Restoring Parkin function: an AAV gene therapy approach for early-onset Parkinson's disease

Biallelic loss-of-function mutations in PRKN gene (encoding Parkin protein) cause early-onset Parkinson's disease (EOPD). Parkin is a crucial component of the PINK1-Parkin pathway, which marks damaged mitochondria for degradation via mitophagy. Without functional Parkin, damaged mitochondria accumulate, causing oxidative stress and neurodegeneration. We investigated Parkin gene replacement via AAV gene therapy as a potential treatment for Parkin-dependent EOPD. We validated phosphorylated ubiquitin Ser65 (pUb^Ser65) as an indicator of PINK1-Parkin pathway initiation/activation. We evaluated AAV-mediated PRKN replacement (AAV-Parkin) in a Parkin knockout neuroblastoma cell (SH-SY5Y cells) and feasibility of delivery in mouse and rat models. Our research showed pUb^Ser65 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 restored these pUb^Ser65 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 (also referred as Spk100), finding Ef1a and Synapsin1 (Syn1) were most effective for transducing relevant cell populations, including dopaminergic neurons, in the SN of mice without causing adverse effects. These findings established a short-term well-tolerated vector dose and an optimal capsid-promoter combination. Our results establish foundational feasibility and short-term tolerability, supporting the potential of AAV-Parkin gene therapy as a promising candidate for further preclinical development for Parkin-deficient EOPD.