Gene editing has become a promising strategy for acutely correct a wide range of disease related mutations. Although Homologous Directed Repair (HDR) is the conventional mechanism used to correct specific mutations, in the case of disorders associated with marked defects in this DNA repair mechanism, such as Fanconi anemia (FA), alternative gene therapy approaches need to be implemented. In this context, we have previously shown that NHEJ- CRISPR/Cas9 mediated gene editing can remove/compensate specific mutations in different FA genes.
The description of novel and safer genome editing tools open the possibility to potentially correct most of the mutations described in FA patients with the final goal of generating precise medicines for FA patients.
To this aim we have optimized the use of Base (BE) and Prime editing (PE) to correct different mutations in FA hematopoietic stem and progenitor cells (HSPCs). 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, we also confirmed the efficiency of PE to precisely correct different mutations in FA lymphoblastic cell lines. Altogether, our results indicate the optimization of base and prime editing in HSPCs will pave the way for the development of personalized medicines for FA patients