245 hematopoietic stem cell gene delivery: high efficiency gene transfer to HSC in vivo with long term expression of anti HIV transgenes after intramarrow injection SV40 derived vectors

245 Hematopoietic Stem Cell Gene Delivery High Efficiency Gene Transfer to HSC In Vivo with Long Term Expression of Anti HIV Transgenes after Intramarrow Injection SV40 Derived Vectors 243 Insertion S[.]

243 Insertion Site Analysis Following Gammaretroviral Gene Therapy for Canine Leukocyte Adhesion Deficiency Reveals Lack of Genotoxicity Mehreen Hai,' Thomas R Bauer, Jr,' Laura M Tuschong,' Yuchen Gu,' Xiaolin WU,2 Dennis D Hickstein.' 'Experimental Transplant and Immunology Branch, National Cancer Institute, National Institutes ofHealth, Bethesda, MD; 2LaboratoJY0/ Molecular Technology, SAIC - Frederick, Inc, NCI at Frederick, Frederick, MD Recent successful hematopoietic stem cell gene therapy and gene marking studies using gammaretroviral vectors have typically involved a selective growth advantage for the transduced cells provided by either the therapeutic transgene or a myeloablative conditioning regimen Genotoxicity has occurred in both situations: three patients in thc French X-SCID clinical trial developed leukemia associated with retroviral insertions in the LM02 gene, and a rhesus macaque developed a granulocytic sarcoma accompanied by retroviral insertions in the BCL2-A I gene Providing the transduced cells with a growth advantage may have contributed to the genotoxicity However, in a recent non-myeloablative gene therapy trial for chronic granulomatous disease (CGD), where there was no selective advantage provided by the transduced gene, both patients developed progressive oligoclonality of retroviral marked cells accompanied by multiple retroviral insertions in EVII-MDS I, PROMI6 and SETBPI The development of clonal dominance despite a lack ofa selective growth advantage for the transduced cells in the CGD trial suggests that the vector design and/or transduction conditions may be important for the emergence ofclonal dominance To compare our results with those in the CGD trial, we analyzed the genotoxicity in our study of six dogs with canine leukocyte adhesion deficiency (CLAD), who were successfully treated using ex-vivo retroviral-mediated CDI8 gene transfer into autologous C034+ cells preceded by non-myeloablative conditioning Similar to the transgene in the CGO clinical trial, the therapeutic COl8 transgene does not confer a selective advantage upon the corrected leukocytes Polyclonality oftransduced cells was demonstrated by linear amplification-mediated PCR (LAM-PCR) using peripheral blood leukocyte DNA from all six dogs up to two years following infusion of gene-corrected cells; no predominant clone emerged over time Analysis of retrovira I integration sites (RIS) using ligation-mediated PCR (LM-PCR) identified 398 unique RIS, 53% of which were located within genes (compared to 39% expected from analysis of 10,000 randomly generated insertion sites, p