Efficient Correction of the Sickle Mutation in Human Hematopoietic Stem Cells Using a Cas9 Ribonucleoprotein Complex

Abstract

Sickle Cell Disease (SCD) is a serious recessive genetic disorder caused by a single nucleotide polymorphism (SNP) in the {beta}-globin gene (HBB). Sickle hemoglobin polymerizes within red blood cells (RBCs), causing them to adopt an elongated \"sickle\" shape. Sickle RBCs damage vasculature, leading to severe symptoms, ultimately diminishing patient quality of life and reducing lifespan. Here, we use codelivery of a pre-formed Cas9 ribonucleoprotein complex (RNP) and a singlestranded DNA (ssDNA) oligonucleotide donor to drive sequence replacement at the SCD SNP in human CD34+ hematopoietic stem/progenitor cells (HSPCs). Corrected HSPCs from SCD patients produce less sickle hemoglobin protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When injected into immunocompromised mice, treated HSPCs maintain editing long-term at therapeutically relevant levels. These results demonstrate that the Cas9 RNP/ssDNA donor approach can mediate efficient HSPC gene editing and could form the basis for treatment of SCD by autologous hematopoietic cell transplantation.