Using HUDEP Cells as a Model for Gene Editing for -Thalassemia and Sickle Cell Disease

β-thalassemia (β-thal) and sickle cell disease (SCD) are amongst the most common genetic disorders worldwide affecting >57,000 and 250,000 new patients per year, respectively. Treatments for these hemoglobinopathies are palliative; the only cure is allogenic bone marrow transplantation. Gene therapy in autologous hematopoietic stem cells (HSC) is a promising alternative avoiding immunological complications of the allogeneic bone marrow transplantation. Hematopoietic stem cells from β-thalassemia or sickle cell patients are limited, therefore, a β-thalassemia and sickle cell disease cell lines were produced using zinc finger nucleases (ZFNs) targeting the beta-globin gene in CD34+ human umbilical cord-derived erythroid progenitor (HUDEP) cells. The development of a β-thal or SCD genotype immortalized HUDEP cell line could facilitate testing gene therapy protocols for hemoglobinopathies. We predict the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology can be used to correct these hemoglobinopathies. Allelic disruption was evaluated for multiple CRISPR guide RNAs around the disrupted β-globin gene or the sickle mutation, in the β-thal and SCD cell lines, respectively. Gene correction was assessed when a homologous donor template was co-delivered. Gene correction assessed by high-throughput sequencing in the treated SCD samples demonstrated that 2% and 5% of the correction was by homology-directed repair (HDR), and ~25% and 27% of indels in the samples treated with 1uM and 3uM of the oligo, respectively. However, in the β-thal samples ~22% of allelic disruption was achieved, but gene correction was not detectable. Therefore, detection of adult hemoglobin by HPLC (~5%) was observed on the SCD samples only.