified maleimide chemistry. Phagocytosis
of modified beads in DCs and Mo was evaluated. (1) Kenausis GL (2000)
J. Phys. Chem. B 104:3298; (2) Albert ML (1998) J. Exp. Med. 188:359;
(3) Verdaguer N (1995) EMBO 14:1690
P18// ADENOVIRAL GENE TRANSFER OF A SOLUBLE IL-1
TYPE II RECEPTOR PROLONGS CARDIAC ALLOGRAFT SURVIVAL IN RATS
NFP37 Nr: 055162; Li J, Dudler J, Fleury S, Driscoll R, Vassalli G.
Cardiology, Rheumatology and Gene Therapy Center, University of
Lausanne Medical School, Lausanne
Interleukin (IL)-1 is involved in inflammatory and immune
responses leading to cardiac allograft rejection. We have constructed
an adenoviral vector expressing a soluble IL-1 type II
receptor/IgG-fusion protein (Ad-sIL-1RII). The effect of this vector
on cardiac allograft survival was tested in a rat model of heart
transplantation. F344 rat donor hearts (n = 9) were instilled with
Ad-sIL-1RII (10E10 PFU/mL) containing saline solution immediately
before transplantation into LEW recipients. Control F344 hearts
received either a control vector (Ad-Null; n = 5) or saline alone (n
= 3) before transplantation into LEW recipients. Allograft survival
was as follows: Ad-sIL-1RII = 15.7 + 5.9 days; Ad-Null = 9.8 + 0.8
days; saline = 7.8 + 0.5 days (Ad-sIL-1RII: p = 0.053 vs Ad-Null;
p<0.05 vs saline). Control LEW/LEW isografts (n = 3) survived
>150 days. Additional heart transplants (n = 3 per group) were
explanted at day 6 for immunohistological analysis (in progress). In
conclusion, sIL-1RII gene transfer produces a moderate prolongation
of cardiac allograft survival in a rat transplantation model.
P20// LIVE SALMONELLA VECTORS FOR ANTIGEN
TRANSFER INTO HUMAN DENDRITIC CELLS
NFP37 Nr: 055164; Menno Kok (1), Elke Walter (2), Elisabeth Buehlmann
(3), Laurent P. Nicod (4), and Donatus Dreher (4). (1) Department
of Medical Biochemistry, University of Geneva; (2) Department of
Applied BioSciences, ETH Zuerich; (3) Department of Genetics and
Microbiology, University of Geneva; (4) Division of Pneumology,
University Hospital of Geneva
BACKGROUND: The natural infection pathway of Salmonellae includes
both extracellular and intracellular stages, the latter involving
passages through macrophages and dendritic cells (DC). In these
cells, the bacteria exploit and modify the endosomal compartment for
growth and survival. The equilibrium between microbial growth and
bactericidal activities of the host cells may result in efficient
antigen presentation via both the exogenous and the endogenous
antigen presentation pathways. This feature qualifies S. typhimurium
(ST) as interesting vector for delivery of macromolecules to APC.
METHODS: Human DC were obtained by culture of peripheral blood
monocytes in the presence of GM-CSF and IL-4. The green fluorescent
protein (GPF) was detected by FACS and fluorescent microscopy and
expression of the Mycobacterium tuberculosis (Mtb) antigen Ag85C was
monitored by Western blotting. DC maturation was measured by FACS
analysis of CD83, CD86 and MHCII expression. Presentation of antigen
by the DC to primed CD4+ or CD8+ T lymphocytes was detected in the
autologous mixed leukocyte reaction (AMLR), measuring
interferon-gamma secretion by activated T cells. RESULTS: While
screening a wide variety of bacterial mutants, infection conditions
and plasmids, we incidentally observed expression of GFP in the
cytoplasm of DC (10-20% of cells), indicative of transfection with
bacterial DNA. This only occurred when monocytes had been infected as
early as three days following the onset of induced development to DC
and when bacteria with artificially increased plasmid contents were
used. In spite of this early exposure to LPS, these cells developed
normally to DC. We observed that attenuated ST efficiently infect and
maturate DC, rather than killing them, prompting us to investigate
the capacity of ST to deliver antigen rather than DNA. By fusion to
E. coli MalE, which stabilizes antigens produced by ST, we obtained
efficient synthesis of the Mtb antigen Ag85C by ST. Antigens from
intracellular ST were processed by the DC for both MHC class I and
class II presentation, resulting in significant stimulation of primed
T-cells in vitro.
CONCLUSIONS: Transfer of antigen by ST to human DC is most effective
when the antigen is synthesized inside the DC by the bacteria. In
fact, human DC infected by ST present antigens efficiently to both
CD4+ and CD8+ T cells. On the other hand, DNA transfer ex vivo by ST
to human DC is not a reliable alternative for immunotherapy.
P21// SALMONELLA TYPHIMURIUM SIPB PROTEIN
MODULATES ACTIVATION AND RELEASE OF IL-18 BY ALVEOLAR MACROPHAGES
NFP37 Nr: 055164; Carolina Obregon1, Donatus Dreher1, Menno Kok2,
Laurence Cochand1, and Laurent P. Nicod1. 1 Division of
Pneumology, University of Geneva; 2 Department of Medical
Biochemistry, University of Geneva
Alveolar macrophages (AM) represent the first line of immune
defense in the lung. They have been shown to produce interleukin
(IL)-18, which stimulates T-cells to secrete IFN-gamma that is
essential for protection against intracellular pathogens such as M.
tuberculosis. This cytokine is synthesized as an inactive precursor
that is proteolytically processed to the mature, biologically active
form by the IL-1beta-converting enzyme (ICE, or caspase-1). SipB has
been identified as the cytotoxic factor of the facultative
intracellular pathogen Salmonella that activates ICE. Therefore, we
have investigated the role played by S. typhimurium (ST) SipB in
IL-18 activation and release, and the role of ICE in this process.
METHODS: The capacity of AM to release mature IL-18 after infection
with genetically modified ST was analyzed. In particular, two
bacterial strains were compared, a virulent strain, and its isogenic
mutant in sipB, which lacks the active SipB protein that activates
ICE. Intracellular IL-18 and its release in the supernatant were
analyzed by Western blot to distinguish the mature (18kDa) from the
immature form (24kDa), while the global amount of IL-18 was
quantified by ELISA. For some experiments, AM were treated with a
caspase-1 specific inhibitor (Ac-YVAD-CMK) at a concentration of 50
micromolar, 1 hour before bacterial infection. RESULTS: AM produce
high amounts of IL-18 as compared to other antigen-presenting cells.
The genetic background of ST had a profound impact on the release of
IL-18 by AM. Infection with the wild type strongly stimulated the
secretion of both the unprocessed and the mature forms of IL-18. The
induction of IL-18 was detected as early as 3 hours after infection,
with maximum levels observed at 24 hours. The ICE inhibitor blocked
the release of IL-18 at an early stage. In contrast to the wild type,
infection with the sipB-deficient mutant neither induced maturation
nor secretion of IL-18. CONCLUSION: Understanding the Salmonella
virulence factors will contribute to the improvement of bacterial
live vectors. The maturation and the release of the immunostimulatory
cytokine IL-18 are specifically triggered by the Salmonella virulence
factor SipB. Thus, the genetic modification of live vectors may be
used to regulate their effects on the immune system.
P22// SINGLE-CHAIN ANTIBODY AND ITS DERIVATIVES
DIRECTED AGAINST VASCULAR ENDOTHELIAL GROWTH FACTOR: APPLICATION FOR
ANTIANGIOGENIC GENE THERAPY
NFP37 Nr: 044820; T. Afanasieva, M. Wittmer, A. Vitaliti, L. Wyder
and R. Klemenz; Division of Cancer Research, Dept. of Pathology,
University Hospital Zuerich, Switzerland
At present antiangiogenic therapy seems to be one of the most
promising directions in preventing expansion and metastasizing of
solid tumors. VEGF IS known to be ONE OF THE the major angiogenic
players. Multiple strategies on VEGF inactivation have been shown to
be successful in tumor growth inhibition. Our research interests are
focused on the antiangiogenic potential of genetically engineered
antibodies for cancer cure. Recently, an anti-VEGF single-chain
antibody (scFv) was selected from phage-display library in our
laboratory. This antibody was able to reduce the growth of
subcutaneous tumors in nude mice by approximately 50% (A. Vitaliti et
al., Cancer Research 2000; 60: 4311- 4314). To improve the
antiangiogenic effect, the selected scFv was subjected to several
modifications: 1) to increase the affinity of scFv to VEGF an in
vitro affinity maturation approach was employed; 2) to increase the
half-life of the scFv in the blood, a minibody consisting of the
human IgG CH3 domain fused with scFv, and a scFv-Fc fusion protein
were generated; 3) because production and purification of bulk
quantities of scFvs in E. coli is laborious, we choose to work with a
gene therapy settings based on the recombinant adenovirus system. We
have produced a number of recombinant adenoviruses expressing
different variants of anti-VEGF-scFv. Expression analysis of the
recombinant adenoviruses produced showed that sustained high levels
of the proteins of interest were achieved after systemic
administration of the recombinant viruses to nude mice. Our
preliminary experiments revealed that single injection of recombinant
adenovirus encoding anti-VEGF scFv (4x108 PFU) led to a reduction of
tumor growth comparable with those achieved by daily injections of
purified recombinant scFv protein. Moreover, this effect could be
improved by multiple injections of the virus. Our ongoing experiments
will clarify the potential of genetically modified antibodies for
therapeutic antiangiogenic treatment of solid tumors.
P51// OVEREXPRESSION OF PARKINSON'S
DISEASE-ASSOCIATED ALPHA-SYNUCLEIN(A53T) IN THE SUBSTANTIA NIGRA OF
MICE BY RECOMBINANT AAV DOES NOT INCREASE THE SENSITIVITY OF
DOPAMINERGIC NEURONS TO THE NEUROTOXIN MPTP
NFP37 Nr: guest; Zhizhong Dong, Boris Ferger*, Joram Feldon*, and
Hansruedi Bueeler; Institute of Molecular Biology, University of
Zuerich, and Institute of Behavioral Neurobiology, ETH Zuerich,
Switzerland
Alpha-synuclein is the major component of Lewy bodies, that are a
hallmark of sporadic and alpha-synuclein-associated familial
Parkinson's disease (PD). Alpha-synuclein is nitrated in Lewy bodies
of several human synucleinopathies. Furthermore, the neurotoxin MPTP
has been shown to cause the nitration of alpha-synuclein in mice and
its aggregation in baboons, suggesting that oxidative
stress-dependent post-translational modifications of alpha-synuclein
may underlie Lewy body formation and dopaminergic neuron death. While
upregulation of endogenous alpha-synuclein in surviving dopaminergic
neurons of mice treated with MPTP indicated that (wild-type)
alpha-synuclein may be neuroprotective, mutated alpha-synuclein was
reported to enhance the vulnerability of clonal SH-SY5Y
"dopaminergic" cells to MPP+, the toxic metabolite of MPTP. In order
to study the influence of the PD-associated alpha-synuclein(A53T)
mutant on MPTP-induced neurodegeneration in vivo, we overexpressed
human alpha-synuclein(A53T) (or EGFP as control) at high levels in
the substantia nigra of mice by rAAV-mediated gene transfer, and
treated the mice 5 weeks later subchronically with MPTP (or saline as
control). Determination of dopaminergic neuron survival and striatal
content of dopamine and its metabolites in the different groups of
mice demonstrated that alpha-synuclein(A53T) neither sensitizes nor
protects dopaminergic neurons from MPTP toxicity. Our results argue
against a direct function of (mutant) alpha-synuclein in oxidative
stress pathways, but do not rule out the possibility that
alpha-synuclein aggregation in neurons exposed to oxidative stress
for long periods of time may be neurotoxic.
P52// HSV/AAV HYBRID VECTORS MEDIATE LONG-TERM
GENE EXPRESSION IN CULTURED HUMAN CELLS
NFP37 Nr: guest; Heister T., Ackermann M., Fraefel C.; Institute
of Virology, University of Zuerich, Winterthurerstr. 266a, 8057
Zuerich, Switzerland
HSV-1-based amplicon vectors have a large transgene capacity and
can efficiently infect many different cell types. One disadvantage of
amplicons is the instability of transgene expression. By contrast,
vectors based on adeno-associated virus (AAV) can either persist in
an episomal form or integrate into the host cell genome, thereby
supporting long-term gene expression. AAV expresses four rep genes,
rep68, 78, 40, and 52. Of those, rep68 or rep78 are sufficient to
mediate site-specific integration of the AAV DNA into the host cell
genome. The major disadvantage of AAV vectors is the small transgene
capacity (~4.5 kb). In this study, we constructed HSV/AAV hybrid
vectors that contain, in addition to the standard HSV-1 amplicon
elements, AAV rep68, or rep78, or both rep68 and 78, or all four rep
genes, and the GFP reporter gene that is flanked by the AAV inverted
terminal repeats (ITR). Southern blots of Hirt extracted DNA from
cells transfected with the hybrid vectors and HSV-1 helper DNA
revealed that the entire vector was replicated as an HSV-1 amplicon
and that the ITR-GFP expression cassette alone was replicated in
addition, indicating that the rep genes and the ITRs were functional.
One month after infection of cultured human cells (293) with
pHyRaGFPa, a hybrid vector that contains all four rep genes,
transgene expression was maintained in 32% of the initially
transduced cells, as determined by flow cytometry; standard amplicon
vectors supported only short-term transgene expression (1-2 weeks).
Cell clones that stably (> 8 months) express the transgene could
easily be isolated without chemical selection. Southern analysis of
genomic DNA from the clones using P32-labeled AAVS-1 and GFP probes
revealed co-localization of the ITR-GFP expression cassette and the
AAVS-1 integration site in 4 of 11 clones analyzed indicating
specific integration of GFP sequences in AAVS-1. We are currently
investigating the junctions between vector derived DNA and AAVS-1
sequences in these clones.
P53// ATTENUATED REPLICATION-COMPETENT
ADENOVIRUSES AS TOOLS FOR MELANOMA THERAPY
NFP37 Nr: guest; Peter I, Schaffner W, Hemmi S; Institute of
Molecular Biology, University of Zuerich, Switzerland; Greber UF,
2Institute of Zoology, University of Zuerich, Switzerland
Clinical trials are currently testing replicating viruses, including
adenoviruses, as oncolytic agents for various tumors. Key issue of
these approaches is to obtain high levels of viral replication
specifically in the tumor tissue but not in nearby normal cells.
Adenovirus controls its replication by the major transactivator, the
E1A proteins. Therefore we aimed to tightly restrict E1A expression
to melanoma, a tumor of readily accessible localization. The
combination of four mouse tyrosinase enhancers (TE) fused to the
human tyrosinase promoter (TP), showed up to 1000-fold higher
activity in tyrosinase-expressing melanoma cells than in non-melanoma
using reporter assays. The TETP promoter was introduced into an
E3-deleted adenovirus to drive E1A. E1A expression was further
restricted by deletion of adenoviral enhancer sequences upstream of
E1A (EP). This virus (Ad*EP-TETP) replicated at efficiencies
comparable to wt Ad5 in melanoma cells such as SK-Mel23. But in a
panel of non-melanoma tumor cells and primary human cells,
replication and cytopathic effects were reduced more than 50 fold.
Injection of Ad*EP-TETP into xenotransplanted melanomas but not HeLa
cells led to tumor regression in 5 out of 6 nude mice. These data
demonstrate the selective oncolytic activity of Ad*EP-TETP and its
potential application in melanoma therapy.
P54// PATTERN OF ACTIVITY OF VIRAL AND
HOUSEKEEPING GENE PROMOTERS USING SELF-INACTIVATING LENTIVIRUS VECTOR
DELIVERY INTO THE MOUSE RETINA
NFP37 Nr: guest; Kostic C, Chiodini F, Hornfeld D, Tekaya M, Salmon
P, Deglon N, Aebischer P, Munier F and Arsenijevic Y; Jules ;
Gonin Eye Hospital, Lausanne, Switzerland
Retinal degenerative diseases such as retinitis pigmentosa and
age-related macular degeneration have an important incidence in the
occidental world. The process impaired in this visual decline is a
progressive loss of photoreceptors. No therapy exists so far to cure
these diseases and gene transfer appears to be promising to prevent
photoreceptor loss. In order to design adequate vectors, we explored
the activity of different promoters (cytomegalovirus (CMV),
phosphoglycerate kinase (PGK), elongation factor-1 (EFS) and
rhodopsin (Rho) promoters) in postnatal and adult mice eyes (n=6 to
18). Self-inactivating lentivirus preparations were injected
intravitreously in DBA/2 mice at 3 to 5 days or 8 weeks postnatal,
followed by analysis of each promoter activity 7 days later. Cell
transductions were observed over a 3 mm distance. As previously
described (Miyoshi H. et al. (1997)), the CMV promoter was active in
RPE cells, photoreceptors and cells of the inner nuclear layer (INL),
but more restricted to RPE cells in adult mice eyes. The PGK promoter
expression pattern in newborn injected eyes was principally defined
as strong in RPE and weak in some cells of the INL. Injected adult
eyes showed a predominant expression in RPE cells. The EFS promoter
derived from the elongation factor-1 gene, allows a broad expression
in the retina of newborn injected mice. Different cell types
expressed the GFP reporter gene, such as glial cells, neurons, RPE
and rarely photoreceptors. Finally, as expected, the Rho promoter is
specifically expressed in photoreceptors. Thus, as shown in our study
of CMV and PGK promoters, infection in the developing retina is more
favorable for transgene expression. Additionally, in future attempts
to slow or rescue photoreceptor degeneration by lentiviral delivery
of a secreted survival factor, both CMV and PGK promoters should be
appropriate due to their strong expression in RPE cells which are
adjacent to the targeted photoreceptors. The EFS promoter could be
useful to target neurons of the INL or ganglionic cells that are
damaged in glaucomas, because it drives expression in adjacent glial
cells. Finally, the Rho promoter is more suitable for genes necessary
to be expressed in the photoreceptors themselves.
P55// PROTEASOME ACTIVITY AND A POTENTIALLY
DIFFERENT ENDOSOMAL ROUTING RESTRICT AAV-2 TRANSDUCTION IN
ENDOTHELIAL CELLS
NFP37 Nr: guest; Pajusola K, Bueler H; Institute of Molecular
Biology, University of Zuerich
Adeno-associated virus (AAV) is considered a promising vector for
various gene therapy applications due to its long-lasting transgene
expression in vivo and wide spectrum of target cell specificity. More
recently however, it has become apparent that there are considerable
differences in the efficiency of transduction of different cell types
by AAV and this has important implications in the various
applications of AAV. In this study, the transduction capacity of
AAV-2 in endothelial cells was studied, because AAV-2 could
potentially be a useful tool for the gene therapy of cancer and
cardiovascular diseases. For this purpose the transduction
efficiencies of AAV-2 in the endothelial cell line EAhy-926, the
glioma cell line LN-229 and the cervical carcinoma cell line HeLa,
which is the optimal target cell for AAV-2 transduction, were
compared. The cell lines were infected with equal MOIs of recombinant
AAV-2 expressing EGFP under CMV or Tie promoter, which is specific
for endothelial cells. When the transgene expression induced by
rAAV-2 under CMV promoter was analysed by FACS, it was discovered
that there is 37 times higher overall expression and up to 92 times
higher high-level expression of EGFP in LN-229 and HeLa cells
compared to EAhy-926. To clarify the reason behind the difference in
the transduction efficiencies, the cells were treated with the
proteasome inhibitor MG-132 or Bafilomycin-A1, which inhibits
endosomal acidification known to be important for AAV-2 transduction.
MG-132 treatment increased the AAV-2 mediated transduction by 11 and
5 times in EAhy-926 or LN-229 and HeLa cell lines, respectively, and
the transduction difference between these cell lines could be reduced
to a factor of 3. On the other hand, Bafilomycin-A1 reduced the
transduction in HeLa cells by a factor of 50 whereas in EAhy-926
cells only by a factor of 2. These results indicate that
proteasome-dependent degradation of AAV-2 particles imparts a major
block to AAV-2 transduction in endothelial cells. Moreover, EAhy-926
cells may use an alternative inefficient endocytic pathway, since
bafilomycin-A1 had little effect on AAV-2 transduction in these
cells. However, additional experiments are required to address the
latter possibility.
P56// INTERFERON RESISTANCE OF CUTANEOUS T-CELL
LYMPHOMA DERIVED CLONAL T-HELPER 2 CELLS ALLOWS SELECTIVE VIRAL
REPLICATION
NFP37 Nr: guest; Willers J, Dummer R, Doebbeling U, Geertsen R, Burg
G, Pavlovic J; University Hospital Zuerich, Zuerich, Depts of
Dermatology and Virology
Cutaneous T cell lymphomas (CTCL) comprise a heterogeneous group
of lymphoproliferative disorders that are characterized by an
accumulation of T lymphocytes in the skin and occasionally in blood
(Sezary Syndrome, SS). In most cases the dominant clone displays T
helper 2 cytokines. Since IFN-gamma is a natural inhibitor of T
helper 2 cells and IFN-alpha is frequently used in CTCL, we studied
the impact of IFNs on SS derived purified clonal T helper 2 cells
using anti-Vb antibodies. Moreover, IFNs are known to mediate virus
resistance in normal cells. The isolated clonal CD4+ cells, but not
the non-clonal CD4+ cells appeared resistant to IFN-gamma and
IFN-alpha stimulation in terms of HLA upregulation and MxA induction
caused in part by alterations in STAT1 molecule mRNA and IFNGR1 mRNA
transcription. The IFN resistance of the patient derived clonal cells
was then targeted by vesicular stomatitis virus infection after
IFN-alpha priming resulting in selective viral replication in clonal
cells. In contrast, non-clonal cells of the same patient showed
IFN-dependent MxA expression, which is a major mediator protein of
viral protection. The IFN resistance of the dominant T helper 2 cells
might be important for lymphomagenesis. Interferon signaling
deficiencies can be targeted for purging patients' cells in vitro.
Furthermore, this approach may allow specific molecular interventions
resulting in efficient treatment of CTCL and other IFN-resistant
neoplasms like lung cancer.
P57// LENTIVIRAL-MEDIATED TRANSFER OF CILIARY
NEUROTROPHIC FACTOR RESCUES INJURED RETINAL GANGLION CELLS: A
POTENTIAL NEUROPROTECTIVE STRATEGY FOR GLAUCOMA TREATMENT
NFP37 Nr: guest; B. A. van Adel3, C. Kostic1, N. Deglon2, A.K. Ball3,
and Y. Arsenijevic1; 1Eye Hospital Jules Gonin, Lausanne, 2 CHUV,
Lausanne, Switzerland, 3Dept. of Pathology and Molecular Medicine,
McMaster University, Hamilton, Canada.
Glaucoma is a progressive optic neuropathy that leads to the
degeneration of retinal ganglion cells (RGCs) and thus permanent
visual field loss. Currently, there are no effective treatments to
prevent RGC death after optic neuropathy. Several lines of evidence
suggest that RGC death may be prevented by the continuous supply of
neurotrophic factors. Ciliary neurotrophic factor (CNTF) has recently
been demonstrated to be one of the most promising trophic factors for
both the survival and regeneration of injured RGCs (Weise et al.,
2000). In the present study, we used optic nerve transection as an in
vivo animal model to test the efficacy of lentiviral-mediated gene
transfer of ciliary neurotrophic factor (CNTF) to prevent RGC death.
The optic nerve was transected intraorbitally 2mm behind the eye and
RGCs were labeled by injecting Dextran-FITC into the proximal nerve
ending. A replication-deficient lentivirus encoding CNTF (PGK-CNTF)
was injected intraocularly at the time of axotomy. For control
experiments, no injection was made or a control vector encoding for
b-galactosidase (PGK-lacZ) or PBS was injected into the eye. RGC
densities were determined using confocal microscopy in flatmounts
isolated from experimental and control groups surviving 14 and 21
days post-axotomy (dpa). The percentage of surviving RGCs was
drastically reduced after optic nerve transection to only 15 ± 2
and 8 ± 1 % at 14 and 21 dpa in untreated animals. In contrast,
the percentage of surviving RGCs at 14 and 21 dpa in retinas treated
with PGK-CNTF was 68 ± 6 and 31 ± 7 % respectively. This
corresponded to a RGC rescue rate of 52 ± 5 and 25 ± 7 % at
14 and 21 dpa above control retinas treated with PGK-lacZ. For the
first time a vector is able 1) to prevent optic nerve cell loss when
injected at the time of injury and 2) to protect a population of
ganglion cells 21 days after the axotomy. Therefore, continuous
administration of CNTF could serve as a potential treatment for
retinal disorders involving optic neuropathy and RGC injury such as
in glaucoma.
P58// HARMONIC THERMODYNAMICAL BEHAVIOR OF
TELOMERIC SEQUENCES
NFP37 Nr: guest; Md. Ashrafuzzaman, Inst de physique, UNI
Neuchatel, Switzerland
The interaction energies between the different types of bases of a
single strand of DNA molecule have been calculated. Using these
original values of energies the harmonic behavior of a number of base
patterns of DNA has been studied. In view of the great interest
aroused by the discovery of the role of the telomere segment of the
DNA in the replication process and its possible link with the aging
process, we have investigated, with simple models, the harmonic
behavior of the telomeric pattern of bases as well as the
thermodynamic response in the biological system. With these results a
conclusion on the probable role of the telomeric pattern on aging has
also been drawn.
P59// EFFICIENT TRANSDUCTION OF DENDRITIC CELLS
AND INDUCTION OF A T CELL RESPONSE BY THIRD GENERATION
LENTIVECTORS
NFP37 Nr: guest; Christoph Esslinger and H. Robson MacDonald;
Ludwig Institute for Cancer Research, Lausanne Branch, University
of Lausanne, CH-1066 Epalinges, Switzerland
In order to induce a therapeutic T lymphocyte response
recombinant viral vaccines are designed to target professional
antigen presenting cells such as dendritic cells (DC). A key
requirement for their use in humans is safe and efficient gene
delivery. The present study assesses 3rd generation lentivectors with
respect to their ability to transduce DC and to induce a CD8+ T cell
response in a murine model. We show that 3rd generation lentivectors
transduce DC with a superior efficiency as compared to adenovectors.
The transfer of DC transduced with a recombinant lentivector encoding
an antigenic epitope resulted in a strong specific CD8+ T cell
response. The occurrence of lower proportions of non-specifically
activated CD8+ cells suggests a lower anti-vector immunity of
lentivector as compared to adenovector. Thus, lentivectors, in
addition to their promise for gene therapy of brain disorders might
also be suitable for immunotherapy.
P60// PAIRED-BOX CONTAINING TRANSCRIPTION
FACTORS AS TARGETS FOR THERAPY IN SOLID TUMORS, MELANOMAS AND BRAIN
TUMORS
NFP37 Nr: guest; Beat W. Schaefer, Christiane M. Margue, Florence A.
Scholl, University of Zuerich, Department of Pediatrics, Division
of Clinical Chemistry and Biochemistry, Steinwiesstrasse 75, CH-8032
Zuerich, Switzerland
Paired-box containing transcription factors are crucial regulators of
developmental processes. Interestingly, they also seem to play an
important role in tumor development as implied by their involvement
in specific chromosomal translocations. PAX3 and PAX7 are found as
chimaeric proteins in the pediatric tumor rhabdomyosarcoma (RMS),
expression of PAX5 is upregulated via a translocation in B cell
lymphomas and, as demonstrated just recently, PAX8 is involved as a
fusion partner in thyroid carcinomas. Previous experiments in our
laboratory have shown that one of the possible oncogenic functions of
PAX3/FKHR in RMS is protection from apoptosis. Surprisingly, a
similar function was found for the native PAX3 in these tumor cells.
To begin to investigate the mechanisms underlying this effect, we
searched for potential target genes of PAX3. Here, we demonstrate
that transcription of the anti-apoptotic protein BCL-XL can be
stimulated by both PAX3 and PAX3/FKHR through cotransfection assays
with the Bcl-x gene promoter as well as electrophoretic mobility
shift assays. Also, modulation of both PAX and BCL-XL protein levels
in tumor cells through antisense oligonucleotides shows that both
proteins are functionally important for cell survival. Based on these
experiments we speculated that upregulation of BCL-XL might also
render tumor cells more resistant towards treatment with
chemotherapeutic drugs. Indeed, RMS cells with enhanced expression of
either PAX3 or PAX3/FKHR are more resistant to etoposide and
actinomycinD treatment. Based on the apparently important function of
PAX3 in solid tumors, we screened additional cancers for expression
of this transcription factor. Indeed, expression of PAX3 was detected
by RT-PCR as well as in situ hybridization experiments in about 50%
of brain tumors and about 75% of melanomas. Using a similar antisense
strategy as before, specific downregulation of its expression could
be achieved resulting again in the induction of apoptosis. Hence,
these experiments suggest that PAX proteins might be important
targets for therapy in a range of tumor types.
P107// Stolarczyc et al., abstract not received
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