Multiple Integration Events into Several Putative Oncogenes Was Required To Cause Leukemogenesis in Two Primate Recipients of RCR Contaminated Stem-Cells.

Abstract

Integrating vectors are essential for stem-cell and T cell gene-therapy approaches to enable passage of the transgene to subsequent progeny, and retroviral vectors have been the commonly utilized for clinical applications. However, insertional events can potentially cause malignant transformation of transduced cells. In 1991, adult rhesus macaques received CD34+ cells transduced with a NEO expressing retroviral vector which was inadvertently contaminated with ampho-pseudotyped replication competent retrovirus(RCR). Three primates (88053, 15445 and 88049) developed a rapidly progressive lymphoid neoplasm 6–7 months after transplantation characterized by diffuse lymphadenompathy, hepatosplenomegaly and a large thymic mass. Immunohistochemistry of the neoplasm revealed a CD8+ CD2+ alphabeta TCR+ phenotype strongly positive for RCR. The neoplasm was preceeded by increasing amphotropic envelope detectable in peripheral blood by PCR which was ultimately detectable in 100% of peripheral blood leukocytes. Tissue is available for study from primates 15445 and 88049. Genomic Southern blotting revealed multiple integrants, more in 15445 than in 88049. Using a simplified technique (FLEA-PCR) for generating libraries of Integrant-Host-Junctions (IHJ), we studied leukemia samples from these two (primates 15545 and 88049). So far we have characterized 52 different flanking sequences from primate 15545 of which we could match 43 genomic loci. We have isolated 39 different flanking sequences from primate 88049 of which we could match 29 genomic loci. Of the characterized integration sites from 15445 the following potential oncogenes could be affected: DKK3, CUTL1, RUNX1, RUNX3, TCF7L2, FOXP1, MTIF2, GAB1, CST7, CABIN1 and RHOH. Of the characterized integration sites from 88049 the following potential oncogenes could be affected: LY86, JARID2, TNIK(TRAF2 and NCK interacting kinase), SMT3H2, PTK2B, MSF, FBXL10 and RAPH1. Both primate leukemia samples contain an integration site just 5 to the gene for Neuronal acetylcholine receptor protein (CHRNA9). RNA derived from leukemic bone marrow from both animals and involved thymic tissue from one was subjected to microarray analysis and compared to that of control tissue using the Affymetrix human genome U133 plus 2 array. The gene expression profile was similar among diseased tissues, and efforts to correlate the expression profile with genes potentially dysregulated by integration events is ongoing. Together with some subjects of the recent X-SCID study, these macaques represent the only cases of leukemogenesis caused by vector induced insertional mutagenesis in primates. Our findings confirm the assumption that with an inert transgene (NEO, RCR), multiple integrations effecting different oncogenes are required to result in malignant transformation of transduced cells.