2. Promoter Trapping Reveals Significant Differences in Integration Site Selection between MLV and HIV Vectors

Abstract

Replication defective retroviral gene transfer vectors have been used as an effective method for transduction of hematopoietic stem cells (HSC). Recent advances allowed for gene transfer levels that made gene therapy applicable to use in clinical trials and successfully cured immunodeficient children. Unfortunately two patients in the trial developed T cell leukemia that was found to be associated with insertional mutagenesis caused by retroviral integration in the LMO2 gene (Hacein-Bey-Abina S., et al., Science, 302: 415). This unexpected result has caused a critical evaluation of retrovirus integration in clinically relevant large animal models to assess the likelihood of malignant transformation occurring in gene therapy trials in patients with different diseases and different transgenes. Here we describe the genomic location of more than 100 viral integrations after transplantation of lentivirally or oncoretrovirally transduced stem cells in non-human primates. CD34-enriched cells from G-CSF/SCF primed marrow or mobilized peripheral blood were transduced with oncoretroviral or lentiviral vectors and transplanted into the animals and peripheral blood DNA was prepared for retroviral integration analysis. Integration sites were cloned out of the bulk genomic DNA background by linear-amplification mediated PCR (LAM-PCR). A valid sequence was only scored as an actual integration site if it contained LTR sequence, linker sequence, and matched with at least 90% identity to the July 2003 assembly of the human genome as scored by the BLAT genome analyzing software. For both oncoretroviral and lentiviral integration sites analyzed, 40% mapped into a coding region for a RefSeq gene and that is approximately two times the frequency that was calculated in a computer simulation (Wu X., et al., Science, 300: 1749). This dramatic shift in distribution of integration sites to RefSeq genes indicates a non-random nature to retroviral insertion. Several sites are of interest in that the RefSeq genes are potential proto-oncogenes: melanoma differentiation associate protein-5 (MDA5), neuroblastoma-amplified protein, and RAB37 a member of RAS oncogene family. A variety of genomic locations have insertions, but no gene or region of the genome has been shown to have retroviral integrations in more than one animal. Importantly, all of these animals have been followed from about one to three years and are healthy with no leukemia or other hematologic abnormalities. This evaluation and other recent evidence (Dave U., et al., Science, 303: 333) suggest that retroviral integration into an oncogene is not sufficient for malignant transformation but requires other additional genetic alterations. This analysis and others like it are critical pre-clinical data to address the inherent risks of gene therapy and the effect that retroviral integration has on hematopoietic development.