PMID- 35955545 OWN - NLM STAT- PubMed-not-MEDLINE DCOM- 20220815 LR - 20231018 IS - 1422-0067 (Electronic) IS - 1422-0067 (Linking) VI - 23 IP - 15 DP - 2022 Jul 29 TI - Correction of Fanconi Anemia Mutations Using Digital Genome Engineering. LID - 10.3390/ijms23158416 [doi] LID - 8416 AB - 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. FAU - Sipe, Christopher J AU - Sipe CJ AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Kluesner, Mitchell G AU - Kluesner MG AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. AD - Medical Scientist Training Program, University of Washington, Seattle, WA 98195, USA. FAU - Bingea, Samuel P AU - Bingea SP AUID- ORCID: 0000-0001-7479-8180 AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Lahr, Walker S AU - Lahr WS AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Andrew, Aneesha A AU - Andrew AA AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Wang, Minjing AU - Wang M AUID- ORCID: 0000-0003-4725-8351 AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - DeFeo, Anthony P AU - DeFeo AP AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Hinkel, Timothy L AU - Hinkel TL AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Laoharawee, Kanut AU - Laoharawee K AUID- ORCID: 0000-0001-8216-2131 AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Wagner, John E AU - Wagner JE AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN 55455, USA. FAU - MacMillan, Margaret L AU - MacMillan ML AUID- ORCID: 0000-0002-0755-0852 AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Vercellotti, Gregory M AU - Vercellotti GM AD - Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Tolar, Jakub AU - Tolar J AUID- ORCID: 0000-0002-0957-4380 AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. AD - Division of Blood and Marrow Transplantation, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Osborn, Mark J AU - Osborn MJ AUID- ORCID: 0000-0003-1790-0841 AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN 55455, USA. FAU - McIvor, R Scott AU - McIvor RS AUID- ORCID: 0000-0001-8726-0902 AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Webber, Beau R AU - Webber BR AUID- ORCID: 0000-0002-3950-8747 AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. FAU - Moriarity, Branden S AU - Moriarity BS AD - Department of Pediatrics, University of Minnesota, Minneapolis, MN 55455, USA. AD - Masonic Cancer Center, University of Minnesota, Minneapolis, MN 55455, USA. AD - Center for Genome Engineering, University of Minnesota, Minneapolis, MN 55455, USA. AD - Stem Cell Institute, University of Minnesota, Minneapolis, MN 55455, USA. LA - eng GR - P01 CA065493/CA/NCI NIH HHS/United States GR - R21 AI163731/AI/NIAID NIH HHS/United States GR - 1/CX/CSRD VA/United States PT - Journal Article DEP - 20220729 PL - Switzerland TA - Int J Mol Sci JT - International journal of molecular sciences JID - 101092791 SB - IM PMC - PMC9369391 OTO - NOTNLM OT - CRISPR-Cas9 OT - Fanconi anemia (FA) OT - Fanconi anemia repair pathway OT - adenine base editing (ABE) OT - base editing OT - base excision repair OT - bone marrow failure OT - cytosine base editing (CBE) OT - digital genome engineering OT - double strand breaks OT - gene therapy COIS- The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. EDAT- 2022/08/13 06:00 MHDA- 2022/08/13 06:01 PMCR- 2022/07/29 CRDT- 2022/08/12 01:12 PHST- 2022/06/16 00:00 [received] PHST- 2022/07/20 00:00 [revised] PHST- 2022/07/25 00:00 [accepted] PHST- 2022/08/12 01:12 [entrez] PHST- 2022/08/13 06:00 [pubmed] PHST- 2022/08/13 06:01 [medline] PHST- 2022/07/29 00:00 [pmc-release] AID - ijms23158416 [pii] AID - ijms-23-08416 [pii] AID - 10.3390/ijms23158416 [doi] PST - epublish SO - Int J Mol Sci. 2022 Jul 29;23(15):8416. doi: 10.3390/ijms23158416.