Restoring Parkin Function: An AAV Gene Therapy Approach for Early-Onset Parkinson's Disease

Basu, S.; Demarest, T. G.; Gattone, N. J.; Gilsrud, A. J.; Wicks, B.; Khatiwada, A.; Nayal, M.; Gentzel, R.; Cohen, D.; Kostuk, E. W.; Narendra, D. P.; Alegre, P. G.; Biferi, M.-G.; Ramsburg, E. A.

doi:10.64898/2026.07.09.737487

bioRxiv · 10.64898/2026.07.09.737487

Restoring Parkin Function: An AAV Gene Therapy Approach for Early-Onset Parkinson's Disease

Basu, S.·Demarest, T. G.·Gattone, N. J.·Gilsrud, A. J.·Wicks, B.·Khatiwada, A.·Nayal, M.·Gentzel, R.·Cohen, D.·Kostuk, E. W.·Narendra, D. P.·Alegre, P. G.·Biferi, M.-G.·Ramsburg, E. A.

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Abstract

BackgroundBiallelic loss-of-function mutations in PRKN gene (encoding Parkin protein) cause early-onset Parkinsons 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. ObjectiveInvestigate Parkin gene replacement via AAV gene therapy as a potential treatment for Parkin-dependent EOPD. MethodsWe initially validated phosphorylated ubiquitin Ser65 (pUbSer65) as an indicator of Parkin-mediated mitophagy initiation. We evaluated AAV-mediated PRKN replacement (hereafter, AAV-Parkin) in a Parkin knockout neuroblastoma cell line (SH-SY5Y cells) and feasibility of delivery in mouse and rat models. ResultsOur research showed pUbSer65 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 PRKN gene replacement successfully restored these pUbSer65 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. ConclusionsOur results support the potential of AAV-Parkin gene therapy as a disease-modifying approach for Parkin-deficient EOPD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=132 SRC="FIGDIR/small/737487v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@16dd13corg.highwire.dtl.DTLVardef@c3dfcdorg.highwire.dtl.DTLVardef@19a310dorg.highwire.dtl.DTLVardef@a66f2_HPS_FORMAT_FIGEXP M_FIG C_FIG

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Basu, S., Demarest, T. G., Gattone, N. J., Gilsrud, A. J., Wicks, B., Khatiwada, A., Nayal, M., Gentzel, R., Cohen, D., Kostuk, E. W., Narendra, D. P., Alegre, P. G., Biferi, M.-G., Ramsburg, E. A.. 2026-07-13. Restoring Parkin Function: An AAV Gene Therapy Approach for Early-Onset Parkinson's Disease. https://doi.org/10.64898/2026.07.09.737487

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