S852 IN VIVO SELECTION FOR CORRECTED BETA‐GLOBIN ALLELES AFTER CRISPR/CAS9 EDITING IN HUMAN SICKLE HEMATOPOIETIC STEM CELLS ENHANCES THERAPEUTIC POTENTIAL
Authors: Walters M. , Magis W. , DeWitt M. , Wyman S. , Vu J.T. , Romero Z.G. , Campo Fernandez B. , Kohn D. B. , Boffelli D. , Corn J.E. , Martin D.I. 
Published: June 2019

Background:Sickle Cell Disease (SCD) is a devastating disorder caused by a single base change in the ß‐globin gene (HBB) that is one component of the adult hemoglobin tetramer (HbA, which contains 2 a‐globin and 2 ß‐globin proteins). SCD is a recessive disorder – if one of the 2 alleles of HBB is normal, the disease is not present; thus gene correction needs to correct only one of the two HBB alleles in any given stem cell.Aims:To correct the sickle allele in human hematopoietic stem cellsMethods:To correct the sickle mutation, we developed a process that directly introduces a preassembled Cas9 ribonucleoprotein (RNP) complex (composed of recombinant Cas9 protein and single guide RNA) along with a single‐stranded DNA oligonucleotide donor template (ssODN) for homology‐directed repair (HDR). We studied gene correction after long‐term xenografting of human sickle HSCs. After electroporation with RNP and ssODN, sickle human HSCs were injected into immunodeficient NBSGW mice, which permit engraftment of human HSCs and erythroid differentiation in the bone marrow.Results:Sixteen to 20 weeks after injection, we analyzed engrafted human cells in the bone marrow of 43 mice in 4 cohorts. We quantified editing in xenografted cells, and found an average of 23.4% of HBB alleles with the corrected genotype, and 65.2% with insertion/deletion mutations (indels). To assess the distribution of HBB alleles within populations of xenografted cells, we inferred HBB genotypes using RNA‐Seq data from 359 individual clonal BFU‐E colonies derived from marrow CD34+ cells (figure 1A). This reveals that a substantial proportion of corrected (HDR) alleles are heterozygous with indel alleles equivalent to ß‐thalassemia mutations, confirming that corrected (wild type) HBB alleles in edited HSCs are distributed such that many erythrocytes have only a single wild type HBB allele. Data from the colony analysis, extrapolated to the entire experimental group, indicates that the 23.4% of corrected HBB alleles are distributed among >30% of HSCs, producing a comparable proportion of functional erythrocytes. SCD is a recessive disorder; based upon observations of mixed chimerism after allogeneic transplantation, this proportion of normal cells is consistent with cure.From engrafted NBSGW mice, we immunoselected human erythroid cells with anti‐CD235a (GlycophorinA), carried out RNA‐Seq, and inferred HBB genotypes in the erythroblast population. For each individual mouse, we compared the proportion of corrected HBB alleles in the xenografted bone marrow, in CD34+ cells immunoselected from marrow, and in CD235a+ erythroblasts. To control for enrichment of alleles corrected by HDR, we distinguished two types of HDR: “PAM‐only” HDR events mutate the Cas9 PAM without the conversion tract reaching the sickle mutation, whereas “sickle‐corrected” HDR events mutate the Cas9 PAM and also correct the sickle mutation. We observe a marked enrichment of “sickle‐corrected” HDR alleles in erythroblasts when compared to marrow or CD34+ cells from the same mouse, but no enrichment of “PAM‐only” HDR alleles (figure 1B).Summary/Conclusion:In summary, we have developed a Cas9‐mediated gene editing protocol that reproducibly yields a cell product in which the sickle mutation is corrected to wild type at a level that is sufficient to cure SCD. Evidence we have presented indicates that cells carrying the corrected ß‐globin gene are enriched during in vivo erythroid differentiation. These results support moving this therapeutic approach to the clinic.image