Efficient manufacturing and engraftment of CCR5 gene-edited HSPCs following busulfan conditioning in nonhuman primates

Hematopoietic stem cell gene therapy has been successfully used for a number of genetic diseases and is also being explored for HIV. However, toxicity of the conditioning regimens has been a major concern. Here we compared current conditioning approaches in a clinically relevant nonhuman primate model. We first customized various aspects of the therapeutic approach, including mobilization and cell collection protocols, conditioning regimens that support engraftment with minimal collateral damage, and cell manufacturing and infusing schema that reflect and build on current clinical approaches. Through a series of iterative in vivo experiments in two macaque species, we show that busulfan conditioning significantly spares lymphocytes and maintains a superior immune response to mucosal challenge with simian/human immunodeficiency virus, compared to total body irradiation and melphalan regimens. Comparative mobilization experiments demonstrate higher cell yield relative to our historical standard, primed bone marrow and engraftment of CRISPR-edited hematopoietic stem and progenitor cells (HSPCs) after busulfan conditioning. Our findings establish a detailed workflow for preclinical HSPC gene therapy studies in the nonhuman primate model, which in turn will support testing of novel conditioning regimens and more advanced HSPC gene editing techniques tailored to any disease of interest. [Display omitted] Kiem, Peterson, and colleagues describe a workflow for autologous hematopoietic stem and progenitor cell (HSPC) transplantation in nonhuman primates, reflective of the most up-to-date clinical practices. As this approach represents a path to cure of HIV-1 infection, the authors’ results are contextualized within this disease model. After comparing conditioning and mobilization regimens, they show engraftment of up to 40% CCR5 CRISPR-edited HSPCs and persistence of CCR5-edited progeny in busulfan-conditioned animals. These results are relevant for ongoing HIV cure studies as well as clinical gene therapy approaches for cancer and myriad genetic diseases.