In vivo gene editing of human hematopoietic stem and progenitor cells using envelope-engineered virus-like particles (PHH cells)

Engineered virus-like particles (VLPs) are a promising technology for in vivo gene editing of human hematopoietic stem and progenitor cells (HSPCs). Here we design and test two different VLP envelopes for human HSPC editing in vitro and in vivo. The first is an optimized version of the baboon envelope BaEVTR, which efficiently transduces human HSPCs in vitro. We show that the optimized BaEVTR VLP enables in vivo editing of ß2 microglobulin in long-term human HSPCs (31% at 8 weeks after dosing) and editing of two hemoglobinopathy-relevant loci, BCL11A and HBG1/2 (26% and 7.5%, respectively, at 5 days after dosing), inducing fetal hemoglobin. Our second VLP design uses a CD133-targeted envelope designed to reduce the transduction of mature blood cells and achieves higher in vivo specificity for HSPCs compared to the optimized BaEVTR VLP. As avoiding delivery in filter organs such as the liver would enhance efficiency and safety, we also demonstrate that both VLPs avoid human hepatocytes in a humanized liver model. Overall design: We investigated the changes in nature of PHHs upon thawing and in vitro culture prior to vector exposure becase we observed negligible transduction in the humanized liver of the FRG mice. Gene expression profiling analysis of RNA-seq data was compared between freshly thawed PHH and PHH cultured in vitro for 24h (time of vector dosing for the in vitro titration experiments) or human hepatocytes freshly isolated from FRG mice prior to vector dosing.