Gene editing mediated by non-homologous end-joining: a versatile approach for the gene therapy of hematopoietic stem cells from fanconi anemia patients

Abstract

Preclinical as well as clinical studies suggest that gene therapy based on the infusion of autologous hematopoietic progenitor and stem cells (HSPCs) previously corrected with lentiviral vectors may constitute a future alternative for treating Fanconi anemia (FA) patients. In this context, gene editing appears to be a new step in the development of safe and precise gene therapy approaches. Since non-homologous end-joining (NHEJ) is the preferred mechanism to repair DNA double-strand breaks in quiescent cells, such as HSPCs, and given that this pathway has been reported to be enhanced in FA cells, we have tested the feasibility of using NHEJ to generate compensatory mutations that may restore the function of FANCA protein, thus mimicking the spontaneous reversions reported in some FA mosaic patients. Our hypothesis was initially tested in two FA-A patient-derived lymphoblastic cell lines carrying different mutations in FANCA using the CRISPR/Cas9 system. Analyses of the targeted sites by next generation sequencing (NGS) revealed the presence of cells harbouring potentially therapeutic repair events whose frequency increased over time. Functional analyses confirmed the re-expression of a new functional FANCA protein capable of correcting the FA cell phenotype. Importantly, transplantation of healthy donor HSPCs after NHEJ-editing in immunodeficient (NSG) mice showed the feasibility of efficiently targeting long-term repopulating HSCs. Moreover, when FA-A patient HSPCs were targeted by our CRISPR/Cas9 nuclease, therapeutic NHEJ-repair events were identified by NGS, showing up to 50-fold in vitro expansion after only 9 days in culture. Furthermore, the transplantation of limited numbers of FA-edited hCD34+ cells into an NSG mouse showed a remarkable in vivo expansion of corrected cells. Additionally, corrected cells showed the reversion of the hypersensitivity to mitomycin C, defined as a hallmark of FA cells. All together, these results demonstrate for the first time the NHEJ-mediated phenotypic correction of FA HSPCs. The NGS analyses of the topfive in silico predicted off-target loci in edited FA HSPCs showed no unspecific activity, confirming the safety of this new approach. Moving forward to in vivo applications of NHEJ-mediated repair approaches, serotype 6 adeno-associated viral vectors (AAVs) were tested in vitro and also in vivo, demonstrating the possibility of transducing hematopoietic progenitor cells in both settings. Moreover, the delivery of the CRISPR/Cas9 system via an all-in-one AAV confirmed the feasibly of these vectors to edit human HSPCs, opening the possibility of future in vivo NHEJ-mediated gene editing approaches in FA