Auto-expansion of in vivo HDAd-transduced hematopoietic stem cells by constitutive expression of tHMGA2

We developed an in vivo hematopoietic stem cell (HSC) gene therapy approach that does not requirecell transplantation. To achieve therapeutically relevant numbers of corrected cells, we are testingapproaches that do not require in vivo selection with cytotoxic drugs for cell enrichment. Weconstructed HSC-tropic HDAd5/35++ vectors expressing a 3UTR truncated HMGA2 gene and a GFPreporter gene. A SB100x transposase vector was used to mediate random integration of thetHMGA2/GFP transgene cassette. HSCs in mice were mobilized by subcutaneous injections of G-CSF andAMD3100/Plerixafor and intravenously injected with the integrating tHMGA2/GFP vector. This resultedin a slow but progressive, competitive expansion of GFP + PBMCs, reaching about 50% by week 44 withfurther expansion in secondary recipients. Expansion occurred at the level of HSCs as well as at thelevels of myeloid, lymphoid, and erythroid progenitors within the bone marrow and spleen. Importantly,based on genome-wide integration site analyses, expansion was polyclonal, without any signs of clonaldominance. The results were validated in humanized mice. This is the first demonstration that simpleinjections of mobilization drugs and HDAd vectors can trigger auto-expansion of in vivo transduced HSCsresulting in transgene-marking rates that, theoretically, are curative for hemoglobinopathies.