Beta-Globin Gene Targeting in Human Hematopoietic Stem Cells Using Cas9 Ribonucleoprotein and rAAV6

Theb-hemoglobinopathies including sickle cell disease (SCD) andb-thalassemia (b-thal) affect millions of people worldwide. SCD andb-thal are caused by mutations in theb-globin gene (HBB) resulting in either abnormal sickling or severely reduced protein production, respectively. A curative strategy for theb-hemoglobinopathies would be ex vivo HBB gene correction in patient-derived hematopoietic stem and progenitor cells (HSPCs) followed by autologous hematopoietic stem cell transplantation (auto-HSCT). We report the first CRISPR/Cas9 gene-editing platform for achieving homologous recombination (HR) at the HBB gene in long-term repopulating HSCs derived from mobilized peripheral blood. We combine electroporation of Cas9 protein complexed with chemically modified sgRNAs and delivery of a HR donor by recombinant adeno-associated viral vectors, serotype 6 (rAAV6). Notably, by including a reporter gene in the HR donor, we are able to identify and purify a population of HSPCs with >90% of cells having targeted integration at the HBB gene. These cells can be identified because HR-mediated integration causes the reporter gene to be expressed at log-fold higher levels than the non-integrated reporter. When transplanted into immunodeficient mice, the purified population gives rise to engraftment of HBB-edited human cells in primary and secondary recipients, confirming the presence of long-term repopulating hematopoietic stem cells (LT-HSCs). Importantly, we show efficient correction of the SCD-causing E6V mutation in SCD patient-derived CD34⁺ HSPCs by either editing the nucleotide mutation or knocking in an anti-sicklingb-globin cDNA. Edited SCD CD34⁺ cells were shown to express adultb-globin (HbA) mRNA after HSPCs were differentiated into erythrocytes in vitro, confirming intact transcriptional regulation of the edited HBB allele.Collectively, these preclinical studies outline a CRISPR-based methodology for targeting HSCs for HR at the HBB locus to advance the development of next generation therapies forb-hemoglobinopathies.