{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,9]],"date-time":"2026-05-09T09:33:53Z","timestamp":1778319233417,"version":"3.51.4"},"reference-count":40,"publisher":"MDPI AG","issue":"20","license":[{"start":{"date-parts":[[2023,10,16]],"date-time":"2023-10-16T00:00:00Z","timestamp":1697414400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"Wellcome Trust","award":["205174\/Z\/16\/Z"],"award-info":[{"award-number":["205174\/Z\/16\/Z"]}]},{"name":"Moorfields Eye Charity","award":["205174\/Z\/16\/Z"],"award-info":[{"award-number":["205174\/Z\/16\/Z"]}]},{"name":"Santen Pharmaceutical","award":["205174\/Z\/16\/Z"],"award-info":[{"award-number":["205174\/Z\/16\/Z"]}]},{"name":"National Institute for Health and Care Research (NIHR) Biomedical Research Centre","award":["205174\/Z\/16\/Z"],"award-info":[{"award-number":["205174\/Z\/16\/Z"]}]},{"name":"Moorfields Eye Hospital NHS Foundation Trust","award":["205174\/Z\/16\/Z"],"award-info":[{"award-number":["205174\/Z\/16\/Z"]}]},{"name":"UCL Institute of Ophthalmology","award":["205174\/Z\/16\/Z"],"award-info":[{"award-number":["205174\/Z\/16\/Z"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold\/matrix attachment region (S\/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS\/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chmru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS\/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS\/MAR-CHM at the one-cell stage. Injected chmru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S\/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.<\/jats:p>","DOI":"10.3390\/ijms242015225","type":"journal-article","created":{"date-parts":[[2023,10,16]],"date-time":"2023-10-16T05:43:49Z","timestamp":1697435029000},"page":"15225","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":5,"title":["Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia"],"prefix":"10.3390","volume":"24","author":[{"given":"Lyes","family":"Toualbi","sequence":"first","affiliation":[{"name":"Development, Ageing and Disease, UCL Institute of Ophthalmology, London EC1V 9EL, UK"},{"name":"Ocular Genomics and Therapeutics, The Francis Crick Institute, London NW1 1AT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria","family":"Toms","sequence":"additional","affiliation":[{"name":"Development, Ageing and Disease, UCL Institute of Ophthalmology, London EC1V 9EL, UK"},{"name":"Ocular Genomics and Therapeutics, The Francis Crick Institute, London NW1 1AT, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Patrick Vingadas","family":"Almeida","sequence":"additional","affiliation":[{"name":"cDNA Vector Research, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Richard","family":"Harbottle","sequence":"additional","affiliation":[{"name":"cDNA Vector Research, German Cancer Research Center (DKFZ), 69120 Heidelberg, Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1688-5360","authenticated-orcid":false,"given":"Mariya","family":"Moosajee","sequence":"additional","affiliation":[{"name":"Development, Ageing and Disease, UCL Institute of Ophthalmology, London EC1V 9EL, UK"},{"name":"Ocular Genomics and Therapeutics, The Francis Crick Institute, London NW1 1AT, UK"},{"name":"Department of Genetics, Moorfields Eye Hospital NHS Foundation Trust, London EC1V 2PD, UK"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2023,10,16]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"e004015","DOI":"10.1136\/bmjopen-2013-004015","article-title":"A Comparison of the Causes of Blindness Certifications in England and Wales in Working Age Adults (16\u201364 Years), 1999\u20132000 with 2009\u20132010","volume":"4","author":"Liew","year":"2014","journal-title":"BMJ Open"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"307","DOI":"10.1016\/S0959-437X(00)00195-7","article-title":"Molecular Genetics and Prospects for Therapy of the Inherited Retinal Dystrophies","volume":"11","author":"Bessant","year":"2001","journal-title":"Curr. Opin. Genet. Dev."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"53","DOI":"10.1016\/j.preteyeres.2017.03.003","article-title":"Unravelling the Genetics of Inherited Retinal Dystrophies: Past, Present and Future","volume":"59","author":"Broadgate","year":"2017","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"702","DOI":"10.1016\/j.oret.2022.03.011","article-title":"The Current State of Genetic Testing Platforms for Inherited Retinal Diseases","volume":"6","author":"Mustafi","year":"2022","journal-title":"Ophthalmol. Retina"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"378","DOI":"10.1016\/j.molmed.2022.02.011","article-title":"Choroideremia: Molecular Mechanisms and Therapies","volume":"28","author":"Sarkar","year":"2022","journal-title":"Trends Mol. Med."},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Abdalla Elsayed, M.E.A., Taylor, L.J., Josan, A.S., Fischer, M.D., and MacLaren, R.E. (2023). Choroideremia: The Endpoint Endgame. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241814354"},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"410","DOI":"10.1097\/ICU.0000000000000392","article-title":"Choroideremia","volume":"28","author":"Dimopoulos","year":"2017","journal-title":"Curr. Opin. Ophthalmol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1016\/j.ajo.2019.06.002","article-title":"Spectrum of Disease Severity and Phenotype in Choroideremia Carriers","volume":"207","author":"Jauregui","year":"2019","journal-title":"Am. J. Ophthalmol."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"101190","DOI":"10.1016\/j.preteyeres.2023.101190","article-title":"Female Carriers of X-Linked Inherited Retinal Diseases\u2014Genetics, Diagnosis, and Potential Therapies","volume":"96","author":"Gocuk","year":"2023","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"24420","DOI":"10.1074\/jbc.270.41.24420","article-title":"Deficient Geranylgeranylation of Ram\/Rab27 in Choroideremia","volume":"270","author":"Seabra","year":"1995","journal-title":"J. Biol. Chem."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1016\/S1471-4914(01)02227-4","article-title":"Rab GTPases, Intracellular Traffic and Disease","volume":"8","author":"Seabra","year":"2002","journal-title":"Trends Mol. Med."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"100975","DOI":"10.1016\/j.preteyeres.2021.100975","article-title":"Early and Late Stage Gene Therapy Interventions for Inherited Retinal Degenerations","volume":"86","author":"Botto","year":"2022","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"661","DOI":"10.1007\/s40291-021-00558-y","article-title":"Molecular Therapy for Choroideremia: Pre-Clinical and Clinical Progress to Date","volume":"25","author":"Kalatzis","year":"2021","journal-title":"Mol. Diagn. Ther."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"849","DOI":"10.1016\/S0140-6736(17)31868-8","article-title":"Efficacy and Safety of Voretigene Neparvovec (AAV2-hRPE65v2) in Patients with RPE65-Mediated Inherited Retinal Dystrophy: A Randomised, Controlled, Open-Label, Phase 3 Trial","volume":"390","author":"Russell","year":"2017","journal-title":"Lancet"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"3855","DOI":"10.2147\/OPTH.S231804","article-title":"Voretigene Neparvovec in Retinal Diseases: A Review of the Current Clinical Evidence","volume":"14","author":"Gao","year":"2020","journal-title":"Clin. Ophthalmol."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"MacLaren, R.E., Fischer, M.D., Gow, J.A., Lam, B.L., Sankila, E.-M.K., Girach, A., Panda, S., Yoon, D., Zhao, G., and Pennesi, M.E. (2023). Subretinal Timrepigene Emparvovec in Adult Men with Choroideremia: A Randomized Phase 3 Trial. Nat. Med., in press.","DOI":"10.1038\/s41591-023-02520-3"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.ajo.2018.06.011","article-title":"Two-Year Results After AAV2-Mediated Gene Therapy for Choroideremia: The Alberta Experience","volume":"193","author":"Dimopoulos","year":"2018","journal-title":"Am. J. Ophthalmol."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1507","DOI":"10.1038\/s41591-018-0185-5","article-title":"Beneficial Effects on Vision in Patients Undergoing Retinal Gene Therapy for Choroideremia","volume":"24","author":"Xue","year":"2018","journal-title":"Nat. Med."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"1887","DOI":"10.1056\/NEJMoa1414221","article-title":"Long-Term Effect of Gene Therapy on Leber\u2019s Congenital Amaurosis","volume":"372","author":"Bainbridge","year":"2015","journal-title":"N. Engl. J. Med."},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1089\/humc.2015.164","article-title":"Safety and Biodistribution Evaluation in Cynomolgus Macaques of rAAV2tYF-PR1.7-hCNGB3, a Recombinant AAV Vector for Treatment of Achromatopsia","volume":"27","author":"Ye","year":"2016","journal-title":"Hum. Gene Ther. Clin. Dev."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"1331","DOI":"10.1136\/bjophthalmol-2021-321023","article-title":"Development of Retinal Atrophy after Subretinal Gene Therapy with Voretigene Neparvovec","volume":"107","author":"Reichel","year":"2023","journal-title":"Br. J. Ophthalmol."},{"key":"ref_22","doi-asserted-by":"crossref","unstructured":"Roychowdhury, T., and Chattopadhyay, S. (2020). Chemical Decorations of \u201cMARs\u201d Residents in Orchestrating Eukaryotic Gene Regulation. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.602994"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"645","DOI":"10.1093\/hmg\/ddn394","article-title":"Differential Nuclear Scaffold\/Matrix Attachment Marks Expressed Genes","volume":"18","author":"Linnemann","year":"2009","journal-title":"Hum. Mol. Genet."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"143","DOI":"10.1016\/j.stemcr.2021.11.011","article-title":"An Episomal DNA Vector Platform for the Persistent Genetic Modification of Pluripotent Stem Cells and Their Differentiated Progeny","volume":"17","author":"Urban","year":"2022","journal-title":"Stem Cell Rep."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"eabf1333","DOI":"10.1126\/sciadv.abf1333","article-title":"A Nonviral, Nonintegrating DNA Nanovector Platform for the Safe, Rapid, and Persistent Manufacture of Recombinant T Cells","volume":"7","author":"Bozza","year":"2021","journal-title":"Sci. Adv."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"349","DOI":"10.1093\/embo-reports\/kvf070","article-title":"An Episomally Replicating Vector Binds to the Nuclear Matrix Protein SAF-A in Vivo","volume":"3","author":"Jenke","year":"2002","journal-title":"EMBO Rep."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"1632","DOI":"10.1093\/hmg\/ddt013","article-title":"S\/MAR-Containing DNA Nanoparticles Promote Persistent RPE Gene Expression and Improvement in RPE65-Associated LCA","volume":"22","author":"Koirala","year":"2013","journal-title":"Hum. Mol. Genet."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"745","DOI":"10.1016\/j.jconrel.2013.08.299","article-title":"Persistence of Non-Viral Vector Mediated RPE65 Expression: Case for Viability as a Gene Transfer Therapy for RPE-Based Diseases","volume":"172","author":"Koirala","year":"2013","journal-title":"J. Control Release"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"823","DOI":"10.1016\/j.omtn.2022.08.026","article-title":"Effective Gene Therapy of Stargardt Disease with PEG-ECO\/pGRK1-ABCA4-S\/MAR Nanoparticles","volume":"29","author":"Sun","year":"2022","journal-title":"Mol. Ther. Nucleic Acids"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e53","DOI":"10.1093\/nar\/gku082","article-title":"S\/MAR Sequence Confers Long-Term Mitotic Stability on Non-Integrating Lentiviral Vector Episomes without Selection","volume":"42","author":"Verghese","year":"2014","journal-title":"Nucleic Acids Res."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"1049","DOI":"10.1016\/0092-8674(92)90253-9","article-title":"Purification of Component A of Rab Geranylgeranyl Transferase: Possible Identity with the Choroideremia Gene Product","volume":"70","author":"Seabra","year":"1992","journal-title":"Cell"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5289","DOI":"10.1074\/jbc.271.10.5289","article-title":"Protein Prenyltransferases","volume":"271","author":"Casey","year":"1996","journal-title":"J. Biol. Chem."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"3416","DOI":"10.1093\/hmg\/ddw184","article-title":"Functional Rescue of REP1 Following Treatment with PTC124 and Novel Derivative PTC-414 in Human Choroideremia Fibroblasts and the Nonsense-Mediated Zebrafish Model","volume":"25","author":"Moosajee","year":"2016","journal-title":"Human. Mol. Genet."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"3009","DOI":"10.1167\/iovs.08-2755","article-title":"Single Choroideremia Gene in Nonmammalian Vertebrates Explains Early Embryonic Lethality of the Zebrafish Model of Choroideremia","volume":"50","author":"Moosajee","year":"2009","journal-title":"Invest. Ophthalmol. Vis. Sci."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"4600","DOI":"10.1073\/pnas.0605818104","article-title":"Noncell-Autonomous Photoreceptor Degeneration in a Zebrafish Model of Choroideremia","volume":"104","author":"Krock","year":"2007","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"807","DOI":"10.1080\/14712598.2018.1484448","article-title":"Choroideremia: Molecular Mechanisms and Development of AAV Gene Therapy","volume":"18","author":"Barnard","year":"2018","journal-title":"Expert. Opin. Biol. Ther."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"100915","DOI":"10.1016\/j.preteyeres.2020.100915","article-title":"Immune Responses to Retinal Gene Therapy Using Adeno-Associated Viral Vectors\u2014Implications for Treatment Success and Safety","volume":"83","author":"Bucher","year":"2021","journal-title":"Prog. Retin. Eye Res."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"3987","DOI":"10.1093\/hmg\/ddn302","article-title":"Translational Bypass of Nonsense Mutations in Zebrafish Rep1, Pax2.1 and Lamb1 Highlights a Viable Therapeutic Option for Untreatable Genetic Eye Disease","volume":"17","author":"Moosajee","year":"2008","journal-title":"Hum. Mol. Genet."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1007\/978-3-319-75402-4_11","article-title":"Antisense Oligonucleotide-Based Splice Correction of a Deep-Intronic Mutation in CHM Underlying Choroideremia","volume":"1074","author":"Garanto","year":"2018","journal-title":"Adv. Exp. Med. Biol."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1865","DOI":"10.1093\/hmg\/ddz028","article-title":"Nonsense-Mediated mRNA Decay Efficiency Varies in Choroideremia Providing a Target to Boost Small Molecule Therapeutics","volume":"28","author":"Sarkar","year":"2019","journal-title":"Hum. Mol. Genet."}],"container-title":["International Journal of Molecular Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/20\/15225\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T21:07:37Z","timestamp":1760130457000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1422-0067\/24\/20\/15225"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,10,16]]},"references-count":40,"journal-issue":{"issue":"20","published-online":{"date-parts":[[2023,10]]}},"alternative-id":["ijms242015225"],"URL":"https:\/\/doi.org\/10.3390\/ijms242015225","relation":{},"ISSN":["1422-0067"],"issn-type":[{"value":"1422-0067","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,10,16]]}}}