Gene Therapy for Non-Motor Defects in Synucleinopathy

Following Alzheimer's disease, synucleinopathies such as Parkinson’s disease (PD) and related conditions are the second most common group of neurodegenerative disorders, characterized by aggregates containing the small synaptic protein α-synuclein (α-Syn). The pervasive involvement of α-Syn in PD and related conditions provides a strong rationale for focusing on α-Syn pathology as a possible target for therapeutic interventions. Diagnosis of PD relies on cardinal motor system abnormalities. However, non- motor symptoms are common and may present for years before motor symptom onset and about half of patients with PD present with α-Syn pathology in the gut before the brain. Constipation is reported by ≥80% of patients with PD and can precede PD diagnosis by decades. Both patients with and mouse models of synucleinopathy (including the transgenic [Tg] L61 model) present with α-Syn pathology throughout the gastrointestinal (GI) system, including robust α-Syn immunopositivity in the enteric nervous system (ENS), associated with constipation-indicative symptoms, suggesting that α-Syn deposition in the gut may contribute to GI functional impairments. In addition, a mouse model of synucleinopathy can be generated by injecting α-Syn preformed fibrils (PFF) into the gut, indicating that α-Syn pathology and neurodegenerative processes may spread from the gut to the brain. Experimental strategies for inhibiting α-Syn oligomer and protofibril formation or promoting clearance include the highly promising active and passive immunization approaches. Single-chain variable fragments (scFvs) are antibody derivatives that are six times smaller than a complete IgG while retaining the same binding specificity, allowing recombinant genes encoding scFVs to be encapsulated into self- complementary adeno-associated viral vectors (scAAVs) for use in gene therapy approaches. In this translational R21, we propose the clinically transferrable strategy of targeting the ENS with a scAAV encoding an scFv against α-Syn, extending our ongoing efforts to (1) identify the biological mechanisms underlying non-motor symptoms of synucleinopathy, such as GI dysfunction, and (2) develop treatments for PD and related conditions. We will treat well-validated Tg L61 and α-Syn PFF mouse models of synucleinopathy with the AAV-MaCPNS2 (known to transduce ENS) expressing either a secreted or non- secreted anti–α-Syn scFv and assess the effects of this approach. In AIM 1, we will evaluate the effects of virally encoded anti–α-Syn scFvs on the ENS in Tg L61 mice by characterizing GI function and histopathology. In AIM 2, we will assess the effects of virally encoded anti–α-Syn scFvs on motor and cognitive functions and brain pathology in mice injected in the gut with α-Syn PFF. In both AIMs, non-regulatory blood toxicology and key organs histopathology will also be done. The successful completion of one of the first in vivo studies assessing the potential value of passive immunization using ENS-targeted anti–α-Syn scFvs will clarify whether α-Syn gut pathology is a prodromal PD symptom amenable to intervention. Project Number: 1R21AG093148-01A1 | Fiscal Year: 2026 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: SERGE PRZEDBORSKI | Institution: COLUMBIA UNIVERSITY HEALTH SCIENCES, NEW YORK, NY | Award Amount: $452,375 | Activity Code: R21 | Study Section: Chronic Dysfunction and Integrative Neurodegeneration Study Section[CDIN] View on NIH RePORTER: https://reporter.nih.gov/project-details/11360748