Adenine base editing is an efficient approach to restore function in FA patient cells without double-stranded DNA breaks

Abstract

Fanconi Anemia (FA) is a debilitating genetic disorder with a wide range of severe symptoms including bone marrow failure and predisposition to cancer. CRISPR-Cas genome editing manipulates genotypes by harnessing DNA repair and has been proposed as a potential cure for FA. But FA is caused deficiencies in DNA repair itself, preventing the use of editing strategies such as homology directed repair. Recently developed base editing (BE) systems do not rely on double stranded DNA breaks and might be used to target mutations in FA genes, but this remains to be tested. Here we develop a proof of concept therapeutic base editing strategy to address two of the most prevalent FANCA mutations in patient cells. We find that optimizing adenine base editor construct, vector type, guide RNA format, and delivery conditions lead to very effective genetic modification in multiple FA patient backgrounds. Optimized base editing restored FANCA expression, molecular function of the FA pathway, and phenotypic resistance to crosslinking agents. ABE8e mediated editing in primary hematopoietic stem and progenitor cells from an FA patient was both genotypically effective and restored FA pathway function, indicating the potential of base editing strategies for future clinical application in FA.

In Vivo Hematopoietic Stem Cell Genome Editing: Perspectives and Limitations

“…Adenine base editors have also been applied for the targeting of the FA-55 and FA-75 mutations in the FANCA gene resulting in the restoration of expression and phenotypic correction in HSPCs derived from a Fanconi anemia patient [ 77 ]. Finally, ABEs have been recently used for the targeted correction of CD3D C202T, a mutation causing CD3δ severe combined immunodeficiency (SCID) in Jurkat T-cells and in CD34+ HSPCs leading to a more efficient CD3 repair compared to a CRISPR-Cas9 correction via homologous recombination [ 78 ].…”