• This study is the first to show that genome-editing approaches can modify multilineage, long-term repopulating cells in a large animal model. • We demonstrate that the persistence of genome-edited hematopoietic stem cells can be tracked in vivo in a mutation-specific manner.Genome editing in hematopoietic stem and progenitor cells (HSPCs) is a promising novel technology for the treatment of many human diseases. Here, we evaluated whether the disruption of the C-C chemokine receptor 5 (CCR5) locus in pigtailed macaque HSPCs by zinc finger nucleases (ZFNs) was feasible. We show that macaque-specific CCR5 ZFNs efficiently induce CCR5 disruption at levels of up to 64% ex vivo, 40% in vivo early posttransplant, and 3% to 5% in long-term repopulating cells over 6 months following HSPC transplant. These genome-edited HSPCs support multilineage engraftment and generate progeny capable of trafficking to secondary tissues including the gut. Using deep sequencing technology, we show that these ZFNs are highly specific for the CCR5 locus in primary cells. Further, we have adapted our clonal tracking methodology to follow individual CCR5 mutant cells over time in vivo, reinforcing that CCR5 gene-edited HSPCs are capable of long-term engraftment. Together, these data demonstrate that genome-edited HSPCs engraft, and contribute to multilineage repopulation after autologous transplantation in a clinically relevant large animal model, an important step toward the development of stem cell-based genome-editing therapies for HIV and potentially other diseases as well. (Blood. 2016;127(20):2416-2426

We have focused on gene therapy approaches to induce functional cure/remission of HIV-1 infection. Here, we evaluated the safety and efficacy of the clinical grade anti-HIV lentiviral vector, Cal-1, in pigtailed macaques (Macaca nemestrina). Cal-1 animals exhibit robust levels of gene marking in myeloid and lymphoid lineages without measurable adverse events, suggesting that Cal-1 transduction and autologous transplantation of hematopoietic stem cells are safe, and lead to long-term, multilineage engraftment following myeloablative conditioning. Ex vivo, CD4+ cells from transplanted animals undergo positive selection in the presence of simian/human immunodeficiency virus (SHIV). In vivo, Cal-1 gene-marked cells are evident in the peripheral blood and in HIV-relevant tissue sites such as the gastrointestinal tract. Positive selection for gene-marked cells is observed in blood and tissues following SHIV challenge, leading to maintenance of peripheral blood CD4+ T-cell counts in a normal range. Analysis of Cal-1 lentivirus integration sites confirms polyclonal engraftment of gene-marked cells. Following infection, a polyclonal, SHIV-resistant clonal repertoire is established. These findings offer strong preclinical evidence for safety and efficacy of Cal-1, present a new method for tracking protected cells over the course of virus-mediated selective pressure in vivo, and reveal previously unobserved dynamics of virus-dependent T-cell selection.

Summary Myeloid-differentiated hematopoietic stem cells (HSCs) have contributed to a number of novel treatment approaches for lysosomal storage diseases of the central nervous system (CNS), and may also be applied to patients infected with HIV. We quantified hematopoietic stem and progenitor cell (HSPC) trafficking to 20 tissues including lymph nodes, spleen, liver, gastrointestinal tract, CNS, and reproductive tissues. We observed efficient marking of multiple macrophage subsets, including CNS-associated myeloid cells, suggesting that HSPC-derived macrophages are a viable approach to target gene-modified cells to tissues. Gene-marked cells in the CNS were unique from gene-marked cells at any other physiological sites including peripheral blood. This novel finding suggests that these cells were derived from HSPCs, migrated to the brain, were compartmentalized, established myeloid progeny, and could be targeted for lifelong delivery of therapeutic molecules. Our findings have highly relevant implications for the development of novel therapies for genetic and infectious diseases of the CNS.