Safe and efficient in vivo hematopoietic stem cell transduction in nonhuman primates using HDAd5/35++ vectors

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 MGMT^P140K-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.