NFP37 SOMATIC GENE THERAPY ANNUAL MEETING Oct 7 and 8 1999 «Inherited versus acquired disorders»

ABSTRACTS

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ABSTRACTS MAIN LECTURES

L01// GENE THERAPY: NEW HOPES, OLD REALITIES
Inder Verma. Salk Institute, San Diego CA, USA
Keywords: Adeno-associated vectors, Lentivectors, hereditary disorders, acquired disorders

L02// GENE CORRECTION AND REPROGRAMMING THROUGH SPLICEOSOME-MEDIATED TRANS-SPLICING
Lloyd Mitchell. Intronn LLC, Durham NC, USA
Keywords: RNA repair, suicide gene therapy, gene regulation, spliceosome-mediated trans-splicing

L03// GENE CORRECTION OF SINGLE POINT MUTATIONS THROUGH CHIMEROPLASTS
Clifford Steer. Univ. Minnesota, Minneapolis MN, USA
Keywords: RNA/DNA oligonucleotide, anionic liposomes, lactosylated PEI, receptor-mediated endocytosis, in vivo hepatic gene delivery, Gunn rat, Crigler-Najjar syndrome type I, Hemophilia B, sickle cell disease, DNA mismatch repair, liver metabolic disorders

L04// GENE THERAPY FOR CANCER - THE CURRENT POSITION
Hardev Pandha. Hammersmith Hospital., Oncology
Keywords: cancer, immunomodulation, prodrug activation, antisense, gene replacement, tumor targeting, systemic vectors

L05// GENE THERAPY CLINICAL BIOANALYSIS
Michael Grace. Schering Plough, Union NJ, USA.
Keywords: use of flow cytometry and laser scanning cytometry for the analysis of human clinical surrogate markers in gene therapy trials

L06// VP22 PROTEIN-MEDIATED DELIVERY APPLICATIONS IN GENE THERAPY
P. O' Hare. Marie Curie Res. Institute, Surrey, UK
Keywords: GFP. intercellular protein trafficking, herpes virus VP22, chimeric proteins

L07// ANGIOGENIC GENE THERAPY WITH INTRAMUSCULAR NAKED DNA
DW Losordo. St. Elisabeth Medical Center, Boston, MA, USA
Keywords: VEGF, angiogenesis, endothelium, ischaemic heart disease, myocardium

ABSTRACTS 20 MIN TALKS

M01// LEGAL AND BIOSAFETY BASIS OF HUMAN GENE TRANSFER
NFP37 Nr: guest;
Karoline Dorsch-Häsler. EFBS, Buwal, 3003 Bern.
In Switzerland, a specific gene technology law has not been established. Gene therapy is regulated by several laws, ordinances and regulations. In addition, a law on medicinal products (Heilmittelgesetz) is in preparation, which will also cover gene therapy. This law should come into force by the year 2000. In this presentation the current regulatory aspects regarding gene therapy applications in Switzerland, with a special emphasis on manufacture of gene therapy products, will be discussed.

M02// PERCEPTIONS OF HEALING NEEDS:SOMATIC GENE THERAPY, DISABILITY AND IDENTITY
NFP37 Nr: 4037-053073;
Scully JL, Rehmann-Sutter C. Arbeitstelle fuer Bioethik, Institut fuer Geschichte und Ethik der Medizin, Universitaet Basel, Schoenbeinstr. 20, 4056 Basel
This project aims to explore how some of the interest groups who will be affected by somatic gene therapy (SGT) perceive the associated ethical problems and, if there are differences, where they arise. While bioethics has succeeded in achieving a broad consensus on the ethical permissibility of SGT, many of the potential 'consumers' remain more ambivalent. Although cancer and AIDS are universally considered to be disease states, other conditions that would be possible future candidates for SGT occupy more ambiguous positions. We hypothesize that this may reflect differences in (1) the perception of a given condition as requiring remediation at all, (2) the degree to which a given state is perceived as reflecting the individual's identity, and (3) the degree that a gene-based intervention is perceived as altering that identity, possibly more than a conventional therapy would. Our aim is to compare the moral frames of reference in an interdisciplinary way. Our project uses a variety of hermeneutic and analytical tools to consider the ethical descriptions of SGT provided by two main target groups: people with disabilities and chronic illnesses, and clinicians and basic scientists involved in gene therapy research. Using source texts, questionnaire responses and structured interviews, we consider whether factors such as assumptions about the experience of suffering associated with disability, or of the relative importance of the agents involved, can produce a differential moral accounting. By acknowledging the importance of the individual perspective and differences within and between groups, and examining the connections between perceived healing needs, self identity, and the permissibility of gene therapeutic interventions, we will gain insight into the ways in which bioethical issues in SGT and other forms of gene-based medicine are formulated, and how to optimise future public information and understanding.

M03// LENTIVIRUS-MEDIATED GENE TRANSFER OF GP91phox CORRECTS CHRONIC GRANULOMATOUS DISEASE PHENOTYPE IN HUMAN X-CGD CELLS
NFP37 Nr: 4037-044826;
Saulnier S*, Michel K, Steinhoff D, Dinauer MC, Grez M, Seger RA and Hossle JP. (* formerly Wasser S.) . Div. of Immunology/Haematology, Univ. Children's Hospital, Zürich; Georg-Speyer-Haus, Frankfurt, Germany
Chronic granulomatous diseases (CGD) are caused by impaired antimicrobial activity in phagocytes, due to absence or malfunction of the respiratory burst NADPH oxidase. Two thirds of the patients have mutations in their X-linked CGD gene encoding gp91phox, the largest subunit of the NADPH oxidase. In our previous work we demonstrated the development of a clinically applicable retroviral vector (SPsLdS) for the correction of X-CGD after transduction of human X-CGD CD34+ bone marrow (BM) cells (Becker et al. 1998, HGT 9:1561). In the current study, aimed at gene therapy of X-CGD already at the earliest level of hematopoietic stem cells, we generated an HIV-1 -based vector with self-inactivating (SIN) features containing the therapeutic gp91phox gene. In this vector transgene expression is exclusively driven by an internal CMV promoter. The green fluorescent protein (GFP) served as reporter for evaluation of transfer and expression of the carried transgene in the human myeloid PLB985 X-CGD cell line. The X-CGD cells were efficiently transduced by the VSV-G pseudotyped lentivirus constructs (74% GFP+ cells at 3 days post transduction) and CMV driven expression was stable for at least 3 weeks after transduction and persisted after granulocytic differentiation of the target cells. Using the lentivector with the gp91phox transgene, 26-48% of the X-CGD cells expressed gp91phox at day 2 and 20 after co-culture with 293T producer cells, respectively. Upon granulocytic differentiation of the transduced X-CGD cells with dimethylformamide (DMF) for 7 days, functional reconstitution of oxidase activity was determined by dihydrorhodamine (DHR) FACS analysis. Up to 52% (mean: 48%, n = 3) of the cells were found to be reconstituted at a mean fluorescence intensity reflecting the levels of superoxide-production of approx. 25% compared to wt PLB985. Therefore, lentiviruses expressing gp91phox are able to at least partially correct human myeloid X-CGD cells and will now be tested for the introduction of the therapeutic gene into very primitive quiescent bone marrow stem cells from X-CGD patients.

M04// IMMUNEMODULATION IN CANCER USING DNA INOCULATION
NFP37 Nr: 4037-048767;
Heinzerling L, Schultz J, Nawrath M, Pavlovic J, Moelling K. Institute of Medical Virology, University of Zürich, Gloriastrasse 30
DNA vaccines are a promising therapy modality for prevention and therapy of infectious diseases and cancer. Gene products which exhibit high biological potency even at low levels of expression are the most promising candidates. One of them is the cytokine interleukin 12 (IL-12) whose anti-metastatic effect was analyzed here. We show that intramuscular injections of plasmid DNA coding for both subunits of IL-12 abolished the establishment of pulmonary metastases of the malignant melanoma cell line B16F10 in a syngeneic mouse model. This anti-metastatic effect of IL-12 DNA was observed during early phases of metastases formation. Pre-established metastases were not affected by the treatment with plasmid DNA. The protective effect is long-lasting and dependent upon the dose of DNA injected. CD8+ T-cells and Natural Killer (NK) cells contribute to the anti-metastatic effect. In contrast, the anti-metastatic effect is not mediated by interferon-g or tumor necrosis factor a (TNF-a) as demonstrated by using IFN-g receptor and TNF-a receptor knock out mice. Depletion of NK cells with antibodies completely abrogated the anti-metastatic effect. A combination of the IL-12 encoding DNA with DNA coding for a melanoma associated antigen gp100, could not further enhance the anti-metastatic effect. An anti-tumor effect of the treatment with IL-12 encoding plasmid DNA could not been shown. Our results suggest that plasmid DNA coding for IL-12 might have potential value as gene medicine against the initiation of metastasis formation in patients diagnosed with high risk melanoma.

M05// A REGULATORY NETWORK FOR THE EFFICIENT CONTROL OF TRANSGENE EXPRESSION
NFP37 Nr: 4037- 044744;
Imhof MO, Chatellard P, Mermod N. Lab. of Molecular Biotechnology, Center for Biotechnology UNIL-EPFL, and Inst. of Animal Biology, UNIL 1015 Lausanne
Expression of heterologous genes in mammalian cells or organisms for therapeutic or experimental purposes often requires tight control of transgene expression. This control should allow no background gene activity in the off-state, high gene expression in the on-state, be consistent in long term experiments, and permit multiple switching between the on- and off-states. Here, we describe a genetic switch system for controlled transgene transcription using chimeric repressor and activator proteins functioning in a novel regulatory network. In the off-state, the target transgene is actively silenced by a chimeric protein consisting of multimerized eukaryotic transcriptional repression domains fused to the DNA-binding tetracycline repressor. In the on-state, the inducer drug doxycycline effects both the derepression of the target gene promoter and activation by the GAL4-VP16 transactivator, which in turn is under control of an autoregulatory feedback loop. The hallmark of this new system is the efficient transgene silencing in the off-state as demonstrated by the tightly controlled expression of the highly cytotoxic diphtheria toxin A gene. Addition of the inducer drug allows for robust activation of transgene expression. In stably transfected cells this control is granted even after months and repeated cycling between the repressed and activated state of the target genes.We conclude that this system permits tight and long term transgene regulation when stably introduced into cell lines,abd thus should have general applications in biotechnology and gene therapy.

M06// LENTIVIRAL VECTOR TRANSDUCING AN ANTISENSE U7 snRNA GENE THAT EFFICIENTLY CORRECTS ABERRANT PRE-mRNA SPLICING CAUSED BY BETA-THALASSEMIC MUTATIONS
NFP37 Nr: 4037-044704;
Reber U, Tomasini R, Suter D, Zufferey R (1), Kole R (2) , Schümperli D. Abt. fuer Entwicklungsbiologie, Univ. Bern
We developed derivatives of U7 snRNA, a natural antisense RNA involved in histone RNA 3' processing, to modulate the splicing of specific pre-mRNAs. Our model are three thalassemic mutations, IVS2-654, -705 and -745, that create new 5' splice sites (ss) in intron 2 of the human beta-globin gene; they activate a common cryptic 3' ss, an intron segment is included in the mRNA and no functional beta-globin is made. We showed previously that antisense U7 snRNA targeted to the cryptic 3' ss corrected pre-mRNA splicing in transiently or stably transfected tissue culture cells expressing the IVS2-705 mutant globin gene. Splicing correction was even more efficient for IVS2-745, but less efficient for IVS2-654, showing that the accessibility of the cryptic 3' ss to antisense RNA depended on downstream parts of the aberrant exon that differed between the three mutations. We now report that double target U7 snRNAs, able to bind to two target sequences, induce considerably more efficient splicing correction for all three thalassemic mutations, including the highly resistant IVS2-654. The fact that such constructs also correct splicing when one or both antisense targets are not ss of the mutant gene analysed indicates a new type of mechanism involving a looped structure of the aberrant exon. This approach has many potential applications, from restoring gene functions in similar types of mutations, over chosing between alternatively spliced products, to gene inhibition by non-productive splicing. The most active double target construct was introduced into a self-inactivating, VSV G-pseudotyped lentiviral vector. Preliminary experiments indicate that the U7 gene is active and corrects aberrant splicing in mutant beta-globin-expressing HeLa cells. Moreover, the activity of a single integrated copy of the U7 gene can be boosted by superinfection. This vector should permit efficient transduction of bone marrow cells from mice transgenic for the IVS2-654 mutation or from human thalassemia patients or heterozygotes. (1) Dept. de Genetique et Microbiologie, Univ. Genève, (2) Lineberger Compr. Cancer Center, Univ. North Carolina, Chapel Hill

M07a// EFFECTS OF GENE TRANSFER WITH DOMINANT NEGATIVE MUTANTS OF RAF-1 AND C-MYC ON ACTIVATION AND APOPTOSIS IN RHEUMATOID ARTHRITIS SYNOVIAL FIBROBLASTS
NFP37 Nr: 4037-055152;
Pap T, , Nawrath M, Hummel KM, Sainsbury I (2), Fernihough JK (2), Gay RE, Billingham M (2), Moelling K (3), Gay S. Ctr Exp Rheum, Dept Rheum, Univ Hosp, CH-8091 Zuerich, Switzerland
Previously, it was shown that inhibition of the Ras-Raf-MAPK pathway with dominant negative (dn) mutants of Raf-1reduces the invasion of RA-SF into cartilage. In this context, phosphorylation of c-Jun and formation of the AP-1 complex represent critical steps in the upregulation MMPs. In view of evidence that c-myc is also involved in the activation of RA-SF, we studied the effects of gene transfer with dn mutants of raf-1 (dn-raf-1) and c-myc (dn-c-myc) on the expression of phospho-c-Jun (p-c-Jun) and MMPs as well as on cell cycle progression and apoptosis in RA-SF. Constructs encoding for dn-Raf-1 and dn-c-Myc were cloned into the retroviral LXSN vector, and cultured RA-SF were transduced with dn-raf-1, dn-c-myc or both. Transduction with the LXSN vector alone was used as control (mock). RA-SF were then co-implanted together with normal human cartilage under the renal capsule of SCID mice. After 60 days, the implants were removed, and immunohistochemistry (IHC) with a monoclonal antibody was used to investigated the expression of p-c-Jun in the RA-SF at sites of invasion into the cartilage. Expression of MMP-1 and MMP-13 was also investigated by IHC. Using FACS technique, we performed cell cycle analysis and studied the apoptosis though staining with annexin V and propidium iodide. While mock transduced RA-SF showed abundant staining for p-c-Jun, particularly at sites of cartilage destruction, a marked reduction in the expression of p-c-Jun was seen in the dn-raf-1 transduced cells. This was accompanied by a decreased expression of MMP-1 and MMP-13. Transduction of RA-SF with dn-raf-1 or dn-c-myc alone did not result in the induction of apoptosis. However, RA-SF that were transduced with both dn-raf-1 and dn-c-myc rapidly underwent apoptosis as seen both by a characteristic pre G1 peak and staining with annexin V and propidium iodide. The data demonstrate that in RA-SF, Raf-1 is involved in signaling cascades that upregulate disease relevant MMPs though the activation of c-Jun. However, the data suggest that induction of apoptosis through the Ras-Raf-MAPK pathway requires the inhibition of both Raf-1 and c-Myc. (2) OSCOR Facility, Univ Vet School, Bristol, BS2 8EJ, UK, (3)Inst Med Virology, CH-8028 Zuerich, Switzerland

M07b// INHIBITION OF ENDOGENOUS P53 BY GENE TRANSFER INCREASES THE INVASIVENESS OF FIBROBLAST-LIKE SYNOVIOCYTES IN THE SCID MOUSE MODEL OF RHEUMATOID ARTHRITIS (RA).
NFP37 Nr: 4037-055152;
Pap T, Aupperle KR (2), Jeisy E, Gay RE, Firestein GS (2), Gay S. Ctr Exp Rheum, Dept Rheum, Univ Hosp, CH-8091 Zuerich, Switzerland.
Recent data not only indicate that the tumor suppressor p53 is critically involved in the regulation of fibroblast-like synoviocyte (FLS) proliferation and apoptosis but also suggest that abnormalities in p53 expression may contribute to the invasiveness of RA-FLS. Here, we tested the hypothesis that inhibition of endogenous p53 with the papilloma virus 18 E6 protein results in an increased cell growth and invasiveness of FLS in vivo. Using retroviral gene transfer, 2 RA and 1 normal FLS cultures were transduced with a LXSN vector encoding for the papilloma virus 18 E6 protein or with the LXSN vector alone (mock). After selection with G418, transduced FLS were co-implanted together with normal human cartilage under the renal capsule of severe combined immunodeficient (SCID) mice. Non-transduced, parental cells from each patient were implanted as additional controls. After 60 days, the implants were removed and the invasion of FLS into the cartilage was studied histologically and assessed by a semiquantitative scoring system. All experiments were performed in duplicate. Mock transduced RA-FLS and parental, non-transduced RA-FLS showed a marked invasion into the cartilage (means scores 2.4±0.4 and 2.1±0.7, respectively). In E6 transduced RA-FLS, a significant increase in the invasiveness could be observed (mean score 3.1±0.9, p<0.05). Inhibition of p53 also resulted in a marked increase of cell growth as seen by a higher density of FLS in the implants. Mock transduced and parental normal FLS did not exhibit significant invasion (mean score: 1.6±0.3), but transduction of these cells with E6 resulted in distinct invasion (mean score: 2.3±0.4) as well as an increase in cell growth. These data suggest that inhibition of p53 leads to increased invasiveness of RA-FLS and may also transform normal FLS to display an aggressive behavior. Therefore, abnormalities such as somatic mutations in the tumor suppressor p53 may contribute to synovial hyperplasia and invasion in RA. (2) Div Rheum, Allergy & Immun, USCD School of Medicine, San Diego, CA, USA.

M08// EFFECTS OF EX VIVO HUMAN ENDOTHELIAL NITRIC OXIDE SYNTHASE GENE TRANSFER ON VASCULAR FUNCTION OF HUMAN SAPHENOUS VEIN BYPASS VESSELS.
NFP37 Nr: 4037-055166;
Tanner FC, Largiader T, Greutert H, Yang Z, Lüscher TF. Cardiology, University Hospital Zürich; Physiology, University Zürich-Irchel; Zürich; Switzerland.
Coronary bypass graft disease is characterized by an increased platelet aggregation and neointima formation due to proliferation and migration of smooth muscle cells (VSMC). Therefore, the effect of human endothelial nitric oxide synthase (NOS) gene transfer on VSMC outgrowth from and thrombocyte aggregation to isolated human SV was investigated. Proliferation of human SV VSMC was inhibited in a concentration dependent manner by the NO donor diethylenetriamine-NONOate (DETANO 10-5 - 10-3 M). The cell number after an observation period of 4 days was 165'406 ± 27'649 under control conditions and 5'750 ± 793 in the presence of maximal concentrations of DETANO. Similarly, migration of SV VSMC was inhibited in a concentration-dependent manner by the NO donor (DETANO 10-5 -10-3) M. The number of migrated cells after an incubation period of 5 hours was 26 ± 2 under control conditions and 10 ± 1 in the presence of maximal concentrations of DETANO. Human SV explants were transfected with NOS by a recombinant adenoviral vector (AdeNOS). After a time period of 21 days the number of explants with outgrowing cells was 90% under control conditions, 69% in control virus transfected explants, 48% in NOS transfected explants. Similarly, deposition of human thrombocytes on SV segments was inhibited by transfection of NOS. Platelet deposition was decreased by 43% as compared to control virus transfected segments as well as to control conditions. This study demonstrates that adenoviral transfection of isolated human SV with NOS impaired VSMC outgrowth. Consistent with this observation, NO inhibited proliferation and migration of cultured SV VSMC. Further, transfection of endothelial NOS inhibited platelet deposition on human SV segments. NOS gene transfer may improve patency rates of SV bypass grafts.

M09a// INCREASED MOTOR NEURON SURVIVAL AND IMPROVED NEUROMUSCULAR FUNCTION IN TRANSGENIC ALS MICE AFTER INTRASPINAL INJECTION OF AN ADENO-ASSOCIATED VIRUS ENCODING BCL-2.
NFP37 Nr: guest;
Azzouz M. 1, Hottinger A. 1 Paterna J.-C.2, Zurn A.1, Aebischer P.1 and Bueler H.2. (1) Division of Surgical Research and Gene Therapy Center, CHUV, 1011 Lausanne, Switzerland. (2) Institute of Molecular Biology, University of Zürich, 8057 Zürich, Switzerland.
Transgenic mice overexpressing a mutated form of human Cu/Zn superoxide dismutase develop a severe, progressive motoneuron disease that closely resembles familial amyotrophic lateral sclerosis (FALS). We investigated the potential of recombinant adeno-associated virus to transfer neuroprotective molecules in this model of FALS. Injection of an adeno-associated viral vector carrying the gene for green fluorescent protein bilaterally into the lumbar spinal cord of normal mice led to expression of the reporter gene in 35% of the motoneurons surrounding the injection site. Injection of a viral vector encoding the anti-apoptotic protein bcl-2 in transgenic FALS mice resulted in bcl-2 expression in motoneurons and increased motoneuron survival at the end-stage of disease. In addition, the compound muscle action potential amplitude elicited by nerve stimulation and recorded by electromyographic measurements was higher in the bcl-2 treated group than in the controls. Local expression of bcl-2 in spinal motoneurons delayed the appearance of signs of motor deficiency but was not sufficient to prolong the survival of the transgenic mice. These studies support the use of adeno-associated viral vectors for the delivery of protective genes to spinal cord motoneurons as a possible way to enhance motoneuron survival and repair.

M09b// EFFECTS OF PROMOTER AND VIRAL POST-TRANSCRIPTIONAL REGULATORY ELEMENTS ON ADENO-ASSOCIATED VIRUS-MEDIATED TRANSGENE EXPRESSION IN THE BRAIN
NFP37 Nr: guest;
Paterna J.-C., Moccetti T., Mura A*. and Bueler, H. Institute of Molecular Biology, University of Zuerich, and *Institute of Toxicology, ETH Zuerich, Switzerland.
Adeno-associated virus (AAV) is a non-pathogenic human parvovirus that depends on a helper virus for productive infection. Recombinant AAV (rAAV) have been developed in the last few years as vectors for gene transfer and gene therapy. We are employing rAAV vectors for gene transfer to the substantia nigra in a rat model of Parkinson's disease to identify molecules that have the potential to inhibit or delay the degeneration of dopaminergic neurons. Towards this end, we have developed an adenovirus-free method for the production of high-titer rAAV vectors. Using this method, we show that neuron-specific AAV vectors carrying the woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) confer efficient and sustained gene expression in dopaminergic neurons of the substantia nigra and their projections in the striatum. Data on transgene expression from a comparative analysis of vectors carrying the CMV or the neuronal PDGF-beta chain promoter and on the function of the WPRE element will be presented.

POSTERS

P01a// BLOCKADE OF THE B7/CD28 AND CD154/CD40 PATHWAYS IMMUNOMODULATES THE TRANSPLANTATION OF ERYTHROPOIETIN-SECRETING C2C12 MYOBLASTS.
NFP37 Nr: 4037-044718;
Schneider B.L., Peduto, G., Déglon, N., and Aebischer, P. Division of Surgical Research & Gene Therapy Center, CHUV; Lausanne, Switzerland
Transplantation of genetically engineered myoblasts is an attractive alternative to the treatment of diseases requiring repetitive administration of a therapeutic protein. However, the outcome of cell transfer is limited by the hurdle of the host immune defences. When 3 million native C2C12 myoblasts are injected in the hind limb muscles of syngeneic C3H mice (H-2k), they form tumors after 40 days. In contrast, most C2C12 cells transfected with an expression plasmid coding for mouse erythropoietin (EPO) are rejected within 15 days post-implantation, as indicated by a transient increase in the hematocrit level. By comparison, a similar experiment performed in FK506-immunosuppressed mice has resulted in sustained elevated hematocrit levels during 35 days. These observations suggest an immune response of the syngeneic recipients against the EPO-releasing myoblasts, which presumably express minor antigens. When implanted in allogeneic DBA/2J mice (H-2d), mouse EPO-secreting C2C12 cells induce only a one-week increase in host hematocrit, which reflects an acute rejection process associated with major histoincompatibility. Recently, administration of molecules blocking costimulatory pathways has been proposed in order to prevent T cell-mediated rejection. Therefore, we have tested whether the fusion protein CTLA4Ig and antibodies binding CD154 may prevent the killing of EPO-secreting myoblasts in both syngeneic and allogeneic recipients. C2C12 cell lines have been modified to secrete both mouse EPO and CTLA4Ig in a constitutive manner. Following intramuscular injection in syngeneic hosts, CTLA4Ig expression has significantly prolonged the survival of mouse EPO-secreting myoblasts in at least 50% of the animals, as demonstrated by their sustained hematocrit level. Nevertheless, in allogeneic DBA/2J mice, the constant release of CTLA4Ig has only induced a one-week delay in completion of the rejection process. In contrast, the conjunction of CTLA4Ig expression with injections of anti-CD154 antibodies on day -1, 0 and +3 has allowed the long-term acceptance (at least 35 days) of allogeneic EPO-secreting myoblasts. This demonstrates a synergistic effect of both strategies in cell allotransplantation. These results suggest that immunomodulating protocols combining CD154/CD40 and B7/CD28 blockades significantly improve the survival of C2C12 myoblasts expressing murine EPO in both syngeneic and allogeneic hosts.

P01b// NIGRO-STRIATAL EXPRESSION OF GDNF BASED ON LENTIVIRAL VECTORS ENHANCE STRIATAL DOPAMINERGIC INNERVATION IN AGED PRIMATE
NFP37 Nr: 4037-044718;
N. Déglon (1), J. Bloch (1), J. Kordower (2) and P. Aebischer (1). (1) Division of Surgical Research and Gene Therapy Center, CHUV, Lausanne, Switzerland and (2) Rush Presbyterian Hospital, Chicago, USA
Glial cell line-derived neurotrophic factor (GDNF) exerts neuroprotective and regenerative properties on the nigro-striatal dopaminergic system. Localized expression of GDNF whuch is required for its clinical application in Parkinson's disease may be achieved by in vivo gene transfer. The aim of the present study was investigate the ability of lentiviral vectors coding for GDNF to enhance dopaminergic striatal expression in aged primates. Stock solutions of lentiviral vectors carrying the cDNA for GDNF (n = 4) (p24 of 450.000) or b-Galactosidase (b-Gal) (n = 4) (p 24 of 240.000) were injected bilaterally at the level of the substantia nigra (SN) (1 track) and the striatum (putamen : 3 tracks ; caudate : 2 tracks) of aged primates. Fluoro-dopa PET-scan were performed 1 month prior to the lentiviral vector injections and 3 months post-injection. Following the last PET-scans, the animals were sacrificed and their brains processed for morphological analysis. Fluoro-dopa PET scans showed a significant enhanced uptake post-injection in the GDNF animals. In contrast, no such enhancement was observed in the b-Gal animals. Immunohistochemistry studies revealed large area ( 1 cm in diameter) of GDNF expression at the level of the caudate nucleus, putamen and substantia nigra. A significant enhanced dopamine content (as measured by HPLC), tyrosine hydroxylase immunoreactivity (TH-ir) (as measured by optical densities) and TH mRNA was observed in the GDNF versus the b-Gal primates. Finally the lenti-GDNF animals had a 700% increase in the number of TH-ir intrinsic striatal neurons relative to the lenti-b-Gal treated primates. These data indicate that lentiviral vectors encoding GDNF are able to significantly enhance dopaminergic striatal innervation in aged primates confirming their potential clinical relevance for the treatment of neurodegenerative diseases upon resolving associated safety issues. Supported by the Swiss National Science Foundation and the PNR 37 program on gene therapy.

P01c// LENTIVIRAL MEDIATED EXPRESSION OF GLIAL CELL LINE DERIVED NEUROTROPHIC FACTOR
NFP37 Nr: 4037-044718; Hottinger, AF, Azzouz, M, Deglon, N, Aebischer, P, Zurn, AD Division of Surgical Research and Gene Therapy Center, Pavillon 4, CHUV, 1011 Lausanne, Switzerland
Axotomy of the facial nerve in adult BalbC mice leads to an approximately 50% loss of motoneurons one month after axotomy. We evaluated the ability of glial cell line derived neurotrophic factor (GDNF) to prevent this cell death by administering the neurotrophic factor directly into the facial nucleus. A lentiviral vector (LV) encoding the GDNF gene under the control of the PGK promoter was injected stereotaxically into the right facial nucleus of 4 month-old BalbC mice. Control animals received injections of either lentiviral vectors coding for a mutated inactive form of GDNF, the reporter protein beta-galactosidase, or a saline solution. Four weeks later, a right facial nerve axotomy was performed. One month after this procedure, the mice were sacrificed and their brains analyzed. Evaluation of the expression pattern of the reporter gene LacZ showed that approximately 25'000 cells were infected in the facial nucleus and the surrounding brainstem. Both neurons and astrocytes were transduced. Axotomy induced the loss of 54.2±3% of facial motoneurons in the LV-mutated GDNF controls, while there was no motoneuron loss in LV-GDNF treated animals (111.9±9.2% survival compared to the non-lesioned side) (n=9, p<0.0001). This indicates that GDNF is capable of protecting motoneurons against axotomy induced cell death for extended periods of time when delivered close to the cell bodies of the motoneurons. Facial nerve lesion in adult mice presents an excellent tool to evaluate the effects of neurotrophic factors that might be beneficial in adult-onset motoneuron diseases. Supported by the Swiss National Science Foundation and Roche Research Foundation.

P01d// DEVELOPMENT OF A CELL-BASED SYSTEM FOR THE EXOGENOUSLY REGULATED DELIVERY OF ERYTHROPOIETIN
NFP37 Nr: 4037-044718;
Sommer B (1), Dalle B (2), Rinsch C (1), Beuzard Y (2), Déglon N (1) and Aebischer P (1) . (1) Division of Surgical Research & Gene Therapy Center, CHUV, Lausanne, Switzerland. (2) Laboratoire Expérimental de Thérapie Génique, Hôpital Saint-Louis, Paris, France
An ex vivo gene therapy approach for the continuous delivery of therapeutic proteins has been developped based on the encapsulation and implantation of genetically engineered C2C12 cells. These murine myoblasts are of special interest for this application because they can differentiate into post-mitotic myotubes avoiding cell overgrowth within the capsule. As a first step towards the development of an exogenously regulated erythropoietin (Epo) expression, we have analyzed the potency of the Tet-system described by Gossen and Bujard in C2C12 cells. We observed high background levels of transgene expression in the non-activated state resulting in only moderate induction rates ranging from 10 to 25-fold. The use of an additional tetracycline-dependent repressor rtTR was one strategy to overcome these limitations. Tightly regulated C2C12 cell clones producing 0.5 UI mouse Epo / 106 cells / 24 h in the induced state were obtained. Current experiments focus on the generation of repressor/activator clones with increased Epo production, for example by introduction of different dimerization domains for tTA1 and rtTR, respectively. This will prevent the heterodimerization and thus the inactivation of the two engineered transcription factors. In a second approach, C2C12 cells were stably transfected with an autoregulatory Tet-Off system. The resulting clones expressed 25 UI mouse Epo / 106 cells / 24h, an amount sufficient for the regulation of the hematocrit in vivo. After subcutaneous implantation of encapsulated cells into DBA/2J mice, the hematocrit of the animals could be modulated by the presence or absence of doxycycline (dox) in the drinking water. Removal of dox resulted in significantly increased hematocrit levels up to 90% reflecting a sustained Epo expression. In contrast, by addition of dox the expression of the transgene could be switched off even after several weeks of Epo production at high levels. Present studies analyze the long-term expression characteristics and the survival of these cells in vivo.

P01e// DEVELOPING A TOLERANCE TO ENCAPSULATED GENETICALLY ENGINEERED XENOGENEIC MYOBLASTS BY TRANSIENT IMMUNOSUPPRESSION
NFP37 Nr: 4037-044718;
Rinsch, C.L., Peduto, G., Schneider, B.L., and Aebischer P. Division of Surgical Research and Center of Gene Therapy, CHUV Pavillon 4,1011 Lausanne, Switzerland.
Immunoisolating cells by surrounding them with a semipermeable membrane prevents the immune destruction of allogeneic cells in the subcutaneous site as well as allogeneic and xenogeneic cells in the central nervous system. On the other hand, when immunoisolated xenogeneic cells are subcutaneously implanted, they are quickly destroyed. Murine C2C12 myoblasts, engineered to secrete mouse erythropoietin (mEpo), were employed as a test cell to understand the nature of this rejection and to evaluate strategies of preventing it. Encapsulated C2C12 mEpo cells were rapidly eliminated in immunocompetent Fischer rats but were accepted by immunodeficient nude rats. Immunosuppressive strategies employing FK506 led to the long-term survival of encapsulated C2C12 mEpo cells in Fischer rats. In particular, short-term immunosuppressive therapy with FK506, for periods lasting either 1, 2, or 4 weeks following implantation, permitted the long-term survival of encapsulated C2C12 mEpo cells. Animals increased their hematocrits to over 70% and maintained these levels for three months, independent of the duration of FK506 treatment. Fischer rats rendered unresponsive to encapsulated xenografts by this technique were challenged with a second implant after immunosuppression had ended to determine if a tolerance to xenografts had been established. Challenging animals initially treated only one week with FK506 induced the rejection of both primary and secondary implants. On the other hand, animals immunosuppressed four weeks accepted second implants, and the initial implants also remained viable. This developed tolerance to xenogeneic myoblasts lasts over extended periods (seven months), in the absence of both immunosuppression and stimulating xenoantigens. Tolerance established to unmodified xenogeneic myoblasts can also transfer to genetically engineered xenogeneic myoblasts, indicating that antigens inherent to myoblasts are responsible for initiating graft rejection. Interestingly, animals tolerized to encapsulated C2C12 mEpo myoblats rejected these cell when they were directly injected intramuscularly at a later date. These findings reveal, for the first time, that host tolerance can be developed to encapsulated xenogeneic myoblasts following transient immunosuppression. These results suggest that, in an immunosuppressed background, continual exposure to xenoantigens either shed or secreted out of the capsule leads to the development of a long-term tolerance to xenogeneic myoblasts when encapsulated. Importantly, the period of concomittant exposure to xenoantigens and immunosuppressor was found to be critical for success of tolerization protocols.

P01f// DEVELOPMENT OF AN ANTIOXIDATIVE GENE THERAPY STRATEGY FOR NEURODEGENERATIVE DISORDERS
NFP37 Nr: 4037-044718;
Ridet JL, Bensadoun JC, Déglon N, Aebischer P, Zurn AD. Surgical Research & Gene Therapy Center, CHUV, Lausanne, Switzerland
Reactive oxygen species may be involved in the pathogenesis of many clinical CNS disorders. Oxidative damage in neurodegenerative diseases may result from an increased production of free radicals and/or a failure of antioxidant defenses. It is hypothesized that free radical neurotoxicity contributes to midbrain neuronal death in Parkinson's disease. A major CNS antioxidant enzyme is the enzyme glutathione peroxidase (GPX). We have previously shown (Bensadoun et al., 1998, Eur. J. Neurosci. 10:3231-6) that the dopaminergic neurons of GPX-overexpressing transgenic mice are partially protected against 6-hydroxydopamine (6-OHDA)-induced toxicity. In the present study, we developed a lentiviral vector carrying the human GPX gene. Genomic DNA coding for human GPX (intron 1, exons 1,2) was cloned into the self-inactivating transfer vector (SIN-W-PGK), carrying the phosphoglycerate kinase promoter (PGK) and a post-transcriptional cis-acting regulatory element of the woodchuck hepatitis virus (WHV). Transient transfection with the calcium phosphate method of 293T and human neuroblastoma (SH-SY5Y) cells with the SIN-PGK-GPXg-WHV plasmid resulted in a 2-3 fold increase in GPX activity as compared to normal non-transfected cells or SIN-PGK-LacZ-WHV-transfected cells. Viral particles were produced by transient transfection of 293T cells with a four plasmid system. Viral batches were tested for the absence of replication-competent vectors. The lentiviral vector LV-SIN-PGK-GPX-WHV was used to infect neuroblastoma cells. GPX activity was increased 2-3 fold in LV-SIN-PGK-GPX-WHV-infected cells as compared to non-infected cells and LV-SIN-PGK-GFP*-WHV-infected cells. In an in vitro model of 6-OHDA toxicity, we compared the neuroprotective effects of various lentiviral vectors carrying the GPX, GDNF or bcl-2 human gene. Our results revealed (a) 83% cell survival with LV-SIN-PGK-GPX-WHV vector compared to 22.9% in LV-SIN-PGK-GFP-WHV-infected cells, and (b) antiapoptotic properties similar to those obtained with bcl-2. We will discuss the relevance of such a vector for gene therapy in neurodegenerative disorders, in particular Parkinson's disease. Supported by the Swiss National Science Foundation.

P01g// NEUROPROTECTIVE EFFECT OF A CNTF-EXPRESSING LENTIVIRAL VECTOR IN THE QUINOLINIC ACID RAT MODEL OF HUNTINGTON'S DISEASE
NFP37 Nr: 4037-044718;
Pereira de Almeida L(1,2), Zala D, Tseng JL, Gaspar R(2), Déglon D, and Aebischer P (1)Division of Surgical Res. and Gene Therapy Center, CHUV, Lausanne, Switzerland.
Huntington's disease (HD) is a fatal neurodegenerative disorder of genetic origin characterised by a slow and progressive degeneration of GABAergic neurons in the striatum. No treatment is currently available, but neuroprotective strategies, employing neurotrophic factors, have been proposed to slow down the progression of the disease. Adequate delivery systems are however required to overcome the presence of the blood-brain barrier and to locally deliver these therapeutic agents in the CNS. In the present study, we have used HIV-1 based vectors which efficiently infect and integrate into the genome of post-mitotic neurons leading to long-term and sustained expression of transgenes. The ciliary neurotrophic factor (CNTF), which has been shown to have a protective effect in the quinolinic acid model of HD, was used to validate this gene delivery system. Two microliters of CNTF or LacZ-expressing lentiviral vectors were injected into the striatum of adult Wistar rats. Three weeks later, the animals were subjected to quinolinic acid (QA) lesion (180 nmol). The behavioural rotational asymmetry, induced by apomorphine, was measured every 4-5 days post-lesion. The animals were then sacrificed and the brains processed for immunohistochemistry. LacZ-injected animals displayed apomorphine-induced rotations ipsilateral to the lesion whereas a significant reduction (82%) was observed in the CNTF-treated animals. Glutamic acid decarboxylase-stained sections revealed that CNTF reduces by 40% the QA induced lesioned area. Finally, the number of choline acetyl transferase and of NADPHdiaphorase stained neurons was significantly higher in the CNTF group (4.9 and 4.4 times, respectively). These results further establish the potential use of lentiviral vectors as in vivo gene therapy approach for neurodegenerative diseases. Supported by the Swiss National Science Foundation. (2) Laboratory of Pharmaceutical Technology; Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal

P01h// LENTIVIRAL VECTOR AS A GENE DELIVERY SYSTEM IN THE MOUSE CNS: NEUROPROTECTION IN A 6-OHDA MODEL OF PARKINSON'S DISEASE USING GDNF
NFP37 Nr: 4037-044718;
Bensadoun JC, Deglon N, Tseng JL, Ridet JL, Zurn AD & Aebischer P. Surgical Research Division & Gene Therapy Center, CHUV, Lausanne, Switzerland
Adequate localization and expression of therapeutic molecules represents one of the limiting factors leading to recovery in models of neurodegenerative disorders. In vivo gene transfer using viral vectors represents a powerful strategy to achieve this goal. The aim of the present study was to validate the lentiviral vector as a gene delivery system in the mouse CNS. A preliminary study with a LacZ-encoding vector injected above the substantia nigra of C57BL/6j mice indicated that lentiviral vectors can infect a large number of cells and diffuse over long distances. Based on these results, glial cell line-derived neurotrophic factor (GDNF) was assessed as a neuroprotective molecule in a 6-hydroxydopamine model of Parkinson's disease in mice. A lentiviral vector carrying the cDNA for GDNF or mutated GDNF was unilaterally injected close to the substantia nigra of C57BL/6j mice. Two weeks later, the animals were ipsilaterally lesioned with 6-hydroxydopamine into the striatum. Drug-induced rotation was significantly decreased in the GDNF-injected group as compared to control animals. Moreover, GDNF efficiently protected tyrosine hydroxylase-positive cells in the substantia nigra against 6-OHDA-induced toxicity since 69.5% of the cells remained compared to 33.1% in the muGDNF group. These data indicate that the lentiviral vector is a powerful gene delivery system for the murine CNS. It thus represents an interesting tool for the screening of therapeutic molecules in mouse models of neurodegenerative diseases. Supported by the Swiss National Science Foundation

P01i// ENCAPSULATION OF HUMAN PRIMARY FIBROBLASTS AS A CELL-BASED DELIVERY SYSTEM OF EPO.
NFP37 Nr: 4037-044718;
Schwenter F.1, Déglon N.1, Rolland2 E., Winkel1 L., Bloch1 J., Bouche1 N., Pralong2 W., Baetge2 E. E., and Aebischer1 P. (1) Division of Surgical Res. & Gene Therapy Center, Lausanne Univ Medical School; (2) Modex Thérapeutiques, Lausanne, Switzerland.
Transplantation of encapsulated cells genetically engineered to secrete human erythropoietin (hEPO) may provide a new approach to the treatment of anemia. The aim of the present study was twofold: a) to evaluate the survival of encapsulated human primary fibroblasts in human volunteers; b) to develop gene transfer protocols allowing the expression of clinically significant levels of hEPO from human fibroblasts. As a first step toward human experimentation, the survival of encapsulated human primary fibroblasts was demonstrated in nude mice. A cell bank derived from these cells was then established and shown to be non-tumorigenic and free of pathogens. Cells from this certified bank were encapsulated in a 2 cm-long semi-permeable hollow fibre. Upon obtention of the ethical committee approval, they were implanted subcutaneously for one month in the forearm of seven volunteers under local anaesthesia. All implants were well tolerated and devoid of any side effects. Histological analyzis of the retrieved capsules demonstrated a cell survival of at least 80% in all implants. A Moloney Murine Leukemia Virus (MoMuLV)-based retroviral vector containing the hEPO cDNA was used to infect human primary fibroblasts. Single infection yielded 50 to 70 IU/106 cells/24hrs of EPO whereas multiple infections reached secretion levels of 130 to 160 IU/106 cells/24hrs of EPO. The ability of these cells to increase hematocrit in nude mice is being currently tested. Safety experiments will be conducted prior to implant encapsulated hEPO-secreting human primary fibroblasts for the treatment of chronic anaemic patients.

P02// STRUCTURAL REQUIREMENTS OF SYNTHETIC BONE MARROW TARGETING PEPTIDES (BMTPS) FOR BINDING TO THE MARROW ENDOTHELIUM
NFP37 Nr: 4037-044804;
Bisoffi M., Finger A.N., Wetterwald A., Gautschi E., Thalmann G. N., Stadler Beda M. (1) and Cecchini M.G. (1) Institute of Immunology, University Hospital, CH-3010, Bern, Gene Therapy Laboratory, Department of Clinical Research and Department of Urology, University Hospital, CH-3010, Bern, Switzerland.
A challenging problem in gene therapy concerns in the efficient transfer of therapeutic genes to the appropriate tissue. Endothelial cells (EC) show molecular heterogeneity among the tissues where they reside. Bone marrow (BM) is a typical site of specialised endothelium. We have previously reported the identification by phage library panning in vivo of hexa-peptide ligands specific for the mouse BM endothelium (bone marrow targeting peptides, BMTPs). These peptides may have the potential for targeting therapeutic molecules to this anatomical district. This possibility was further investigated by analysing the structural requirements of one of the BMTPs, namely BMTP-2, for its binding to the receptor molecules on the BM endothelial cells (BMEC). The binding of a BMTP-2 bearing phage clone was further verified in vitro on freshly isolated mouse bone marrow cells and on BMEC lines (STR-4, -10 and -12). The binding to BMEC in vivo and in vitro of a synthetic biotinylated peptide, composed of the BMTP-2 hexa-peptide plus the 11 amino acids of the pIII phage protein, was tested. The binding inhibition of the BMTP-2 bearing phage to BMEC lines by the above synthetic peptide was also analysed. The phage clone bearing BMTP-2, but not control phage clones bearing no insert or an irrelevant hexa-peptide, were shown by immunofluorescence microscopy to bind the mouse bone marrow endothelial cell lines STR-10 and -12, and, to a lesser extent, STR-4. In contrast, the BMTP-2 synthetic peptide was unable to bind either in vivo to BMEC or in vitro to the STR-10 cell line. Furthermore, an excess of free BMTP-2 synthetic peptide did not inhibit the binding of the BMTP-2 bearing phage clone to the STR-10 cell line. The results above seem to indicate that the BMTP-2 is unable to bind as a short mono-meric peptide to BMEC. However, peptide inserts are expressed on the pIII phage protein in a penta-meric configuration. This may suggest a requirement for a structural interaction of the BMTP-2 with the pIII phage protein for binding to BMEC. We are currently addressing the importance of the pIII phage protein domains and/or the multimeric appearance of BMTP-2 for the interaction with the BMEC receptor.

P03// FAS LIGAND AND TOPOTECAN COMBINATION THERAPY OF EXPERIMENTAL GLIOMAS
NFP37 Nr: 4037-044702;
Benjamin B. Ambar, Luca Bernasconi, Ursula Malipiero, and Adriano Fontana. Section of Clinical Immunology, University Hospital, CH-8044 Zürich, Switzerland.
The incedence of primary brain tumors. is 11.7 per 100'000 persons, among these glioblastomas are 8% of all cases and 42% of all malignant primary brain tumors. Since human glioma cells express Fas and undergo Fas-mediated apoptosis, we have generated replication-deficient adenoviruses encoding FasL under the control of the cytomegalovirus (CMV) promoter (rAd-CMV-FasL). The Fas receptor (APO-1/CD95) pathway is one of the best characterized intracellular pathways leading to apoptosis. It is initiated by the binding of Fas ligand (FasL) and leads to apoptosis through the activation of cysteine proteases called caspases. Local expression of FasL using rAd-CMV-FasL leads to significant prolongation of survival of glioma bearing rats, but without the occurrence of long term surviving animals. Topotecan is a derivative of the plant alkaloid campothecan which inhibits topoisomerase (topo) I. Topotecan has shown promising tumoricidal effects in preclinical studies, but had only modest activity against recurrent malignant gliomas in adults (Macdonald et al., 1996, Ann. Oncol., 7:205). Topotecan has been shown to act synergistically with supernatant containing soluble murine membrane FasL on the killing of human glioma cell lines (Winter et al., 1998, J. of. Pharm. And Exper. Therapeutics, 286:1374). In this work we investigate the synergistical effects of rAd-CMV-FasL and topotecan on the killing of human and rat glioma cell lines in vitro. Experiments investigating the synergistical effects of rAd-CMV-FasL and topotecan on the killing of rat glioma cells in vivo are currently ongoing.

P04a// STEROID MEDIATED GENE DELIVERY (SMGD)
NFP37 Nr: 4037-04480;
Rebuffat A (1),Bernasconi AGF (1), Wehrli HU (1),Nawrocki AR (1),Ceppi M (2),Fonte C (2),Frey BM (1), Rusconi S (2), Frey FJ (1). Division of Nephrology University of Berne (1) and Institute of Biochemistry, University of Fribourg, Switzerland.
In the aim of improving the efficiency of gene transfer, numerous attempts to develop appropriate delivery systems based either on viruses or non-viral vectors have been proposed. We have designed an alternative approach, the so- called SMGD, to improve the nuclear uptake of transfected DNA with the help of the natural shuttle for steroid molecules. The rationale is to derivatize steroids and link them to a DNA molecule by covalent, ionic or hydrophobic bond. We anticipate that with an appropriate formulation, the conventionally transfected steroid-DNA complex will be more efficiently and actively translocated into the nucleus via the cognate intracellular receptor shuttle. For that purpose we synthesized 17 glucocorticosteroid conjugates (patent pending) consisting of either cortisol or dexamethasone, a chemical spacer (>C4), and a DNA-binding moiety linked to a reporter gene. The biological stability was established by incubating the conjugates with different biolgical extracts and confirming their structural integrity by nuclear magnetic resonance spectroscopy. The affinity of the glucocorticoid receptor (GR) was assessed and the five conjugates with the highest affinity were further investigated. In order to demonstrate translocation of the conjugates from the cytoplasm to the nucleus by GR, a cell line expressing a GR-GFP chimera protein was used. The conjugates with the highest affinity for the GR and a long spacer showed highest translocation efficiency. The expression of the reporter gene attached to the conjugates was studied in CV-1 GR+ and CV-1 GR- cells. An enhanced expression was observed in GR+ cells (see poster P04b by Ceppi et al.) In conclusion, the present data indicate that steroid decorated DNA-conjugates can be translocated by the naturally occuring steroid receptor shuttle from the cytoplasm to the nucleus. Since steroid receptors exhibit cell specific expression the SMGD is a new promissing strategy for enhancement of gene targeting in vivo.

P04b// ENHANCING GENE DELIVERY WITH STEROID RECEPTOR NUCLEAR SHUTTLES
NFP37 Nr: 4037-044802; M. Ceppi, C. Fonte, A.G.F. Bernasconi*, A. Rebuffat*, E. Lovati*, B. Frey*, F.J. Frey* and S. Rusconi Institute of Biochemistry, Rue du Musée 5, CH-1700 Fribourg; *Div.Nephrology, Dept.Internal Medicine, University Hospital Bern, CH-3010 Bern, Switzerland
We are designing an approach, the so-called steroid mediated gene delivery (SMGD), with the aim to improve the nuclear uptake of transfected DNA with the help of the natural shuttle for steroid molecules. The rationale is to derivatize steroids to link them to a DNA molecule by covalent, ionic or hydrophobic bonds. We have modelled the SMGD principle with the glucocorticoid receptor (GR). To this purpose we have synthesized and tested several steroid derivatives, all composed by a steroid molecule covalently branched to a DNA interacting compound (see Poster P04a by Rebuffat et al). The compounds that retained substantial affinity for the GR were able to induce in vivo the nuclear translocation of a transiently transfected GR-GFP reporter protein. Random decoration by the steroid derivatives mediates a 2- to 5-fold higher expression of a transfected reporter gene if compared with DNA incubated with control molecules. This effect is absent in cells which do not express the GR (monkey CV-1) and is resumed when those latter were supplemented with the glucocorticoid receptor by previous infection with a GR-encoding recombinant adenovirus. We are currently testing the extent of the SMGD effect in quiescent cells, with steroid conjugates that can be covalently linked to DNA, and with other steroid molecules. The final general scope of the SMGD procedure is indeed to offer a selective advantage to the therapeutic treatment of target cells that express a specific steroid receptor.

P05// SENDAI PARAMYXOVIRUS AS A NON-INTEGRATIVE RNA VIRUS VECTOR
NFP37 Nr: 4037-044711;
Jonathan E. Freeman, Laurent Roux & Dan Kolakofsky. CMU, Genetics & Microbiology, Geneva University
Sendai paramyxovirus (SeV) is a natural pathogen of mice; its genome is a negative strand 15,384 base RNA. SeV is pneumotropic and non-integrative, replicating within the cytoplasm of infected cells. Used with transgenic models of human disorders, SeV may be an excellent way to study lung therapy. We have been introducing transgenes into SeV to characterise its use in gene therapy. Two vectors have been made. In a "contiguous" recombinant SeV (rSeV), the transgene is placed within the linear array of endogenous genes. In the "ambisense" rSeV, the right-end promoter is modified to support transcription, and the transgene placed downstream of the viral genes, in the reverse coding sense. Both of these vectors express a luciferase transgene. Contiguous rSeVs give higher expression levels and a more user-friendly has been made. Studies in vivo show that carrying a transgene per se does not block virus viability in the mouse lung. Given its pneumotropism and the availability of mouse models, the first chronic disease to be assessed will be a1 anti-trypsin (a1 AT) deficiency. Viruses expressing a1 AT have been constructed. In addition, an industrial partner is checking whether these rSeVs can deliver immunogens in vivo.

P06// OXIDANTS AND ANTIOXIDANTS INFLUENCE THE EFFICIENCY OF RECOMBINANT ADENOVIRUS INFECTION: H2O2 INHIBITS WHEREAS N-ACETYLCYSTEINE AND ALPHA-LIPOIC ACID ENHANCE GENE TRANSFER TO HUMAN ENDOTHELIAL CELLS.
NFP37 Nr: 4037-044717;
Jornot L, Petersen H, Moix I, Morris MA(2), Pavirani A (3), Mehtali M (3), Rochat T. Respiratory Division, University Hospital, Geneva, Switzerland.
High levels of reactive oxygen metabolites have been shown to accumulate in CF patients, resulting from 1) increased metabolic rate, 2) chronic neutrophil-dominated inflammation, and 3) impaired antioxidant status as a result of fat malabsorption. Furthermore, recombinant adenoviral vectors have been shown to contribute significantly to airway inflammation, especially when used at high titers for efficient transduction. Because over-activity of free radicals can damage plasma membranes and DNA, we hypothesized that oxidants may reduce the efficiency of adenovirus-mediated gene transfer/expression to inflamed tissue. To address this, human umbilical vein endothelial cells (HUVEC) were exposed to various doses of H2O2 for 1h prior to infection with E1E3-deleted adenovirus containing a LacZ reporter gene driven by an RSV promoter (100 IU/cell for 4h). B-galactosidase (Bgal) activity, measured 48h after infection, decreased by 18% and 50% in cells pretreated with 100 and 200 uM H2O2 respectively. This decrease was not related to a direct inhibitory effect of H2O2 on Bgal synthesis and/or activity, since H2O2 treatment after adenovirus infection did not alter Bgal activity. Addition of the antioxidant N-acetylcysteine (NAC) not only prevented the inhibitory effect of H2O2 but also greatly enhanced Bgal expression in infected cells not exposed to H2O2. Cells pretreated for 24h with 10 mM NAC showed a 2 + 0.2 fold increase in Bgal activity, compared to untreated cells. When NAC was also present during the infection and recovery periods, Bgal activity was further enhanced to a 14.9 + 3.5 fold increase. There were concomitant intracellular increases of 2.7 + 0.5 fold in vector DNA (assessed by Southern blot) and of 1.7 + 0.1 fold in GSH. When NAC was present only during the recovery period, Bgal expression was increased 4.6 + 1.7 fold but no difference in vector DNA level was detected. A similar but smaller effect was observed with the antioxidant a-lipoic acid (LA; 100 uM). We conclude that, in HUVEC: 1) adenovirus-mediated gene transfer is modulated by oxidants and antioxidants; 2) NAC has a stimulatory effect on adenovirus entry and/or processing of the virus; and 3) NAC directly enhances the transcription, translation and/or activity of the Bgal transgene. (2) Division of Medical Genetics, University Hospital, Geneva, Switzerland, *(3) Transgene SA, Strasbourg, France.

P07a// CHARACTERIZATION OF CD24 PROMOTER AS A TOOL FOR DIRECTING TISSUE SPECIFIC GENE EXPRESSION TO SMALL CELL LUNG CANCER
NFP37 Nr: 4037-044824;
Wegmann B., Di Paolo C., Sigrist J., Stahel R. Division of Oncology, Departement of Internal Medicine, University Hospital Zürich, CH-8044 Zürich, Switzerland
One way for targeting transgene expression of therapeutic genes specifically to cancer cells while leaving normal tissue unaffected is the usage of tissue specific promoters. Good candidates for cis-controlling of therapeutic genes in small cell lung cancer (SCLC) are those that are overexpressed in SCLC like the CD24 antigen. Several other promoters which may be predestined to direct the expression of therapeutic genes in SCLC depend on the neuroendocrine properties of SCLC but most of them were not shown to be able to drive therapeutic genes selectively to tumor cells. We have isolated 3.4 kb of the 5'UTR of CD24, an antigen highly expressed on the surface of small cell lung cancer (SCLC) cell lines and SCLC tissue. Its characterization revealed that a 269bp deletion fragment shows stronger promoter activity than the whole 3.4kb fragment while preserving tissue specificity as shown by comparison of promoter activity in SCLC to that in normal bronchial epithelial cells. Measuring promoter activity after disruption of consensus binding sites of putative transcription factors by introduction of specific point mutations into the CD24 promoter revealed that the cAMP responsive element (CRE) and two binding sites for MZF1 seem to play a pivotal role for promoter activity. Albeit, we could not show an increase of CD24 promoter activity in the presence of 8-Br-cAMP. In order to assess the utility of CD24 promoter for directing expression of suicide genes to SCLC we constructed SIN-retroviral vectors carrying HSV thymidine kinase (HSVtk) under the control of CD24 promoter. The killing activity of our CD24 controlled vector upon incubation with ganciclovir is compared to that of control vectors where HSVtk is driven by viral control promoters (CMV, SV40, both in lung cancer cell lines and in normal bronchial epithelial cells.

P07b// BCL-XL ANTISENSE TREATMENT INDUCES APOPTOSIS IN LUNG AND BREAST CANCER CELLS
NFP37 Nr: 4037-044824;
Gautschi O, Simžes-Wuest AP, Leech SH, Olie RA, Tschopp S, Zangemeister-Wittke U, Stahel RA . Division of Oncology, Department of Medical Oncology, University Hospital Zuerich, CH-8044 Zuerich, Switzerland
The anti-apopototic protein Bcl-xL is overexpressed in many human tumors and plays a pivotal role in tumor initiation and the development drug resistance. We have designed a phosphorothioate antisense oligonucleotide targeting a region on the bcl-xL mRNA which is not shared by the pro-apoptotic splice variant bcl-xS. Important functional properties of this deoxy oligonucleotide, such as nuclease resistance and hybridization affinity, were improved by 2'-O-methoxy-ethoxy (2'-MOE) modifications made to riboses of 5' and 3' flanking nucleotides to produce the 2'-MOE-deoxy gapmer oligonucleotide 4259. The effect of 4259 on bcl-xL expression and the induction of apoptosis was investigated in the non-small cell lung cancer cell lines A549 and NCI-H125, and the breast cancer cell lines MCF7, T-47D, ZR-75-1, and MDA-MB-231. As shown by real-time PCR and Western blot analysis, treatment of tumor cells with oligonucleotide 4259 at a concentration of 600 nM for 20 h efficiently inhibited bcl-xL expression at the mRNA and protein level. In all cell lines inhibition of bcl-xL expression resulted in the induction of apoptosis as revealed by the loss of mitochondrial transmembrane potential, release of cytochrome c from mitochondria, and activation of caspase-3-like proteases. Our data provide strong evidence that Bcl-xL is an important survival factor for lung and breast cancer cells, and suggest that oligonucleotide 4259 deserves attention as a therapeutic compound in lung and breast cancer, as well as other malignancies in which bcl-xL is overexpressed.

P08// REVERSIBLE IMMORTALIZATION OF HUMAN PRIMARY CELLS USING A CRE/LOXP LENTIVECTOR SYSTEM
NFP37 Nr: 4037-046196;
Salmon P, Oberholzer J, Triponez F, Lou J, Occhiodoro-Scott T, Morel P, Trono D. Dept of Genetics and Microbiology and Division of Surgical Res., Univ. of Geneva
Reversible immortalization is a powerful means to obtain unlimited supplies of human primary cells. Since lentiviral vectors can infect non-dividing cells, we have developed a HIV vector-based system for the reversible expression of immortalizing genes in human primary cells. To validate this approach, we constructed a HIV-based vector that contains the Egfp (Enhanced Green Fluorescent Protein) gene, the ECMV IRES sequence driving the HSV1 thymidine kinase gene, and a LoxP sequence in the 3' LTR of the vector plasmid. After integration, this vector is designed to govern expression of both Egfp and HSV1-TK from a proviral cassette flanked by two LoxP sites in the LTRs. Hela cells and 293T cells infected with this vector stably express Egfp, as assessed by FACS analysis, and become sensitive to nucleoside analogs like Acyclovir (ACV) and Ganciclovir (GCV). When these cells are transduced with adenoviral vectors expressing the Cre protein, most cells become Egfp negative. When the remaining cells are treated with ACV or GCV, the remaining Egfp-positive cells are eliminated. Human pancreatic islet cells were transduced with such lentiviral vectors expresing Egfp at various multiplicities of infection (MOI). After 4 days, direct fluorescence analysis of intact islets showed that the majority of cells were transduced. This system can be used to deliver immortalizing genes in human primary cells to induce their proliferation. HIV vectors expressing transgenes such as the SV40 large T antigen and the catalytic subunit of telomerase are being tested for their capacity to induce proliferation of human primary cells. After sufficient cell proliferation, the immortalizing genes can be excised by introducing Cre. The presence of HSV1-TK in the excisable cassette will provide an additional safety feature, allowing selective destruction with low-toxicity drugs such as ACV or GCV of cells that have not excised the transgene and are still proliferating. Further phenotypic and functional analyses will be performed on immortalized and "reverted" cells.

P09a// TOWARDS MEASLES-BASED VIRUSES SELECTIVELY ENTERING HIV-INFECTED CELLS M.
NFP37 Nr: guest;
Rager(1), U. Schneider (1,2) , and R. Cattaneo (1,2). (1) Institut fuer Molekularbiologie, Univ. Zuerich, Winterthurerstr. 190, 8057 Zuerich, Switzerland; (2) Molecular Medicine Program, Guggenheim 18, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA
Measles virus (MV) is a human negative strand RNA virus for which a safe and effective live attenuated vaccine exists. That MV particles may accommodate multiple genomes, and that no RNA recombination was ever observed seem auspicious for the stable propagation of MV-based vectors. We aim at constructing MV-based particles targeting human immunodeficiency virus (HIV) infected cells. In these novel viruses the HIV receptor CD4 and one of its co-receptors, e.g. Bonzo (STRL33/TYMSTR), should substitute the two MV glycoproteins fusion (F) and hemagglutinin (H). To favour incorporation into MV particles we replaced the cytoplasmic tails (t) of CD4 and Bonzo, or both the cytoplasmic tail and transmembrane domain (tm+t) of CD4, by the corresponding domains of the MV F protein. MVs efficiently expressing the CD4/Ft, Bonzo/Ft and CD4/Ftm+t hybrid proteins, either separately or CD4/Ft and Bonzo/Ft together, in addition to the standard envelope proteins were rescued. Whereas incorporation of CD4 hybrid proteins into viral particles is efficient, hardly any Bonzo/Ft can be detected in purified virus. We are now attempting to improve coreceptor incorporation and to rescue viruses with CD4 and Bonzo substituting MV F and H. These viral particles are expected to infect only cells presenting the HIV envelope protein gp120 at their surface. This principle has already been established in rhabdoviruses. Nevertheless, our MV has the advantage of being an approved human vaccine strain.

P09b// DEVELOPMENT OF A NEW CLASS OF REPLICATING VIRAL VECTORS FOR CYTOLYTIC GENE THERAPY U.
NFP37 Nr: guest;
Schneider, F. Bullogh, S.J. Russell and R. Cattaneo. Molecular Medicine Program, Mayo Clinic, Rochester MN
Nonsegmented negative strand RNA viruses of the family Paramyxoviridae have two distinctive advantages as replicating vectors for gene therapy. They do not recombine and their particles, being pleomorphic, do not have physical constraints limiting the amount of genetic material incorporated. Using a reverse genetics system we attempted the production of vaccine based measles viruses (MV) displaying foreign domains on their attachment protein (hemagglutinin, H), restricting cell tropism. We show here that epidermal growth factor (EGF, 54aa) or insulin-like growth factor-I (IGF, 70aa) can be attached to the C-terminus of the H protein and that recombinant viruses expressing H/EGF or H/IGF in place of H can be rescued. Both H-chimeras remained stable over several passages. Viral replication was analysed on cells with different receptor profiles. On two cell lines expressing low levels of the EGF receptor the H/EGF virus reached similar titers as standard MV. Titers of cell free and cell associated H/EGF virus were 100-500 fold lower on A431 cells expressing high amounts of the EGF receptor, whereas titers of the H/IGF virus were only 10-50 fold reduced. This suggests that the reduced titers of the H/EGF virus were at least in part due to EGF receptor dependent sequestration. Furthermore, protease pretreatment of a virus with a factor Xa cleavage site between H and EGF yielded a slight increase in infectivity on A431 cells. The titer of cell associated H/IGF virus was slightly reduced on 3 cell lines expressing moderate amounts of the IGF receptor. This and a pronounced change in syncytia morphology argue for an interaction between IGF and its receptor. This work demonstrates that novel MV like viruses with restricted propagation in specific tissues can be produced.

P10// RE-TARGETING OF ADENOVIRUS: CAR-Fc FUSION PROTEIN ALLOWS EFFICIENT TRANSGENE EXPRESSION IN Fcg-RECEPTOR-POSITIVE CELLS
NFP37 Nr: guest;
Ebbinghaus, C.(1), Operschall, E.(2), Peter, I.(1), Hemmi, S.(1). (1) Institute of Molecular Biology I, University of Zürich, Winterthurerstr. 190, CH-8057 Zuerich, Switzerland; (2) Institute of Medical Virology, University of Zürich, Gloriastr. 30, CH-8028 Zuerich, Switzerland
Modification of the host range of adenoviruses can in principal be accomplished by two means: genetic alterations of the adenovirus fiber protein or the use of bifunctional reagents. We constructed a CAR-Fc fusion protein consisting of the extracellular domain of the human coxsackievirus and adenovirus receptor (CAR) and the human IgG immunoglobulin Fc domain. This fusion protein was found to efficiently block transgene expression of a recombinant adenovirus expressing the green fluorescence protein (GFP) in A549 human lung carcinoma cells, presumably by blocking binding of virus to its primary receptor. The CAR-Fc protein was also tested for its ability to re-direct AdCMV-GFP to cells devoid of CAR expression, which however express one of the Fcg-receptors (CD16, CD32 and CD64). Upon addition of CAR-Fc, we found an at least 100 fold increase of GFP-expression when analyzing the human monocyte-like cell line THP-1, which expresses the high affinity FcgRI (CD64). The CAR-Fc bispecific protein is currently tested using additional cell lines of the myeloid/lymphatic cell type.

P11// SOMATIC GENE THERAPY USING NAKED PLASMID DNA ENCODING VASCULAR ENDOTHELIAL GROWTH FACTOR-C (pVEGF-C) IN PATIENTS WITH CHRONIC CRITICAL LIMB ISCHEMIA.
£NFP37 Nr: 4037-055161;
Baumgartner I., Müller M., Skjelsvik C., Vogt A. Inselspital, Bern
Chronic critical limb ischemia (CLI) is a severe manifestation of peripheral arterial occlusive disease that can necessitate partial or complete amputation of the affected limb and is often accompanied by skin changes that does not improve without clinical intervention. Therapeutic angiogenesis seeks to employ natural mechanisms of angiogenesis to promote new blood vessel growths in a controlled manner. VEGF's are a recently identified family of angiogenic growth factors that are essential for the development and maintenance of normal vasculature. Previous studies of intramuscular pVEGF-C gene therapy in the rabbit ischemic hindlimb model indicate that pVEGF-C is safe as well as effective in promoting a robust angiogenic response. On basis of compelling experimental data in animal models, a Phase I randomized, double-blind, placebo-controlled multicenter study of intramuscular VEGF-C gene therapy in patients with CLI was initiated. The plasmid is a 5283 base pair plasmid that contains the human VEGF-C coding sequence. Expression from the VEGF-C gene is modulated by the presence of enhancer sequences (from CMV) and promoter sequences (from RSV). Ribonucleic acid processing signals (rat pre-proinsulin polyadenylation and 3'splice sequences) are also present to enhance VEGF-C messenger RNA stability. The plasmid also contains a gene that confers kanamycin resistance to host cells. In the phase I trial (6/99-8/99) 10 patients have received intramuscular VEGF-C gene therapy at doses of 4mg and 8 mg at our study center (another 14 patients were treated at four centers in the United States). At these doses, evidence of bioactivity (e.g., decreases of rest pain intensity, decrease in number of rest pain episodes and augmented ischemic ulcer healing) has been seen. No adverse events other than occasional edema and injection site pain have been attributed to this investigational treatment. (table with intermediate results will be presented at the poster). A Phase II randomized, double-blind, placebo-controlled multicenter study of multiple doses of intramuscular VEGF-C gene therapy in patients with CLI is planned to start in November 1999. The hypothesis will be tested, whether increased delivery of the pVEGF-C to ischemic tissues may lead to new blood vessel growth that is not only more robust but more evenly distributed than with a single treatment alone. The primary objective of this study is to assess the effect of multiple doses of pVEGF-C on rest pain, exercise tolerance and ulcer healing in patients with CLI.

P12// GENETIC BACKGROUND OF SALMONELLA HAS PROFOUND INFLUENCE ON PHAGOCYTOSIS, MATURATION AND CYTOKINE PRODUCTION IN HUMAN DENDRITIC CELLS.
NFP37 Nr: 4037-055164;
D. Dreher, L. Cochand, M. Kok, L.P. Nicod. Div. Pneumologie, Hôpital Cantonal Universitaire, 1211 Genève
Background : Genetic vaccination may offer more effective immunization against intracellular pathogens, such as tuberculosis. By transfer of DNA coding for TB proteins into antigen presenting cells (APC), specific activation of T-lymphocytes could be achieved. Study aim : To develop a live vector for delivery of mycobacterial antigens to human APC, we compared the infectious and immunological properties of several genetically modified Salmonella typhimurium (Stm) strains in human dendritic cells (DC). Methods : Stm is capable of plasmid transfer to mammalian cells and can be easily engineered genetically to influence its interactions with APC. Here, we present results with the following strains: 1) wt : non-attenuated wild-type; 2) phoP : missing major virulence regulator gene phoP; 3) phoQ : mutation in phoQ leading to misregulation of virulence genes; 4) aroA : incapable of producing aromatic compounds and to survive in endosomes. Human DC were differentiated from peripheral blood monocytes. Results : Infection of human DC was efficient for all strains, with reproducibly highest rates, over 50%, being obtained with phoQ. This observation was specific for DC as invasion of epithelial cells by phoQ was inefficient. Intracellular survival of phoQ was shorter than for the other strains. Killing of the host cell was attenuated in all modified strains. Maturation of DC was clearly promoted only by phoQ. Inflammatory cytokine production by DC was strongly stimulated by all strains. PhoQ induced a Th1 cytokine pattern with markedly higher IL-12 and lower Il-10 production than the other strains. Conclusions : Genetic engineering of Stm not only controls the infectious properties of Stm, but also activates DC and favors the release of cytokines leading to a Th1 response. The properties of the phoQ strain, which are: (i) specifically high infectiousity for DC, (ii) short intracellular survival, (iii) promotion of DC maturation, and (iv) induction of a Th1 cytokine pattern, appear particularly useful for a TB vaccine vector.

P13// A PHOTOSENSITISING ADENOVIRUS FOR PHOTODYNAMIC THERAPY.
NFP37 Nr: 4037-055140;
J Gagnebin, M Brunori, M Otter, L Juillerat-Jeanneret, P Monnier and R Iggo. ISREC, CH-1066 Epalinges, Switzerland.
We have developed a new approach to photodynamic therapy based on adenoviral transduction of the rate-limiting enzyme in heme synthesis. Conventional phototherapy uses porphyrin-based chemical photosensitisers, including 5-aminolaevulinic acid (ALA) which is converted to protoporphyrin IX (PpIX) by the enzymes of the heme biosynthetic pathway. The lack of a specific mechanism for targeting chemical photosensitisers and PpIX to tumour cells means that therapeutic irradiation can damage normal tissue and exposure to sunlight following treatment can cause severe burns. The rate limiting enzyme in PpIX synthesis is ALA-synthase (ALA-S). We have developed a new yeast vector system for manipulation of the adenovirus genome and used it to construct a virus expressing a mutant form of ALA-S lacking the iron response elements which regulate ALA-S translation and the heme regulatory motifs which regulate import of ALA-S into mitochondria. The virus induces a large increase in PpIX expression and confers photosensitivity on cultured cells. Unlike conventional photodynamic therapy, a viral approach makes it possible to restrict photosensitivity by biological rather than purely physical or chemical means. Like HSV thymidine kinase, ALA-S expression is a general mechanism for sensitisation to a therapeutic agent which can easily be adapted to whatever means of gene delivery is most effective. LJJ, PM: University Hospital (CHUV), 1011 Lausanne, Switzerland

P14// TRUNCATED FAK PROTEINS TARGETED TO FOCAL ADHESIONS INHIBIT CELL MOTILITY AND INDUCE APOPTOSIS/CELL DEATH IN GLIOBLASTOMA CELLS
NFP37 Nr: 4037- 055167;
Graham Jones and Adrian Merlo. Molecular Neuro-Oncology, Schanzenstrasse 46, Kantonsspital Basel, 4031, Basel.
Anaplastic gliomas are highly invasive tumors which are resistant to chemotherapeutic intervention. Cell motility and survival requires an interaction between the tumor cell and the extracellular matrix (ECM), which is mediated by integrin receptors at the cell membrane. The extracellular matrix associated with invading glioma cells appears to differ from that of normal brain tissue and is correlated with the expression of specific integrin receptors. Upon activation of integrin receptors by their ligands, focal adhesions are formed at the leading edge of the migrating cell which requires phosphorylation of the focal adhesion kinase (FAK). Naturally occurring inhibitors of FAK, the FAK-related non-kinase proteins (FRNK), can arise either as distinct isoforms of FAK or from proteolytic cleavage following activation of apoptosis. FRNK is targeted to the focal adhesions via a focal adhesion targeting (FAT) domain at the C-terminus. We first tested whether the inhibition of FAK activity by FRNK inhibits the invasive phenotype of glioblastoma cells. Expression of recombinant FRNK in human glioblastoma cell lines resulted in a significant reduction of motility which was accompanied by a reduction in the phosphorylation of endogenous FAK and a loss of phosphotyrosine from focal adhesions. Further experiments showed the FAT domain within FRNK was sufficient for this activity. In parallel assays for cell survival, both vitronectin and fibronectin protected glioblastoma cells from apoptosis. The protective effect of these ECM components was abolished by expression of the FAT domain alone. These results suggest that disruption of signalling by FAK in glioblastoma cells can reduce both cell motility and cell survival and that vectors expressing truncated FAK proteins potentially represent a novel approach to target brain tumors by gene therapy (NFP 37 4037/055167/1).

P15// DISRUPTION OF INTEGRIN-DEPENDENT ENDOTHELIAL CELL ADHESION USING ADENOVIRAL CONSTRUCTS WITH DOMINANT-NEGATIVE EFFECT ON INTEGRIN FUNCTION
NFP37 Nr: 4037-055150;
Oguey D, Werffely-George P and Rüegg C. Laboratory of the Centre Pluridisciplinaire d'Oncologie, c/o ISREC, 1066 Epalinges
Tumor angiogenesis is an essential step in tumor progression and metastasis formation, and suppression of tumor angiogenesis results in the inhibition of tumor growth. Recent work has implicated integrin-dependent endothelial cell adhesion in the induction and maintenance of tumor angiogenesis. MAbs or antagonistic peptides directed against aVb3, an integrin which is highly expressed on angiogenic but not on quiescent endothelial cells, cause selective suppression of angiogenesis and induce tumor regression. In this study, we explored the possibility to use a genetic approach to inhibit integrin-mediated endothelial cell adhesion in tumors. To this purpose, we constructed recombinant adenoviruses (Ad) expressing the cytoplasmic and transmembrane domains of integrin b1 (CH1) or b3 (CH3) connected to the extracellular domain of the murine CD4 molecule (used as a reporter of expression). The genes were placed under the control of the ubiquitous CMV promoter (AdCMV) or the endothelial cell specific Tie-1 promoter (AdTie). An Ad expressing the CD4 molecule without the integrin cytoplasmic domain was used as a control (CH2). All constructs were expressed in a dose and time dependent manner in human umbilical cord vascular endothelial cells (HUVEC) with expression in over 90% of cells 24 h. post-infection with the AdCMVs and 72 h. with the AdTies. Confluent monolayers of HUVEC infected with CH1 and CH3 detached from the substrate in a time and dose dependent manner (paralleling the level of expression of the constructs) with over 95% of the AdCMV-infected HUVEC being detached after 48 hours. AdTie-infected cells detached 3 to 4 days post-infection. Before cell detachment, AdCMVCH1 provokes a disruption of focal adhesions and cytoskeletal reorganization. There was a rapid loss of viability of the detached cells by apoptotic cell death. When tested in adhesion assays AdCH1- or AdCH3-infected cells failed to attach to all matrix proteins tested, without, however, any specificity toward b1 or b3 integrin-mediated adhesion. The control construct CH2 had no effect on the adhesion of HUVEC. Current efforts are aimed to test these constructs in vivo and to improve the integrin specificity (b3 versus b1). These results demonstrate the possibility of using dominant negative constructs targeting integrin to disrupt endothelial cell monolayers and may represent a basis to the development of a gene therapeutic approach to disrupt angiogenic vessels in vivo.

P16// MELANOMA SPECIFIC IMMUNOTHERAPY WITH RECOMBINANT VACCINIA VIRUS; FROM LAB VECTOR TO BEDSIDE VACCINE.
NFP37 Nr: 4037-055151;
P.Zajac, W.R.Marti, G.Spagnoli, C.Noppen, E.Padovan, M.Zuber, U.Luescher, T.Kocher, F.Harder, M.Heberer and D.Oertli. Kantonsspital Basel, Research unit of Surgery dept, Hebelstrasse 20, CH-4031 Basel
Active specific tumor immunotherapy requires the generation of highly immunogenic reagents able to induce vigorous immune response against cancer cells. In this context, we designed and constructed a recombinant vaccinia virus encoding HLA-A2 restricted epitopes from human tumor associated antigen. At first, we demonstrated in-vitro that these viral constructs, even after replication inactivation, efficiently generate specific CD8+ CTL from healthy donors' PBMC (Int.J.Cancer, 1997). We then showed that the immunogenicity of this reagent could be further enhanced by co-expressing two costimulatory molecules B7.1 and B7.2 (Cancer Res.,1998). Based on this recombinant virus together with synthetic peptides boost strategy, we have then designed a phase I/II clinical protocol for human therapy. With the approval of the local and national Swiss health authorities and the financial support of the "National Fund Project 37", we addressed the necessary logistic required in order to transform a laboratory research vector into a suitable clinical reagent for human experimentation. Among the many steps this "product development" required were; the selection of a production site with cGMP standards, the design and validation of a production protocol, the design, validation and implementation of sterility, safety, identity and potency tests 3 years, after the initial cloning steps, we are now ready to enter the clinical part of this project.

P17// RECOMBINANT ADENO-ASSOCIATED VIRUS (rAAV) VECTORS CONFER LONG-TERM GENE EXPRESSION IN MURINE CARDIOMYOCYTES IN VIVO
NFP37 Nr: 4037-055162;
Vassalli G (1,2), Bueler H (3), Sadeghi C (1), von Segesser L (2), Kappenberger L (2), Aebischer P (1). (1) Centre de therapie genique; (2) Cardiologie et chirurge cardiaque CHUV 1011 Lausanne; (3) Institut fuer Molekularbiologie Univ. ZH Irchel, 8957 Zuerich.
OBJECTIVE: Gene delivery to the heart and arteries has been carried out with plasmid DNA or recombinant adenovirus (rAd) vectors. However, the low efficiency of uptake of naked DNA and the short duration of transgene expression with rAd vectors have limited these approaches. The aim of the present study was to compare the efficiency of rAAV and rAd-mediated in vivo gene transfer into both murine cardiomyocytes and arterial endothelial cells (ECs), and to determine the duration of transgene expression. METHODS: A rAAV vector containing a green fluorescent protein (EGFP) marker gene under the transcriptional control of a cytomegalovirus promoter was produced by a helper-dependent technique. We injected 2.5 X 10E6 TU of this vector into the left ventricular (LV) wall of CD-1 mice (n = 12). Additional mice were injected with either 5 X 10E7 PFU of a rAd vector containing a CMV-EGFP expression cassette (n = 10), an AAV control vector (n = 3) or dilution buffer (n = 3). Hearts were harvested at different time points (3 days ; 1, 2, 8, 26, and 52 weeks) after gene transfer. EGFP expressing cardiomyocytes were counted on heart sections by fluorescence microscopy. Arterial gene transfer was carried out by infusion of either rAAV or rAd vectors into mouse carotids (n = 3 each). Arteries were explanted 3 and 14 days after rAd and rAAV infusion, respectively, and EGFP expressing ECs were counted on vessel sections. RESULTS: In the rAAV group, EGFP expression in cardiomyocytes was first detected 2 weeks, peaked at 2 to 8 weeks (2.5%; range: 1-4% of LV myocytes), and persisted for more than one year after gene transfer. In contrast, EGFP expression peaked at 3 days (4%; 3-14%) and was detectable only for 2 weeks in the rAd group. Transduction rates of ECs were higher with rAd (13%; 8-30%) than with rAAV (2%; 0-3%). CONCLUSIONS: rAAV vectors efficiently and stably transduce cardiomyocytes in vivo. These vectors may be very useful to deliver therapeutic genes in patients with chronic heart disease.

P18a// TRANSLOCATION OF RIBONUCLEIC ACIDS ACROSS MEMBRANES - THE CASE OF MITOCHONDRIAL tRNA IMPORT
NFP37 Nr: 4037-055154;
Tan T., Schneider A. . Institute of Zoology, Pérolles, CH-1700 Fribourg
Transport of nucleic acids across biological membranes is of great interest for many gene therapeutic approaches. 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. In nature one finds some natural occuring processes where nucleic acis are transported across biological membranes. A particularly interesting example is import of tRNAs into mitochondria, since the mitochondrial inner membrane represents a very tight barrier which is semipermeable even for protons. Trypanosoma brucei represents an excellent model system to study mitochondrial tRNA import since all organellar tRNAs need to be imported from the cytosol. The focus of our research is to elucidate the mechanism of this process, in order to understand how hydrophilic RNAs can cross the lipid bilayers of mitochondria. In the long term, this knowledege may help to devise novel protocols to transport nucleic acids across biological membranes. Furthermore, many mitochondrial cytopathies exist which are not amenable to classic protocols of gene therapy, since they are caused by mutations in mitochondrially encoded tRNAs. Nuclear transfection of the affected cells with the corresponding wild type tRNA genes will therefore only be of benefit if the tRNA can be imported into mitochondria. A detailed knowledege of a natural occuring mitochondrial RNA import systems is clearly a prerequisite to achieve this.

P18b// SELECTION OF RNA MOLECULES FOR RIBOFECTION
NFP37 Nr: 4037-055154;
Anne Genilloud, Sandro Rusconi. Institute of Biochemistry, UNIFR, Pérolles 1700 Fribourg, Switzerland
We have designed a procedure to generate in vitro a library of randomized RNA molecules with a complexity of about 10x(exp+14). The RNA oligo nucleotides are engineered to allow reverse trans cription and PCR amplification. The in vitro prod uced RNA are selected for their capacity of pene trating mammalian cells. Selected RNAs are reverse transcribed and used to generate templates for a sub sequent in vitro transcription. We have established conditions in which random molecules are reduced by four logarithms at each selection cycle. We estimate that few selection-amplification cycles shall yield first generation RNA oligos with impro ved autonomous translocation capacity across mam malian cell membranes. Further refinements of these 'ribofectors' are discussed.

P19// EFFICIENT LENTIVIRAL GENE TRANSFER OF GREEN FLUORESCENT PROTEIN TO HEMATOPOIETIC PROGENITOR CELLS FROM CORD BLOOD OF PRETERM HUMAN FETUSES.
NFP37 Nr: 4037-055157;
Luther-Wyrsch A, Costello E(1) , Thali M(1), Buetti E(1), Surbek D(2), Holzgreve W(2), Nissen C and Wodnar-Filipowicz A . Research Department, University Hospital Basel; Switzerland
Preterm cord blood (CB) hematopoietic stem cells (HSC) are considered as targets for autologous somatic gene therapy of inborn defects before birth of an affected child. Recently, HIV-derived lentiviral vectors have been shown to efficiently transduce repopulating human HSC. We analysed the content and the growth properties of HSC in preterm CB from 2nd and early 3rd trimester of pregnancy and used them as targets for lentiviral transfer of the enhanced green fluorescent protein (GFP) gene. The frequency of CD34+ and CD34+38- cells was about 4-fold higher in preterm than in term CB. Progenitors giving rise to colonies in CFU and LTC-IC assays were enriched about 2.5-fold, and could be expanded in 7-day stroma-free liquid cultures supplemented with growth factors as efficiently as progenitors from term CB. Various gene transfer protocols were tested by flow cytometry and CFU/LTC-IC assays for GFP+ cells and colonies. Integration of the GFP gene in GFP+ colonies was confirmed by PCR. In liquid cultures, 19.8 to 55.0% of CD34+ cells were expressing GFP at two weeks after transfection, and this expression persisted for at least five weeks. Best results were obtained by exposing cells to viral supernatant, which was supplemented with protamine sulfate, in 3h spinoculation (3000rpm) on 2 subsequent days. Prestimulation with IL-3, IL-6, SCF and flt-3 ligand for 3 days prior to transfection increased the efficiency of GFP gene transfer to committed (22.8 ± 3.0% vs. 7.4 ± 1.4% GFP+), but not to primitive progenitors scored in LTC-IC assays (11.3 ± 3.4% vs. 19.0 ± 4.9% GFP+). The frequency of transduced CD34+ cells and colony-forming cells from preterm and term CB was comparable. These results demonstrate the feasibility of efficient and persistent lentiviral gene transfer into immature HSC from term and preterm CB. This may help to develop gene therapy strategies for in utero treatment of disorders amenable to stem cell transplantation. (1) Institute for Microbiology, University Hospital Lausanne; Switzerland (2) Obstetrics Department, University Hospital Basel; Switzerland

P20a// POLY(DL-LACTIDE-CO-GLYCOLIDE)-ENCAPSULATED DNA: STABILITY AND RELEASE CHARACTERISTICS
NFP37 Nr: 4037-055144;
Elke Walter1, Karin Moelling2, Jovan Pavlovic2, and Hans P. Merkle1. (1) Department of Applied BioSciences, ETH Zürich, Winterthurerstrasse 190, CH-8057 Zürich. (2) Institute of Medical Virology, Gloriastrasse 30, 8028 Zürich, Switzerland
Direct inoculation of plasmid DNA into muscle has previously been explored as a realistic option for novel, efficacious vaccines [1]. However, the activation of T-cell responses requires presentation of antigen by professional antigen presenting cells (APCs). As several types of APCs phagocytose particulate material in the micron to sub-micron-range, microencapsualtion of plasmid DNA could be an interesting approach for the specific targeting of APCs. Much attention has focused on biodegradable polymers such as poly(DL-lactide-co-glycolide) (PLGA) for the encapsulation of proteins and peptides [2]. Although the preparation methods for PLGA microspheres have been well established [3], encapsulation of highly hydrophilic therapeutic agents with large molecular masses such as plasmid DNA have not been investigated in detail so far. The processes commonly used to produce PLGA microspheres include phase separation, solvent evaporation and spray-drying [3]. Concerns regarding DNA stability are due to exposure to organic solvents and higher shear forces during encapsulation. The extremely hydrophilic character of the DNA could lead to low entrapment levels. In this study, we explored microencapsulation of DNA by spray-drying. We have chosen three different types of DNA including circular plasmid DNA (5 kbp), DNA from salmon testes (~20 kbp) and denatured DNA from calf thymus (~0.65 kbp). Different analytical methods were tested for their suitability to detect intact DNA. Double-stranded DNA is extremely stable under physiological conditions in vitro but is rapidly degraded under acidic conditions and high shear forces. The presence of ions during sonication provides protection against mechanical degradation of DNA. Microencapsulation of DNA with PLGA results in encapsulation efficiencies of 40-70% depending on the formulation parameters. DNA is found inside the core of the microparticles, and extracted DNA has demonstrated to be intact to a major extent. Stability of DNA is maintained during the burst release phase, but massive degradation occured during the second release phase possibly due to acidic catalyzed decomposition. The results stress the need for efficient DNA stabilization inside PLGA. REFERENCES: (1.) Shiver JW, Adv. Drug Del. Rev. 21 (1996) 19. (2) Putney SD, Nature Biotech. 16 (1998) 153. (3) Thomasin C, J. Control. Rel. 41 (1996) 131

P20b// EVALUATION OF SURFACE-DEPENDENT PARTICLE UPTAKE BY MONOCYTE-DERIVED DENDRITIC CELLS IN VITRO
NFP37 Nr: 4037-055144;
L. Thiele, B. Rothen-Rutishauser, H. Wunderli-Allenspach, H.P. Merkle and E. Walter. Dept. of Applied BioSciences, ETH Zürich, Irchel Campus, CH-8057 Zürich
Dendritic cells (DC) play a central role in controlling immunity [1]. They belong to the group of antigen presenting cells (APC). Their extraordinary ability to present antigen (Ag) for primary specific T-lymphocyte responses in vitro and in vivo distinguishes them from other APC and makes them a logical target for vaccines [2], including DNA vaccines. Furthermore, DC are able to elicit strong cytotoxic T cell (CTL) response by Ag presentation via MHC class I which is important to fight viral infections. Microspheres were reported to be a potent Ag delivery system [3]. When loaded with specific epitopes microspheres elicit CTL response in mice [4]. In contrast to other APC, DC are less frequent and have reduced phagocytotic activity. Therefore, efficient targeting of DC requires a suitable delivery system to enhance Ag uptake. Here we present a method to assess the targeting of coated microparticles to monocyte-derived DC in vitro. A) Fluorescence activity and quenching of fluorescent particles. Polystyrene (PS) particles differing in size and location of the fluorescent dye are compared with respect to their fluorescence intensity. PS and bioparticles (lyophilized fluorescent staphylococcus aureus) are labeled with FITC exclusively at the surface, so-called yellow green (YG) particles are stained throughout the particle. To assess the phagocytotic ingestion of particles, we quenched extracellular fluorescence by adding trypan blue (TB) at pH 4.4. B) Phagocytosis of bioparticles by DC. Fluorescence activity of bioparticles is completely quenchable upon direct contact with TB. Bioparticles internalized by viable cells are not in contact with TB and therefore remain fluorescent. They are taken up at high numbers and are thus used as positive control for phagocytosis and cell viability. C) Confocal laser scanning microscopy (CLSM) studies. To control the reliability of our phagocytosis assay we additionally studied the uptake of differently coated YG particles by CLSM. External particles can be clearly distinguished from internalized particles. D) Influence of surface properties on particle uptake. Depending on (i) the protein attached to the surface of PS particles and (ii) on the incubation media, most particles were internalized though with different efficiency. Throughout the uptake studies the amount of particles seen in the quenching assays correlated well with CLSM studies. Conclusion: For unequivocal assessment of particle ingestion by viable cells, surface-labeled particles have to be utilized when examined by fluorescence microscopy in combination with TB. The fluorescent label has to be positioned at the particle surface to be able to interact with TB; incorporated fluorescence cannot be quenched. Our method provides a semi-quantitative approach to measure particle uptake by DC. Uptake of particles by DC is clearly dependent on size and surface properties. In further experiments we will use this technique to design optimized systems for DNA delivery into DC. REFERENCES: (1) Banchereau J., Nature 392 (1998) 245. (2) Hart D., Blood 90 (1997) 3245. (3) Thomasin C. et al., J. Control.Rel. 41 (1996) 131. (4) Men Y. et al., Vaccine 15 (1997) 1405

P21// TOWARDS GENE THERAPY FOR MHC CLASS II DEFICIENCY J.
NFP37 Nr: 4037-055159;
Villard, M Peretti and W. Reith. Department of Genetics and Microbiology, University of Geneva Medical School, Centre Medical Universitaire (CMU), 1 rue Michel-Servet, CH-1211 Geneva 4
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. We have now isolated the genes encoding all four of these transacting factors. In particular, we have very recently identified the one (RFXANK) that is mutated most frequently in the disease. This has completed our genetic and molecular definition of MHC-II deficiency, and has provided us with the tools that are required for developing gene therapy for this disease. An investment in the development of gene therapy for MHC-II deficiency is justified both by the number of patients that could benefit from it and by the lack of an efficient therapeutic alternative to bone marrow transplantation, which has a poor success rate in this disease. Moreover, many of the features of MHC-II deficiency render it an excellent model system of more general interest for the field of somatic gene therapy. We are therefore pursuing a project that will contribute to the development of gene therapy for MHC-II deficiency. Bicistronic lentiviral based constructs are being used as delivery vectors for the genes encoding the four regulatory factors that are defective in MHC-II deficiency. These vectors are being used for three different but related aspects of the project. First they are being used to set up a simple, rapid and reliable system for determining which of the four MHC-II regulatory genes is defective in newly identified MHC-II deficiency patients. Second, they will be used to set up and optimize gene therapy protocols in two mouse models (RFX5-/- and CIITA-/- mice) for MHC-II deficiency. Finally, the same vectors will be used for pre-clinical studies involving the correction of cells, including hematopoietic stem cells, isolated from MHC-II deficiency patients. Our preliminary results show that: 1. The lentiviral vectors are able to correct B cell lines derived from MHC class II deficiency patients belonging to all four complementation groups. 2. The bicistronic approach is very efficient for the purification of transduced. 3. The lentiviral vectors are able to transduce c-Kit+ mouse bone marrow stem cells.

P22// A STRATEGY FOR GENE THERAPY OF PHENYLKENTONURIA
NFP37 Nr: guest;
Laufs S, Blau N, Thony B. Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zürich, Steinwiesstrasse 75, CH-8032 Zürich
Classical phenylketonuria (PKU) is an autosomal recessive disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). Dietary phenylalanine (phe) restriction, if initiated in the neonatal period, reduces plasma phe levels and can largely prevent the neurological impairment characteristic of this disease. Despite these successes, there are still some limitations associated with dietary therapy, including a decline in mental or behavioral performance in adulthood. Furthermore, pregnant women with PKU must restrict their phe intake to prevent damage of their offspring ('maternal PKU'). An alternative to dietary therapy for PKU is somatic gene therapy, whereby a functional PAH gene is introduced into the liver or other tissues. Recently, Harding et al. (Gene Therapy, 1998; 5:677-683) showed that circulating toxins such as high levels of plasma phe may be cleared by expression of PAH in skeletal and cardiac muscle. In their experiment, they used a PKU mouse model and generated transgenic progeny that expressed PAH constitutively under the control of a mouse muscle creatine kinase promoter. Unfortunately, these mice were still hyperphenylalaninemic under normal dietary conditions, but plasma phe levels decreased significantly when the mice were supplemented orally with tetrahydrobiopterin (BH4), the required cofactor for PAH. In this work, our aim is to generate a gene transfer vector for the expression of PAH together with cofactor-biosynthetic enzymes in muscle tissue. Sufficient production of BH4 should then allow PAH to clear toxic serum phe levels. The biosynthesis of BH4 requires three enzymes, GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase (SR). Since we have previously shown in our laboratory that coordinate expression of three enzymes from a multi-IRES containing retroviral vector allowed the reconstitution of BH4 biosynthesis in primary human cells (Laufs et al., in preparation), we wanted to apply a similar strategy for PKU gene therapy. We thus determined in a mouse myoblast cell line (C2C12) that GTPCH is completely absent, PTPS may be limiting, and SR is sufficiently present for BH4 biosynthesis. Next, we generated stable expressing GTPCH, or GTPCH and PTPS myoblasts, and shall transfect PAH and assay its activity. Depending on the BH4-enzymes required, coordinate expression of PAH under the control of a muscle specific promoter on a multi-IRES containing vector will then be applied.

P23// FAITHFUL REGULATION OF BTK GENE, FOR XLA THERAPY.
NFP37 Nr: 4037-044812;
Maria Chiara Bassi & Jovan Mirkovitch. Swiss Institute for Experimental Cancer Research Ch. Des Boveresses 155, Lausanne CH.
A prerequisite for effective gene therapy is to achieve adequate tissue-specific and quantitative expression of the transferred gene. In most cases, this is easiest to accomplish at the level of transcription initiation. Our goal was to construct an expression cassete for the precise tissue-specific expression of Btk gene for XLA gene therapy. Mutations in the Btk gene result in the XLA immunodeficiency. As the gene codes for a tyrosine kinase that seems essential for B cell differentiation, its expression should be stringently controled. The major result of this proposal was to provide the transcription regulatory tools that would drive high levels of tissue-specific transcrition of the BTK gene. Regular approaches or promoter analyses have not succeded into obtaining a strong tissue-specific expression from Btk control elements.We therefore started a chromatin analysis to identify regions implicated in Btk transcriptional regulation by DNaseI hypersensitivity which should map remote enhancers and putative control regions. Two such site specific for B cell were been identified. To test in detail the function of these regions, we are employing EBV episomes.Our laboratory has recently demonstrated that these episomes provide a milieu in which transcription can be tightly regulated, and which faithfully reproduces the DNA-protein structures of the original gene. WE have identify a new transcription regulatory element for the Btk gene downstream of the transcription start site. The inclusion of this elements along with the promoter should provide the transcription regulatory sequnece which could direct correct tissue specific expression of the Btk human tissues. This may then allow the precise correction of the Btk function in the context of gene therapy trials.

P24// ANOXIA REDUCES EXPRESSION OF ANGIOGENESIS INHIBITOR THROMBOSPONDIN-1 IN GLIOBLASTOMA CELLS
NFP37 Nr: 4037-044729;
Tenan M (1), Fulci G (1), Albertoni M (1), El Atifi-Borel M.(2), Feige J.J.(3), Pepper M.S.(4), Desbaillets I.(1) and Van Meir E.G.(1,5). University Hospital, 1011 Lausanne; Switzerland and Laboratory of Molecular Neuro-Oncology, Emory University, Atlanta.
Glioblastoma is the most malignant and vascularized stage of astrocytoma progression. Both the physiological and genetic changes occurring during astrocytoma progression induce angiogenesis by disrupting the balance between stimulatory and inhibitory factors. In pseudopalisading cells lining necrotic areas, oxygen deprivation upregulates the mRNA levels of angiogenic stimulators vascular endothelial growth factor (VEGF) (Plate, 1992) and interleukin-8 (IL-8) (Desbaillets, 1997). Loss of p53 function in astrocytoma can affect stimulators and inhibitors of angiogenesis. Wild-type p53 reduces basic fibroblast growth factor (bFGF) gene expression, while mutant p53 activates it in vitro (Ueba, 1994). WTp53 may also negatively regulate VEGF expression in glioma cells (Mukhopadhyay, 1995). In fibroblasts p53 upregulates the expression of thrombospondin-1 (TSP-1), a natural inhibitor of angiogenesis (Dameron, 1994). In glioblastoma cells, WTp53 can stimulate the release of an as yet unidentified angiogenesis inhibitor called GD-AIF (Van Meir, 1994), and it is unknown whether GD-AIF is related to TSP-1. In glioma, TSP-1 is also under the control of an unknown tumor suppressor gene on chromosome 10 (Hsu, 1996). Here, we evaluated the regulation of TSP-1 in glioblastoma cells. We found that anoxia reduces expression of TSP-1 in glioblastoma cells independently of p53. This suggests that reduced oxygen tension as occurs in tumors can stimulate angiogenesis not only by promoting the synthesis of inducers, such as VEGF or IL-8, but also by reducing the production of inhibitors. This mechanism should prove to be relevant not only in tumor development but also in other physiological and pathological conditions linked to oxygen depletion. (1)Laboratory of Tumor Biology and Genetics, Neurosurgery Department, University Hospital (CHUV), 1011 Lausanne; Switzerland. (2)Laboratory of Biochemistry A, University Hospital (CHRG), F-38043 Grenoble, France. (3)INSERM Unit 244, Department of Molecular and Structural Biology, CEA/G F-38054, Grenoble, France. (4)Department of Morphology, University of Geneva Medical Center, 1211 Geneva 4; Switzerland. (5)Laboratory of Molecular Neurooncology, Neurosurgery Department and Winship Cancer Center, Emory University, Atlanta, GA 30322, USA.

P25// INHIBITION OF TUMOR ANGIOGENESIS
NFP37 Nr: 4037-044820;
Vitaliti A, Wyder L, Wittmer M, Ajmo M, Klemenz R. Division of Cancer Research, Dept. Pathology, University Hospital, CH-8091 Zürich.
Neovascularization of tumors is a prerequisite for their expansion. VEGF is a crucial mediator of tumor vascularization and its neutralization should prevent tumor growth. It has previously been shown by several investigators that the sequestration of VEGF with a monoclonal antibody can reduce tumor growth substantially. Since it is technically very demanding to produce sufficient amounts of monoclonal antibodies for cancer patient treatment we will instead evaluate gene therapeutic strategies. Monoclonal antibodies are heterodimeric proteins and as such not very suitable for gene therapy. Therefore, we have selected anti VEGF single chain antibodies (scFv) from a phage display library. Two of the isolated anti-VEGF scFvs were shown to inhibit angiogenesis in the chicken chorioallantois membrane model. Daily intraperitoneal injection of bacterially produced anti-VEGF scFv decreased the growth of subcutaneous tumors in nude mice twofold. Two antiangiogenic scFvs have been affinity matured and will now be used for gene therapeutic studies. Adenoviruses were generated which contain cDNAs encoding anti-VEGF scFv as well as derivatives thereof which are fused to different parts of a human IgG constant region were. We expect these fusion proteins to be more stable in the blood stream than unmodified scFv. I.v. injection of these recombinant viruses into tumor bearing animals will be performed in the near future.

P26// DEVISING AN APPROACH TO MODULATE THE GRAFT VERSUS HOST DISEASE IN PROTOCOLS OF ADOPTIVE IMMUNOTHERAPY.
NFP37 Nr: 4037-055105;
Herrera PL, Fuhrmann E, and Vassalli JD. Department of morphology, Universite de Genève, 1, rue michel-servet, 1211 Genève 4
Graft versus Host Diseases (GvHDs) are at this time the major limiting factor in both bone marrow transplantation (BMT) and adoptive immunotherapy protocols. The GvHD is due to the presence of donor T cells within the bone marrow graft. Nevertheless, the graft of T cell-depleted marrows to leukemic individuals has also severe drawbacks. It has thus become urgent to develop the therapeutic tools that should allow modulating these GvHDs. We have presently planned the construction of a suicide gene that could be helpful in controlling the T lymphocytes that elicit the graft-versus-host diseases. The project proposed in this application takes advantage of a version of the Cre / loxP system in which the activity of Cre recombinase is inducible: the hydroxy-tamoxifen (4-OHT)-dependent Cre-ERT fusion enzyme. We suggest herein that flanking the Cre-ERT gene by two loxP sites could be used to interrupt another gene of interest whose expression should occur only at a particular time point. Thus, the floxed Cre-ERT coding region would simultaneously be both the block of the transcription of a gene placed downstream and the inducible and irreversible activator of the transcription of this very same gene. More precisely, we propose the construction of a suicide transgene that should encode the A subunit of the diphtheria toxin, but only after administration of 4-OHT, the ligand (or "activator") of Cre-ERT. This transgene will be introduced ex vivo into donor T cells that will afterwards be used in BMT trials. When required, the patients will be given 4-OHT. If the expected results are obtained in vitro and in vivo (in mice), we will then start such clinical tests in collaboration with the Hôpital Universitaire de Genève.

P27// POXVIRUS AS A VECTOR TO TRANSDUCE HUMAN DENDRITIC CELLS: ABORTIVE INFECTION BUT REDUCED APC FUNCTIONS
NFP37 Nr: 4037-044813;
Huegin AW, Jenne L, Arrighi J-F, Hauser . Department of Dermatology and Department of Immunology and Allergology, University Hospital, 1211 Geneva, Switzerland
Dendritic cells (DC) are the most potent antigen presenting cells. Ongoing preclinical and clinical studies exploit this capacity for the immunotherapy of tumors. To induce an antitumoral immunity in patients, autologous DC are loaded with tumor cells, tumor derived cell extracts, c-DNA, m-RNA, proteins or peptides. Various methods, including vira vectors have been tested to transduce DC with genes of tumor "specific" antigens. We tested vaccinia virus (VV) as a vector to transduce human DC by analysing virological parameters and immunological phenotypic and functional parameters after VV infection. Blood-derived monocytes were cultured for 5 days in the presence of GM-CSF and IL-4 to obtain immature DC and in GM-CSF, IL-4, IL-1, IL-6, TNF-alpha and PGE2 to obtain mature DC. These cells were infected with 1) GFP expressing recombinant VV to analyse infection rates and virus replication in DC and the effect of infection on DC surface markers as analysed by two color FACS, and 2) recombinant VV expressing beta-galactosidase under the control of viral early, intermediate and late promoters to analyse the poxvirus driven gene expression. The following results were obtained: While the infection rate in DC was comparable to a permissive fibroblast cell line, viral beta-galactosidase gene expression was limited to early promoters. Genes under the control of virus late promoters were not expressed by VV in DC. This indicates an abortive infection due to a block in viral DNA replication. Although the VV infection was abortive in DC, the expression of DC maturation marker CD83 and of costimulators (CD80 and CD83) was reduced to various degrees by the infection. In line with this finding, there was a pronounced reduction in the capacity of VV infected DC to stimulate allogeneic cells in a mixed lymphocyte reaction. These results indicate that vectors may have complex effects on their target cells. In the case of DC used for immunotherapy, this may be detrimental to their function as a potent MHC class II antigen presenting cell and thus their capacity to induce T helper cells.

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