A simplified, G-CSF-free procedure allows for in vivo HSC gene therapy of sickle cell disease in a mouse model.

We have reported direct repair of the sickle cell mutation in vivo in a disease model using vectorized prime editors after hematopoietic stem cell (HSC) mobilization with G-CSF/AMD3100. The use of G-CSF for HSC mobilization would be a hurdle for the clinical translation of the approach. Here, we tested a G-CSF-free mobilization regimen using WU-106, a PEG-conjugated inhibitor of integrin VLA-4 (4β1), 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 pro-inflammatory cytokine IL-6 in serum were minimal. The combination of WU-106+AMD3100 mobilization and intravenous injection of an HDAd-PE5 vector together with in vivo selection resulted in a SCD mutation editing (T>A correction) rate of ~23% in bone marrow and peripheral blood cells of CD46/Townes mice. The treated mice demonstrated phenotypic correction, reflected by normalized blood parameters and spleen size. Editing rates were significantly increased (29%) in secondary recipients indicating preferential mobilization/transduction of long-term repopulating HSCs. Using this approach, we found <1% of undesired indels and no detectable off-target editing at 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 intravenous injections with a good safety profile. The same-day mobilization regimen makes in vivo HSC gene therapy more attractive for the resource-poor settings where SCD does the most damage.