In vivo gene editing of CCR5 in bone marrow using improved lentiviral vectors

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Project Summary/Abstract: While research efforts to develop highly-active antiretroviral therapy have greatly extended healthy lifespan for HIV-1 infected individuals receiving treatment, the virus poses unique challenges for the development of a long-sought after cure. Part of the viral lifecycle involves the permanent insertion of the HIV genome into a host cell's genetic material, establishing a long term population of HIV infected cells. New strategies to prevent spread from these cells must be developed. Unlike currently available antiviral drugs, anti-HIV gene therapy holds the potential for the treatment and even cure of HIV-1 infection. The gene therapy-mediated knockout of the HIV co-receptor CCR5 in HIV susceptible cells has shown potential in greatly reducing HIV viral loads in both mouse models of HIV disease and early phase human clinical trials. Surprisingly, in rare trial participants, this drop in viral load is sustained even when antiretroviral drug therapy is halted. However, all current anti-HIV gene therapy strategies rely on the separation and ex vivo manipulation of HIV susceptible cells, followed by re-infusion of these newly HIV-resistant cells back into patients. This approach requires access to advanced cell culture and bone marrow transplant facilities and is currently prohibitively expensive for the majority of HIV infected individuals living where the HIV epidemic continues to worsen. To address these technical, economic, and biological challenges, new approaches to inexpensive gene therapy must be developed. CD34+ hematopoietic stem and progenitor cells (HSPCs) are an attractive target for gene therapy, given their status as predecessors to all cells susceptible to HIV. However, these cells have shown resistance to genetic modification by currently available lentiviral vectors. In new data introduced in this application, I demonstrate previously unforeseen levels of gene delivery to CD34+ HSPCs in vitro, achieved by pseudotyping lentiviral vectors with the measles virus hemagglutinin and fusion glycoproteins. Mechanistic insights from this work has guided mutagenesis of vesicular-stomatitis virus glycoproteins which will be used to identify restriction factors in CD34+ HSPCs. In this application, I propose using these novel lentiviral vectors to evaluate the potential of in vivo transduction as a potential anti-HIV strategy, using a humanized mouse model of HIV infection. By direct intrafemoral injection of lentiviral vectors carrying the CRISPR/Cas9 system targeting CCR5, I will evaluate if this approach can achieve sufficiently high levels of gene knockout to protect from HIV challenge. If successful, this approach holds the potential to radically reduce the cost and technical difficulty of anti-HIV gene therapy.