In vivo HSC transduction in humanized mice mediated by novel capsid-modified HDAd vectors

We developed an <i>in vivo</i> hematopoietic stem cell (HSC) gene therapy approach consisting of HSC mobilization and intravenous injection of helper dependent adenovirus (HDAd) vectors. While we have demonstrated safety and efficacy of the <i>in vivo</i> approach in CD46-transgenic mice and rhesus macaques, studies in mice with a humanized hematopoietic system could facilitate its potential clinical translation for the treatment of hemoglobinopathies and HIV. Using mild myelo-conditioning in NSGW41 mice and cryopreserved human CD34⁺ cells from healthy donors we achieved ∼10% human chimerism in peripheral blood. Engrafted primitive human CD45⁺/CD34⁺/CD90⁺-HSCs efficiently mobilized by different approaches involving AMD3100 in combination with granulocyte colony-stimulating factor G-CSF, truncated Groβ (tGroβ), or WU106/tGroβ. At the peak of mobilization, integrating HDAd-GFP vectors were injected intravenously followed by O⁶BG/BCNU <i>in vivo</i> selection. Long-term stable GFP expression was shown for HDAd5/35 and the new vector platforms HDAd6/3 and HDAd5/35_lam, a fiber/penton-modified vector. Two months post transduction, GFP marking in the periphery were 22.38% (8.17%), 41.12% (10.62%), and 32.15% (4.49%) for HDAd5/35, HDAd6/3, and HDAd5/35_lam, respectively. GFP levels in bone marrow were 33.53% (8.96%), 53.51% (6.95%), and 33.29% (5.21%) and in spleen 32.6% (9.25%), 33.75% (5.47%), and 20.79% (6.15%). Our study describes a new animal model for <i>in vivo</i> HSC transduction with HDAds, with implications for studies with other vectors.