343. Repair of the Sickle-Cell Disease Mutation in Human Stem Cells Using CRISPR/Cas9 and Single-Stranded Oligodeoxynucleotides

Abstract

Sickle cell disease is a hereditary anemia caused by a homozygous point mutation in the β-globin gene resulting in a glutamic acid to valine change at position 6 in the globin β-chain. Association of the mutated β-chain with α-chain results in the formation of hemoglobin S, which polymerizes intracellularly thus causing erythrocyte sickling. One promising therapy relies on the transplantation of autologous hematopoietic stem cells transduced with a lentiviral vector encoding a wild-type or mutant β-globin gene. However, one of the main concerns associated with the use of recombinant retroviruses to deliver therapeutic genes in stem cells is the risk of insertional mutagenesis following semi-random retroviral DNA integration. Alternatively, gene repair by homology-directed repair (HDR) is recognized as a potentially less genotoxic approach to repair mutations. Although HDR is intrinsically inefficient in human cells, enzymes that create specific DNA double-strand breaks can efficiently increase HDR frequency. Our long-term goal is to develop an efficient HDR approach to repair the sickle cell anemia mutation in stem cells. We have previously evaluated meganucleases and TALEN-based approaches to edit the β-globin gene in a human induced pluripotent stem (iPS) cell line carrying a homozygous sickle cell mutation. Here, we investigate the use of CRISPR/Cas9 system in combination with single-stranded oligodeoxynucleotides (ssODNs) to repair the sickle mutation in the above mentioned sickle iPS line (sciPS). Based on gene alignment analysis with the highly homologous Δ-globin gene, we have designed two β-globin-gene-specific CRISPR guide RNAs (gRNAs). Using these gRNAs, along with Cas9 and ssODNs, we show repair of the sickle cell mutation in sciPS cells. In the short term, we expect to translate these genome editing technologies to repair the sickle mutation in patient-specific cells, such as CD34+ hematopoietic cells, an approach that holds promise for future autologous stem cell therapies.