Expanding the potential of novel gene editing tools to correct hematopoietic stem cells: therapeutic applicability in Fanconi anemia

Abstract

Gene editing has become a realistic therapeutic alternative for the specific repair of many genetic disorders, with a special role in hematological diseases. More precisely, the use of CRISPR/Cas9 technology has been widely exploited for the generation of targeted double-stranded breaks (DSBs), which allow the generation of precise genetic modifications through the use of the homology directed repair (HDR) or non-homologous end-joining (NHEJ) strategies. Considering that the NHEJ pathway is the preferred repair mechanism in quiescent cells, such as hematopoietic stem cells (HSCs), in previous studies we exploited the error-prone nature of this mechanism for the generation of compensatory insertion and deletion (indel) events by CRISPR/Cas9, resembling spontaneous reversions described in Fanconi anemia (FA) mosaic patients. In this doctoral thesis, NHEJ-mediated gene editing efficiency has been optimized to demonstrate its efficacy to target HSCs and to expand the application of NHEJ-mediated gene editing to different FA complementation groups. Considering that this repair mechanism is only applicable to certain FA mutations, and taking into account that the HDR system is the conventional strategy used to correct specific mutations, we have also implemented an improved HDR-mediated gene therapy approach to enhance gene editing efficiencies by the use of a dual cut donor template, carrying wild-type (WT) exon 4 sequence of FANCA to specifically repair the most prevalent FA mutation in Spain (c.295 C>T). To extend the application of gene editing to the vast majority of mutations described in FA patients, we have also implemented the recently described base and prime editing strategies, DSB-free genome editing tools by which the majority of small insertions, as well as small deletions and point conversions described in FA could be corrected, avoiding the use of donor templates and reducing the risks associated to the generation of DSBs. Our results have demonstrated that adenine base editor ABE8e is an efficient tool to target long-term repopulating HSCs (LT-HSCs) from healthy donors (HDs) and can be applied to repair the c.295 C>T mutation in FA lineage negative (Lin-) and CD34+ enriched hematopoietic cells, making base editing the most efficient gene editing strategy to target FA hematopoietic progenitors described so far. Additionally, studies conducted in this doctoral thesis have also shown for the first time the high efficiency, specificity and safety of prime editing for the correction of challenging mutations in patient-derived lymphoblastic cell lines (LCLs) to WT sequence, leading to the restoration of the FA pathway. Overall, we have implemented a gene editing platform to efficiently correct the vast majority of the mutations described in FA patients, taking a step further towards precision medicine for this and other hematological disorders