735. VITA, a Method for the Analysis of Lentiviral Integration Patterns
Authors: Julie Mantovani, Nathalie Holic, Olivier Danos, Javier Perea
Journal: Molecular Therapy (2006)

Retroviral vectors integrate into the genome, providing an efficient, stable and durable transgene expression in diverse cellular types. However, integration can result in cis-acting molecular interactions that deregulate the expression of either the transgene or neighboring host cell genes. In particular, integration of some MLV derived vectors close to protooncogenes has been shown to induce tumorigenesis in mouse and, more worryingly, in humans.The analysis of retroviral integration patterns has recently been approached by several groups who have shown that integration is not random. Both retroviral and lentiviral vectors integrate preferentially into transcribed regions, RV close to the transcription start and LV along the transcription unit. The transcriptional activity of targeted cells can also influence the integration pattern. Previous knowledge of integration site profiles of gene therapy vectors under the various metabolic conditions of the target cells seems to be an essential criterion in the choice of the vectors and the conditions of gene transfer.In addition to integration sites-transcription units correlation, other less obvious biases with other genomic features may exist and could be discovered with a larger integrome analysis.We have developed a new high-throughput method for the rapid analysis of high numbers of lentiviral integration sites called Vector Integration Tag Analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites which are used as signatures of the integration events. We use the capacity of the tagging MmeI enzyme to cleave DNA at a defined distance of its recognition site, in order to generate 21 and 22-bp long tags. This length is sufficient, in most of cases to identify without ambiguity a unique position in the human genome. Concatenation, cloning and sequencing of tags allow to obtain information about 20–25 insertion sites in a single sequencing reaction.Using VITA, 1349 different insertion sites of a lentiviral vector in HeLa cells were mapped from only 487 sequencing reactions. Accuracy of VITA was tested in a cellular clone with 8 well known lentiviral insertions. Less than 2 % of obtained tags were false positives. Retroviral vectors integrate into the genome, providing an efficient, stable and durable transgene expression in diverse cellular types. However, integration can result in cis-acting molecular interactions that deregulate the expression of either the transgene or neighboring host cell genes. In particular, integration of some MLV derived vectors close to protooncogenes has been shown to induce tumorigenesis in mouse and, more worryingly, in humans. The analysis of retroviral integration patterns has recently been approached by several groups who have shown that integration is not random. Both retroviral and lentiviral vectors integrate preferentially into transcribed regions, RV close to the transcription start and LV along the transcription unit. The transcriptional activity of targeted cells can also influence the integration pattern. Previous knowledge of integration site profiles of gene therapy vectors under the various metabolic conditions of the target cells seems to be an essential criterion in the choice of the vectors and the conditions of gene transfer. In addition to integration sites-transcription units correlation, other less obvious biases with other genomic features may exist and could be discovered with a larger integrome analysis. We have developed a new high-throughput method for the rapid analysis of high numbers of lentiviral integration sites called Vector Integration Tag Analysis (VITA). VITA is based on the identification of Genomic Tags associated to the insertion sites which are used as signatures of the integration events. We use the capacity of the tagging MmeI enzyme to cleave DNA at a defined distance of its recognition site, in order to generate 21 and 22-bp long tags. This length is sufficient, in most of cases to identify without ambiguity a unique position in the human genome. Concatenation, cloning and sequencing of tags allow to obtain information about 20–25 insertion sites in a single sequencing reaction. Using VITA, 1349 different insertion sites of a lentiviral vector in HeLa cells were mapped from only 487 sequencing reactions. Accuracy of VITA was tested in a cellular clone with 8 well known lentiviral insertions. Less than 2 % of obtained tags were false positives.