AbstractWe have reported the direct repair of the sickle cell mutation in vivo in a disease model using vectorized prime editors after hematopoietic stem cell (HSC) mobilization with granulocyte colony-stimulating factor (G-CSF)/AMD3100. The use of G-CSF for HSC mobilization is a hurdle for the clinical translation of this approach. Here, we tested a G-CSF-free mobilization regimen using WU-106, a polyethylene glycolconjugated inhibitor of integrin very late antigen 4 (α4β1), plus AMD3100 for in vivo HSC prime editing in sickle cell disease (SCD) mice (CD46/Townes). Mobilization with WU-106 + AMD3100 in CD46/Townes mice was rapid and efficient. In contrast to the G-CSF/AMD3100 approach, mobilization of activated granulocytes and elevation of the key proinflammatory cytokine interleukin-6 (IL-6) in the serum were minimal. The combination of WU-106 + AMD3100 mobilization and IV injection of a helper-dependent adenovirus-PE5 (HDAd-PE5) vector together with in vivo selection resulted in an SCD mutation editing (T > A correction) rate of ∼23% in the bone marrow and peripheral blood cells of CD46/Townes mice. The treated mice demonstrated phenotypic correction, as reflected by normalized blood parameters and spleen size. Editing rates were significantly increased (29%) in secondary recipients, indicating the preferential mobilization/transduction of long-term repopulating HSCs. Using this approach, we found <1% undesired insertions/deletions and no detectable off-target editing at the top-scored potential sites. Our study shows that in vivo transduction to treat SCD (including HSC mobilization and HDAd injection) can now be done within 2 hours involving only simple IV injections with a good safety profile. The same-day mobilization regimen makes in vivo HSC gene therapy more attractive for resource-poor settings, where SCD does the most damage.