Multiplex Engineering of Human CD34+ HSPCs Enables Dual Gene Knock-out While Maintaining High Engraftment Potential and Safety

Genome engineering of allogeneic hematopoietic stem cell transplants (HSCT), in which leukemia-specific cell surface antigens are removed by editing from the donor graft, may provide high therapeutic index and safety in combination with post-transplant targeted therapeutics. This strategy allows the therapeutics to specifically target leukemic cells by sparing the gene-edited graft, and thereby enable the next generation of HSCTs for treatment of acute myeloid leukemia (AML). So far, no single target antigen is known to be expressed in 100% of AML blasts, leukemic stem cells, or even in affected patients. Therefore, use of combinatorial therapies targeting multiple antigens may represent a potential paradigm shift in the standard of care for AML. Multiplex editing of HSCT in which two or more target antigens are removed from the same cell would enable combinatorial targeting while simultaneously protecting the engineered graft from on-target, off-tumor toxicity. Here we report proof-of-concept data that multiplex editing with two independent CRISPR/Cas9 ribonucleoproteins (RNPs) targeting genetic loci on chromosomes 11 and 19 robustly maintains long-term in vivo engraftment potential with persistent gene edits at both target sites, with minimally detectable translocations in bone marrow samples 16 weeks after engraftment in NSG mice. The overall viability of dual-edited input CD34+ HSPCs post gene editing was >75% and the proportion of CD34+CD38-CD45RA-CD90+CD49f+ putative long-term hematopoietic stem cells (LT-HSCs) in input cells were similar (1-2%) between unedited and multiplex-edited cells. In vivo engraftment potential of multiplex-edited cells was maintained long-term, with average chimerism of hCD45+ cells in mice transplanted with dual knock-out (KO) HSPCs similar to those transplanted with unedited HSPCs (53.36±11.80% versus 47.45±18.03%, respectively). Dual KO cells also maintained normal differentiation potential as judged by multilineage reconstitution in vivo and in vitro colony forming unit (CFU) assay, indicating the dual RNP delivery process does not lead to any observable impact on the multipotency of the engineered graft. Quantification of on-target editing by multiplexed NGS amplicon sequencing revealed no reduction in total editing between dual KO input and bone marrow cells, indicating the gene modifications in dual engineered cells persist post-engraftment.