3042 – CAS9-AAV6 GENE CORRECTION OF AUTOLOGOUS HSCS IMPROVES SICKLE CELL DISEASE ERYTHROPOIESIS IN MICE

With the ability to reconstitute the entire hematopoietic system, hematopoietic stem cells (HSCs) are an attractive target for a range of disease therapies. CRISPR/Cas9-mediated beta-globin (HBB) gene correction of Sickle Cell Disease (SCD) patient-derived HSCs in combination with autologous transplantation represents an exciting new paradigm in gene therapy. Although several Cas9-based HBB-correction approaches have been proposed, functional correction of in vivo erythropoiesis has not been investigated to date. Here, we have applied a humanized globin-cluster SCD mouse model to study Cas9-AAV6-mediated HBB-correction in functional HSCs within the context of autologous transplantation using a novel ex vivo HSC culture system (Wilkinson et al., Nature 2019). We have discovered that long-term multipotent HSCs can be gene corrected ex vivo and stable hemoglobin-A production can be achieved in vivo from HBB-corrected HSCs following autologous transplantation back into SCD mice. There was a direct correlation between increased HBB-corrected myeloid chimerism and normalized in vivo RBC features, but even low levels of chimerism resulted in significant HgbA levels. Our results provide a useful framework for evaluating Cas9-based HSC gene therapies and support the clinical investigation of Cas9-AAV6 technologies.