Abstract
We tested a new in vivo hematopoietic stem cell (HSC) transduction/selection approach in rhesus macaques using HSC-tropic, integrating, helper-dependent adenovirus vectors (HDAd5/35++) designed for the expression of human γ-globin in red blood cells (RBCs) to treat hemoglobinopathies. We show that HDAd5/35++ vectors preferentially transduce HSCs in vivo after intravenous injection into granulocyte colony-stimulating factor (G-CSF)/AMD3100-mobilized animals and that transduced cells return to the bone marrow and spleen. The approach was well tolerated, and the activation of proinflammatory cytokines that are usually associated with intravenous adenovirus vector injection was successfully blunted by pre-treatment with dexamethasone in combination with interleukin (IL)-1 and IL-6 receptor blockers. Using our MGMTP140K-based in vivo selection approach, γ-globin+ RBCs increased in all animals with levels up to 90%. After selection, the percentage of γ-globin+ RBCs declined, most likely due to an immune response against human transgene products. Our biodistribution data indicate that γ-globin+ RBCs in the periphery were mostly derived from mobilized HSCs that homed to the spleen. Integration site analysis revealed a polyclonal pattern and no genotoxicity related to transgene integrations. This is the first proof-of-concept study in nonhuman primates to show that in vivo HSC gene therapy could be feasible in humans without the need for high-dose chemotherapy conditioning and HSC transplantation.
Highlights
IntroductionAutologous hematopoietic stem cell (HSC) gene therapy for hemoglobinopathies has shown promising effective cures.[1,2,3,4] Despite the encouraging clinical results, current ex vivo HSC gene therapy protocols have multiple shortcomings throughout the process: (1) harvesting HSCs by leukapheresis or bone marrow aspiration (invasive procedure); (2) myeloablation by chemotherapy (high-dose-chemotherapy-related side effects, infectious disease complications, conditioning-associated genotoxicity), (3) in vitro HSC culture and transplantation (loss of HSC pluripotency during extended ex vivo culture, need for specialized facility/staff); and (4) the cost of the approach
Autologous hematopoietic stem cell (HSC) gene therapy for hemoglobinopathies has shown promising effective cures.[1,2,3,4] Despite the encouraging clinical results, current ex vivo HSC gene therapy protocols have multiple shortcomings throughout the process: (1) harvesting HSCs by leukapheresis or bone marrow aspiration; (2) myeloablation by chemotherapy, (3) in vitro HSC culture and transplantation; and (4) the cost of the approach
HSC targeting through CD46 with HDAd5/35++ vectors We have recently reported that CD46 is expressed at a higher level on human CD34+ cells compared to other mononuclear cells in the bone marrow and peripheral blood,[12] suggesting that CD46 has function in HSCs
Summary
Autologous hematopoietic stem cell (HSC) gene therapy for hemoglobinopathies has shown promising effective cures.[1,2,3,4] Despite the encouraging clinical results, current ex vivo HSC gene therapy protocols have multiple shortcomings throughout the process: (1) harvesting HSCs by leukapheresis or bone marrow aspiration (invasive procedure); (2) myeloablation by chemotherapy (high-dose-chemotherapy-related side effects, infectious disease complications, conditioning-associated genotoxicity), (3) in vitro HSC culture and transplantation (loss of HSC pluripotency during extended ex vivo culture, need for specialized facility/staff); and (4) the cost of the approach. Because of the cost and technical complexity, it is unlikely that ex vivo protocols will be widely applicable, in developing countries where the greatest demand for hemoglobinopathy therapy lies. HDAd5/35++ vectors are easy to manufacture at high yields, can carry a payload of 35 kb, and can efficiently transduce primitive, quiescent HSCs through CD46.12 The vectors’ affinity to CD46 has been increased[19] to allow for in vivo HSC transduction without significant vector uptake by hepatocytes.[20,21] The random integration of HDAd5/35++ vectors
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