Cas9-AAV6 Gene Correction of Beta-Globin in Autologous HSCs Improves Sickle Cell Disease Erythropoiesis in Mice

<jats:title>Abstract</jats:title><jats:p>CRISPR/Cas9-mediated beta-globin (<jats:italic>HBB</jats:italic>) gene correction of Sickle Cell Disease (SCD) patient-derived hematopoietic stem cells (HSCs) in combination with autologous transplantation represents a novel paradigm in gene therapy. Although several Cas9-based <jats:italic>HBB</jats:italic>-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated. Here, we used a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated <jats:italic>HBB</jats:italic>-correction in functional HSCs within the context of autologous transplantation. We discover that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from <jats:italic>HBB</jats:italic>-corrected HSCs following autologous transplantation. We observed a direct correlation between increased <jats:italic>HBB</jats:italic>-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in robust hemoglobin-A levels. Moreover, this study offers a platform for gene editing of mouse HSCs for both basic and translational research.</jats:p>