Crispr Modification of Induced Pluripotent Stem Derived from Diamond-Blackfan Anemia Fibroblasts

Introduction Diamond Blackfan Anemia (DBA) is a congenital bone marrow failure disorder characterized by severe erythroblastopenia. Nearly 70% of patients harbor mutations in ribosomal genes that lead to deficiency in ribosomal protein synthesis and impaired red blood cell production. Primary treatment modalities for DBA are steroids or red blood cell transfusions, while allogeneic hematopoietic stem cell (HSC) transplant remains the only curative option. While gene therapy is currently being utilized in a limited number of diseases, the advent of induced pluripotent stem cell (iPSC) technology and the capacity to manufacture HSCs from one’s own fibroblasts could expand the utility of gene correction strategies in disorders of hematopoiesis. We hypothesized that creation of iPSCs from a DBA patient’s fibroblasts could provide an in vitro model of a disease with defective erythroid development, and that correction of the mutation might alleviate the presumed block in erythropoiesis. Methods and Results Creation of iPSCs from DBA patients has been problematic for investigators due to reasons that are not entirely clear. After multiple attempts, five iPS cell lines (using an integration-free, novel 6-factor episomal construct) were created from a DBA patient carrying a non-sense mutation (R94X) in the third exon of RPS19. Experiments characterizing iPSCs ability to generate HSC (via OP9 mouse stromal cell co-culture) and undergo erythropoiesis demonstrated an approximate decrease of ~8-9x production of both CD71+/CD235+ and CD71-/CD235+in DBA cells compared to controls. Multiple strategies have been developed to target the RPS19 mutation of interest and replace it with corrected sequence. The first strategy relies on large targeting vectors that carry 2-4kb of homologous DNA flanking the mutation of interest. These vectors are engineered to carry drug selectable cassettes within the adjacent intron to permit retrieval of cells that have undergone homologous recombination. The second strategy targets the mutation using CRISPR technology using three key components: an endonuclease (Cas9), a chimeric RNA (crRNA + tracrRNA) capable of recognizing a specific genomic sequence and a correction oligo. Additional novel strategies are also underway that provide an avenue towards increased targeting efficiency. All correction strategies were employed in skin fibroblasts and iPSCs from a DBA patient. CRISPR vectors tested in 293T HEK cells and DBA skin fibroblasts have yielded promising results. Using a combination of the SURVEYOR assay and DNA sequencing to test targeting/cutting efficiency, we found the CRISPR vector cuts the mutated allele (designed to target only the mutated allele) in 13% of alleles sequenced. In comparison, vectors designed to cut either allele (targeting does not incorporate mutation of interest) were able to cut 17% of alleles sequenced, suggesting corrected cells may be retrieved without drug selection and minimal intervention. CRISPR correction has now been performed in DBA iPSCs and early experiments have indicated successful targeting in 3.5% of cells. Conclusions CD34+ and erythroid progenitor cells generated from DBA iPSC’s recapitulate disease characteristics and offer a potential source of cells to study disease characteristics in vitro. CRISPR methodology allowed for correction of this specific mutation and studies are currently underway to determine if the correction has rescued the erythopoietic defect. Futhermore, whole genome sequencing studies are needed to determine the background mutagenicity and safety of this approach. This technology may allow for the correction of specific DBA mutations that result in the generation of autologous ‘gene corrected’ hematopoietic stem cells that are suitable for transplantation. Disclosures Townes:University of Alabama at Birmingham: Employment.