Hematopoietic stem cell transplantation gene therapy for hemophilia a using non-genotoxic ANTI-CD117 immunotoxin conditioning

Abstract

Background & Aim Hematopoietic stem cell transplantation (HSCT) for coagulation factor VIII (fVIII) gene therapy is a potentially curative approach for the treatment of the most common severe bleeding disorder, hemophilia A (HA). Our laboratory has previously established the ability to produce stable therapeutic levels of fVIII in a mouse model of HA following irradiation or chemotherapy and transplantation of Sca-1+ cells, a population containing both HSC and progenitors, genetically modified ex vivo using recombinant lentiviral vector encoding a bioengineered high-expression fVIII variant, termed ET3. Conventional myeloablative regimens for HSCT such as total body irradiation and myeloablative alkylating chemotherapy are associated with adverse side effects, including sterility, hormonal dysregulation and genotoxicity. These risks represent a primary barrier to clinical translation of HSCT gene therapy for HA and many other monogenic diseases. Therefore, the development of targeted, non-genotoxic conditioning agents is critical. Methods, Results & Conclusion We have successfully implemented the use of an immunotoxin bioconjugate comprised of a monoclonal antibody against the stem cell factor receptor c-Kit (anti-CD117) and the ribosome-inactivating protein toxin saporin (sap) for non-genotoxic HSCT conditioning. We achieved consistent depletion of bone marrow LSK (lineage-negative Sca-1+ c-Kit+) and long-term HSC (LSK+ CD150+ CD48−) compartments five days after retro-orbital venous sinus injection of 0.5 mg/kg CD117-sap compared to control (59.2% LSK reduction; 38.9% LT-HSC reduction) in both C57BL/6J wild-type and HA mice. Following CD117-sap conditioning, HA mice were transplanted with 1 × 106 ET3-transduced Sca-1+ cells from GFP+ congenic mice. We observed robust mixed hematopoietic chimerism and myeloid engraftment at two and four weeks post-transplantation in recipients conditioned with CD117-sap compared to control mice (2 wk: 86.4 ± 9.2% vs. 1.2 ± 0.6% GFP+ granulocytes, p = 0.00009; 4 wk: 93.9 ± 5.6% vs. 0.2 ± 0.3% GFP+ granulocytes, p = 0.0018). Moreover, these mice exhibit therapeutic levels of circulating fVIII activity as early as 2 weeks post-transplantation (0.11 IU/ml, p = 0.005). Taken together, these preliminary data are a promising proof-of-concept demonstrating the feasibility of using non-genotoxic immunotoxin conditioning for HA gene therapy.