Non-genotoxic conditioning facilitates hematopoietic stem cell gene therapy for hemophilia A using bioengineered factor VIII

Abstract

Hematopoietic stem and progenitor cell (HSPC) lentiviral gene therapy is a promising strategy toward a lifelong cure for hemophilia A (HA). The primary risks associated with this approach center on the requirement for pre-transplantation conditioning necessary to make space for, and provide immune suppression against, stem cells and blood coagulation factor VIII, respectively. Traditional conditioning agents utilize genotoxic mechanisms of action, such as DNA alkylation, that increase risk of sterility, infection, and developing secondary malignancies. In the current study, we describe a non-genotoxic conditioning protocol using an immunotoxin targeting CD117 (c-kit) to achieve endogenous hematopoietic stem cell depletion and a cocktail of monoclonal antibodies to provide transient immune suppression against the transgene product in a murine HA gene therapy model. This strategy provides high-level engraftment of hematopoietic stem cells genetically modified ex vivo using recombinant lentiviral vector (LV) encoding a bioengineered high-expression factor VIII variant, termed ET3. Factor VIII procoagulant activity levels were durably elevated into the normal range and phenotypic correction achieved. Furthermore, no immunological rejection or development of anti-ET3 immunity was observed. These preclinical data support clinical translation of non-genotoxic antibody-based conditioning in HSPC LV gene therapy for HA.

Innate Immune Response to Viral Vectors in Gene Therapy

“…Preclinical investigations have explored the application of LV vector gene therapy for several diseases prior to clinical implementation. Promising outcomes have been observed in murine models of breast cancer [ 127 ], liver cancer [ 128 ], bladder cancer [ 129 ], hemophilia [ 130 , 131 , 132 , 133 ], and AD [ 134 , 135 , 136 ], following treatment with LV vectors.…”

Worked to the bone: antibody-based conditioning as the future of transplant biology

“…An anti-CD117-saporin ADC was examined in a haemophilia A mouse model [ 58 ]. While progenitor cell depletion was not observed 5 days after treatment, HSCs were markedly reduced.…”

Ligand-based targeting of c-kit using engineered γδ T cells as a strategy for treating acute myeloid leukemia

“…Up to 90% of AML patients have c-kit expression correlating with poor prognosis and resistance to chemotherapy (15,16), making this receptor an attractive target for malignant myeloid cell and leukemic stem cell (LSC) ablation. In fact, we (17) and others (18)(19)(20)(21)(22) have shown the continued use of c-kit as a target for non-genotoxic conditioning due to its expression on hematopoietic stem cells (HSCs). These therapies have proven safe for use in preclinical murine models with toxicities only observed within the hematopoietic compartment (17) despite limited c-kit expression on some organs outside of the hematopoietic compartment, like the cerebellum, female reproductive organs, and lungs.…”

“…In fact, we (17) and others (18)(19)(20)(21)(22) have shown the continued use of c-kit as a target for non-genotoxic conditioning due to its expression on hematopoietic stem cells (HSCs). These therapies have proven safe for use in preclinical murine models with toxicities only observed within the hematopoietic compartment (17) despite limited c-kit expression on some organs outside of the hematopoietic compartment, like the cerebellum, female reproductive organs, and lungs. Clinically, c-kit is the target of monoclonal antibody briquilimab, which has shown promise in treating patients with AML and myelodysplastic syndromes (MDS) in a Phase 1 clinical trial (NCT04429191), Fanconi anemia in a Phase 1/2 clinical trial (NCT04784052), severe combined immunodeficiency (SCID) in a Phase 1/2 clinical trial (NCT02963064), and sickle cell disease in a Phase 1/2 clinical trial (NCT05357482) with no treatment-related adverse events in 130 dosed subjects as of late September 2023 (23,24).…”