733. Integration Site Distribution and Clonal Selection Related to the Insertion of Lentiviral SIN-Vectors

Abstract

Previous observations of retroviral vector induced oncogenesis in preclinical models and in a clinical SCID-X1 gene therapy study have prompted large-scale integration site analysis. In vitro and in vivo studies indicate that lentiviral vectors may lower the risk of insertional mutagenesis compared to common retroviral vectors. Several studies could show that integration site distribution is vector dependent (MLV, HIV, SIV, ASLV) and the type of LTR may play a crucial role. Here, we have analyzed the possible influence of distinct internal promoters and transgenes on integration site distribution and clone survival.To analyze the influence of different vector elements on target site selection of lentiviral vector integration we transduced HeLa cells with three different lentiviral SIN-HIV-based vectors (promoter: CMV, SFFV; transgene: GFP, Mertk; SFFV-vectors contained WPRE) and compared the integration site distribution (971 integration events) at various time points (1d, 30d, 60d).Overall integration site distribution of the three vectors showed no difference in integration frequency in RefSeq genes. 66–69% of the integration events were detected in RefSeq genes, 81–88% were in RefSeq genes including the surrounding 10kb of sequence. Chromosomal distribution analysis showed no significant difference between the vectors. Integration site comparison at 30 days and 60 days after transduction revealed over-representation of genes with specific molecular functions, most significantly with transferase activity, ATP binding or protein serine/threonine kinase activity. Interestingly, these categories showed a strong increase in significance over time, indicating that lentiviral SIN-vectors induce clonal selection in vitro. The clustering of more than 10% of the integrations as common integration sites (CIS) also points to a vector related selective advantage of individual clones. Our findings indicate for the first time that lentiviral SIN-vector architecture, potentially independently of internal vector elements, may influence the clonal growth of targeted cells. The character of such effects and any putative relevance of such genotoxicity for the in vivo situation will have to be investigated in detail for the role of individual vector/cell type configurations. Especially if strong internal promoter/enhancer combinations are used, even lentiviral SIN-vectors may not be completely biologically inert with relation to their genomic environment, and in vitro assays may be highly useful in early detection of such interactions.MS and CvK hold US-patent on LAM-PCR. Previous observations of retroviral vector induced oncogenesis in preclinical models and in a clinical SCID-X1 gene therapy study have prompted large-scale integration site analysis. In vitro and in vivo studies indicate that lentiviral vectors may lower the risk of insertional mutagenesis compared to common retroviral vectors. Several studies could show that integration site distribution is vector dependent (MLV, HIV, SIV, ASLV) and the type of LTR may play a crucial role. Here, we have analyzed the possible influence of distinct internal promoters and transgenes on integration site distribution and clone survival. To analyze the influence of different vector elements on target site selection of lentiviral vector integration we transduced HeLa cells with three different lentiviral SIN-HIV-based vectors (promoter: CMV, SFFV; transgene: GFP, Mertk; SFFV-vectors contained WPRE) and compared the integration site distribution (971 integration events) at various time points (1d, 30d, 60d). Overall integration site distribution of the three vectors showed no difference in integration frequency in RefSeq genes. 66–69% of the integration events were detected in RefSeq genes, 81–88% were in RefSeq genes including the surrounding 10kb of sequence. Chromosomal distribution analysis showed no significant difference between the vectors. Integration site comparison at 30 days and 60 days after transduction revealed over-representation of genes with specific molecular functions, most significantly with transferase activity, ATP binding or protein serine/threonine kinase activity. Interestingly, these categories showed a strong increase in significance over time, indicating that lentiviral SIN-vectors induce clonal selection in vitro. The clustering of more than 10% of the integrations as common integration sites (CIS) also points to a vector related selective advantage of individual clones. Our findings indicate for the first time that lentiviral SIN-vector architecture, potentially independently of internal vector elements, may influence the clonal growth of targeted cells. The character of such effects and any putative relevance of such genotoxicity for the in vivo situation will have to be investigated in detail for the role of individual vector/cell type configurations. Especially if strong internal promoter/enhancer combinations are used, even lentiviral SIN-vectors may not be completely biologically inert with relation to their genomic environment, and in vitro assays may be highly useful in early detection of such interactions. MS and CvK hold US-patent on LAM-PCR.