INDUCTION OF GAMMA-GLOBIN GENE SYNTHESIS BY CRISPR/CAS9 DELETIONS OF THE HPFH REGION AT - 196 OF THE HBG1 GENE IN beta-K562 AND HUMAN beta-THALASSEMIA HEMATOPOIETIC STEM CELLS

Hereditary persistence of fetal hemoglobin (HPFH) is the major genetic modifier of beta-thalassemia and beta chain hemoglobinopathies. Hence, reproducing an HPFH phenotype by genome editing in human hematopoietic stem cells could potentially release the repression of the gamma-globin genes, correct the degree of alpha/non-alpha-chain imbalance and ultimately ameliorate or cure the disease. With the assumption that binding of a repressor causes the HPFH phenotype from mutations in the -196/-202 region of the HBG1 promoter, we used a CRISPR/Cas9-mediated genome editing approach associated with NHEJ repair to disrupt repressor binding and reproduce the effects of the naturally occurring HPFH mutations of the region. In order to select the proper CRISPR target site in the human HBG1/HBG2 promoter sequence, three guide-RNA sequences were selected, cloned into a gRNA/Cas9 vector and tested in the beta-K562 cell line, able to express both beta and gamma-globin chains. By electroporation and limiting dilution we obtained several unselected individual clones with efficiency of genome editing up to 25%. The results obtained in beta-K562 clones supported our hypothesis that small deletions of the HPFH regions were able to increase HbF synthesis. Quantitative RT-PCR and Western blotting analysis in beta-K562 edited clones showed increased HBG mRNA and ?-globin protein levels (from 2 to 6x), with the best of the three guides designed. To further correlate the effects of the globin gene expression with the extent of the base deletions, we analyzed the DNA sequence of all isolated ?-K562 clones and found deletions of variable length, all starting in the predicted cutting site, but extending differently from a minimum of 2 nucleotides to the entire interpromoter region of the HBG genes. We also found in some of our clones unwanted large deletions and chromosomal recombination, due to lack of NHEJ repair or repair with DNA cut in off-target sites. To confirm these results in human erythroid progenitors, we also edited the gamma promoters in CD34+ hematopoietic stem cells derived from patients homozygous for the E39X beta0 -thalassemia common Sardinian mutation. We evaluated the gamma-globin inducing effect of the promoter modifications in patient-derived CD34+ cells by separating the population of transfected cells with FACS sorting of living cells into two sub-populations based on the fluorescent labeling specific for the genome edited cells. We found that the expression of HBG1/2 in edited CD34+ cells was increased about 2/3-fold compared to the editing negative CD34+ population. Our results suggest that the NHEJ gene-editing strategy, aimed at reproducing an HPFH phenotype, is an efficient method to permanently induce HbF synthesis with potential clinical applications in the therapy of any kind of beta-thalassemia, independently from the type of mutation. However, some cytogenetic anomalies induced by the NHEJ repair should caution against the immediate clinical translation and suggest the preferential use of safer genome editing strategies that do not induce double strand DNA breaks.