Efficient Multiplex Gene Editing of CD33 and CLL-1 in Human Hematopoietic Stem Cells Enables the Potential of Next-Generation Transplants for AML Treatment

Acute myeloid leukemia (AML) is a heterogenous disease and the most common form of adult acute leukemia, characterized by abnormal clonal expansion, with median 5-year survival rate <30%. Allogeneic hematopoietic stem cell transplant (HCT) has been the standard of care for high-risk AML patients, yet ~40% of patients relapse post-HCT, highlighting the need for new therapeutic approaches such as immunotherapy. CD33 and CLL-1 are highly expressed in AMLs and we confirmed by flow cytometry, from 26 AML patient samples, that CD33 is expressed on >94% of blasts/leukemic stem cells (LSCs) while CLL-1 is expressed on >85% of blasts/LSCs, with antigen density for both at levels that are targetable by immunotherapies (1283-2260 molecules per blast). This suggests that immuno-targeting both CD33 and CLL-1 can potentially address AML heterogeneity and reduce chances of tumor resistance. However, CD33 and CLL-1 are also highly expressed on normal hematopoietic cells, thus impeding the use of therapies targeting these antigens due to on-target, off-tumor myelotoxicity. Therefore, we propose to delete both CD33 and CLL-1 from hematopoietic stem cell grafts, thereby confining these antigens to AML cells should relapse occur post-HCT and enable subsequent targeted immunotherapy. Mobilized peripheral blood CD34⁺ human hematopoietic stem and progenitor cells (hHSPCs) were sequentially CRISPR/Cas9 gene edited against CD33 and CLL-1 to reduce simultaneous double stranded breaks at both loci. This temporally staggered editing strategy resulted in high levels of biallelic deletion of both genes in >80% cells. Loss of CD33 and CLL-1 proteins did not impact cell viability (>90%) or the distribution of hematopoietic stem cells, multipotent progenitors, myeloid or lymphoid progenitors nor did this impact editing frequencies in each of these compartments. Multiplex-edited hHSPCs showed no defect in myeloid or erythroid potential and could in vitro differentiate into granulocytic or monocytic lineages similar to the control edited cells. The myeloid cells derived from multiplex-edited hHSPCs also preserved their phagocytic function and cytokine secretion ability. Importantly, CD33 and CLL-1 multiplex-edited hHSPCs xenotransplanted into NOD-scid IL2Rγ^null (NSG) mice showed successful long-term engraftment with no impact on multilineage reconstitution, supported by equal distribution of ten hematopoietic lineages between multiplex and control groups. CD33 and CLL-1 antigens were simultaneously absent from >95% of myeloid cells due to genome editing (CD33⁺/CLL-1⁺ monocytes: 0.9±0.4% in multiplex-edited versus 71.5±1.4% in unedited arms; CD33^-/CLL-1^- monocytes: 92.2±2.5% in multiplex-edited versus 1.1±0.3% in unedited arms). This data highlighted that long-term persistence of high levels of biallelic editing at both genes had no impact on myeloid differentiation in vivo. Next-generation sequencing of targeted amplicons revealed persistence of high gene editing frequency in the bone marrow at 16 weeks post-engraftment. Together, these data indicated that gene modifications in dual engineered hHSPCs can persist long-term post-engraftment without counter-selection for these cells or their progenies. Lastly, CD33 and CLL-1 dual edited cells were protected from the cytotoxicity of chimeric antigen receptor (CAR)-T cells targeting CD33 and/or CLL-1. Here we showed that CD33 and CLL-1 multiplex-edited hHSPCs sustain functional hematopoiesis with high levels of editing at both loci and that the dual edited cells were resistant against CD33 and/or CLL-1 immunotherapies, validating the core concept of killing tumor cells while sparing the edited graft. Our findings can enable a next-generation of AML treatment by pairing multiplex-edited hHSPCs with subsequent multi-specific immunotherapy to obviate concerns around tumor heterogeneity and potential escape mechanisms.

Xavier-Ferrucio:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Angelini:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Krishnamurthy:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Patel:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Pettiglio:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Collingsworth:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Halfond:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Etchin:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Keschner:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Mundelboim:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Qiu:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Wang:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ge:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lydeard:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lin:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chakraborty:Vor Biopharma: Current Employment, Current equity holder in publicly-traded company.