Objective: To develop an adeno-associated virus (AAV)-based artificial miRNA (miRNA) system targeting human α-syn.

Background: Synucleinopathies such as Parkinson’s disease (PD) and Multiple System Atrophy (MSA) are a group of neurodegenerative disorder characterized by accumulation of phosphorylated, misfolded, and insoluble α-syn. While the understanding of molecular pathogenesis is still evolving, reduction of soluble α-syn has been widely explored as a potential neuroprotective strategy. Preclinical studies have demonstrated the feasibility of reducing α-syn levels using RNA interference (RNAi) approaches and its neuroprotective effects in disease models. Here, we report the development of an AAV expressing a miRNA targeting human α-syn mRNA and provide evidence of efficacy in a disease-relevant model.

Method: In silico algorithms were used to design small interfering RNA (siRNAs) targeting disease specific α-syn isoforms with predicted high binding efficiency and minimal off-target effects. Selected siRNAs were expressed as miRNAs under a ubiquitous promoter and packaged in a proprietary AAV capsid (AAV-miRNA). Preliminary efficacy of the AAV-miRNA was evaluated after co-injection with an AAV-expressing human mutant α-syn into the striatum or substantia nigra (SN) of naïve mice. Expression of α-syn mRNA and distribution of the AAV-miRNA was evaluated by RT-qPCR. α-syn protein levels were measured through automated western blot and striatal dopamine content by HPLC. miRNA expression and processing were evaluated by small RNA sequencing.

Results: Two lead AAV-miRNA candidates were selected and tested in vivo. The two AAV-miRNAs induced efficient reduction of human α-syn mRNA and protein by up to 90% in striatum and cortex, respectively. Despite the robust knockdown of α-syn mRNA, the therapeutic miRNAs represented only a small percentage of the total miRNome, without disrupting endogenous miRNA expression. RNA-sequencing analysis confirmed that both candidate miRNAs were processed efficiently at the 5’ end. Finally, PD model animals treated with the AAV-miRNA in SN had significantly less loss of dopamine and metabolites vs animals dosed with the control AAV expressing a non-targeting miRNA.

Conclusion: AAV-miRNAs designed to reduce human α-syn levels were successfully tested and warrant further development as a potential RNAi-based therapeutic for synucleinopathies.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/development-of-an-aav-mediated-artificial-mirna-targeting-human-alpha-synuclein-%ce%b1-syn-as-a-potential-gene-therapy-for-synucleinopathies/