414. Design of an HIV Vector for Site-Specific Gene Insertion

Abstract

Top of pageAbstract Pluripotent hematopoietic stem cells (HSCs) give rise to all lineages of mature blood cells, and serve as an important gene therapy target. While vectors derived from murine leukemia virus (MLV) are widely used to deliver genes into HSCs in various human gene therapy trials, their transduction efficiency of HSCs is rather low. This could be partly due to the quiescent nature of HSCs. Vectors derived from human immunodeficiency virus (HIV) are developed to circumvent the problem of low transduction efficiency of quiescent cells by the MLV vector. However, since both vectors integrate randomly into the host genome, it can lead to gene disruption or unanticipated gene activation through the enhancer or the promoter element present in the vector. The leukemia incidence induced by MLV vector integration near an oncogene in severe combined immunodeficiency (SCID) patients and recent discoveries of preferential integration by MLV and HIV near or within active genes underscore the importance of designing strategies for site-specific gene insertion. To address this issue, we have incorporated the Cre-loxP system into an HIV vector. We generated a human cell line containing a unique loxP site in the genome to serve as a target for gene insertion. A targeting vector containing the loxP site was produced from 293T cells and used to transduce the loxP-containing cell line. Transduced cells were pooled and the genomic DNA was prepared. PCR analysis of the genomic DNA showed site-specific insertion of the gene carried by the targeting vector into the loxP site in the host genome. From the number of the transduced cells, the efficiency of site-specific gene insertion was determined to be in the range between 0.04% and 0.004%. If we enriched for the site-specific gene insertion event by using an HIV vector deficient in integrase, the efficiency was increased to 0.7%. Our approach demonstrates the first step of designing HIV vectors for therapeutic gene targeting.