rologie i - European Congress of Virology

5 th European Congress of Virologyreprogramming with AdV was inefficient. In our experience, RV systemrepresented an affordable in house method for reprogramming, leading toiPSCs generation with sufficient efficiency although with the risk of mutagenesisdue to vector integration. The SV based protocol was an efficient,virus free method, with consistent capacity of reprogramming, but with thedisadvantage of being more expensive than the other methods. AdV couldrepresent the method of choice, being integration free and easy to produce,but more efforts should be done to improve reprogramming conditions.REF 013Lentiviral and fluorescent reporter vectors for genome editing usingzinc finger nucleasesNadia GUETTARI, Monika MINNER, Qiaohua KANG, YongmeiFENG, Gregory D. DAVISSigma Aldrich Biotechnology, St. Louis, MO, USAZinc finger nuclease (ZFN) design and manufacturing at Sigma Aldrichhas successfully addressed a wide variety of genome editing applicationsranging from gene knockout to more challenging, site restrictedprojects requiring targeted double strand breaks near disease SNPs andother small genetic elements. Despite major advances in ZFN engineering,ZFN delivery and expression remain challenging for particular celltypes. Genetic tools such as shRNA and microRNA have benefited greatlyfrom lentiviral gene expression, enabling manipulation of primary cells,non dividing cells, and a variety of other difficult cell types. Lentiviraltiters are known to be negatively impacted by large genetic payloads, andsubcloning of repetitive sequences into lentiviral vectors has often resultedin vector recombination and instability (Holkers et al., 2013). Due to theircompact protein structure and low content of repetitive sequence, ZFNshave been successfully expressed and implemented using lentiviral vectors(Lombardo et al., 2007). Here we present data showing the use of lentiviraldelivery methods in genome editing applications using CompoZr ZFNs. Asan additional alternative approach to overcoming low delivery and expressionlevels in particular cell types, we have created vectors in which ZFNexpression is linked to fluorescent reporters. This reporter linked expressionformat enables enrichment of cell populations that have undergoneboth efficient transduction and subsequent high level expression of ZFNtransgenes and can substantially increase genome editing frequenciesREF 014Measles virus glycoprotein pseudotyped lentiviral vectors transduceprestimulated and resting long-term repopulation hCD34+ cells at anefficiency without precedentCamille LEVY, Anais GIRARD, Fouzia AMIRACHE, CeciliaFRECHA, Caroline COSTA, Didier NEGRE, François-Loïc COSSETand Els VERHOEYENCIRI, EVIR team, Lyon, FRANCEHematopoietic stem cell (HSC) based gene therapy holds promise for thecure of many inherited and acquired diseases. The field is now movingtowards the use of lentiviral vectors (LVs): clinical trials now use VSV-G-LVs at high doses combined with strong cytokine-cocktails to obtaintherapeutic transduction levels with the risk of compromising ‘HSC’ character.Previously, we have shown that measles virus (MV) glycoproteindisplaying LVs (MV-LVs) were able to transduce efficiently stimulatedand resting T and B cells. We now evaluated these MV-LVs for hCD34+cell transduction after SCF+TPO prestimulation in order to better preservethe ’HSC’ characteristics. After a single application at a low vector doses,MV-LVs stably transduced 100% of stimulated hCD34+ cells, where VSV-G-LVs reached 5-10%. Moreover, these MV-LVs allowed also efficienttransduction of 50-70% quiescent hCD34+-cells, an efficacy without precedent.We found that this high transduction level was correlated withthe expression of the MV receptor, CD46. Importantly, reconstitution ofNSG mice with MV-LV transduced hCD34+ cells resulted in transductionlevels close to 100% for all analyzed myeloid and lymphoid cell lineagesincluding CD34+ cells in the bone marrow. Moreover, this transductionpattern was confirmed in secondary mice engraftments. Together, theseresults strongly suggest that the MV-LVs efficiently transduce true HSCsat extremely high levels. This paves the way to HSC-based gene therapyof multiple diseases such as Fanconi Anemia, for which high level HSCcorrection is needed to be successful.REF 015Baboon retrovirus envelope pseudotyped LVs allow efficient transductionof progenitor T cells, thymocytes and adult T and B cellsAnais GIRARD, Fouzia AMIRACHE, Caroline COSTA, Camille LEVY,Dimitri LAVILLETTE, Didier NEGRE, François-Loïc COSSET and ElsVERHOEYENCIRI EVIR team, Lyon, FRANCEEfficient gene transfer into quiescent T and B lymphocytes for gene therapyor immunotherapy purposes may allow the treatment of severalgenetic dysfunctions of the hematopoietic system, such as immunodeficiencies,and the development of novel therapeutic strategies for cancersand acquired diseases. Previously, we have shown that measles virus gpsLVs (MV-LVs) were able to transduce efficiently stimulated or resting Tand B cells. We now showed that the newly engineered Baboon retrovirusenvelope pseudotyped LVs (BAEVgp-LVs) allowed in addition to hematopoieticstem cell transduction, high level transduction of activated T andB cells and resting B cells, where VSVG-LVs fail. Moreover, both naiveand memory cells were efficiently transduced and their phenotypes conserved.Since the thymus plays a key role in the induction of self-toleranceand by intra-thymus injection of an LV in situ correction of a geneticimmunodeficiency might be envisaged, it is important to develop vectorsfor efficient thymocyte transduction. BAEVgp-LVs allowed high leveltransduction of the thymocytes in the early stages of development with preferencefor early thymic progenitor T cells while MVgp-LVs, showed anequivalent transduction level of all thymic subpopulations. BAEVgp-LVsare especially superior over VSVG-LVs for the transduction of the veryimmature T cell progenitor subpopulation, which is long-lived in vivoand might confer a permanent therapeutic effect. Currently, the vectorsare tested for intra-thymic transduction in human immune system micemodel.S122 Virologie, Vol 17, supplément 2, septembre 2013