333. Preferential Targeting of Transcriptional Start Sites after Retroviral-Mediated T-Cell Gene Therapy for Adenosine Deaminase Deficiency

Abstract

Integrating gene transfer vectors based on murine oncoretroviruses have been widely used in clinical gene therapy trials to attempt correction of genetic diseases and provide novel therapeutic approaches for HIV-1 infection and cancer. However, the recent occurrence of uncontrolled lymphoproliferation in patients treated with retroviral gene transfer for X-linked severe combined immune deficiency has substantiated the risk of insertional oncogenesis associated with the clinical use of such vectors. Data on safety of retroviral T-cell-directed gene transfer is lacking, therefore we set out to analyze the pattern of retroviral integrations in a patient affected with adenosine deaminase (ADA) deficiency who received eleven infusions retroviral vector-transduced cells between 1990 and 1992. No adverse events have been observed in this patient who still carries |[sim]|15|[ndash]|20% of gene-corrected peripheral blood lymphocytes. Genomic DNA was prepared from stored lymphocyte samples dating 1991, 1992, 1995, 1998, 2000, and 2003. By inverse PCR and ligation-mediated PCR, we have identified |[sim]|760 bona fide insertion sites. Search for homology within the human genome using BLAT returned |[sim]|275 unique hits that involved a variety of genes, including transcription factors and oncogenes (e.g. RUNX1, STAT5, FYN). We found high prevalence of integrations within 10 kb upstream and downstream of genes that was significantly different (p<0.001) from the distribution of 2000 randomly generated data sets. We also observed a particularly high prevalence of integrations within a 10 kb range around the 5' region of reference sequences (RefSeq). In addition, DNA regions targeted by retroviral integration were found to show higher sensitivity to DNase compared to random regions of the genome.