Gene Editing of the Human Pklr Gene in Human Hematopoietic Progenitors to Correct Pyruvate Kinase Deficiency

Pyruvate Kinase Deficiency (PKD) is a rare erythroid metabolic disease caused by mutations in the PKLR gene. This gene encodes the erythroid specific Pyruvate Kinase (RPK) enzyme, implying that this defective enzyme fails to produce normal levels of ATP and consequently, erythrocytes from PKD patients show an energetic imbalance. Hematopoietic stem cell gene therapy using gene editing will be the safest approach to treat PKD patients. However, its direct application in hematopoietic progenitor cells (HPCs) is challenging. Different gene editing approaches are being explored to correct PKD, either by Knock-In of an optimized cDNA sequence in the first introns of the gene or by site-specific correction using ssODN. While Knock-In strategy will allow the treatment of most PKD mutations, an ssODN-mediated gene editing will correct specific PKD patient-mutations. In our Knock-In system, a recombination matrix carrying an exon 3-11 partial codon optimized version of RPK cDNA and a puromycin selection cassette is inserted in the second intron of the PKLR gene assisted by TALEN^® nucleases. We have previously shown that this approach was effective to correct PKD phenotype in PKDiPSC lines. Here, we have tested the feasibility of our approach in hematopoietic progenitors. Thus, the therapeutic matrix together with specific TALEN^® for the second intron of PKLR was electroporated in purified cord blood CD34⁺ from healthy donors. Cells were then expanded and puromycin selected to enrich the population for gene edited ones. Up to 96% of the colony forming units that survived the puromycin selection showed the specific integration of the donor cassette. After transplantation into immunodeficient NSG mice, a low though detectable percentage of gene edited cells was . Additionally, CRISPR-Cas9 site-specific correction is being developed by combining guide RNAs directed against PKD patient's mutation and precise ssODN to restore RPK protein. Overall, our data show that gene editing in engraftable HPCs is feasible, although further improvements are needed to increase efficiency of this gene therapy appraoch prior to consider its therapeutic application in PKD patients.