391. Effective Gene Therapy with Non-Integrating Lentivirus Vectors

Abstract

Gene therapy strategies need to achieve efficient transgene expression whilst minimizing toxic effects. Retrovirus vectors are very effective in several systems, but they have been shown to cause malignancies through insertional mutagenesis, one of the most serious side-effects identified to date in pre-clinical studies and clinical trials. The risk of insertional mutagenesis has been considered unavoidable because no gene expression has been demonstrated in vivo in the absence of retroviral integration. However, retrovirus and lentivirus vectors can be rendered integration-defective by mutation in the integrase coding sequence. Following reverse transcription, mutant vectors fail to integrate and instead accumulate in transduced cells as double-stranded DNA circles. These circular forms are non-replicating but very stable and will persist if not diluted by cell division. Contrary to the results from previous in vivo studies, recent reports have shown efficient transgene expression in vitro from integration-deficient lentivirus vectors in post-mitotic and growth-arrested cells. These vectors also led to transient transgene expression or, in combination with episomal systems, to stable expression in proliferating cells in vitro. Given these developments we decided to re-evaluate the proficiency of integration-defective lentiviral vectors for in vivo expression in rodents. We now show that integration-deficient HIV-1 vectors lead to highly efficient, long-term marker gene expression in post-mitotic ocular and brain tissues. We also demonstrate efficient rescue in two rodent models of retinal degeneration, the Rpe65rd12/rd12 mouse and the Mertk-deficient Royal College of Surgeons (RCS) rat. The relevance of these studies for clinical application is highlighted by the fact that we have been able to transduce excised human retinal pigment epithelium with integration-deficient vectors. In conclusion, we demonstrate that integration-deficient HIV-1 vectors transduce post-mitotic tissues in vivo as efficiently as their integrating counterparts. For clinical applications in quiescent tissues these non-integrating vectors should virtually abolish the risk of insertional mutagenesis-induced malignancy.