Abstract

Current clinical studies show the therapeutic potential of lentiviral-mediated gene therapy in patients with Fanconi anemia (FA). In an attempt to develop alternative gene therapy approaches in FA, we have previously shown that NHEJ-CRISPR/Cas9 mediated gene editing can remove/compensate specific mutations in different FA genes. To improve the safety and the efficacy of gene editing in FA HSPCs, we have now focused on the use of Base (BE) and Prime editing (PE) strategies to edit FA genes, since they do not generate doublestrand breaks (DSBs) in the cell genome and do not require a donor template. As a proof of concept we focused on a frequent mutation present in FA patients from Spain, which results in a stop codon in exon 4 of FANCA (c.295 C>T). Using BE technology we generated a therapeutic base conversion in >50% BM HSPCs from FA patients that harbor this mutation. The efficacy of the genetic conversion was confirmed by the correction of the characteristic phenotype of FA cells. Using an optimized Prime editing approach, combining the use of a PEmax architecture in synergy with the PE3 system, the FA mutated sequence was precisely corrected to the WT sequence in up to 40% of cells from a FA-A lymphoblastic cell line. As expected,corrected cells showed a marked proliferative advantage together with an evident phenotypic correction. Importantly, we have also confirmed the potential of PE to edit CD34+ from healthy donors Altogether, our results suggest that BE and PE may constitute alternative therapeutic approaches for the treatment of bone marrow failure in FA patients carrying specific mutations.