Correction of Fanconi Anemia Mutations Using Digital Genome Engineering

• Published in: International journal of molecular sciences Vol. 23; no. 15; p. 8416
• Main Authors: Sipe, Christopher J, Kluesner, Mitchell G, Bingea, Samuel P, Lahr, Walker S, Andrew, Aneesha A, Wang, Minjing, DeFeo, Anthony P, Hinkel, Timothy L, Laoharawee, Kanut, Wagner, John E, MacMillan, Margaret L, Vercellotti, Gregory M, Tolar, Jakub, Osborn, Mark J, McIvor, R Scott, Webber, Beau R, Moriarity, Branden S
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 29.07.2022; MDPI
• Subjects: Adenine; adenine base editing (ABE); Adenosine; Alkylation; Anemia; Autografts; base editing; Bone marrow; Cell cycle; Clinical trials; CRISPR; CRISPR-Cas9; Crosslinking; Cytosine; cytosine base editing (CBE); Deoxyribonucleic acid; DNA; DNA damage; DNA repair; Double-strand break repair; FANCA protein; Fanconi anemia (FA); Fanconi syndrome; Fibroblasts; Gene therapy; Genes; Genetic disorders; Genome editing; Genomes; Graft-versus-host reaction; Hematopoietic stem cells; Histocompatibility antigen HLA; Homology; Immunology; Leukemia; Leukocytes; Mitomycin C; Mutation; Nuclease; Patients; Progenitor cells; Stem cells; Surveillance; Ubiquitination; bone marrow failure; cytosine base editing (CBE); digital genome engineering; Fanconi anemia (FA); double strand breaks; CRISPR-Cas9; Fanconi anemia repair pathway; base excision repair; gene therapy; adenine base editing (ABE); base editing
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms23158416

Fanconi anemia (FA) is a rare genetic disease in which genes essential for DNA repair are mutated. Both the interstrand crosslink (ICL) and double-strand break (DSB) repair pathways are disrupted in FA, leading to patient bone marrow failure (BMF) and cancer predisposition. The only curative therapy for the hematological manifestations of FA is an allogeneic hematopoietic cell transplant (HCT); however, many (>70%) patients lack a suitable human leukocyte antigen (HLA)-matched donor, often resulting in increased rates of graft-versus-host disease (GvHD) and, potentially, the exacerbation of cancer risk. Successful engraftment of gene-corrected autologous hematopoietic stem cells (HSC) circumvents the need for an allogeneic HCT and has been achieved in other genetic diseases using targeted nucleases to induce site specific DSBs and the correction of mutated genes through homology-directed repair (HDR). However, this process is extremely inefficient in FA cells, as they are inherently deficient in DNA repair. Here, we demonstrate the correction of FANCA mutations in primary patient cells using ‘digital’ genome editing with the cytosine and adenine base editors (BEs). These Cas9-based tools allow for C:G > T:A or A:T > C:G base transitions without the induction of a toxic DSB or the need for a DNA donor molecule. These genetic corrections or conservative codon substitution strategies lead to phenotypic rescue as illustrated by a resistance to the alkylating crosslinking agent Mitomycin C (MMC). Further, FANCA protein expression was restored, and an intact FA pathway was demonstrated by downstream FANCD2 monoubiquitination induction. This BE digital correction strategy will enable the use of gene-corrected FA patient hematopoietic stem and progenitor cells (HSPCs) for autologous HCT, obviating the risks associated with allogeneic HCT and DSB induction during autologous HSC gene therapy.