5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>Saturday 14 th September 2013, 8h30 – 10h30WORKSHOP 2: “ONCOLYTIC VIRUSES AND GENETHERAPY”Chairpersons: Penelope MAVROMARA (Athens, GREECE) &Dirk NETTELBECK (Heidelberg, GERMANY)Room Tête d’OrKEYNOTE:New lentiviral vector pseudotypes <strong>of</strong>fer exciting perspectives for T, B,DC and HSC mediated immuno and gene therapyEls VERHOEYENCIRI, EVIR team, INSERM U1111, Lyon, FRANCEImportant target cells for immuno- and gene therapy are human T cells, Bcells, dendritic cells (DCs) and hematopoietic stem cells (HSCs). We haveengineered two new lentiviral vector (LV) pseudotypes that allow highlevel transduction <strong>of</strong> these targets treatment <strong>of</strong> several genetic dysfunctions<strong>of</strong> the hematopoietic system. One LV displaying the measles virusglycoproteins at their surface (MV-LVs), which was able to transduce efficientlystimulated and resting human T, B cells and DCs and thymocytes.A second LV displaying the Baboon retrovirus envelope (BAEV-LVs)allowed high level transduction <strong>of</strong> T and B cells and thymocytes. Forboth LVs this characteristic is unique since VSVG-LVs fail to transducethese resting target cells. Each <strong>of</strong> these LV pseudotypes demonstrates adifferent transduction pattern <strong>of</strong> thymocyte subpopulations. Even morestriking was that these new pseudotypes allowed unexpectedly at lowvector doses efficient transduction <strong>of</strong> HSCs. Importantly, reconstitution<strong>of</strong> NOD/SCID gamma-c-/- mice with MV-LV or BAEVTR transducedpre-stimulated or resting hCD34+ cells resulted reproducibly in transductionlevels close to 100% or 50-70%, respectively, <strong>of</strong> all analyzedmyeloid and lymphoid engrafted lineages in all hematopoietic tissues,including CD34+ lineage negative cells in the bone marrow. Moreover,these high transduction levels were confirmed in secondary miceengraftments.In conclusion, these new LV technologies <strong>of</strong>fer exciting perspectivesfor multiple Tgene and immuno therapy applications in thefuture.ORAL COMMUNICATIONSinfected cells to concentrate iodide enabling non invasive imaging andcombination radiovirotherapy. Through high resolution in vivo and exvivo imaging we documented homogeneous virus seeding in xenograftsand subsequent spread in perfused tissue. Infection <strong>of</strong> metastases wasmore rapid and intense than infection <strong>of</strong> primary tumors, achieving isotopeuptake within nearly 3 fold the efficiency <strong>of</strong> the thyroid. Virotherapy combinedwith systemic 131 I resulted in more rapid disease regression thaneither therapy alone. We then asked if oncolytic efficacy was dependenton the primary MV receptor signaling lymphocyte activation molecule(SLAM) or the MV attenuation receptor CD46. Strikingly, only SLAMdependent entry sustained efficient viral spread, tumor regression, and prolongedsurvival. While virus entry into cells can occur through multiplereceptors, the efficacy <strong>of</strong> oncolytic protocols may depend on the concurrentactivation <strong>of</strong> cellular responses that support efficient viral replication.Functioning as both an entry receptor and signaling molecule, SLAMexpression on hematological tumors represents a targetable vulnerabilityfor MV based oncolysis in future clinical trials.REF 0130Cell surface receptor targeting expanded: from oncolytic measlesviruses to lentiviral and AAV vectorsAnke RASBACH, Christian BUCHHOLZPaul Ehrlich Institut, Langen, GERMANYRestricting gene transfer to the relevant cell type <strong>of</strong> choice at the level <strong>of</strong>cell entry is among the main challenges for vector improvement in geneand virotherapy. Vectors with a restricted tropism, ideally highly specificjust for the cell type <strong>of</strong> interest for the given therapeutic application,are expected to improve safety and efficacy <strong>of</strong> gene therapy and facilitatein vivo gene transfer strategies. Altering receptor usage <strong>of</strong> a viral vectoris a complex protein engineering task, which was first accomplished foroncolytic measles viruses (MVs) by fusing a tumor antigen specific singlechain antibody fragment (scFv) to the virus attachment protein hemagglutinin(H) and abolishing natural receptor usage through point mutations.Cytoplasmic tail truncations in H and the MV fusion protein (F) thenallowed transfer <strong>of</strong> this approach to lentiviral vectors (LVs). Recently,this strategy has been extended to AAV vectors by mutating the heparansulfate proteoglycan (HPSG) binding site in the capsid proteins andsimultaneously fusing a designed ankyrin repeat protein (DARPin) withhigh affinity for the tumor antigen Her2/neu to the VP2 capsid protein.Thus, three different vector types can now be manipulated in a flexibleand rationally based approach to enter cells via a free chosen cell surfacemolecule. Data on the ex vivo and in vivo application <strong>of</strong> these vectortypes targeted to different cell types such as endothelial cells, lymphocytes,hematopoietic stem cells, tumor cells and putative tumor stem cells will bediscussed.REF 0129Mantle cell lymphoma radiovirotherapy: high resolution in vivo imagingdocuments critical role <strong>of</strong> measles virus entry through thesignaling lymphocyte activation moleculeRoberto CATTANEO, Tanner MIEST, Marie FRENZKEMayo Clinic, Rochester MN, USAWe developed here a vaccine identical measles virus (MV) as an oncolyticagent against mantle cell lymphoma, which is incurable but radiosensitive.We armed the virus with the sodium iodide symporter, which causesREF 0131Preclinical therapy <strong>of</strong> disseminated carcinomas and <strong>of</strong> Gliomas withretargeted oncolytic herpesvirusesGrabriella CAMPADELLI-FIUME 1 , Valentina GATTA 1 , PatriziaNANNI 1 , Laura MENOTTI 2 , Carla DE GIOVANNI 1 , PaoloMALATESTA 3 , Pier Luigi LOLLINI 11 Department <strong>of</strong> Experimental, Diagnostic and Specialty Medicine University<strong>of</strong> Bologna, Bologna, ITALY; 2 Department <strong>of</strong> Pharmacy andBiotechnology University <strong>of</strong> Bologna, Bologna, ITALY; 3 IRCCS San MartinoIST University <strong>of</strong> Genoa, Genoa, ITALYVi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013S95
5 th <strong>European</strong> <strong>Congress</strong> <strong>of</strong> <strong>Virology</strong>Oncolytic viruses aim to selectively kill cancer cells. To generate cancerspecific oncolytic Herpes simplex viruses (oHSVs), we retargeted HSVto Human Epidermal growth factor Receptor 2 (HER2), overexpressed inovary and breast cancers. The HER2 retargeted HSV (R LM249) exclusivelyinfects cells expressing HER2, and exerts antitumor activity whenadministered i.t. Ablation <strong>of</strong> disseminated cancer cells in vivo remains amajor hurdle. Here, we report the efficacy <strong>of</strong> systemically (i.p.) administeredR LM249 against disseminated tumors in mouse models thatrecapitulate tumor spread in ovarian or breast carcinoma patients. Humanovarian carcinoma SK OV 3 cells were implanted i.p. or breast tumor MDAMB 453 cells were injected i.v. in immunodeficient Rag2/;Il2rg/ mice.Repeated i.p. administrations <strong>of</strong> R LM249 caused a 95% reduction in thetotal weight <strong>of</strong> peritoneal neoplastic nodules; 60% <strong>of</strong> treated mice werefree from tumor peritoneal diffusion. R LM249 exhibited also anti metastaticactivity against ovarian metastases and reduced brain metastases.The i.p. administration <strong>of</strong> the HER 2 retargeted oncolytic HSV effectivelyreduced i.p. ovarian carcinomatosis and inhibited both visceral and distantmetastases.HER2 is expressed also by high grade gliomas (HGGs). HER2 retargetedHSV R LM113 was effective against HGG and lengthened the medial survivaltime <strong>of</strong> NOD/SCID or immunocompetent mice carrying orthotopicHGGs.Cumulatively, HER2 retargeted HSVs exerted anticancer activity againstperitoneally diffuse breast and ovary cancers, and against gliomas.REF 0132Gene therapy with a replicative HSV vector expressing LIF limits loss<strong>of</strong> oligodendrocytes and modulates autoimmunity during experimentaldemyelinating diseaseMichaela NYGÅRDAS 1 , Henrik PAAVILAINEN 1 , Nadine MÜTHER 2 ,Claus Henning NAGEL 2 , Matias RÖYTTÄ 3 , Beate SODEIK 2 ,Hukkanen VEIJO 11 Department <strong>of</strong> <strong>Virology</strong>, University <strong>of</strong> Turku, Turku, FINLAND;2 Institute <strong>of</strong> <strong>Virology</strong>, Hannover Medical School, Hannover, GERMANY;3 Department <strong>of</strong> Pathology, Turku, FINLANDHerpes simplex viruses (HSV) have several features making them goodgene therapy vector candidates, especially for the treatment <strong>of</strong> central nervoussystem (CNS) disease. Experimental autoimmune encephalomyelitis(EAE) is an inflammatory autoimmune disease <strong>of</strong> the CNS and is theprimary disease model for multiple sclerosis (MS). Demyelination andinflammation are hallmarks <strong>of</strong> these diseases. Leukemia inhibitory factor(LIF) has neuroprotective properties and limits demyelination and oligodendrocyteloss. Here, we studied the effect on EAE <strong>of</strong> an HSV 1 vectorexpressing LIF.We constructed BAC derived HSV 1 vectors, deleted <strong>of</strong> the neurovirulencegene gamma34.5, encoding LIF or a control gene ZeoR. Female SJL miceinduced for EAE, were treated with 10E7 PFU vector intracranially. ClinicalEAE scores were observed daily. Brain, spinal cord and trigeminalganglion (TG) samples were collected on days 9, 14 and 21 p.i. for microscopicalanalysis and for the study <strong>of</strong> viral and host gene expression withquantitative RT PCR.The HSV LIF significantly alleviated the clinical scores from day 15 p.i.when compared to untreated mice. Virus replication was detected in brain,TGs and spinal cords by virus culture, PCR and IVIS luminometry. Wedetected an increase in oligodendrocytes in the brains <strong>of</strong> HSV LIF treatedmice during recovery. The HSV LIF therapy also lowered IL 17 and inducedIL 10 and TGF beta mRNA expression in the brains, indicating a shiftfrom Th17 towardsaTregulatory cell response. We acknowledge MartinMesserle and Eva Maria Borst for their advice on BAC mutagenesis.REF 0133Efficient generation gf Human Natural Killer Cells by viral transformationBernhard FLECKENSTEIN 1 , Armin ENSSER 1 , Benjamin VOGEL 1 ,Karin TENNERT 1 , Florian FULL 1,21 Institut für Klinische und Molekulare Vi<strong>rologie</strong>, Universitätsklinikum,Friedrich Alexander Universität, Erlangen, GERMANY; 2 Dept. <strong>of</strong> Microbiologyand Immunobiology, Harvard Medical School, Boston, MA, USAThe short lifespan <strong>of</strong> human natural killer (NK) cells is one <strong>of</strong> the majorobstacles in human NK cell research. Few continuous NK like cell linesexist and all except one derive from NK cell leukemia or lymphomapatients.Here we describe the in vitro growth transformation <strong>of</strong> functional humanNK cells by Herpesvirus saimiri (HVS), a primate rhadinovirus. Infection<strong>of</strong> human NK cells was revealed by using a selectable recombinant HVS H2Kk marker virus. This led us to the discovery that HVS infection resultsin continuous expansion <strong>of</strong> NK cells; these growth transformed NK celllines have a strict requirement for exogenous IL 2, they express effectormolecules and exert cytolytic activity similar to freshly isolated NK cells.HVS transformation <strong>of</strong> human NK cells can provide large numbers andspecific populations <strong>of</strong> NK cells for biochemical and functional analysis.Further studies will have to address an eventual therapeutic potential andtheir value in generating cell lines <strong>of</strong> NK cell deficiencies and in studyingvirus interactions with human innate immunity.REF 0134Lentiviral delivery <strong>of</strong> multiple short hairpin RNAS: A gene therapyapproach for HIV 1 infectionFrancesca SPANEVELLO 1 , Arianna CALISTRI 1 , Claudia DELVECCHIO 1 , Barbara MANTELLI 1 , Saverio Giuseppe PARISI 1 , GiorgioPALÙ 1 , Marina CAVAZZANA CALVO 2,3 , Cristina PAROLIN 41 Department <strong>of</strong> Molecular Medicine, University <strong>of</strong> Padova, Padova,ITALY; 2 René Descartes University <strong>of</strong> Paris, Paris, FRANCE; 3 NeckerChildren’s Hospital, Paris, FRANCE; 4 Department <strong>of</strong> Biology, University<strong>of</strong> Padova, Padova, ITALYHIV gene therapy holds considerable promise as an alternative or complementarystrategy to drug based antiretroviral therapy for the treatment<strong>of</strong> HIV 1 infection. Among the antiviral genes designed to inhibit HIV 1replication, short hairpin RNAs (shRNAs) mediating knockdown <strong>of</strong> viralgenes or cellular co factors have proven to be effective. In this study, weobtained lentiviral vectors expressing shRNAs targeting the viral genesvif, tat/rev and the cellular gene CCR5. To account for HIV 1 variability,we adopted combinatorial RNA interference approaches based onthe simultaneous expression <strong>of</strong> the different shRNAs either from distinctpromoters or as an extended shRNA. We tested and compared three differentpromoters for shRNA expression: the human U6, 7SK and H1polymerase III promoters. The safety and the biological activity <strong>of</strong> thedeveloped vectors were investigated both in cell lines and in human primarycells. Our results indicate that shRNA efficacy is strictly dependenton the employed promoter. Moreover, single promoter activity can differconsiderably among the selected combinatorial RNAi platforms. Ourfindings show that the insertion <strong>of</strong> up to three promoter/shRNA cassettesinto a single vector did not negatively affect the inhibitory effect <strong>of</strong> eachindividual shRNA. These results gain insights into the design constrainsand the promoter selection required to express a combination <strong>of</strong> antiviralshRNAs and confirmed the potential <strong>of</strong> RNAi to inhibit HIV 1replication.S96 Vi<strong>rologie</strong>, Vol 17, supplément 2, septembre 2013
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