The long-term objective of this proposal is to achieve highly efficient gene editing and maximize engraftment of genetically engineered HIV-resistant hematopoietic stem/progenitor cells (HSPCs) to achieve HIV cure. HSPC based gene therapy that results in HIV-resistant progenies from engrafted patient derived blood stem cells can provide long-term protection against HIV with the promising possibility of achieving a cure. However, previous HSPC based gene therapy strategies did not provide clear therapeutic effects. Recently, the ease and versatility of the CRISPR/Cas9-mediated gene editing technology has spurred an immense interest in using it to edit the CCR5 gene in HSPC based anti-HIV gene therapy strategies. Nonetheless, a successful cure by an anti-HIV HSPC based gene therapy still has to overcome 2 major barriers: 1) insufficient levels of CCR5 gene modification in HSPC and 2) poor engraftment of gene modified HSPCs. Firstly, to maximize the levels of CCR5 gene modification in HSPCs without the residual genotoxicity associated with DNA based delivery platforms, we developed a novel RNA-based Sendai virus (SeV) vector for highly efficient CRISPR/Cas9-mediated gene editing of human HSPC. Our SeV-Cas9 transduces and edits these HSPCs with unprecedented efficiency (up to ~80% at the CCR5 locus). Secondly, to improve the engraftment of gene modified HSPCs, we have developed a novel in vivo chemoselection strategy that employs 6-thioguanine (6TG), a clinically available prodrug that requires hypoxanthine-guanine phosphoribosyl-transferase (HPRT) for activity. Editing of HPRT allows for pre- conditioning and post-transplant in vivo chemoselection of HPRT-deficient HSPC in the humanized bone marrow, liver and thymus transplanted (hu BLT) mouse model. We hypothesize that efficient SeV delivered CRISPR/Cas9 mediated editing of CCR5 and HPRT genes in HSPC followed by in vivo selection with 6TG will maximize the engraftment of HIV-resistant HSPC. In toto, our strategy functionally maximizes ex vivo HPSC gene modification and in vivo engraftment efficiency, which will continuously provide sufficient numbers of HIV- resistant progenies that might ultimately replace HIV latently infected cells in hu BLT mice. To achieve our overall goal, and to speed the clinical translation of our gene therapy strategy, we propose the following Specific Aims:
AIM 1. Develop a clinically relevant SeV vector for highly efficient CRISPR/Cas9 mediated gene modification of HSPC.
AIM 2. Maximize the efficiency of engraftment by a safe and effective pre-conditioning and selection strategy for CCR5 and HPRT modified HSPC in hu BLT mice.
AIM 3. Investigate HIV inhibition by engraftment of CCR5 and HPRT modified HSPC in hu BLT mice.
Finding a new HIV cure therapy is a major focus of NIH HIV research program. Our Sendai virus vector can genetically modify hematopoietic stem cells to confer HIV-resistance at unprecedented efficiencies. This overcomes the major obstacles in the field for anti HIV stem cell based gene therapy, and allows for the possibility of a HIV cure ? the successful long-term drug-free control of HIV disease.