BioProject

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. More...

Genetic diseases of blood cells are prime candidates for treatment through ex vivo gene editing of CD34+ hematopoietic stem/progenitor cells (HSPCs), and a variety of technologies have been proposed to treat these disorders. Sickle Cell Disease (SCD) is a recessive genetic disorder caused by a single nucleotide polymorphism (SNP) in the ß-globin gene (HBB). Sickle hemoglobin damages erythrocytes, causing vasoocclusion, severe pain, progressive organ damage, and premature death. Here we optimize design and delivery parameters of a ribonucleoprotein (RNP) complex comprising Cas9 protein and unmodified sgRNA together with a single-stranded DNA oligonucleotide donor (ssODN) to enable efficient replacement of the SCD mutation in human HSPCs. Corrected HSPCs from SCD patients produce less sickle hemoglobin RNA and protein and correspondingly increased wild-type hemoglobin when differentiated into erythroblasts. When engrafted in immunocompromised mice, ex vivo treated human HSPCs maintain SCD gene edits throughout sixteen weeks at a level likely to have clinical benefit. These results demonstrate that an accessible approach combining Cas9 RNP with an ssODN can mediate efficient HSPC genome editing, enables investigator-led exploration of gene editing reagents in primary hematopoietic stem cells, and suggests a path towards the development of new gene editing treatments for SCD and other hematopoietic diseases. Less...