{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T12:35:34Z","timestamp":1777984534210,"version":"3.51.4"},"reference-count":25,"publisher":"MDPI AG","issue":"11","license":[{"start":{"date-parts":[[2025,11,1]],"date-time":"2025-11-01T00:00:00Z","timestamp":1761955200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"the Jones Lab in the Department of Chemistry at Illinois State University"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Genes"],"abstract":"<jats:p>Background: Point mutations in mitochondrial DNA (mtDNA) cause a range of neurometabolic disorders that currently have no curative treatments. The m.8993T&gt;G mutation in the Homo sapiens MT-ATP6 gene leads to neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP) when heteroplasmy exceeds approximately 70%. Methods: We engineered a split DddA-derived cytosine base editor (DdCBE), each half fused to programmable TALE DNA-binding domains and a mitochondrial targeting sequence, to correct the m.8993T&gt;G mutation in patient-derived induced pluripotent stem cells (iPSCs). Seven days after plasmid delivery, deep amplicon sequencing showed 35 \u00b1 3% on-target C\u2022G\u2192T\u2022A conversion at position 8993, reducing mutant heteroplasmy from 80 \u00b1 2% to 45 \u00b1 3% with less than 0.5% editing at ten predicted off-target loci. Results: Edited cells exhibited a 25% increase in basal oxygen consumption rate, a 50% improvement in ATP-linked respiration, and a 2.3-fold restoration of ATP synthase activity. Directed neural differentiation yielded 85 \u00b1 2% Nestin-positive progenitors compared to 60 \u00b1 2% in unedited controls. Conclusions: Edits remained stable over 30 days in culture. These results establish mitochondrial base editing as a precise and durable strategy to ameliorate biochemical and cellular defects in NARP patient cells.<\/jats:p>","DOI":"10.3390\/genes16111298","type":"journal-article","created":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T13:22:36Z","timestamp":1762176156000},"page":"1298","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Mitochondrial Base Editing of the m.8993T&gt;G Mutation Restores Bioenergetics and Neural Differentiation in Patient iPSCs"],"prefix":"10.3390","volume":"16","author":[{"given":"Luke","family":"Yin","sequence":"first","affiliation":[{"name":"Center of Student Inquiry and Research, Illinois Mathematics and Science Academy, Aurora, IL 60506, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Angel","family":"Yin","sequence":"additional","affiliation":[{"name":"Weinberg College of Arts and Sciences, Northwestern University, Evanston, IL 60208, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6306-0700","authenticated-orcid":false,"given":"Marjorie","family":"Jones","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Illinois State University, Normal, IL 61761, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2025,11,1]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"135","DOI":"10.1093\/bmb\/ldt017","article-title":"Mitochondrial genetics","volume":"106","author":"Chinnery","year":"2013","journal-title":"Br. Med. Bull."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"753","DOI":"10.1002\/ana.24362","article-title":"Prevalence of nuclear and mitochondrial DNA mutations related to adult mitochondrial disease","volume":"77","author":"Gorman","year":"2015","journal-title":"Ann. Neurol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"1482","DOI":"10.1126\/science.283.5407.1482","article-title":"Mitochondrial diseases in man and mouse","volume":"283","author":"Wallace","year":"1999","journal-title":"Science"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"389","DOI":"10.1038\/nrg1606","article-title":"Mitochondrial DNA mutations in human disease","volume":"6","author":"Taylor","year":"2005","journal-title":"Nat. Rev. Genet."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"878","DOI":"10.1038\/nrg3275","article-title":"Human mitochondrial DNA: Roles of inherited and somatic mutations","volume":"13","author":"Schon","year":"2012","journal-title":"Nat. Rev. Genet."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"33426","DOI":"10.1074\/jbc.274.47.33426","article-title":"Threshold effect and tissue specificity: Implication for mitochondrial cytopathies","volume":"274","author":"Rossignol","year":"1999","journal-title":"J. Biol. Chem."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"631","DOI":"10.1038\/s41586-020-2477-4","article-title":"A bacterial cytidine deaminase toxin enables CRISPR-free mitochondrial base editing","volume":"583","author":"Mok","year":"2020","journal-title":"Nature"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"101","DOI":"10.1016\/j.tig.2017.11.001","article-title":"Mitochondrial genome engineering: The revolution may not be CRISPR-Ized","volume":"34","author":"Gammage","year":"2018","journal-title":"Trends Genet."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"450","DOI":"10.1016\/S0168-9525(97)01266-3","article-title":"Mammalian mitochondrial genetics: Heredity, heteroplasmy and disease","volume":"13","author":"Lightowlers","year":"1997","journal-title":"Trends Genet. TIG"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"102521","DOI":"10.1016\/j.omtn.2025.102521","article-title":"Optimization of mtDNA-targeted platinum TALENs for bi-directionally modifying heteroplasmy levels in patient-derived m.3243A>G-iPSCs. Molecular therapy","volume":"36","author":"Yahata","year":"2025","journal-title":"Nucleic Acids"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1038\/s41587-022-01486-w","article-title":"Precision mitochondrial DNA editing with high-fidelity DddA-derived base editors","volume":"41","author":"Lee","year":"2022","journal-title":"Nat. Biotechnol."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"1764","DOI":"10.1016\/j.cell.2022.03.039","article-title":"Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases","volume":"185","author":"Cho","year":"2022","journal-title":"Cell"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"300","DOI":"10.1038\/nrg.2016.28","article-title":"Defining and improving the genome-wide specificities of CRISPR\u2013Cas9 nucleases","volume":"17","author":"Tsai","year":"2016","journal-title":"Nat. Rev. Genet."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1016\/j.jgg.2013.10.003","article-title":"Very low-level heteroplasmy mtDNA variations are inherited in humans","volume":"40","author":"Guo","year":"2013","journal-title":"J. Genet. Genom. = Yi Chuan Xue Bao"},{"key":"ref_15","first-page":"852","article-title":"Heteroplasmic mtDNA mutation (T\u2192G) at 8993 can cause Leigh disease when the percentage of abnormal mtDNA is high","volume":"50","author":"Tatuch","year":"1992","journal-title":"Am. J. Hum. Genet."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"458","DOI":"10.1002\/emmm.201303672","article-title":"Mitochondrially targeted ZFNs for selective degradation of pathogenic mitochondrial genomes bearing large-scale deletions or point mutations","volume":"6","author":"Gammage","year":"2014","journal-title":"EMBO Mol. Med."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"751","DOI":"10.1042\/bj20021594","article-title":"Mitochondrial threshold effects","volume":"370","author":"Rossignol","year":"2003","journal-title":"Biochem. J."},{"key":"ref_18","first-page":"492","article-title":"mtDNA depletion with variable tissue expression: A novel genetic abnormality in mitochondrial diseases","volume":"48","author":"Moraes","year":"1991","journal-title":"Am. J. Hum. Genet."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"2491","DOI":"10.1073\/pnas.1419651112","article-title":"Extensive tissue-related and allele-related mtDNA heteroplasmy suggests positive selection for somatic mutations","volume":"112","author":"Li","year":"2015","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_20","first-page":"E3622","article-title":"Tissue- and cell-type\u2013specific manifestations of heteroplasmic mtDNA mutation in human induced pluripotent stem cells","volume":"110","author":"Manninen","year":"2013","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1111","DOI":"10.1038\/nm.3261","article-title":"Specific elimination of mutant mitochondrial genomes in patient-derived cells by mitoTALENs","volume":"19","author":"Bacman","year":"2013","journal-title":"Nat. Med."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"1691","DOI":"10.1038\/s41591-018-0165-9","article-title":"Genome editing in mitochondria corrects a pathogenic mtDNA mutation in vivo","volume":"24","author":"Gammage","year":"2018","journal-title":"Nat. Med."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Di Donfrancesco, A., Massaro, G., Di Meo, I., Tiranti, V., Bottani, E., and Brunetti, D. (2022). Gene Therapy for Mitochondrial Diseases: Current Status and Future Perspective. Pharmaceutics, 14.","DOI":"10.3390\/pharmaceutics14061287"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Norota, K., Ishizuka, S., Hirose, M., Sato, Y., Maeki, M., Tokeshi, M., Ibrahim, S.M., Harashima, H., and Yamada, Y. (2025). Lipid nanoparticle delivery of the CRISPR\/Cas9 system directly into the mitochondria of cells carrying m.7778G>T mutation in MtDNA (mt-Atp8). Sci. Rep., 15.","DOI":"10.1038\/s41598-025-03671-8"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"501","DOI":"10.1007\/s11019-015-9656-3","article-title":"The ethical challenges of the clinical introduction of mitochondrial replacement techniques","volume":"18","author":"Appleby","year":"2015","journal-title":"Med. Health Care Philos."}],"container-title":["Genes"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4425\/16\/11\/1298\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,11,3]],"date-time":"2025-11-03T13:27:28Z","timestamp":1762176448000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4425\/16\/11\/1298"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,11,1]]},"references-count":25,"journal-issue":{"issue":"11","published-online":{"date-parts":[[2025,11]]}},"alternative-id":["genes16111298"],"URL":"https:\/\/doi.org\/10.3390\/genes16111298","relation":{},"ISSN":["2073-4425"],"issn-type":[{"value":"2073-4425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,11,1]]}}}