Abstract
Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.
Highlights
INTRODUCTION b-hemoglobinopathies are a common group of genetic blood disorders, encompassing sickle cell disease (SCD) and b-thalassemia, which affect millions of people worldwide.[1]
Optimizing the Delivery of HR at the b-globin (HBB) Cas9/single-guide RNA (sgRNA) RNP into LT-Repopulating HSCs Prior work demonstrated that the Cas9/sgRNA system delivered as a RNP complex by electroporation is the most effective method for creating double-strand break (DSB) and stimulating homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs).[5,6,19,20]
In this study, we present an optimized protocol for using the Cas9/ sgRNA system in combination with recombinant adeno-associated virus serotype 6 (rAAV6) homologous donor vectors to edit and expand HSPCs that have been targeted at the HBB locus
Summary
INTRODUCTION b-hemoglobinopathies are a common group of genetic blood disorders, encompassing sickle cell disease (SCD) and b-thalassemia, which affect millions of people worldwide.[1] SCD is caused by a point mutation at codon 6 in the b-globin gene (HBB), resulting in a glutamate to valine substitution.[2] In the deoxygenated state, hemoglobin containing mutant b-globin polymerizes. This polymerization causes red blood cells (RBCs) to adopt a sickle shape, which leads to reduced RBC lifespan and vaso-occlusion.[3] Vaso-occlusions can lead to strokes, bone pain, kidney damage, and acute chest syndrome, impairing both quality of life and survival.[1] On the other hand, b-thalassemia is caused by multiple mutations in the HBB locus and is characterized by insufficient production of b-globin protein. We identified that low-density culture conditions drives higher frequencies of HR and determined that ex vivo culturing using low-density conditions supplemented with UM171/SR1 supports expansion of targeted LT-HSCs
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