SNCA-targeted transcriptional repression therapy alleviates pathological and behavioral perturbations in a Parkinson's disease mouse model.

Alpha-synuclein (SNCA) overexpression is implicated in Parkinson's disease (PD) pathogenesis, making SNCA downregulation a promising therapeutic strategy. We developed a SNCA-targeted transcriptional repression therapy using an all-in-one lentiviral vector (LV) carrying deactivated CRISPR/(d)Cas9, gRNA targeted at SNCA-intron1, and either the catalytic domain of DNA-methyltransferase3A (DNMT3A), or an engineered repressor molecule, a fusion of MeCP2's transcription repression domain (TRD) and KRAB. Therapeutic efficacy was evaluated following co-administration of the therapeutic and model vectors in a new PD mouse model, generated with an adeno-associated viral vector carrying an engineered minigene comprised of the human (h)A53T-SNCA expressed via the human native regulatory region. Both therapeutic vectors reduced expression of α-synuclein in the substantia nigra (SN), with LV/dSaCas9- KRAB-MeCP2(TRD) demonstrating greater repression. LV/dSaCas9- KRAB-MeCP2(TRD) also significantly reduced pathological α-synuclein aggregation and phosphorylation (Ser 129), and preserved tyrosine hydroxylase expression in the SN and the striatum. Behavioral analysis following LV/dSaCas9-KRAB-MeCP2(TRD) injection, showed significant improvement in motor deficits characteristic of our PD-mouse model. Preliminary safety assessments found normal blood counts, serum chemistry, and weights. Collectively, these findings provide in vivo proof-of-concept for SNCA-targeted transcriptional repression therapy in a PD-mouse model and support its further preclinical development toward investigational new drug enablement.