706. Retrovirus Integration Site Distribution in Long-Term Gene-Corrected Hematopoiesis of the SCID-X1 Gene Therapy Clinical Trial

Abstract

An amphotropic pseudotyped Simian Immunodeficiency Virus (SIV) based lentiviral vector developed previously efficiently transduces rhesus macaque cells (Blood 102: 989a, 2003) in contrast to HIV-based lentiviral vectors, which are blocked from optimal transduction of monkey cells due to species-specific factors. Due to current interest in using lentiviral vectors for gene therapy applications, this SIV vector system can be useful as a pre-clinical model for evaluation of safety and efficiency of such gene therapy protocols. Three animals previously reconstituted with transduced G-CSF+SCF mobilized peripheral blood CD34+ cells, exhibited genetically modified cells in multiple lineages. We have now extended the period of observation and these animals still have stable marking levels of 5–20% in different peripheral blood lineages at 6 to 9 months after transplant. The recent report of insertional mutagenesis within the LMO2 proto-oncogene following hematopoietic stem cell directed retroviral gene therapy prompted us to investigate the distribution pattern of the SIV vector genome in cells of the hematopoietic system. We used a modified LAM-PCR methodology to retrieve the insertion sites in circulating granulocytes and mononuclear cells of these animals at steady state, at least 6 months after transplant. The LAM-PCR generated products were sequenced. A sequence was considered a genuine insertion site if it contained the SIV-LTR and the linker sequences, and showed at least 90% identity to the NCBI build 34 of the human genome with a unique best hit in the BLAT ranking. So far we have sequenced a total of 241 insertion sites from these three animals (Table). 79 insertions sites were found to be in repetitive elements (Line, Sine, etc.) of the genome so the exact location of these insertion sites can not be ascertained. Of the remaining 162 insertion sites, 36 (22%) are located in non-coding regions of the genome and 126 (78%) are found to be located inside coding regions of the RefSeq set of genes, with 5 genes being independently interrupted twice. Four of the genes interrupted twice occurred in two different animals (EYA3: eyes absent 3; Scap1:Src family associated phosphoprotein 1; PACS1:phosphofurin acidic cluster sorting protein 1; and ITGAL:integrin alpha L precursor) and one occurred twice in the same animal (STK38:serine/threonine kinase 38). We are currently in the process of sequencing and analyzing additional insertion sites, and comparing the pattern to the over 400 insertion sites we have generated from rhesus macaques previously transplanted with CD34+ cells transduced with MLV-derived onco-retroviral vectors. These findings advance our understanding of lentiviral vector insertional mutagenesis risk in hematopoietic stem and progenitor cells of nonhuman primates, a highly relevant pre-clinical model, and ultimately will have significant impact on the design of lentiviral gene therapy trials.