{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T08:50:45Z","timestamp":1775983845215,"version":"3.50.1"},"posted":{"date-parts":[[2025,3,29]]},"group-title":"Neuroscience","reference-count":57,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,3,29]],"date-time":"2025-03-29T00:00:00Z","timestamp":1743206400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,3,29]]},"abstract":"<jats:title>\n                  <jats:bold>Abstract<\/jats:bold>\n                <\/jats:title>\n                <jats:p>MECP2 duplication syndrome (MDS) is a rare X-linked neurodevelopmental disorder caused by duplications of the dosage-sensitive methyl-CpG-binding protein 2 (MECP2) gene. Developing effective therapies for MDS is particularly challenging due to the variability in MECP2 expression among patients and the potential risk of inducing Rett syndrome through excessive pharmacological intervention. Reducing dosage to optimize silencing levels often compromises durability and necessitates increased dosing frequency. We present here a series of fully chemically modified small interfering RNAs (siRNAs) designed for both isoform-selective and total MECP2 silencing. Among these, we identify six lead siRNA candidates across two distinct chemical scaffolds, achieving targeted total MECP2 expression reductions ranging from 25% to 75%, sustained for at least four months following a single administration. The efficacy and safety of human ortholog silencing were evaluated using two mouse models with distinct levels of human MECP2 transgene expression. In the severe duplication model, a single dose of the total isoform-silencing siRNA fully rescued early mortality and behavioral impairments. Additionally, we show that the isoform-selective targeting strategy may be safer in mild cases of MDS where exaggerated pharmacology may lead to Rett Syndrome. Overall, this study introduces a series of preclinical candidates with the capacity to address the varying levels of MECP2 duplication encountered in clinical settings. Furthermore, it establishes a target selection strategy that may be applied to other dosage-sensitive gene imbalances.<\/jats:p>\n                <jats:sec>\n                  <jats:title>One Sentence Summary<\/jats:title>\n                  <jats:p>Therapeutic siRNAs provide safe and durable modulation of MECP2 for the treatment of mild and severe MECP2 Duplication Syndrome.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2025.03.26.645328","type":"posted-content","created":{"date-parts":[[2025,3,30]],"date-time":"2025-03-30T02:37:45Z","timestamp":1743302265000},"source":"Crossref","is-referenced-by-count":5,"title":["Single-dose administration of therapeutic divalent siRNA targeting MECP2 prevents lethality for one year in an MECP2 duplication mouse model"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-9624-1244","authenticated-orcid":false,"given":"Vignesh N.","family":"Hariharan","sequence":"first","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Ashley","family":"Summers","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Amy E.","family":"Clipperton-Allen","sequence":"additional","affiliation":[{"name":"The Jackson Laboratory; Bar Harbor, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jillian","family":"Caiazzi","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Samuel R.","family":"Hildebrand","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniel O\u2019","family":"Reilly","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Qi","family":"Tang","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Zachary","family":"Kennedy","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dimas","family":"Echeverria","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Nicholas","family":"McHugh","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Cooper","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jacqueline","family":"Souza","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Chantal","family":"Ferguson","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laurent","family":"Bogdanik","sequence":"additional","affiliation":[{"name":"The Jackson Laboratory; Bar Harbor, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Monica","family":"Coenraads","sequence":"additional","affiliation":[{"name":"The Rett Syndrome Research Trust; Trumbull, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anastasia","family":"Khvorova","sequence":"additional","affiliation":[{"name":"RNA Therapeutic Institute, University of Massachusetts Chan Medical School; Worcester, United States of America"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2026041201264022000_2025.03.26.645328v1.1","doi-asserted-by":"publisher","DOI":"10.1016\/S1474-4422(20)30217-9"},{"key":"2026041201264022000_2025.03.26.645328v1.2","doi-asserted-by":"crossref","unstructured":"K. Allison , M. Maletic-Savatic , D. Pehlivan , MECP2-related disorders while gene-based therapies are on the horizon. Front Genet 15, 1332469 (2024).","DOI":"10.3389\/fgene.2024.1332469"},{"key":"2026041201264022000_2025.03.26.645328v1.3","doi-asserted-by":"crossref","unstructured":"Y. Shao , Y. Sztainberg , Q. Wang , S. S. Bajikar , A. J. Trostle , Y. W. Wan , P. Jafar-Nejad , F. Rigo , Z. Liu , J. Tang , H. Y. Zoghbi , Antisense oligonucleotide therapy in a humanized mouse model of MECP2 duplication syndrome. Sci Transl Med 13, (2021).","DOI":"10.1126\/scitranslmed.aaz7785"},{"key":"2026041201264022000_2025.03.26.645328v1.4","doi-asserted-by":"publisher","DOI":"10.1038\/528S123a"},{"key":"2026041201264022000_2025.03.26.645328v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s42003-024-06990-0"},{"key":"2026041201264022000_2025.03.26.645328v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-019-0205-0"},{"key":"2026041201264022000_2025.03.26.645328v1.7","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa670"},{"key":"2026041201264022000_2025.03.26.645328v1.8","doi-asserted-by":"publisher","DOI":"10.1089\/nat.2022.0004"},{"key":"2026041201264022000_2025.03.26.645328v1.9","doi-asserted-by":"publisher","DOI":"10.1016\/j.bjae.2019.11.002"},{"key":"2026041201264022000_2025.03.26.645328v1.10","doi-asserted-by":"publisher","DOI":"10.1111\/ner.13325"},{"key":"2026041201264022000_2025.03.26.645328v1.11","doi-asserted-by":"crossref","unstructured":"V. Belov , J. Appleton , S. Levin , P. Giffenig , B. Durcanova , M. Papisov , Large-Volume Intrathecal Administrations: Impact on CSF Pressure and Safety Implications. Front Neurosci 15, 604197 (2021).","DOI":"10.3389\/fnins.2021.604197"},{"key":"2026041201264022000_2025.03.26.645328v1.12","doi-asserted-by":"publisher","DOI":"10.1111\/epi.17625"},{"key":"2026041201264022000_2025.03.26.645328v1.13","doi-asserted-by":"publisher","DOI":"10.1111\/cge.13521"},{"key":"2026041201264022000_2025.03.26.645328v1.14","doi-asserted-by":"crossref","unstructured":"A. Martinez de Paz , L. Khajavi , H. Martin , R. Claveria-Gimeno , S. Tom Dieck , M. S. Cheema , J. V. Sanchez-Mut , M. M. Moksa , A. Carles , N. I. Brodie , T. I. Sheikh , M. E. Freeman , E. V. Petrotchenko , C. H. Borchers, E. M. Schuman, M. Zytnicki, A. Velazquez-Campoy, O. Abian, M. Hirst, M. Esteller, J. B. Vincent, C. E. Malnou, J. Ausio , MeCP2-E1 isoform is a dynamically expressed, weakly DNA-bound protein with different protein and DNA interactions compared to MeCP2-E2. Epigenetics Chromatin 12, 63 (2019).","DOI":"10.1186\/s13072-019-0298-1"},{"key":"2026041201264022000_2025.03.26.645328v1.15","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0090645"},{"key":"2026041201264022000_2025.03.26.645328v1.16","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M111.309864"},{"key":"2026041201264022000_2025.03.26.645328v1.17","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddt640"},{"key":"2026041201264022000_2025.03.26.645328v1.18","doi-asserted-by":"publisher","DOI":"10.1136\/jmg.2004.026161"},{"key":"2026041201264022000_2025.03.26.645328v1.19","doi-asserted-by":"publisher","DOI":"10.1016\/j.ejmg.2005.11.002"},{"key":"2026041201264022000_2025.03.26.645328v1.20","doi-asserted-by":"crossref","first-page":"1019","DOI":"10.1002\/ajmg.a.32776","article-title":"Novel exon 1 mutations in MECP2 implicate isoform MeCP2_e1 in classical Rett syndrome","volume":"149A","year":"2009","journal-title":"Am J Med Genet A"},{"key":"2026041201264022000_2025.03.26.645328v1.21","doi-asserted-by":"publisher","DOI":"10.1038\/ejhg.2011.145"},{"key":"2026041201264022000_2025.03.26.645328v1.22","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0610593104"},{"key":"2026041201264022000_2025.03.26.645328v1.23","doi-asserted-by":"publisher","DOI":"10.1126\/science.1138389"},{"key":"2026041201264022000_2025.03.26.645328v1.24","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0401626101"},{"key":"2026041201264022000_2025.03.26.645328v1.25","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkh349"},{"key":"2026041201264022000_2025.03.26.645328v1.26","doi-asserted-by":"publisher","DOI":"10.1038\/s41573-024-00912-9"},{"key":"2026041201264022000_2025.03.26.645328v1.27","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky037"},{"key":"2026041201264022000_2025.03.26.645328v1.28","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa595"},{"key":"2026041201264022000_2025.03.26.645328v1.29","doi-asserted-by":"publisher","DOI":"10.1002\/path.2993"},{"key":"2026041201264022000_2025.03.26.645328v1.30","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2017.12.021"},{"key":"2026041201264022000_2025.03.26.645328v1.31","doi-asserted-by":"crossref","unstructured":"K. Yamada , V. N. Hariharan , J. Caiazzi , R. Miller , C. M. Ferguson , E. Sapp , H. H. Fakih , Q. Tang , N. Yamada , R. C. Furgal , J. D. Paquette , A. Biscans , B. M. Bramato , N. McHugh , A. Summers , C. Lochmann , B. Godinho , S. Hildebrand , S. O. Jackson , D. Echeverria , M. R. Hassler , J. F. Alterman , M. DiFiglia , N. Aronin , A. Khvorova , Enhancing siRNA efficacy in vivo with extended nucleic acid backbones. Nat Biotechnol, (2024).","DOI":"10.1038\/s41587-024-02336-7"},{"key":"2026041201264022000_2025.03.26.645328v1.32","doi-asserted-by":"publisher","DOI":"10.1016\/j.omtn.2022.06.009"},{"key":"2026041201264022000_2025.03.26.645328v1.33","doi-asserted-by":"publisher","DOI":"10.1016\/j.ymthe.2022.04.019"},{"key":"2026041201264022000_2025.03.26.645328v1.34","doi-asserted-by":"publisher","DOI":"10.1038\/s41467-023-42714-4"},{"key":"2026041201264022000_2025.03.26.645328v1.35","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddh282"},{"key":"2026041201264022000_2025.03.26.645328v1.36","doi-asserted-by":"publisher","DOI":"10.1038\/nbt936"},{"key":"2026041201264022000_2025.03.26.645328v1.37","doi-asserted-by":"publisher","DOI":"10.1038\/nprot.2007.278"},{"key":"2026041201264022000_2025.03.26.645328v1.38","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gky745"},{"key":"2026041201264022000_2025.03.26.645328v1.39","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkx818"},{"key":"2026041201264022000_2025.03.26.645328v1.40","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/23.11.2019"},{"key":"2026041201264022000_2025.03.26.645328v1.41","first-page":"5008","article-title":"siRNA carrying an (E)-vinylphosphonate moiety at the 5\u0384 end of the guide strand augments gene silencing by enhanced binding to human Argonaute-2","volume":"45","year":"2017","journal-title":"Nucleic Acids Res"},{"key":"2026041201264022000_2025.03.26.645328v1.42","doi-asserted-by":"publisher","DOI":"10.1089\/nat.2024.0027"},{"key":"2026041201264022000_2025.03.26.645328v1.43","doi-asserted-by":"publisher","DOI":"10.1101\/gad.345397.120"},{"key":"2026041201264022000_2025.03.26.645328v1.44","doi-asserted-by":"publisher","DOI":"10.1002\/emmm.201303744"},{"key":"2026041201264022000_2025.03.26.645328v1.45","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddi016"},{"key":"2026041201264022000_2025.03.26.645328v1.46","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pgen.1006780"},{"key":"2026041201264022000_2025.03.26.645328v1.47","doi-asserted-by":"crossref","unstructured":"G. Kreiner , Compensatory mechanisms in genetic models of neurodegeneration: are the mice better than humans? Front Cell Neurosci 9, 56 (2015).","DOI":"10.3389\/fncel.2015.00056"},{"key":"2026041201264022000_2025.03.26.645328v1.48","doi-asserted-by":"publisher","DOI":"10.1093\/hmg\/ddp366"},{"key":"2026041201264022000_2025.03.26.645328v1.49","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkac539"},{"key":"2026041201264022000_2025.03.26.645328v1.50","doi-asserted-by":"crossref","unstructured":"S. J. Barker , M. B. Thayer , C. Kim , D. Tatarakis , M. J. Simon , R. Dial , L. Nilewski , R. C. Wells , Y. Zhou , M. Afetian , P. Akkapeddi , A. Chappell , K. S. Chew , J. Chow , A. Clemens , C. B. Discenza , J. C. Dugas , C. Dwyer , T. Earr , C. Ha , Y. S. Ho , D. Huynh , E. I. Lozano , S. Jayaraman , W. Kwan , C. Mahon , M. Pizzo , Y. Robles-Colmenares , E. Roche , L. Sanders , A. Stergioulis , R. Tong , H. Tran , Y. Zuchero , A. A. Estrada , K. Gadkar , C. M. M. Koth , P. E. Sanchez , R. G. Thorne , R. J. Watts , T. Sandmann , L. A. Kane , F. Rigo , M. S. Dennis , J. W. Lewcock , S. L. DeVos , Targeting the transferrin receptor to transport antisense oligonucleotides across the mammalian blood-brain barrier. Sci Transl Med 16, eadi2245 (2024).","DOI":"10.1126\/scitranslmed.adi2245"},{"key":"2026041201264022000_2025.03.26.645328v1.51","doi-asserted-by":"publisher","DOI":"10.1016\/j.jconrel.2015.12.041"},{"key":"2026041201264022000_2025.03.26.645328v1.52","doi-asserted-by":"publisher","DOI":"10.1016\/j.addr.2015.01.008"},{"key":"2026041201264022000_2025.03.26.645328v1.53","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-021-00972-x"},{"key":"2026041201264022000_2025.03.26.645328v1.54","doi-asserted-by":"crossref","unstructured":"C. Mazur , B. Powers , K. Zasadny , J. M. Sullivan , H. Dimant , F. Kamme , J. Hesterman , J. Matson , M. Oestergaard , M. Seaman , R. W. Holt , M. Qutaish , I. Polyak , R. Coelho , V. Gottumukkala , C. M. Gaut , M. Berridge , N. J. Albargothy , L. Kelly , R. O. Carare , J. Hoppin , H. Kordasiewicz , E. E. Swayze , A. Verma , Brain pharmacology of intrathecal antisense oligonucleotides revealed through multimodal imaging. JCI Insight 4, (2019).","DOI":"10.1172\/jci.insight.129240"},{"key":"2026041201264022000_2025.03.26.645328v1.55","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/gkaa1235"},{"key":"2026041201264022000_2025.03.26.645328v1.56","doi-asserted-by":"crossref","unstructured":"C. M. Ferguson , B. M. Godinho , J. F. Alterman , A. H. Coles , M. Hassler , D. Echeverria , J. W. Gilbert , E. G. Knox , J. Caiazzi , R. A. Haraszti , R. M. King , T. Taghian , A. Puri , R. P. Moser , M. J. Gounis , N. Aronin , H. Gray-Edwards , A. Khvorova , Comparative route of administration studies using therapeutic siRNAs show widespread gene modulation in Dorset sheep. JCI Insight 6, (2021).","DOI":"10.1172\/jci.insight.152203"},{"key":"2026041201264022000_2025.03.26.645328v1.57","doi-asserted-by":"publisher","DOI":"10.1089\/nat.2015.0578"}],"container-title":[],"original-title":[],"link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/2025.03.26.645328","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,4,12]],"date-time":"2026-04-12T08:26:52Z","timestamp":1775982412000},"score":1,"resource":{"primary":{"URL":"http:\/\/biorxiv.org\/lookup\/doi\/10.1101\/2025.03.26.645328"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,3,29]]},"references-count":57,"URL":"https:\/\/doi.org\/10.1101\/2025.03.26.645328","relation":{},"subject":[],"published":{"date-parts":[[2025,3,29]]},"subtype":"preprint"}}