{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,4,8]],"date-time":"2026-04-08T04:40:36Z","timestamp":1775623236055,"version":"3.50.1"},"posted":{"date-parts":[[2023,11,15]]},"group-title":"Molecular Biology","reference-count":40,"publisher":"openRxiv","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2023,11,15]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:p>\n                  A system enabling the expression of therapeutic proteins specifically in diseased cells would be transformative, providing greatly increased safety and the possibility of pre-emptive treatment. Here we describe \u201cTDP-REG\u201d, a precision medicine approach primarily for amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which exploits the cryptic splicing events that occur in cells with TDP-43 loss-of-function (TDP-LOF) in order to drive expression specifically in diseased cells. In addition to modifying existing cryptic exons for this purpose, we develop a deep-learning-powered algorithm for generating customisable cryptic splicing events, which can be embedded within virtually any coding sequence. By placing part of a coding sequence within a novel cryptic exon, we tightly couple protein expression to TDP-LOF. Protein expression is activated by TDP-LOF\n                  <jats:italic>in vitro<\/jats:italic>\n                  and\n                  <jats:italic>in vivo<\/jats:italic>\n                  , including TDP-LOF induced by cytoplasmic TDP-43 aggregation. In addition to generating a variety of fluorescent and luminescent reporters, we use this system to perform TDP-LOF-dependent genomic prime editing to ablate the\n                  <jats:italic>UNC13A<\/jats:italic>\n                  cryptic donor splice site. Furthermore, we design a panel of tightly gated, autoregulating vectors encoding a TDP-43\/Raver1 fusion protein, which rescue key pathological cryptic splicing events. In summary, we combine deep-learning and rational design to create sophisticated splicing sensors, resulting in a platform that provides far safer therapeutics for neurodegeneration, potentially even enabling preemptive treatment of at-risk individuals.\n                <\/jats:p>\n                <jats:sec>\n                  <jats:title>One-Sentence Summary<\/jats:title>\n                  <jats:p>We engineer TDP-43-regulated cryptic exons, enabling exceptionally precise activation of gene therapies in diseased neurons.<\/jats:p>\n                <\/jats:sec>","DOI":"10.1101\/2023.11.15.565967","type":"posted-content","created":{"date-parts":[[2023,11,15]],"date-time":"2023-11-15T22:35:15Z","timestamp":1700087715000},"source":"Crossref","is-referenced-by-count":2,"title":["Creation of de novo cryptic splicing for ALS\/FTD precision medicine"],"prefix":"10.64898","author":[{"given":"Oscar G.","family":"Wilkins","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Max Z.Y.J.","family":"Chien","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Josette J.","family":"Wlaschin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maria","family":"Pisliakova","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"David","family":"Thompson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Holly","family":"Digby","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rebecca L.","family":"Simkin","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Juan Antinao","family":"Diaz","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Puja R.","family":"Mehta","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matthew J.","family":"Keuss","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Matteo","family":"Zanovello","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Anna-Leigh","family":"Brown","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peter","family":"Harley","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Annalucia","family":"Darbey","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Rajvinder","family":"Karda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Elizabeth M.C.","family":"Fisher","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Tom J.","family":"Cunningham","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Claire E.","family":"Le Pichon","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jernej","family":"Ule","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pietro","family":"Fratta","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"54368","reference":[{"key":"2023111806050808000_2023.11.15.565967v1.1","doi-asserted-by":"publisher","DOI":"10.1007\/s13311-015-0338-x"},{"key":"2023111806050808000_2023.11.15.565967v1.2","doi-asserted-by":"publisher","DOI":"10.1111\/bpa.12424"},{"key":"2023111806050808000_2023.11.15.565967v1.3","doi-asserted-by":"publisher","DOI":"10.1093\/brain\/awz099"},{"key":"2023111806050808000_2023.11.15.565967v1.4","doi-asserted-by":"publisher","DOI":"10.1126\/science.aab0983"},{"key":"2023111806050808000_2023.11.15.565967v1.5","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-018-0300-4"},{"key":"2023111806050808000_2023.11.15.565967v1.6","doi-asserted-by":"publisher","DOI":"10.1038\/s41593-018-0293-z"},{"key":"2023111806050808000_2023.11.15.565967v1.7","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-04436-3"},{"key":"2023111806050808000_2023.11.15.565967v1.8","doi-asserted-by":"publisher","DOI":"10.1038\/s41586-022-04424-7"},{"key":"2023111806050808000_2023.11.15.565967v1.9","doi-asserted-by":"publisher","DOI":"10.1038\/s41556-022-00988-8"},{"key":"2023111806050808000_2023.11.15.565967v1.10","doi-asserted-by":"publisher","DOI":"10.1126\/science.abq5622"},{"key":"2023111806050808000_2023.11.15.565967v1.11","doi-asserted-by":"crossref","unstructured":"H. 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