Promoters and insulators: creating a safer integrating vector

UnrestrictedThe permanent integration of genes into the cellular genome using retroviral vectors holds promise for the correction of diseases caused by single gene defects. However, the semi-random pattern of integration is complicated by the risk of transactivation of cellular proto-oncogenes in close proximity to the integration site. Adverse events resulting from vector-mediated insertional mutagenesis have been documented in clinical and preclinical gene therapy studies and clearly indicate the need for improved safety mechanisms.; We have chosen to investigate two cis-regulatory elements, whose incorporation into an integrating vector may reduce vector-mediated genotoxicity. As the strong enhancer-promoter of the retroviral long terminal repeat (LTR) has been implicated in the process of insertional mutagenesis, we sought to identify promoters with low transactivation potential. Results from reporter gene expression assays revealed that the risk of transactivation is positively correlated with the transcriptional strength of a promoter, making it difficult to identify an element which may improve vector safety without sacrificing expression of the therapeutic transgene.; A second cis-regulatory element with the potential to improve vector safety is the insulator, which is defined by enhancer-blocking activity. The incorporation of an insulator into an integrating vector may isolate the neighboring cellular genome from any strong enhancer-promoter elements contained within the integrated provirus. However, direct evidence for the effectiveness of insulators within vectors is limited and no systematic comparison has been performed to identify the most active elements. Our studies have identified two insulators, the murine Igf2/H19 imprinting control region and the murine T-cell receptor alpha/delta HS1' element, with the capability to abolish enhancer-promoter interactions within a standard gene expression assay. However, we caution that these insulators are complex, relatively uncharacterized, elements which must be further analyzed in the context of an integrating vector.; Finally, using zinc finger nuclease technology to achieve site-specific integration within the cellular genome, we have begun to re-examine the transactivation potential of LTRs within the native genomic environment. Our goal is to utilize this system as a screening tool for the assessment of vector-mediated transactivation and the identification of insulators, or other elements, which may minimize vector genotoxicity