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Splicing can be therefore modulated for therapeutic purposes, and splicing-switching molecules are in clinics for some diseases. Here, conscious that over 10% of all pathogenic mutations occurs at 5\u2019ss, we aimed at characterizing and rescuing nine 5\u2019ss mutations in three models of defective <jats:italic>F<\/jats:italic>8 exons whose skipping would lead to factor VIII (FVIII) deficiency (Hemophilia A), the most frequent coagulation factor disorder.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Methods<\/jats:title>\n            <jats:p>HEK293T cells were transfected with <jats:italic>F8<\/jats:italic> minigene variants, alone or with engineered U1 small nuclear RNAs (U1snRNAs), and splicing patterns analysed via RT-PCR.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Results<\/jats:title>\n            <jats:p>All 5\u2019ss mutations induced exon skipping, and the proportion of correct transcripts, not predictable by computational analysis, was consistent with residual FVIII levels in patients. For each exon we identified a unique engineered U1snRNAs, either compensatory or Exon Specific (ExSpeU1), able to rescue all mutations. Overall, ExSpeU1s were more effective than compensatory U1snRNAs, particularly in the defective exons 6 and 22.<\/jats:p>\n          <\/jats:sec>\n          <jats:sec>\n            <jats:title>Conclusions<\/jats:title>\n            <jats:p>Data highlight the importance of splicing assays to elucidate genotype-phenotype relationships and proved the correction efficacy of ExSpeU1s for each targeted defective <jats:italic>F8<\/jats:italic> exon, thus expanding their translational potential for HA.<\/jats:p>\n          <\/jats:sec>","DOI":"10.1186\/s10020-025-01176-8","type":"journal-article","created":{"date-parts":[[2025,3,27]],"date-time":"2025-03-27T15:33:23Z","timestamp":1743089603000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Rescue of a panel of Hemophilia A-causing 5\u2019ss splicing mutations by unique Exon-specific U1snRNA variants"],"prefix":"10.1186","volume":"31","author":[{"given":"Laura","family":"Peretto","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claudia","family":"D\u2019angiolillo","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Paolo","family":"Ferraresi","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dario","family":"Balestra","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mirko","family":"Pinotti","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,3,27]]},"reference":[{"issue":"17","key":"1176_CR23","doi-asserted-by":"publisher","first-page":"4809","DOI":"10.1093\/hmg\/ddv205","volume":"24","author":"D Balestra","year":"2015","unstructured":"Balestra D, Barbon E, Scalet D, Cavallari N, Perrone D, Zanibellato S, et al. 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All the other authors declare no competing interests.","order":4,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"121"}}