{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,7,16]],"date-time":"2026-07-16T13:14:41Z","timestamp":1784207681091,"version":"3.55.0"},"posted":{"date-parts":[[2026,7,13]]},"group-title":"Neuroscience","reference-count":21,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T00:00:00Z","timestamp":1783900800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2026,7,13]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background<\/jats:title>\n                  <jats:p>\n                    Biallelic loss-of-function mutations in\n                    <jats:italic>PRKN<\/jats:italic>\n                    gene (encoding Parkin protein) cause early-onset Parkinson\u2019s disease (EOPD). Parkin is a crucial component of PINK1-Parkin pathway, which marks damaged mitochondria for degradation via mitophagy. Without functional Parkin, damaged mitochondria accumulate, causing oxidative stress and neurodegeneration.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Objective<\/jats:title>\n                  <jats:p>Investigate Parkin gene replacement via AAV gene therapy as a potential treatment for Parkin-dependent EOPD.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Methods<\/jats:title>\n                  <jats:p>\n                    We initially validated phosphorylated ubiquitin Ser65 (pUb\n                    <jats:sup>Ser65<\/jats:sup>\n                    ) as an indicator of Parkin-mediated mitophagy initiation. We evaluated AAV-mediated\n                    <jats:italic>PRKN<\/jats:italic>\n                    replacement (hereafter, AAV-Parkin) in a Parkin knockout neuroblastoma cell line (SH-SY5Y cells) and feasibility of delivery in mouse and rat models.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Results<\/jats:title>\n                  <jats:p>\n                    Our research showed pUb\n                    <jats:sup>Ser65<\/jats:sup>\n                    signal was reduced in Parkin-KO SH-SY5Y cells when compared to wild-type cells after mitochondrial stress, indicating deficiency in initiation of mitophagy. AAV-mediated human\n                    <jats:italic>PRKN<\/jats:italic>\n                    gene replacement successfully restored these pUb\n                    <jats:sup>Ser65<\/jats:sup>\n                    levels in knockout cells. We saw restoration in patient-derived fibroblasts following AAV-Parkin overexpression. We developed a translatable gene therapy approach using rodents. We demonstrated the feasibility of delivering AAV-Parkin directly into the substantia nigra (SN) of wild-type rats. Using an AAV1 capsid with Ef1a promoter, we achieved dose-dependent Parkin expression and identified a well-tolerated dose. We also evaluated multiple promoters in a proprietary Spark100 capsid, finding Ef1a and Synapsin1 (Syn1) were most effective for transducing dopaminergic neurons in the SN of mice without causing adverse effects. These findings established a well-tolerated vector dose and an optimal capsid-promoter combination.\n                  <\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusions<\/jats:title>\n                  <jats:p>Our results support the potential of AAV-Parkin gene therapy as a disease-modifying approach for Parkin-deficient EOPD.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"737487v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.64898\/2026.07.09.737487","type":"posted-content","created":{"date-parts":[[2026,7,14]],"date-time":"2026-07-14T05:55:19Z","timestamp":1784008519000},"source":"Crossref","is-referenced-by-count":0,"title":["Restoring Parkin Function: An AAV Gene Therapy Approach for Early-Onset Parkinson\u2019s Disease"],"prefix":"10.64898","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-1281-1543","authenticated-orcid":false,"given":"Sambuddha","family":"Basu","sequence":"first","affiliation":[{"name":"Spark Therapeutics, Philadelphia, PA, USA"},{"name":"Roche Innovation Center (RICP), Philadelphia, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tyler G","family":"Demarest","sequence":"additional","affiliation":[{"name":"Spark Therapeutics, Philadelphia, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicholas J.","family":"Gattone","sequence":"additional","affiliation":[{"name":"Spark Therapeutics, Philadelphia, PA, USA"},{"name":"Roche Innovation Center (RICP), Philadelphia, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alexandra J","family":"Gilsrud","sequence":"additional","affiliation":[{"name":"National Institutes of Health (NIH), National Institute of Neurological Disorders and Stroke (NINDS), Bethesda, MD, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Brittany","family":"Wicks","sequence":"additional","affiliation":[{"name":"Spark Therapeutics, Philadelphia, PA, USA"},{"name":"Roche Innovation Center (RICP), Philadelphia, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Apeksha","family":"Khatiwada","sequence":"additional","affiliation":[{"name":"Spark Therapeutics, Philadelphia, PA, USA"},{"name":"Roche Innovation Center (RICP), Philadelphia, PA, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mohammad","family":"Nayal","sequence":"additional","affiliation":[{"name":"Spark Therapeutics, 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