NFP37 SOMATIC GENE THERAPY ANNUAL MEETING October 6, 2000 «Yin-Yang between industry and academia in gene transfer research»

ABSTRACTS

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ABSTRACTS MAIN LECTURES

L01// USING PNA-DEPENDENT GENE CHEMISTRY TO IMPROVE GENE DELIVERY
Philip Felgner, Gene Therapy Systems, San Diego, CA., USA
Keywords: PNA, triple helix, transfection, non-viral gene delivery

L02// ADENOVECTORS AS VEHICLES FOR THERAPEUTIC GENES: NOVEL APPROACHES TO GENE THERAPY
Imre Kovesdi, Genvec Inc., Gaithersburg, MD, USA
Keywords: replication defective adenovirus, in vivo delivery, cell and tissue specific targeting, preclinical disease models, angiogenesis

L03// UTILITY OF THE GENOME INITIATIVE FOR PRE-SYMPTOMATIC GENE THERAPY
Tom Caskey, Cogentech, Houston, TX, USA
Keywords: coronary artery disease, colon cancer, breast cancer

ABSTRACTS 20 MIN TALKS

M01// FROM 'GENE MYTH? TO HEALTH INSURANCE' - GENE THERAPY BETWEEN ETHICS AND LAW
NFP37 Nr: 044714; Thomas Luchsinger, Blumenrain 2, 4051 Basel
Die Studie ist Teil der Bemühungen des nationalen Forschungsprogramms 37 "Somatische Gentherapie", nebst den naturwissenschaftlichen Fragen auch die gesellschaftlichen Auswirkungen der neuen Technologien untersuchen lassen. Die rechtliche Regelung der Gentherapie wird im politischen und ethischen Umfeld vorgestellt. Öffentliche Debatte, Utopien, aber auch Schreckensszenarien üben einen starken Einfluss auf die Rechtsetzung aus. Vorgestellt werden deshalb sowohl die aktuellen und absehbaren Einsatzmöglichkeiten der Gentechnik wie somatische Gentherapie oder Impfungen als auch die spekulativen Methoden wie Keimbahnbehandlungen und Klonen. Zu jeder Methode werden der Stand der Technik, die Einsatzmöglichkeiten und die ethischen Fragestellungen kurz umrissen, die Forderungen an Politik und Rechtsetzung untersucht und die rechtliche Reaktion besprochen, von der Forschungsfreiheit über die Vorschriften zur Biosicherheit bis hin zur Integration möglicher Behandlungen in die Sozialversicherungen. Die Kernfrage bleibt, ob es möglich ist, die in der Öffentlichkeit sichtbar gewordenen Ängste mit rechtlichen Mitteln zu beruhigen oder die technische Entwicklung zu steuern. Es zeigt sich, dass es kein rechtliches "Wundermittel" gibt. Kein Rechtsbegriff kann alle Fragen im voraus beantworten. Jede technische Neuentwicklung wird eine neue, unvoreingenommene Güterabwägung notwendig machen. Es ist aber möglich, fallweise Kompromisse zwischen den verschiedenen Interessen zu finden, doch werden diese Lösungen in verschiedenen Gesellschaften auch verschieden ausfallen. Besprochen werden deshalb auch die international und in ausgewählten Ländern getroffenen Regelungen, mit einem Überblick über die anwendbaren Vorschriften für Praktiker.

M02// SELECTIVELY ENHANCED TRANSFER OF STEROID-DECORATED TRANSGENES IN NUCLEAR RECEPTOR-POSITIVE CELLS
NFP37 Nr: 044802 and 049003; A. Bernasconi (1), A. Rebuffat (1), H. Wehrli (1), B. Frey (1), F.J. Frey (1), M. Ceppi (2), S. Brenz Verca (2), I. Merdol (3); S. Rusconi (2). (1) Nephrology Dept., Inselspital, CH-3010 Bern; (2) Biochemistry Institute, UniFr, Pérolles, CH-1700 Fribourg, Switzerland; (3) Histology Inst. UniFr, Pérolles, 1700 Fribourg
The idea behind the steroid-mediated gene delivery (SMGD) strategy is to facilitate the nuclear uptake of transfected DNA with the help of the shuttling action of nuclear receptors. We modelled the SMGD with the glucocorticoid receptor (GR). To this purpose, we synthesized and tested a large number of bifunctional steroid derivatives and ultimately selected the compound that we shortly denominate DR8NP for the experiments presented in this work. This compound links via a a relatively soluble chemical spacer (8 atoms) a high affinity GR ligand (dexamethasone) with a crosslinkable DNA intercalator (psoralen). We show that GR efficiently translocates into the nucleus upon exposure to 100 nanomolar amounts of DR8NP. We describe how DR8NP can be crosslinked to plasmid DNA and how the decorated plasmids can be easily purified from the excess unreacted ligands. Finally we demonstrate the following crucial properties of steroid decorated reporter plasmids: (a) the expression of DR8NP-decorated reporters is reproducibly enhanced in presence of GR; (b) this effect is seen only in cells expressing the GR; (c) the enhancement is independent from the transactivation potential of GR; (d) the effect correlates with a measurable preferential nuclear accumulation of decorated plasmid DNA. The SMGD approach can be extended to other ligands that interact with nuclearly-shuttling intracellular receptors and offers thereby an additional selective advantage to the gene transfer-based treatment of somatic tissues that express specific steroid receptors. Supported by the Cantons of Berne and Fribourg and by grants from Swiss Natl Res. Foundation. nr: 4037-044802 and 4037-049003, NFP37 program.

M03a// MONITORING BONE METASTASIS DEVELOPMENT IN LIVING MICE BY BIOLUMINESCENT REPORTER IMAGING.
NFP37 Nr: 044804; Wetterwald A (1), Karperien M (3), Gautschi E (1), Stadler BM (2), Thalmann GN (1), van der Pluijm G (3), Lowik CWGM (3) and Cecchini MG (1). (1) Gene Therapy Laboratory, Department of Clinical Research and Urology Clinic; (2) Institute of Immunology and Allergology, University Hospital, CH 3010 Bern, Switzerland and (3) Department of Endocrinology, Leiden University Medical Centre, Leiden, The Netherlands.
The development of novel strategies for cancer treatment, such as gene therapy, requires the elaboration of non-invasive in vivo assays to monitor their effectiveness. Breast and prostate carcinomas frequently metastasise to bone marrow and osteolytic lesions can be detected by X-ray. However micro-metastasis are not readily detectable and methods that are more sensitive are needed to detect minimal disease states. We describe here the use of a CCD camera connected to the Argus-20 image processor (C-2400-47/VIM, Hamamatsu) to monitor in vivo the formation of bone metastasis by tumour cells transfected with the luciferase reporter gene.
The human mammary carcinoma cells MDA-MB-231 were stably transfected with the pCMV plasmid containing the firefly luciferase gene (MDA-231/Luc+). BALB/c nu/nu mice were injected into the left cardiac ventricle with MDA-231/Luc+ cells and monitored at weekly intervals for the development of bone metastasis. Anaesthetised mice were injected intra-peritoneally with D-Luciferin and photon emission was integrated over 5 minutes.
Distinct photon emission localised in the hind legs and spine was first detected 24 days after the intra-cardiac injection. At the same time point, no osteolytic lesions were detectable by radiography. Instead, first metastatic bone lesions were seen on radiographs not earlier than 35 days after injection of tumour cells. In order to determine the minimal number of cells detectable by photon imaging, different amounts of cells were injected directly into the femoral marrow cavity and light emission was immediately recorded. The lowest detection limit was 10'000 cells. For comparison, as little as 1000 cells were detected after subcutaneous detection.
Advantages of bioluminescent reporter imaging are early detection of minimal bone metastatic lesions, possibility to follow the kinetics of tumor growth in the same animal, and quantification of the tumor burden for each metastatic site. This method will be extremely useful to follow development of metastasis in living animals, to monitor expression of targeted gene vectors, and to verify the efficacy of novel therapeutic approaches based on gene delivery.

M03b// GENE EXPRESSION PROFILING OF TUMOUR-DERIVED ENDOTHELIAL CELLS
NFP37 Nr: 044804; Schmid MC (1), Bisoffi M (1), Wetterwald A (1), Gautschi E (1), Thalmann GN (1), Stadler BM (2), Mitola S (3), Bussolino F (3) and Cecchini MG (1): (1) Gene Therapy Laboratory, Department of Clinical Research and Urology Clinic;(2) Institute of Immunology and Allergology, University of Bern, Switzerland and (3) Institute for Cancer Research and Treatment, 10060 Candiolo, Italy.
Angiogenesis is a key process in a variety of human diseases, including cancer. The ability to target selectively the tumor neo-vasculature is potentially useful for the diagnosis and treatment of cancer. Still, little information is available regarding markers that are restricted to the endothelial cells (EC) of tumor neo-vessels. The cDNA array technology allows simultaneous analysis of relative expression levels of a broad spectrum of genes in two related cell populations. We have used this technology with the aim to identify markers specific for the tumor EC (TEC).
TEC were isolated by immuno-magnetic separation from tumours induced by subcutaneous injection of NF9006 breast carcinoma cells into syngeneic mice. Control EC (CEC) were isolated from lactating mammary glands. The endothelial nature of isolated cells was confirmed by reverse-transcriptase polymerase chain reaction (RT-PCR) amplification of the CD31 transcript and by uptake of Dil-labelled acetylated low-density lipoprotein. 32P-labelled cDNA probes generated by reverse transcription from total RNA were hybridised to mouse-specific gene array membranes. Eight genes consistently showed differential expression between TEC and CEC. However, for only two of these genes expression was restricted exclusively to EC and not to non-EC contaminating cells. Especially, the insulin-like growth factor binding protein 3 (IGFBP-3) was highly over-expressed in TEC. Quantitative RT-PCR (TaqMan) has substantiated a 28 to 32-fold differential expression of IGFBP-3. These results above relate for the first time IGFBP-3 over-expression to TEC, at least in this tumor model in mouse. The elucidation of its role in angiogenesis may open the possibility that IGFBP-3 may represent a therapeutic target.

M04// HUMAN PHASE I/II MELANOMA TRIAL BASED ON UV-INACTIVATED RECOMBINANT VACCINIA VIRUS
NFP37 Nr: 055151; Zajac P, Marti WR, Adamina M, Kocher T, Noppen C, Padovan E, Spagnoli GC, Heberer M, Oertli D. Research unit of Surgery Dept, University Hospital of Basle, Switzerland
Based on a combined immunization protocol using psoralen-UV inactivated recombinant vaccinia virus and synthetic peptides injected s.c. together with a GM-CSF treatment, a phase I/II human clinical was initiated with stage III/IV metastatic melanoma patients. Taking advantage of the recent development in tumor immunology research and applied field, our recombinant vaccinia virus is co-expressing 3 endoplasmic reticulum targeted HLA-A2 melanoma associated antigen epitopes (Melan-A/Mart-1 27-35, GP100 280-288 and Tyrosinase 1-9) plus both human B7 (CD80 and CD86) molecules.
Since November 1999 a total of 10 patients have been enrolled into the trial. Clinically, no major adverse effect occured. Concerning the specific anti-tumor response, the preliminary results obtained so far using limiting dilution indicates an expansion of specific CTL toward all 3 epitopes, although to different degrees, mostly detectable after each viral injection. A CTL response against MelanA/Mart-1, gp100, and tyrosinase was observed in 6/9 (67 %), 5/7 (71 %), and in 5/7 (71 %) evaluable patients, respectively. Taken together, these indications underline the immunogenic quality and the complete safety of a UV-inactivated vaccinia based heterologous protocol.

M05a// INTRAVASCULAR DELIVERY OF A RECOMBINANT ADENOVIRUS EXPRESSING ISOLATED B INTEGRIN CYTOPLASMIC DOMAINS THAT INHIBIT INTEGRIN FUNCTION RESULTS IN VASCULAR ENDOTHELIAL CELL DAMAGE
NFP37 Nr: 055150; Vassalli G (1,2), Oguey D (3,5), Bamat J (4), Paroz C (3) and Rüegg C (3). (1) Centre de therapie genique, (2) Cardiologie, (3) et Centre Pluridisciplinaire d'Oncologie, School of Medicine, University of Lausanne, CH-1011 Lausanne, Switzerland, (4) Swiss Institute for Experimental Research, CH-1066 Epalinges (5) Present address: F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland
We have recently reported that adenovirus (Ad) -mediated expression of chimeric proteins consisting of the cytoplasmic and transmembrane domains of the integrin beta1 or beta3 subunits (CH1 or CH3), connected to the extracellular domain of the L3T4 molecule, results in the disruption of integrin-mediated endothelial cell adhesion trough a dominant negative effect (Gene Ther. 2000, 7, 1292-1303). To collect initial evidence on the effectiveness of this approach in disrupting vascular endothelial cell adhesion in vivo, AdCMVCH1 and the control vector AdCMVCH2 (expressing L3T4 without the integrin cytoplasmic domain) were instilled into the lumen of rat common carotid arteries using established catheter technique. After a 20 min-dwelling time of the vectors in the arterial lumen, blood flow was reestablished through the internal carotid artery. Transduced vessels were explanted at 8, 24, 48 hours and 6 days post-infection and frozen-sectioned over the entire length. Chimeric protein expression was analyzed by double immunofluorescence staining using antibodies directed against L3T4 and anti-von Willenbrand Factor. Following AdCMVCH2 administration, L3T4-expressing endothelial cells were observed at all time points and infected cells retained their flattened morphology. Upon AdCMVCH1 administration, L3T4-expressing cells were observed in arteries removed 6 and 24 hours post-infection. A large proportion of the CH1-expressing endothelial cells lost their normal flattened morphology, rounded up and detached from the vessel wall. No CH1-expressing cells were detected 48 hours and 6 days post infection, suggesting that all CH1-expressing cells may effectively and rapidly detach from the arterial wall. Endothelial cell transduction was patchy yet widespread with both vectors (transduction rates: ~10-75%). Only rare non-endothelial cells of the vessel wall stained positive for L3T4. Arteries were also double stained with anti-L3T4 antibody and with the TUNEL technique to detect apoptotic cells. TUNEL-positive cells were observed within CH1-expressing endothelial cells 24 hours post-infection., while no TUNEL-positive endothelial cells were observed upon AdCMVCH2 infection.
These data constitute first evidence that Ad-mediated gene transfer of a dominant-negative integrin construct effectively disrupts adhesion and induces apoptosis of quiescent endothelial cells in vivo. We are currently exploring potential applications of this approach to modulate pathophysiological processes that involve integrin-dependent cell adhesion events (i.e. adhesion, migration and survival) including tumor angiogenesis and arterial restenosis post-angioplasty.

M05b// INDUCTION OF ENDOTHELIAL CELL DETACHMENT AND APOPTOSIS BY ADENOVIRUS-MEDIATED EXPRESSION OF ISOLATED BETA INTEGRIN CYTOPLASMIC DOMAINS
NFP37 Nr: 055150; Oguey D1, Foletti A, Werffeli George P, Paroz C and Ruegg C. Centre Pluridisciplinaire d'Oncologie, School of Medicine, University of Lausanne, CH-1011 Lausanne, Switzerland. (1) Present address: F. Hoffmann-La Roche Ltd, CH-4070 Basel, Switzerland
Integrin-dependent endothelial cell adhesion is essential for the induction and the maintenance of tumor angiogenesis. Administration of integrin antagonists (i.e. function blocking monoclonal antibodies or peptides) results in suppression of tumor angiogenesis and tumor progression in several animal models. We explored the possibility of using a genetic approach to inhibit integrin-mediated endothelial cell adhesion and survival. We constructed recombinant adenoviruses (Ads) expressing chimeric proteins consisting of the cytoplasmic and transmembrane domains of integrin beta 1 (CH1), beta 3 (CH3) or the beta 1 transmembrane domain alone (CH2) connected to the extracellular domain of L3T4 (murine CD4) placed under the control of the CMV promoter (AdCMV) or the endothelial cell specific Tie-1 promoter (AdTie). All constructs were expressed in a dose- and time-dependent manner with over 90% of cells expressing the constructs within 24 hours (AdCMVs) or 72 hours (AdTies) post-infection. Confluent monolayers of HUVEC expressing the CH1 or the CH3 constructs detached from the substrate in a dose-dependent manner, while expression of the CH2 construct had no effect on cell attachment. Cell detachment was preceded by the disruption of focal adhesions and reorganization of the actin cytoskeleton. The cell surface density of beta 1 integrins remained unchanged. Detached cells failed to re-adhere to different matrix proteins, without, however, any specificity toward beta 1 or beta 3 integrin-mediated adhesion (trans-dominant negative effect). The lack of integrin specificity is likely due to the high degree of identity of the juxtamembrane-portion of the cytoplasmic beta 1 and beta 3 domains used. These regions may interfere with the function of the natural endogenous integrin in a interchangeable manner.
Although these results demonstrate that dominant negative inhibition of integrin function is an effective approach to disrupt endothelial cell adhesion and survival in vitro, the lack of specificity toward alphaV beta 3 make these constructs unsuitable for targeting angiogenic endothelial cells/vessels in vivo. We are currently evaluating tree different approaches to solve this problem: 1, expression of the current constructs from promoter specific for angiogenic endothelial cells; 2, expression of shorter cytoplasmic constructs consisting of the most variable region of the beta 3 cytoplasmic; 3, expression of high affinity intracellular antibodies binding to the alphaV or beta 3 cytoplasmic domain.

M06// REPLICATING ADENOVIRUSES TARGETING WNT SIGNALLING DEFECTS FOR COLON CANCER THERAPY.
NFP37 Nr: 055140; Michele Brunori (1), Maddalena Malerba (1), Haruhiko Kashiwazaki1(2) and Richard Iggo (1). (1)Swiss Institute for Experimental Cancer Research (ISREC), 1066 Epalinges, Switzerland. (2)Institute for Genetic Medicine, Hokkaido University, Sapporo, Japan. Correspondence should be addressed to Richard.Iggo@isrec.unil.ch
Despite important advances in understanding the molecular basis of cancer, few treatments have been devised which rationally target known causal oncogenic defects in tumour cells. Selectively replicating viruses have a major advantage over non-replicating viruses to target these defects because the therapeutic effect of the injected virus is augmented by virus produced within the tumour. An E1B mutant replicating adenovirus thought to target p53 defects has shown promise in early clinical trials, although the basis for its tumour selectivity has been questioned. To permit rational targeting of colon tumours, we have developed replicating adenoviruses that express the viral replication (E2) genes from a promoter controlled by the Tcf4 transcription factor. Tcf4 is constitutively activated by mutations in the adenomatous polyposis coli (APC) and b-catenin genes in virtually all colon tumours, and constitutively repressed by Groucho and CtBP in normal tissue. Viruses with Tcf4 sites in the E2 promoter show greater than 100-fold selectivity of viral replication for cells with APC and b-catenin mutations. Insertion of Tcf4 sites in the E1B promoter of these viruses has only small effects on replication in vitro, but significantly reduces the inflammatory response in a rodent lung model in vivo. The viruses we have developed with Tcf regulation of both E1B and E2 transcription are potentially useful for the treatment of liver metastases from colorectal tumours.

M07// CELL SIGNALING BY INCOMING ADENOVIRUS ENHANCES VIRAL MOTILITIES AND MICROTUBULE DEPENDENT NUCLEAR TARGETING
NFP37 Nr: guest; U.F. Greber (1), M.Y. Nakano, K. Boucke, S. Keller & M. Suomalainen . (1) Institute of Zoology, University of Zurich, Winterthurerstr. 190, 8057 Zuerich
Infectious adenovirus particles are transported to the nucleus by the minus end directed, microtubule-dependent dynein/dynactin motor complex in competition with a plus end directed motor. We demonstrate here that external stimuli of an adenovirus infection transiently activate two distinct signaling pathways specifically enhancing shuttling motions and long range transport of cytosolic particles to the nucleus. The first pathway activates integrins and downstream cyclic AMP dependent protein kinase (cAPK). If cAPK activation is blocked, cytosolic viruses are less motile and move along microtubules towards the cell periphery. The second pathway activates the p38/MAP kinase target MAPKAP kinase 2 (MK2), independent of integrins and cAPK. Specific inhibitors of p38, but not extracellular signal regulated kinase ERK1,2 slow viral motilities and promote microtubule dependent virus transport to the periphery consistent with an attenuated inhibition of a plus end directed motor. MK2-/- cells do not support long range adenovirus transport, but nuclear transport is rescued by an overexpressed constitutively active MK2 target, the small heat shock protein hsp25 stimulating actin metabolism. This is the first evidence showing that microtubule dependent and independent transport machineries are integrated into cellular signaling pathways triggered by external stimuli of an incoming virus.

M08// LENTIVIRAL VECTORS FOR LIVER-DIRECTED GENE THERAPY: EVALUATION OF THE EX VIVO APPROACH.
NFP37 Nr: 046196; Zufferey R, Birraux J, Triponez F, Klages N, Trono D. Departments of Genetics and Microbiology, Pediatrics, and Surgery, Faculty of Medicine, 1 rue Michel-Servet 1211 Geneva 4.
Hepatocytes are important targets for gene therapy. Beside numerous inborn metabolic diseases affecting primarily hepatocytes, these cells are naturally designed for synthesis and blood secretion of therapeutic proteins (e.g. Factor VIII).
Although lentiviral vectors transduce efficiently non-dividing cells such as neurons in vivo, attempts to transduce hepatocytes in vivo using the same vectors have met with limited success. Surprisingly, induction of cell cycling by partial hepatectomy was required for hepatocytes transduction by lentivectors as previously demonstrated for MLV-based vectors. These observations prompted us to evaluate lentiviral vectors in an ex vivo approach.
The ex vivo approach consists in hepatocytes isolation, in vitro culture and transduction, followed by reimplantation. Our studies aim at the optimization of each of these steps using the Gunn rat as an animal model. Gunn rats have an abnormally high plasmatic concentration of unconjugated bilirubin because they are deficient in UDP-glucuronyl bilirubin transferase (UGBT), a liver-specific enzyme. We transduce hepatocytes with vectors encoding the Green Fluorescent Protein (GFP) for marking studies or UGBT for the correction of the metabolic deficiency. The gene therapy efficacy is easily quantified by monitoring the reduction of bilirubin concentration in blood.
In contrast to the in vivo studies, we have found that transduction of hepatocytes cultured in vitro does not require cell cycling. The basis of this difference is currently under investigation. In vitro, quiescent hepatocytes were transduced efficiently (up to 60%) by HIV-1-based vectors but not at all by MLV-based vectors. The presence or absence of HIV-1 accessory proteins had no effect on transduction efficacy. As other primary cells, hepatocytes are efficiently transduced only at a high multiplicity of infection (MOI of 5 to 10). Vector production by transient transfection of 293T cells would be a limiting factor for our in vivo studies requiring large vector batches (108 transducing units/animal). This problem was solved by the generation of stable packaging cell lines producing third generation lentiviral vectors in large amounts. Since transduction rates were similar for cells cultured under a variety of conditions (e.g. plastic or collagen coated plates) culture conditions resulting in high engraftment rates and long term cell survival can be selected. Transduced hepatocytes are injected in the spleen of recipient Gunn rats. To monitor the intrasplenic survival of the injected hepatocytes, we have found that the nuclear magnetic resonance imaging (MRI) after administration of a contrast agent is a useful, non-invasive method.

M09a// NATURAL KILLER CELLS OVEREXPRESSING ACTIVATORY RECEPTORS: TOWARDS A POTENTIAL IMMUNOTHERAPY FOR RESIDUAL LEUKEMIA
NFP37 Nr: 055157; Kalberer CP, Siegler U, Luther-Wyrsch A, Nissen C and Wodnar-Filipowicz A. Research Department, University Hospital Basel; Switzerland.
The cytotoxic activity of natural killer (NK) cells is governed by integrating signals obtained from activatory and inhibitory cell surface receptors. Inhibitory receptors recognizing self MHC class I molecules on target cells protect against the initiation of a cytotoxic cascade. Inhibitory signals are greatly reduced or absent whenever syngeneic target cells have lost the MHC class I expression or allogeneic MHC class I molecules are not recognized. In such situations, activatory receptors recognize specific ligands, whose molecular nature is not yet well understood, and trigger a cytotoxic NK cell response. A recent report showed that shifting the balance towards activatory signals by blocking the interaction of MHC class I molecules and inhibitory receptors leads to an increased NK cell mediated killing of leukemic cells in vitro and in tumor bearing mice (Bennet et al., 1999). Thus, we want to test whether shifting the balance by overexpressing activatory receptors in human NK cells would increase their anti-leukemic cytotoxic activity.
To this aim we cloned cDNAs encoding activatory NK cell receptors into lentiviral vectors (kindly provided by P.Salmon and D.Trono, Univ. of Geneva). Vectors carrying a bicistronic construct with NKG2D or NKp46 sequences and GFP as a marker gene were prepared. Expression of the NK cell receptors in 293T cells was confirmed by RT-PCR and Western blotting. Virus generated from these vectors will be used to infect NK cell lines as well as hematopoietic stem cells which will be differentiated in vitro into NK cells in the presence of flt-3 ligand and IL-15. Activation of NK cells by receptor cross-linking will be measured in vitro by IFN-g release and cytotoxicity assays. In order to analyze the long-term expression and the activity of NK cell receptors in vivo, the NOD/SCID transplantation model has been established in our laboratory. As a final step, we plan to test the anti-tumor activity of NK cells overexpressing activatory receptors in NOD/SCID mice which had been transplanted with human leukemic blasts.

M09b// STABLE LENTIVIRAL VECTOR-MEDIATED GENE TRANSFER TO HUMAN FETAL CORD BLOOD PROGENITOR CELLS WITH HIGH SELF-RENEWAL CAPACITY
NFP37 Nr: 055157; Luther-Wyrsch A, Costello E (1) , Thali M (2), Buetti E(3), Surbek D (4), Holzgreve W (4), Nissen C and Wodnar-Filipowicz A. Research Department, University Hospital Basel; Switzerland; (1) Department of Surgery, University of Liverpool, Liverpool; Great Britain; (2) Department of Microbiology and Molecular Genetics, University of Vermont, Burlington; Vermont, USA; (3) Institute for Microbiology, University Hospital Lausanne; Switzerland; (4) Obstetrics Department, University Hospital Basel; Switzerland
Umbilical cord blood (CB) from the early gestational human fetus is recognized as a rich source of hematopoietic stem cells (Wyrsch et al., Exp Hematol 27:1338). To examine the value of fetal CB for gene therapy of inborn immunohematopoietic disorders, we tested the feasibility of genetic modification of CD34+ cells from CB at weeks 24 to 34 of pregnancy, using lentiviral vector-mediated transfer of the green fluorescent protein (GFP) gene. The transduction rate of CD34+ cells was 42±9%, resulting in GFP expression in 23±4% of colonies derived from colony-forming units (CFUs) and 11±1% from primitive long-term culture-initiating cells (LTC-ICs). Cell cycle analysis demonstrated that freshly isolated fetal CD34+ cells were largely residing in G0 phase and, upon cytokine exposure, rapidly entered G1. Upon transduction of cytokine-stimulated cells, GFP expression was found in a small proportion of residual dormant G0 cells, and the bulk expression was evenly distributed among cells which progressed to G1 and actively dividing S/G2/M phase cells. Transduced fetal CD34+ cells could be expanded 1000-fold in long-term cultures supplemented with megakaryocyte growth and development factor along with flt3 ligand. At week 10, expression of GFP was observed in 40.5±11.7% of CFU-derived colonies. While prestimulation of CD34+ cells with cytokines prior to transduction increased the efficiency of GFP transfer two- to threefold, long-term maintenance of GFP-expressing CFUs occurred only in the absence of prestimulation. The GFP gene was found integrated into the genomic DNA of 35% of LTC-IC-derived colonies initiated at week 10, but GFP expression was not detectable, suggesting downregulation of transgene activity during the extended culture period. These results indicate that human fetal CB progenitors are amenable to genetic modification by lentiviral vectors and may serve as a target for gene therapy of hematopoietic disorders by prenatal autologous transplantation.

M10// LENTIVIRAL DELIVERY OF GDNF IN MPTP-TREATED MONKEYS: EFFECTS ON BEHAVIOR AND FD PET UPTAKE
NFP37 Nr. 044718; P. Aebischer (3), M.E. Emborg (1)*, J. Bloch (3), S. Ma (1), Y. Chu (1), L. Leventhal (1), S. Palfi (1, 5), J. McBride (1), J.Stansell (1), P. Carvey (1,2), P. Hantraye (5), J. Holden (6), D. Brown (6), M.Taylor (6), N. Déglon (3), and J. H. Kordower (1). (1) Dep. Neurol. Sci., and Res. Ctr. for Brain Repair, (2) Dept Pharmacol. Rush Univ., Chicago IL 60612; (3) Div. Surg. Res. and Gene Therapy Ctr, Lausanne Univ. Med. Sch., Switzerland; (4) Dep. Neurosurg. Univ. IL Med. Ctr., Chicago IL 60612; (5) CEA CNRS URA 2210 Serv. Hosp. F.Joliot, CEA, DSV, DRM, Orsay cedex, France; (6) Dep. Radiop. Univ. WI, Madison,WI.
The present report examined whether lentiviral delivery of glial cell line-derived neurotrophic factor (GDNF) can reverse the functional consequences of MPTP-induced nigrostriatal degeneration in nonhuman primates. Eight young Rhesus monkeys (male, 5-8yrs, 5.5-8.5 kg) received a unilateral intracarotid (ica) infusion of MPTP-HCL (3 mg). One week later, monkeys received MRI-guided-stereotaxic injections of lentivirus encoding for GDNF (lenti-GDNF, n=4) or þ-galactosidase (lenti-þGal, n=4) into the caudate nucleus (n=2; 5 µl and 10 µl), putamen (n=3; 10 µl, 10 µl and 5 µl) and substantia nigra (n=1; 5 µl). Monkeys were assessed by a blinded observer for performance on an operant pick-up task (PUT) and motor deficits using a clinical rating scale (CR). Assessments were made before treatment and for 3 months post injection. Just prior to sacrifice, all monkeys received a fluorodopa (FD) PET scan. þGal treated animals displayed robust deficits on the PET and clinical rating following MPTP that lasted the 3 mo. post-operative period. Lenti-GDNF treated monkeys displayed significant (55% faster; p<.05) improvements on the PET with the hand contralateral to the MPTP infusion and significantly lower (21.1%; p<.04) scores on the CR scale. Lenti-GDNF treated monkeys also displayed enhanced (332.8%; p<.05) FD uptake in the striatum on PET on the treated side. These results indicate that site specific delivery of GDNF in nonhuman primates by lentiviral vectors can reverse MPTP-induced parkinsonian signs and may be a suitable therapy for treatment of PD.
Supported by the Swiss National Science Foundation.

POSTERS ABSTRACTS

P01a// EVALUATION OF IN VITRO AND IN VIVO GENE TRANSFER INTO CARDIAC MYOCYTES AND ENDOTHELIAL CELLS USING MULTIPLY ATTENUATED, SELF-INACTIVATING LENTIVIRAL VECTORS
NFP37 Nr: 055162; Simeoni E, Fleury S, Driscoll R, Deglon N, Vassalli G. Cardiology and Gene Therapy Center, CHUV, Lausanne
Naked DNA and adenovirus vectors used in gene therapy studies are limited by low efficiency and unstable gene expression, respectively, as well as failure to integrate into the host genome. Murine retrovirus vectors do integrate, but they are limited by inefficient transduction of nondividing cells. In contrast, lentivirus vectors transduce both dividing and nondividing cells, while retaining the ability to integrate. Multiply attenuated HIV vectors deleted in the genes that encode the accessory proteins and containg a self-inactivating (SIN) system have a markedly improved safety profile compared to first-generation HIV vectors. These vectors have not been tested for cardiovascular gene transfer.
Methods: Multiply attenuated SIN lentiviral vectors pseudotyped with the vesicular stomatitis virus (VSV)-G envelope protein contained a lacZ marker gene under the control of either a PGK or a CMV promoter. They also contained a post-transcriptional regulatory sequence from the woodchuck hepatitis virus (WHV). In vitro gene transfer was studied both in neonatal and in adult mouse and rat cardiac myocytes (CM) and in human umbilical cord endothelial cells (HUVEC). Ex vivo gene transfer was studied by transducing neonatal CM in vitro and cell transplant into rat hearts in vivo. In vivo gene transfer was studied in mouse and rat hearts and carotid arteries.
Results: Dose-dependent in vitro gene transfer was achieved both in neonatal (transduction rate: ~80%) and adult CM (~20%) and in HUVEC (~50%). Transduction of neonatal CM was inhibited by n-butyrate but not aphidicoline, which block cell cycle progression at the early and late G1 stage, respectively. Transduced CM were detected one week after cell transplant into rat hearts. Direct in vivo gene transfer into mouse and rat CM and arterial EC was detected, albeit at a very low level (<0.1% as compared to ~10% with an adenoviral vector). A non-attenuated lentiviral vector containing the accessory protein genes did not improve CM transduction in vivo.
Conclusions: Lentiviral vectors efficiently transduce CM and EC in vitro; however, in vivo gene transfer into the myocardium and arterial endothelium is inefficient. The inhibitory effect of cell cycle blockers suggests that metabolically active cells are more susceptible to lentiviral transduction. Lentiviral vectors may be useful in ex vivo gene therapy of cardiovascular diseases. Their unique capability of chromosomal integration in nondividing cells provides a potential for permanent genetic modification of cardiovascular tissues.

P01b// EFFICIENT ADENOVIRUS-MEDIATED GENE TRANSFER INTO TRANSPLANTED RAT HEARTS: AN EXPERIMENTAL MODEL FOR GENE THERAPY OF HEART TRANSPLANTATION
NFP37 Nr: 055162; Fleury S, Dudler J, Driscoll R, Vassalli G. Cardiology and Gene Therapy Center, CHUV, Lausanne
Local expression of protective genes within the transplanted heart may be useful in preventing allograft rejection, while minimizing systemic side-effects. Access to the donor heart at the time of harvest provides a unique opportunity for genetic manipulation of this organ before transplantation.
Methods: In a first study, we have assessed the ability of recombinant adenovirus and adeno-associated virus (AAV) vectors to direct efficient and stable in vivo gene transfer into mouse hearts. Intramyocardial vector injection into the LV wall was used in these experiments. In a second study, we have tested recombinant adenovirus vectors for gene transfer into transplanted rat hearts. An E1/E3-deleted adenovirus vector (Ad.CMV-EGFP; 109 plaque forming units) containing a beta-galactosidase reporter gene was infused into the aortic root of explanted Lewis (LEW) rat hearts during a 20 min cold ischemia time. Donor hearts were then transplanted heterotopically into syngeneic recipients. Cardiac isograft were explanted 5 and 40 days after gene transfer for analysis of transgene expression.
Results: In vivo gene transfer into nontransplanted mouse hearts was efficient both with adenovirus and AAV vectors (6% and 2% of all LV myocytes, respectively, with regional transduction rates of up to 20%). Transgene expression lasted for 2-4 weeks with adenovirus vectors and for more than 1 year with AAV vectors. The onset of gene expression was early with adenovirus but late (14 days) with AAV vectors. After intracoronary adenovirus-mediated gene transfer into transplanted hearts, transduction rates of cardiac myocytes were ~15%. Significant transgene expression in transplanted hearts was detectable at 40 days. Preliminary results in cardiac allografts (LEW/F344) suggest that adenovirus-mediated gene transfer of a soluble IL-1 receptor might provide prolonged allograft survival (> 1 month vs. 20 days in controls).
Conclusions: Adenovirus-mediated gene transfer into donor hearts is efficient. Ex vivo intracoronary perfusion with adenovirus-containing solution during the cold ischemia time achieves widespread transduction of the grafted organ. Transgene expression in cardiac isografts lasts for more than 40 days. Local gene transfer of immunomodulating factors may be useful to prolong allograft survival and, eventually, to induce allograft tolerance.

P02// TOWARDS GENE THERAPY FOR MHC CLASS II DEFICIENCY
NFP37 Nr: 055159; J.Villard, F. Matheux, W. Reith; Department of Genetics and Microbiology, University of Geneva Medical School; Centre Médical 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. The genetic and molecular definition of MHC-II deficiency, 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. The RFX5 lentiviral vector is being used to set up and optimize gene therapy protocols in a mouse model (RFX5-/- mice) for MHC-II deficiency. This mouse model closely reproduce the human disease and this step is therefore very essential to validate gene therapy as an alternative therapy for this disease. 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 results show that the bicistronic mouse RFX5/GFP lentiviral vectors is able to correct B cell lines derived from MHC class II deficiency patients but is less efficient than the monocistronic mouse RFX5 lentiviral vector. In our conditions, the lentiviral vectors have low efficacy to transduce mouse bone marrow derived hematopoietic cells in an ex-vivo system and seems to require high MOI. Weave therefore also initiated experiments in parallel with a retroviral vector system (MIGRI) optimized for the use in mouse stem cells. Mouse bone marrow transplantation with transduced hematopoietic cells from RFX5-/- mice are under way.

P03// INTERACTION BETWEEN DNA-LOADED POLY(DL-LACTIDE-CO-GLYCOLIDE) MICROSPHERES AND HUMAN ANTIGEN-PRESENTING CELLS
NFP37 Nr: 055144; Elke Walter (1), Donatus Dreher (2), Menno Kok (3), Lars Thiele 1), Stephen Gitahi Kiama (4), Peter Gehr (4), and Hans Peter Merkle (1). (1)Department of Applied Biosciences, Swiss Federal Institute of Technology Zuerich (ETH), Winterthurerstr. 190, 8057 Zuerich (CH) // (2)Department of Genetics and Microbiology, University of Geneva, 1 rue Michel-Servet, 1211 Geneva 4 (CH) // (3)Division of Pneumology, University Hospital of Geneva, 24 rue Micheli-du-Crest 24, 1211 Geneva 14 (CH) // (4)Institute of Anatomy, University of Bern, Buehlstr. 26, 3000 Bern 9 (CH)
The encapsulation technology and the type and molecular weight of the polymer employed has been demonstrated to effect the release of DNA from microspheres. Moreover, surface hydrophobicity has been reported to be one of the major factors regulating phagocytosis of microspheres by phagocytic cells. We studied the influence of different PLGA type polymers on the encapsulation and release of DNA to improve the properties of the microspheres with regard to microencapsulation of DNA and its delivery into human antigen-presenting cells (APC) for genetic vaccination. Hydrophobicity and molecular weight of the PLGA-polymers had profound influence on both the encapsulation efficiency of DNA and its release kinetics in vitro: the hydrophilic polymers showed higher encapsulation efficiency and faster DNA release than the hydrophobic ones. Microsphere preparations consisting of different PLGA-type polymers, even the microspheres prepared from the most hydrophilic polymer RG502H, were efficiently phagocytosed by primary human macrophages (MF) and dendritic cells (DC). Surprisingly, uptake of PLGA microspheres by DC was almost as efficient as uptake by the highly phagocytic MF. The PLGA microspheres did not affect the maturation of DC. Intracellular degradation of microspheres was in good agreement with in vitro studies, microspheres consisting of the most hydrophilic PLGA polymer RG502H being eliminated more rapidly than the more hydrophobic ones. None of the microsphere preparations induced significant apoptosis or necrotic cell death in the DC. These results suggest that the PLGA polymer RG502H might have improved characteristics for DNA delivery to human APC.

P04// A REGULATORY NETWORK FOR THE EFFICIENT CONTROL OF TRANSGENE EXPRESSION
NFP37 Nr: 044744; Markus O. Imhof (1), Philippe Chatellard (1), Mickaël Bettan (2), Veronika Pochanke (1), Yann Karlen (1), Abder Mahfoudi (2), Daniel Scherman (2), and Nicolas Mermod (1). (1) Laboratory of Molecular Biotechnology, University of Lausanne, 1015 Lausanne, (2) Unité mixte de recherche 7001 CNRS/ENSCP/Aventis, Centre de Recherche de Vitry-Alfortville, France
We have constructed a genetic switch system for doxycycline-regulated gene expression in somatic gene therapy using a network of repressor and activator proteins. The hallmark of this network is efficient transgene silencing in the off-state as demonstrated by the tight control of the highly cytotoxic diphtheria toxin A gene. Using the erythropoietin gene, we show that addition of the inducer drug allows for robust activation of transgene expression, and this control is granted even after months and repeated cycling between the repressed and activated state in stably transfected cell lines. We also found that several factors limit the maximal expression of the therapeutic gene. One limitation may be the toxicity of the activator protein when highly expressed. This was addressed by generating new and potentially less toxic chimeric activators of transcription. Furthermore, we employed the regulatory system to express cofactors for the transactivator proteins. Finally, ex vivo gene therapy procedures, such as the implantation of encapsulated xenogeneic cells expressing a desired protein, are limited by the number of cells or capsules that can be implanted in an animal or human host. Therefore, we turned to an alternative in vivo gene transfer approach: the electrotransfer of DNA in muscles in situ. Results obtained with these various approaches will be presented, including first data on the network-mediated regulation of a test gene electrotransferred in the tibialis muscle of mice.

P05// THE "PEPTABODY" AS A TOOL TO VERIFY BINDING SPECIFICITY OF A PEPTIDE LIGAND WITH AFFINITY TO MOUSE BONE MARROW ENDOTHELIAL CELLS
NFP37 Nr: 044804; Clément G (1), Bisoffi M (1), Finger AN (1), Wetterwald A (1), Gautschi E (1), Thalmann GN (1), Stadler BM (2) and Cecchini MG (1). (1) Gene Therapy Laboratory, Department of Clinical Research and Urology Clinic, and; (2) Institute of Immunology and Allergology, University Hospital, CH 3010 Bern, Switzerland.
Abstract not available

P06// INDUCIBLE AND IRREVERSIBLE CONTROL OF GENE EXPRESSION USING A SINGLE TRANSGENE.
NFP37 Nr: 055105; Fuhrmann-Benzakein E, GarcÌa-Gabay I*, Pepper MS, Vassalli JD, Herrera PL. Departments of Morphology and Pathology (*), University of Geneva Medical School 1 Rue Michel-Servet, CH-1211 Geneva 4, Switzerland
Experimental or therapeutic designs involving the conditional expression of genes often require the use of two different transgenes; this can represent a major undertaking. One of these systems takes advantage of inducible recombinases. Here we show a novel use of such enzymes, in that an inducible recombinase-encoding sequence can function to both block the transcription of a gene placed downstream and, subsequently, irreversibly activate the transcription of this very same gene. This double function, which circumvents the need for two transgenes, can be achieved by flanking the inducible recombinase gene by two of its target sequences. In our design, as inducible recombinase gene we used the Cre-ERT gene, flanked by two loxP sites. This cassette was placed between a mouse PGK promoter and the EGFP coding sequence. Massive EGFP gene expression in BHK cells bearing this transgene was observed upon administration of 4-OHT, the inducer of the recombinant activity of Cre-ERT. In the absence of 4-OHT, EGFP transcription was prevented. Because of its simplicity (only a single transgene needs to be used), this strategy is of obvious interest in certain protocols of gene or cell therapy and in a variety of experimental designs in which conditional expression of genes is required.

P07// DOUBLE GENE TRANSFER WITH IL1-RA AND IL-10 BUT NOT WITH IL-1RA ALONE REDUCES CARTILAGE DEGRADATION IN THE SCID MOUSE MODEL OF RHEUMATOID ARTHRITIS (RA)
NFP37 Nr: 055152; Pap T (1), Mueller-Ladner U (2), Neumann E (2), Gabay C (3), Arend WP (4), Mima T (4), Seemayer CA (1), Evans CH (5), Robbins PD (5), Gay RE (1), Gay S (1). (1) Center for Experimental Rheumatology and WHO Collaborating Center for Molecular Biology and Novel Therapeutic Strategies for Rheumatic Diseases, Univ. Hospital, Zuerich, Switzerland; (2) Dept of Internal Medicine I, Univ. of Regensburg, Germany; (3) Div. de Rhumatologie, Hopital Cantonal Univ., Geneva, Switzerland; (4) Dept of Medicine, Univ. of Colorado Health Sciences Center, Denver, CO, USA; (5) Univ. of Pittsburgh, Pittsburgh, Pennsylvania, USA
Objective: Several lines of evidence indicate that the delivery of interleukin-1 receptor antagonist (IL-1Ra) and interleukin (IL)-10 may have beneficial and complementary effects on cartilage destruction in RA. While IL-1Ra appears to reduce the perichondrocytic degradation rather than the invasiveness of synovial fibroblasts (SF), IL-10 has been shown to decrease the aggressive potential of RA-SF in the SCID mouse model for RA. Here, we investigated whether double gene transfer of IL-1Ra and IL-10 results in an additive effect in the SCID mouse model of RA as compared to gene transfer of different IL-1Ra isoforms alone. In addition, we analyzed the molecular alterations following retrovirus-based double gene transfer of IL-1Ra and IL-10.
Methods: RA synovial fibroblasts were transduced using retroviral IL-10- or IL1Ra-encoding MFG vectors. Transduced cells were then mixed, cultured together for one week, and then co-implanted with human cartilage for 60 days into SCID mice. IL-1Ra and IL-10 protein expression was monitored throughout the experiment. RNA of transduced cells was isolated and the expression of proto-oncogenes and signaling molecules before and after IL1ra and IL10 double gene transfer was analyzed using a combination of RNA arbitrarily primed PCR (RAP-PCR) and cDNA expression array (Clontech"). For control, RA-SF were transduced with retroviral pLXSN based constructs encoding for intracellular IL-1Ra isoforms (icIL-1Ra1 and 3) or the secreted IL-1Ra (sIL-1Ra) alone. Again, these cells were co-implanted with normal human cartilage into SCID mice for 60 days, and the invasiveness of the RA-SF was determined.
Results: As seen in previous experiments, gene transfer of the IL-1Ra alone did not result in a significant decrease of the invasiveness of RA-SF, and no differences could be observed between the IL-1Ra isoforms. In spite of some reduction in the perichondrocytic degradation, IL-1Ra over-expressing cells exhibited considerable invasion into the co-implanted cartilage. RA-SF overexpressing icIL-1Ra1 and 3 did not differ in their invasiveness from sIL-1Ra expressing RA-SF. However, combination of IL-1Ra and IL-10 overexpression showed an additive effect resulting in a decrease of perichondrocytic cartilage degradation and nearly a complete reduction of the invasiveness of the RA-SF, which was already achieved by the single gene transfer with IL-10 alone. IL-1Ra and IL-10 double gene transfer resulted also in distinct alteration of gene expression of molecules involved in cell activation and metabolism. Of these, up-regulation of transcription factor STAT2 reflected cellular activation, whereas reduced invasive growth might be attributed to growth inhibitor p33ING1. Of interest, double gene transfer was followed by down-regulation of cdc2-related protein kinase, an effect that was not observed in single gene transfer.
Conclusions: Our data indicate, that double gene transfer of cartilage-protective molecules is a feasible approach resulting in a reduction of both RA-SF invasiveness perichondrocytic cartilage degradation. Moreover, double gene transfer is accompanied by distinct alterations in gene expression that result in the inhibition of pathways involved in cartilage degradation and cellular activation.

P08a// APOPTOSIS IN DENDRITIC CELLS: CONSEQUENCES FOR IMMUNOTHERAPY WITH SALMONELLA VECTORS
NFP37 Nr: 055164; Donatus Dreher (1), Menno Kok (2), Stephen G. Kiama (3), Laurence Cochand (1), Peter Gehr (3), and Laurent P. Nicod (1). (1) Division of Pneumology, University Hospital of Geneva; (2) Department of Genetics and Microbiology, University of Geneva; (3) Institute of Anatomy, University of Bern, Switzerland
BACKGROUND: A paradigm of gene therapy is that the vector should not kill its target cell. However, for genetic immunotherapy, this concept was recently questioned, because apoptotic bodies could enhance the induction of antigen-specific cytotoxic T lymphocytes (Nature Biotech 18, 974-9).
METHODS: The bacterial vector Salmonella typhimurium (ST) can be genetically modified to control its capability to induce apoptosis. We used such mutants to investigate the role of apoptosis in the behavior of human monocyte-derived dendritic cells (DC), in particular with respect to the production of apoptotic bodies and the secretion of IL-18, a potent Th1 cytokine that stimulates cellular immune responses. Apoptosis was documented by flow cytometry with annexin-V, and by electron microscopy (EM).
RESULTS: Mutations affecting the ST gene sipB almost completely abolished the induction of apoptosis in the DC. EM revealed several changes in the morphology of DC following infection: 1. membrane ruffling; 2. small spherical structures loosely connected to the cells ("iccosomes", J Immunol 140, 341-53); 3. apoptotic bodies, and 4. intracellular inclusions. Mutants that did not induce apoptosis still caused the production of "iccosomes", and the accumulation of intracellular inclusions. Therefore, and because of their morphological aspect, we assume that these inclusions were "iccosomes", taken up from the other DC, rather than apoptotic bodies. The determination of IL-18 by ELISA showed that all ST strains stimulated the production of IL-18 inside the DC, but only the strains capable of inducing apoptosis triggered the release of the IL-18 into the culture medium.
CONCLUSIONS: Induction of apoptosis does not seem to be necessary for the production and uptake of "iccosomes" that may amplify the immune response after infection with ST. The release of IL-18 is triggered by the apoptosis induced by ST. Taken together, our findings suggest that the induction of apoptosis by the gene vectors could influence the immune response qualitatively rather than quantitatively.

P08b// NEW OPTIONS FOR GENE TRANSFER INTO HUMAN DC: LIVE SALMONELLA AND PLGA MICROSPHERES
NFP37 Nr: 055164; Menno Kok (1), Elke Walter (2), Elisabeth Bühlmann (1), Laurent P. Nicod (3), and Donatus Dreher (3). (1) Department of Genetics and Microbiology, University of Geneva; (2) Department of Applied BioSciences, ETH Zurich; (3) Division of Pneumology, University Hospital of Geneva
BACKGROUND: Salmonella typhimurium (ST) recombinants have been suggested as gene vectors for immunotherapy (Cell 91, 765-75), but their efficiency for gene transfer into human dendritic cells (DC) has not been investigated previously.
Whereas ST invades many cell types by inducing phagocytosis, poly(lactide-co-glycolide) (PLGA)-microspheres only enter professional phagocytes. With these inert particles, which can be loaded with plasmid DNA, some properties of ST might be mimicked.
METHODS: Recombinant ST were used to test gene transfer to human DC derived from monocytes by culture during 6 days in GM-CSF/IL-4. Gene transfer was monitored by expression of GFP from SV40 and CMV promoters.
To trigger transfer of the contents of PLGA microspheres into the cytosol of DC, anionic liposomes were co-encapsulated in polymer, and translocation of FITC-dextran from the phagosome into the cytosol was followed by confocal microscopy.
RESULTS: Cells could only be transfected at day 3 of their development into DC by ST with an artificially amplified plasmid content. A systematic search for other protocols to facilitate gene transfer into DC never resulted in efficient gene transfer at day 6, when cells become capable of antigen acquisition. Antigen production by the transfected cells was considerably less efficient than antigen delivery by recombinant bacteria inside the DC. The co-encapsulation of anionic liposomes into the PLGA microspheres resulted in efficient translocation of macromolecules from the phagosome-trapped microspheres into the cytosol.
CONCLUSIONS: At this stage, two possibilities appear feasible for immunotherapy using ST as a live vector for human DC: first, antigen delivery by the intracellular bacteria, and, second, gene transfer into the precursors of the DC.
As our data indicate that the second possibility might also be achieved by artificial microspheres, which have a perfect safety profile, we will also further explore gene transfer by PLGA-encapsulated DNA.

P09// GENE THERAPY USING PLASMID DNA ENCODING VEGF-C IN PATIENTS WITH CRITICAL LIMB ISCHEMIA.
(1H-MR spectroscopy of deoxymyoglobin (DMG) to quantitatively evaluate tissue perfusion, and clinical results of a randomized, placebo-controlled, phase I trial)
NFP37 Nr: 055161; I. Baumgartner, R. Kreis (1), C. Skjelsvik, C. Boesch (1), J. Cloetta. Swiss Cardiovascular Center, Division Angiology, University Hospital Bern, and (1) Department for Clinical Research (MR Spectroscopy & Methodology), University Bern, Switzerland
In animal models of peripheral ischemia it has been shown that application of angiogenic growth factors or plasmid DNA encoding an angiogenic growth factor augments natural collateral artery development (therapeutic angiogenesis). Initial encouraging clinical experience derived from a non-randomized, open labeled gene therapy trial using naked plasmid DNA encoding VEGF-A in patients with chronic critical limb ischemia (CLI) [Baumgartner, 1998]. Objectives of the present study was, 1. to assess safety and clinical efficiency (e.g., decreases of rest pain, ulcer healing) of plasmid DNA encoding the human VEGF-C gene* in patients with CLI compared to placebo (3:1), 2. to validate 1H-MR spectroscopy of DMG as a tool to quantitatively evaluate tissue perfusion. Between 6/99 and 9/99, 10 patients with CLI were enrolled and underwent single-dose intramuscular pVEGF-C gene transfer (4mg/8 mg). Open-labeled, multiple-dose, cross over rescue therapy was performed in 6 patients with insufficient clinical improvement. Serious adverse events were reported in 6 patients, non associated with the study drug. No adverse events other than occasional edema, injection site pain, and deterioration of neurodermitis have been attributed to gene therapy.

Table 1 intermediate results of clinical trials

Pa	CLI	Single-D	Clinical response	M-D	Clinical response
Rest pain
LU	1/99	6/99: Ge	no change	Y	RESPONDER no rest pain
RM	3/99	7/99: Ge	no change	Y	NON RESPONDER
TK	12/98	7/99: Pl	less rest pain	N	PLACEBO, less rest pain
RW	5/99	7/99: Ge	worse new lesions	Y	RESPONDER, lesions healed
SH-P	1998	8/99: Pl	forefoot amputation	N	PLACEBO, amputation
Gangrene
AP	10/98; di I Le	6/99: Ge	healing	N	RESPONDER healed
JM	12/98 di I Le	7/99: Ge	healing	N	RESPONDER improved
OH	1/99 di I Ga	7/99: Ge	worse osteomyelitis	Y	(RESPONDER?) limited amputation
NI	11/98; di I/II Ne, Fo Le	8/99: Ge	dig.I/II amp. healing	Y	RESPONDER improved
SO	6/99 di II/III+ He Le	8/99: Pl	worse	Y	(NON RESPONDER?) low output failure, died

Legend: Pa, Patient initials; di, digit; Le, lesion(s); Ga, gangrene; Ne, necrosis; Fo, Foot; He, Heel; Ge, gene, Pl, placebo, M-D, Multiple dosis, Y, yes; N, No

1H-MRS of DMG in combination with application of a pressure cuff to induce a complete ischemia was shown to give reliable (r=0.92), quantitative information on local tissue perfusion. Measurement of the recovery time (DMG to myoglobin) upon release of pressure cuff was most sensitive to determine perfusion differences (controls 6±3 sec, patients 71±66, p=0.0008).

Table 2

H-MRS of DMG before and after pVEGF-C gene transfer or placebo
Pa	Recovery time in seconds (calf level)
	Baseline	Single dose	Multiple dose	Occlusion level
RM	8.1	8.4	7.4	Foot arteries
AP	12.9	5.2		Distal calf arteries
TK	188.9	180.0		Calf arteries
RW	143.2	231.0	53.9	Thigh/calf arteries

A phase II, multiple dose, placebo-controlled, multicenter trial is under consideration
* 5283 base pair plasmid encoding human VEGF-C; expression 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) to enhance VEGF-C messenger RNA stability; kanamycin resistance

P10// GLIOBLASTOMA MULTIFORME GENE THERAPY
NFP37 Nr: 055155; Bernasconi L., Malipiero U., Fontana A.; Section of Clinical Immunology, Departement of Internal Medicine, University Hospital Zuerich, Switzerland
Glioblastoma multiforme is a highly malignant tumor, which infiltrates the brain extensively. It is responsible for 29 percent of all primary intracranial gliomas. Despite advances in conventional therapy, the prognosis for most glioma patients remains dismal. The average survival of patients affected by this disease ranges from six to twelve months.
The topoisomerase-1 inhibitor topotecan has been found to prolong survival of experimental animals with glioma. To assess its effect on F98 rat glioma cells when using the drug in combination with adenovirus-Fas Ligand (Ad-FasL), we performed an in vitro experiment using F98 rat glioma cells. The in vitro data demonstrate a synergistic effect on glioma cells. The in vivo experiments confirmed our preliminary results showing an increase of the mean survival of the rats if compared with the animals treated with a single compound. Unfortunately the combined treatment never led to the complete remission of the disease.
Interferon gamma inducible protein (IP-10) has been reported to act in vivo as an anti-angiostatic factor and also to play an important role as chemoattractant for lymphocytes, neutrophils and monocytes. In a mouse model we injected tumour-forming glioma 261 cells 3 days before treatment with adenovirus IP-10. No effect was seen on tumor growth. We are now planning to perform experiments using the combination of IP-10 as an angiostatic and chemotactic principle with the proapoptotic cytokine Fas Ligand in the rat system.

P11// IDENTIFICATION OF AN RGD-CONTAINING PEPTIDE LIGAND THAT MEDIATES BINDING TO KIDNEY CORTICAL COLLECTING DUCT CELLS BUT NOT TO PROXIMAL CONVOLUTED TUBULE CELLS
NFP37 Nr: 044802; Odermatt A (1), Audige A (1), Frick C (1), Vogt B (1), Frey BM (1), Frey, FJ (1), and Mazzucchelli L (2). (1) Division of Nephrology and Hypertension and (2)Institute of Pathology, Department of Clinical Research, University of Berne, Switzerland. Correspondence to: alex.odermatt@dkf2.unibe.ch
We have developed a novel method to identify receptor ligands for defined renal tubular segments. Ex vivo screening of phage-display peptide libraries on isolated intact rat proximal convoluted tubules (PCT) and cortical collecting ducts (CCD) allowed the direct access of phage to the basolateral surface of tubular epithelial cells.Two distinct peptide motifs were selected for CCD and PCT, suggesting molecular heterogeneity of the basolateral surface of kidney tubules. Using the linear peptide motif ELRGD(R/M)AX(W/L), recovered from freshly isolated rat CCD, mediated 16-fold selectivity of phage binding to CCD compared to PCT. Binding to CCD was 39-fold higher than that of a random control phage. Binding and subsequent internalization of phage, most likely by an integrin-mediated endocytosis pathway, was abolished by the addition of the corresponding synthetic peptide. Furthermore, the results demonstrate that presentation and flanking amino acids determine the specific binding properties of RGD ligands to their putative integrin receptors. Our results emphasize the need of a native cell system for the identification of renal epithelial cell surface ligands. Such ligands are of potential relevance for the analysis of interactions between extracellular matrix and kidney tubules or for the development of improved vectors for kidney-specific drug delivery or gene transfer.

P12// MUTANT SENDAI VIRUS INFECTION CAN PROLONG THE LIFE OF MATURE DENDRITIC CELLS.
NFP37 Nr: 044711; Dominique Garcin, Salomé Landmann, Walter Reith, and Daniel Kolakofsky. ; Dept. of Genetics and Microbiology, University of Geneva.
Sendai virus (SeV) infections are generally cytopathic. However, there are mutant viruses that are particularly cytopathic, and others that are non-cytopathic and that lead automatically to long-term persistent infections. Among the latter are GP42 and GP48, which appear to be actively anti-apoptotic (this property is dominant in mixed infections), and this infection also protects cells from apoptosis induced by serum starvation (but not from that induced by chemicals such as ectoposide).
A large effort is already underway to use dendritic cells (DCs) loaded with tumor antigen peptides ex vivo as anti-tumor agents. If the DCs also express an anti-apoptotic gene like bxl (in a mouse model), their anti-tumor activity is much improved. We have therefore examined the effects of SeV infection on human DCs generated from CD14+ monocyte precursors, and found that:
1. SeV infection very efficiently induces DC maturation.
2. Consistent with its ability to interfere with IFN action, SeV infection of DCs prevents the IFN-induced up-regulation of Stat1 (SeV infection thus probably induces maturation via a different pathway than IFN). More importantly, the viral interference with IFN action prevents the induction of an antiviral state, and the infection is strong and sustained. However, mature DC death, although delayed, occurs.
3. Mature DCs are naturally eliminated by apoptosis. SeV-GP48 infected DCs also efficiently induced maturation and led to a sustained, strong infection. However, the GP48-infected DCs remained remarkably healthy as determined by light microscopy as compared to the IFN-treated or wild-type SeV-infected cultures. By 15 days, there were 10-15 times more viable cells than in the controls.
Dendritic cells infected with a GP48 virus carrying the tumor antigen as a transgene, and perhaps other transgenes like IL-12 and CD40, could represent a very potent anti-tumor agent. We hope to repeat this experiment in a mouse. Dendritic cells infected with a GP48 virus carrying HIV-1 genes may be of interest for AIDS immune therapy.

P13// INHIBITION OF FAK BY EXPRESSION OF THE FOCAL ADHESION TARGETING DOMAIN INDUCES APOPTOSIS AND INCREASES CHEMOSENSITIVITY IN GLIOMA CELLS
NFP37 Nr: 055167Jones GJ and Merlo AM; Departments of Research and Surgery, Kantonsspital Basel, Switzerland
Glioblastoma are highly invasive tumours which are resistant to chemo- and radiotherapeutic treatments and successful surgical resection remains the best prognostic factor for patient survival. The observation that glioma cells exhibit altered expression of integrin receptors, and secrete a specialised extracellular matrix, suggests an important role for the focal adhesion kinase (FAK). The phosphorylation and activation of FAK is inhibited by a C-terminal isoform of FAK, the FAK related non-kinase (FRNK). The FRNK isoform includes the focal adhesion targeting domain (FAT), which is required to target FAK to focal adhesions. The potential of the FAT domain to inhibit FAK signalling was investigated by generating stable transfectants of the LN-401 and LN-229 glioma cell lines. In LN-401 cells, which express neither PTEN nor p53, expression of the FAT domain competed with FAK for localisation at focal adhesions and the phosphorylation of FAK was inhibited. Expression of the FAT domain also reduced the invasive potential of LN-401 cells. Furthermore, apoptosis induced under adherent and non-adherent culture conditions revealed that survival signals from both the extracellular matrix and from serum-dependent pathways involved FAK but were apparently independent of PKB/Akt. The LN-229 cell line, although lacking p53, does express PTEN. In addition, robust expression of Bcl-2 was seen in LN-229 cells, but not in LN-401 cells. Despite the presence of PTEN, FAK was highly phosphorylated in a fibronectin adhesion assay, and was inhibited by expression of the FAT domain. Under adherent conditions, withdrawal of serum induced a moderate increase in apoptosis only in those cells which expressed the FAT domain. Apoptosis increased a further two fold under non-adherent conditions and was accompanied by a decrease in the levels of Bcl-2. Furthermore, incubation of these cells with either cisplatin, camptothecin or BCNU revealed an increased cytotoxicity of each drug associated with expression of the FAT domain. Significantly, although administration of cisplatin or camptothecin was accompanied by increased caspase-3 activity, the cytotoxic effects of BCNU was independent of caspase-3. Therefore, our results suggest that FAK is an important mediator of both cell migration and survival in human glioblastoma.

P14// LENTIVIRUS-MEDIATED GENE TRANSFER OF GP91phox FOR CORRECTION OF CHRONIC GRANULOMATOUS DISEASE
NFP37 Nr. 055165; Saulnier SO(1), Steinhoff D(1), Wiznerowicz M(3), Salmon P(3), Trono D(3), Dinauer MC(2), Seger RA(1) and Hossle JP(1). (1)Div. of Immunology/Hematology, Univ. Children's Hospital, Zurich, Switzerland; (2)Herman B Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN, USA; 3Department of Genetics and Microbiology, University of Geneva Medical School, Geneva, Switzerland.
Chronic granulomatous diseases (CGD) are inherited disorders of phagocytes, in which the microbicidal activity of the respiratory burst NADPH oxidase is defective, leading to recurrent, life-threatening infections. Two thirds of the patients have mutations in their X-linked CGD gene encoding gp91phox, the largest subunit of the NADPH oxidase. As a therapeutic application for X-CGD, we demonstrated in our previous work correction of X-CGD after transfer of the human gp91phox gene in human X-CGD CD34+ bone marrow (BM) cells using the bicistronic retroviral vector SPsLdS (Becker et al. 1998, HGT 9:1561). In order to evaluate long-term vector function in a pre-clinical setting, we are currently testing SPsLdS in a murine model of X-CGD. Aimed at gene therapy of X-CGD at the level of resting pluripotent hematopoietic stem cells, we generated several HIV-1-based vectors containing the gp91phox gene under the control of either cytomegalovirus (CMV) or elongation factor-1 (EF-1) promoter. Gene transfer and expression were evaluated in the human myeloid PLB985 X-CGD cell line. The X-CGD cells were efficiently transduced (74%-99% green fluorescent protein (GFP)+ cells). CMV and EF-1 driven expression were stable for at least 3 weeks after transduction and persisted after granulocytic differentiation of the target cells. After granulocytic differentiation, as determined by dihydrorhodamine (DHR) FACS analysis up to 63% of the X-CGD cells transduced with the CMV-gp91phox construct were found to be functionally reconstituted with levels of superoxide-production of average 31% (n=7) compared to wild-type (wt) PLB985 cells. The EF-1-gp91phox lentivector further improved functional reconstitution with up to 95% positive cells and levels of superoxide-production of 52% (n=2) compared to wt cells. Therefore, lentiviruses expressing gp91phox are able to at least partially correct human myeloid X-CGD cells and are now being tested for the introduction of the therapeutic gene into very primitive quiescent bone marro stem cells from X-CGD patients.

P15a// LENTIVIRAL MEDIATED DELIVERY OF MUTATED HUNTINGTIN FRAGMENTS AS A NEW STRATEGY TO DEVELOP ANIMAL MODELS OF HUNTINGTON'S DISEASE
NFP37 Nr: 044718; L.Pereira de Almeida(1,2), D.Zala1, R.Gaspar(2), P.Aebischer(1) and N.Deglon(1). (1) Division of Surgical Research and Gene Therapy Center, CHUV, Lausanne, Switzerland; (2) Pharm. Tech. Lab. of the Faculty of Pharmacy and Center for Neurosciences, University of Coimbra, Portugal.
We have evaluated the potential of lentiviral vectors as a gene delivery system to create genetic models of Huntington's disease (HD) in rats. A truncated cDNA coding for 171 AA of Huntingtin gene (Htt171) with either 19 (wild type) or 82 CAG repeats (mutant) was cloned into a lentiviral vector. These viruses were injected into the right and left striatum of adult rats, respectively. The animals were sacrificed at 1-12 weeks post-injection and the brains processed for immunohistochemistry. Pathological features were only observed on the Htt171-82 CAG injected hemisphere and appeared as a cascade of successive events. One week post-injection a large number of cells exhibited accumulations of Huntingtin protein, followed one week later by appearance of ubiquitinated neuronal inclusions. The presence of a lesion was evident at 4 weeks with a loss of DARPP-32, NeuN, and ChAT immunoreactivity around the injection site. These pathological features progressed over 2 months as shown by shrinkage of the striatum. Apoptotic cell death was identified by TUNEL assay at 2 and 3 months. Experiments are under way to further assess the impact of huntingtin fragment length and polyglutamine size on the kinetics and specificity of the pathology.
Supported by the Swiss National Science Foundation; L.P.A. is a recipient of a PRAXIS XXI grant

P15b// TIGHTLY REGULATED LONG-TERM DELIVERY OF ERYTHROPOIETIN IN VIVO USING ENCAPSULATED C2C12 CELLS
NFP37 Nr: 044718; Sommer B(1), Dalle B(2), Rinsch C(1), Beuzard Y(2), Deglon 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 a exogenously regulated erythropoietin (Epo) expression, we have analyzed the potency of the Tet-system described by Gossen and Bujard in C2C12 cells. High background levels of Epo expression and only moderate induction were observed in stable clones reflecting the cell type specificity of the original Tet-system. To overcome these limitations, C2C12 cells were transfected with an autoregulated Tet-Off system. Stable clones producing 25 IU mouse Epo / 106 cells / 24h were obtained. In the presence of doxycycline (dox), the Epo expression was down-regulated up to 200 fold in a dose-dependent manner. After subcutaneous implantation of encapsulated cells into DBA/2J mice, the hematocrit of the animals could be modulated by the presence or absence of dox in the drinking water. Removal of dox resulted in significantly increased hematocrit levels up to 90% reflecting a sustained Epo expression. Six to eight weeks after the administration of dox the hematocrit returned to normal levels. A tightly regulated expression of Epo with up to three on/off cycles was observed during 40 weeks. Encapsulation and implantation of tetracycline-regulated C2C12 cells thus represents an interesting method for the controlled long-term delivery of proteins in vivo.
Supported by the Swiss National Science Foundation

P15c// SECRETION OF HUMAN ERYTHROPOIETIN BY ENCAPSULATED GENETICALLY ENGINEERED HUMAN PRIMARY FIBROBLASTS
NFP37 Nr. 044718; F. Schwenter, N. Déglon and P. Aebischer. Division of Surgical Research and Gene Therapy Center, Lausanne University Medical School, Switzerland
A variety of acquired and inherited disorders such as anemia, hemophilia or diabetes are treated by repeated intravenous or subcutaneous injections of recombinant proteins. A gene therapy approach for the systemic continuous delivery of therapeutic proteins would represent an attractive alternative. The aims of the present project are twofold, 1) to develop a gene transfer protocol based on a cell encapsulation technology allowing to express clinically relevant levels of erythropoietin (EPO) from human cells ; 2) to investigate the immune response against these encapsulated xenogeneic cells in different animal models.
As a first step, the survival of encapsulated human primary foreskin fibroblasts was assessed in nude mice and DBA/2J mice receiving or not an immunosuppressive treatment with FK506. One month post-implantation in the subcutaneous tissue, the histological analysis of the retrieved capsules demonstrated a cell survival of at least 80% in all animals. Based on these data, human fibroblasts were transduced with a murine leukemia retroviral vector containing the human EPO cDNA under the control of the phosphoglycerate kinase (PGK) promoter. A pool of infected fibroblasts producing approximately 200 IU/106 cells/day of EPO was used for implantation in mice as previously described. The animals had increased hematocrit levels over 65% for at least one month. These results indicate that polymer encapsulation of genetically engineered human primary fibroblasts is a promising approach for delivery of bioactive molecules and might be relevant for human application.
Supported by the Swiss National Science Foundation.

P16// MODIFIED ANTISENSE U7 SNRNA, STABLY EXPRESSED AFTER LENTIVIRAL TRANSDUCTION, CORRECTS ABERRANT SPLI-CING OF B-GLOBIN PRE-MRNA IN AN ERYTHROID CELL LINE
NFP37 Nr: 044704; Reber U.(1), Zufferey R.(2), Trono D.(2), Liu S.-K.(1), Bühler M.(1), Schümperli D.(1) // (1) Institut für Zellbiologie, Universität Bern, Switzerland; (2) Département de génétique et de microbiologie, Université de Genève, Switzerland
We are studying mutations at positions 654 and 705 in intron 2 of the b-globin gene which create new 5' splice sites. These mutations also activate a cryptic 3' splice site at position 579, thus introducing an aberrant miniexon with a premature stop codon. Normal splicing can be restored in model cell lines (HeLa and NIH3T3) by trans-fection of antisense oligonucleotides3) or genes encoding antisense U7 snRNA derivatives4) targeted against the aberrant splice sites. The 62-nucleotide U7 snRNA forms stable ribonucleoprotein parti-cles in the nucleoplasm and normally functions in 3' end processing of histone pre-mRNA. By replacement of the natural antisense sequence recognising histone pre-mRNA, it can be induced to bind to other pre-mRNAs and to act as site-specific inhibitor for splicing factors.
For an efficient in vivo application, the modified U7 gene(s) must be introduced and expressed in hematopoietic cells of bone marrow or peripheral blood. We have analysed gene delivery by retroviral vectors, especially using a HIV-derived system5) that allows for transduction of nondividing (hematopoietic) cells. HeLa and erythroleukemic K562 cells stably expressing the IVS2-654 b-globin gene under a CMV promoter were transduced with a lentiviral construct containing an active antisense U7 gene, and provirus integra--tion, expre-ss-ion of the introduced U7 gene and splicing correc-tion of b-globin mRNA were detected. Furthermore, non-thalassemic, but erythroleukemic MB02 cells were also transduced success-fully. These results indicate the feasibility of trans-ferring the thera-peutic U7 gene into cells of hematopoietic lineages.
3) Sierakowska H. et al. 1996. Proc Natl Acad Sci USA 93, 12840.
4) Suter D. et al. 1999. Hum Mol Genet. 8, 2415.
5) Zufferey R. et al. 1998. J Virol. 72, 9873.

P17// FUNCTIONAL AND SELECTIVE TARGETING OF ADENOVIRUS TO HIGH AFFINITY FCg RECEPTOR I POSITIVE CELLS USING A BISPECIFIC HYBRID ADAPTER
NFP37 Nr: guest; Ebbinghaus C, Al-Jaibaji A, Operschall E (1), Schoeffel A, Peter I, Greber U (2), and Hemmi S. Institute of Molecular Biology, University of Zuerich, Winterthurerstr. 190, CH-8057 Zuerich, Switzerland ; (1) Institute of Medical Virology, University of Zuerich, Gloriastr. 30, CH-8028 Zuerich, Switzerland; (2) Institute of Zoology, University of Zuerich, Winterthurerstr. 190, CH-8057 Zuerich, Switzerland
Adenovirus (Ad) efficiently delivers its DNA genome into a variety of cells and tissues provided that these cells express appropriate receptors, including the coxsackie-adenovirus receptor (CAR) which binds to the terminal knob domain of the viral capsid protein fiber. To render CAR negative cells susceptible to Ad infection we have produced a bispecific hybrid adapter protein consisting of the aminoterminal extracellular domain of the human CAR protein (CARex) and the Fc region of the human immunoglobulin (Ig) G1 protein, comprising the hinge and the CH2 and CH3 regions. CARex-Fc was purified from COS7 cell supernatants and mixed with Ad particles thus blocking Ad infection of CAR positive, but Fc receptor negative cells. Functionality of the CARex domain was further confirmed by successful immunization of mice with CARex-Fc followed by selection of a monoclonal anti-human CAR antibody (E1-1) which blocked Ad infection of CAR positive cells. When mixed with Ad expressing eGFP, CARex-Fc mediated an up to 250-fold increase of transgene expression in CAR negative human monocytic cell lines expressing the high affinity Fcg receptor I (CD64), but not in cells expressing the low affinity Fcg receptor II (CD32) or III (CD16). These results open new perspectives for Ad-mediated cancer cell vaccination, including the treatment of acute myeloid leukemia (AML).

P18// A NOVEL ANTISENSE OLIGONUCLEOTIDE TARGETING SURVIVIN-EXPRESSION INDUCES APOPTOSIS AND SENSITIZES LUNG CANCER CELLS TO CHEMOTHERAPY
NFP37 Nr: guest; Olie RA, Simoes-Wuest AP, Baumann B, Leech SH, Fabbro D (1), Stahel RA, Zangemeister-Wittke U.; Division of Oncology, Department of Internal Medicine,University Hospital Zuerich, Haeldeliweg 4, CH-8044 Zuerich, (1) Department of Oncology Research, Novartis Pharma AG, CH-4002 Basel.
Survivin, an inhibitor of apoptosis protein, deserves attention as a selective target for cancer therapy, since it lacks expression in differentiated adult tissues but is expressed in a variety of human tumors. We designed 20-mer phosphorothioate antisense oligonucleotides targeting different regions of the survivin mRNA and investigated their ability to downregulate survivin mRNA and induce apoptosis in the lung adenocarcinoma cell line A549. Oligonucleotide 4003, which targets nucleotides 232 to 251 of the survivin mRNA, was identified as the most potent compound. As measured by real-time PCR, 4003 downregulated survivin mRNA in a dose-dependent manner with an IC50 of 200 nM. Its maximum effect was achieved at a concentration of 400 nM at which mRNA was downregulated by 70%. As revealed by increased caspase-3-like protease activity, nuclear condensation and fragmentation, and trypan blue uptake treatment with 4003 induced apoptosis and sensitized tumor cells to the chemotherapeutic agent etoposide. Oligonucleotide 4003 did not reduce the viability of normal blood leukocytes with marginal levels of survivin mRNA.

P19// GENE THERAPY OF DISEASES INVOLVING TETRAHYDROBIOPTERIN AS A LIMITING FACTOR SUCH AS PHENYLKETONURIA (PKU) AND CARDIOVASCULAR DISEASE
NFP37 Nr: guest; Elzaouk L, Blau N and Thony B. Division of Clinical Chemistry and Biochemistry, Department of Pediatrics, University of Zurich, Steinwiesstrasse 75, CH-8032 Zurich
We are generating gene transfer vectors as therapeutic tools to develop treatment for diseases that specifically involve tetrahydrobiopterin (BH4) as a limiting factor. The BH4 cofactor is required for the activation of the hepatic phenylalanine hydroxylase (PAH) necessary for phenylalanine degradation, and the tyrosine and tryptophane hydroxylases responsible for catecholamine and serotonin biosynthesis. Furthermore, BH4 is an essential cofactor in the synthesis of NO by nitric oxide synthase (NOS). Diseases involving BH4 as a limiting factor include inborn errors of metabolism such as hyperphenylalaninemias, and coronary bypass graft disease where sufficient NO supply plays a crucial role.
The BH4 cofactor is synthesized from GTP via sequential enzyme reactions catalyzed by three enzymes. Our laboratory is involved in screening and treatment, but also with the molecular analysis of defects in the BH4 metabolism. Recently, we showed successful reconstitution of the BH4-biosynthetic pathway in deficient primary patient cells with triple-cistronic IRES-containing retroviral vectors. Consequently, in order to supply sufficient BH4 for treatment of disorders with limiting BH4 availability, similar multiple-cistronic vectors will be generated carrying BH4-biosnythesis genes along with PAH (A) or NOS (B).
(A) Autosomal recessive mutations in the hepatic PAH lead to the classical hyperphenyl-alaninemia and phenylketonuria (PKU). It was previously shown that PAH expression in muscle can lower serum phenylalanine levels if the tissue is supplied exogenously with sufficient BH4. We are attempting muscle specific, ectopic expression of BH4 genes along with PAH to clear phenylalanine from the circulating blood in a mouse model for PKU.
(B) A complication after coronary bypass graft disease is the increased platelet aggregation and neointima formation due to proliferation and migration of smooth muscle cells (VSMC). Increased endothelial NOS function was shown to directly inhibit such VSMC outgrowth, if sufficient BH4 is supplied. We will test adenovirus-directed ectopic and transient expression of BH4 enzymes with NOS to inhibit such secondary damage by human VSMC outgrowth. Gene therapeutic approaches for both diseases will be presented.

X01// SELECTION OF SPONTANEOUSLY TRANSLOCATING NUCLEIC ACIDS
NFP37 Nr: 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 oligoDNA molecules. Depending on the primer pairs used to amplify the initial single stranded template, double stranded products bearing or not a stabilising 3'-hairpin can be generated. Starting with a population of randomized, denatured dsDNA molecules with a complexity of 1014, the ss-desoxy-oligonucleotides are selected for their accelerated capacity of penetrating mammalian cells. Selected DNAs are PCR-amplified and subjected to subsequent cycles of selection-amplification.
We expect to identify candidate oligos with improved autonomous translocation capacity across mammalian cell membranes with either strategy (with or without the stabilising 3'-hairpin). We anticipate that the sequences and properties of lead-oligonucleotides emerging from each approach will be different.

X02// IN VITRO IMPORT OF RNA INTO MITOCHONDRIA OF TRYPANOSOMA BRUCEI
NFP37 Nr: 055154; Tan T., Schneider A. Department of Biology, Unit of Zoology, Perolles, 1700 Fribourg, Switzerland
The long term goal of the project is to establish an in vitro import system for tRNAs into mammalian mitochondria with the idea to provide the tools for gene therapeutic approaches for diseases linked to mutations in mitochondrial-encoded tRNAs. Trypanosomatids import all mitochondrial tRNAs and are therefore the optimal positive control for mammalian mitochondria where in vivo import of any type of RNA is not well documented. We have established an in vitro system for import of small RNA pieces (up to 20 nucleotides) into mitochondria of Trypanosoma brucei. All tested RNA fragments irrespectively of their primary sequence were imported as long as they were short. Unexpectedly, all imported RNAs were converted to larger fragments after import. Preliminary results indicate that this might be due to the activity of a mitochondrial RNA ligase. Furthermore, more recent experiments have shown that full length tRNA can be imported into isolated mitochondria of T. brucei provided RNA-free cytosolic fraction is added to the assay. Cytosol-dependent in vitro import was only 1-2% of the expected in vivo efficiency but it was dependent on ATP and on an intact membrane potential. Future studies will concentrate on the identification of the putative cytosolic import-directing activity.

X03// PERCEPTIONS OF HEALING NEEDS: SOMATIC GENE THERAPY, DISABILITY AND IDENTITY
NFP37 Nr: 053073; Scully JL, Rippberger C, Löw C, Rehmann-Sutter C; Arbeitsstelle für Bioethik, Institut für Geschichte und Ethik der Medizin, Universität Basel, Schönbeinstrasse 20, 4056 Basel
This project analyses differences in the way the ethical issues associated with somatic gene therapy are perceived by different interest groups. The potential consumers of gene therapy are known to display a much broader range of opinions towards it than do medical professionals or ethicists, but how this reflects their ethical standpoints is unclear. We have hypothesized that this variety reflects differences not simply in how these groups perceive a condition, but how they see the relationship between the condition and identity, and the effect on identity of a gene-based intervention. Our approach uses qualitative research methodologies to analyse data from questionnaires, interviews and a range of written materials produced by the medical profession and by lay self-help groups. Our results so far suggest that there are substantial differences between professionals and the potential users in terms of what are considered to be the gravest ethical issues in gene therapy. However, the differences between persons with different conditions, which up to now have tended to be conflated into a single homogeneous category, are also notable, and these appear to be correlated with a person's sense of identification with the condition.
These data form part of our interest in the differential effects of the program and system models of gene action on ideas of identity and embodiment (CR-S), and a comparative ethical approach to marginalised groups in gene-based medicine (JLS, CR, CL). We have been able to present some of this work at meetings and a forthcoming paper in Human Gene Therapy in which we challenge the validity of the distinction between therapy and enhancement in somatic gene therapy.

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