1:CIEMAT; 2:CIBERER; 3:IIS-FJD, UAM

Pyruvate kinase deficiency (PKD) is an autosomal recessive hemolytic anemia caused by mutations in the PKLR gene. PKD is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. Lentiviral gene therapy of Autologous Hematopoietic Stem Cell Transplantation (HSCT) has demonstrated curative properties (NCT#04105166). To develop a precise therapy, we developed a knock-in gene editing strategy by combining RNP electroporation and recombinant adeno-associated viral vector (rAAV6) donors. We obtained stable integration in up to 40% of colony forming units (CFUs) and efficient engraftment in primary and secondary NSG mice, in the absence of toxicity. Nevertheless, clinically relevant correction requires additional developments.

Different alternatives to optimize our original gene editing protocol have been explored in order to reach therapeutic levels. Firs, we assessed the addition of different compounds involved in AAV transduction (Bleomycin, Bortezomib, Teniposide, Daunorubicin and Polyvinyl alcohol), homology repair pathway (BRCA1 and MRE11A proteins) or in the maintenance of the stem compartment (SR1) during gene editing process. Some compounds gave moderate increases. However, best results were obtained when pre-stimulation of HSPC was increased to 48 hours and cell density during the editing process was reduced, reaching editing frequencies up to 70% in clonogenic progenitors. Edited HSPCs under this optimized protocol were infused into immunodeficient mice and the specific integration was detected in up to 25% of the human cells three months after transplant.

These results confirm the feasibility PKLR gene editing in human HSPCs as a potential clinical application for the treatment of PKD.