LB: Immune Tolerance to Microdystrophin by Hepatic Expression Results in Enhanced Muscle Expression in Dystrophin-Deficient mdx Mice

Topic:

Pre-Clinical Research

Poster Number: 488 LBT

Author(s):

Madhurima Saha, PhD, University of Florida, Radhika Suresh Bhake, MS, University of Florida, Kirsten E Coleman, MBA, University of Florida, Prasad Trivedi, MS, University of Florida, Denise Cloutier, BS, University of Florida, Megan B Wichman, BS, University of Florida, Daniil Shabashvili, PhD, University of Florida, Claudia Mercado-Rodriguez, PhD, University of Florida, Moanaro Biswas, PhD, Indiana University, Roland Herzog, PhD, Indiana University, Manuela Corti, PT, PhD, Univ, Barry Byrne, MD, PhD, University of Florida

Introduction:
Systemic AAV-mediated microdystrophin (µDys) gene therapy is a promising therapeutic approach for Duchenne muscular dystrophy (DMD); however, immune responses against the AAV capsid and dystrophin transgene limit efficacy and durability. While current clinical immunosuppression (IMS) protocols effectively reduce anti-capsid responses, they fail to control anti-dystrophin immunity. Liver-directed AAV gene therapy is known to induce antigen-specific regulatory T cells (Tregs) and promote immune tolerance in diseases like hemophilia and Pompe disease. We hypothesized that liver-directed µDys expression would induce systemic immune tolerance, thereby enhancing and sustaining therapeutic transgene expression in extrahepatic tissues.

Methods:
Dystrophin-deficient mdx mice (n=4-5/group) were assigned to four treatment groups: (1) AAV9-Des-UFµDys2 (muscle-directed therapeutic vector; 1×10¹⁴ vg/kg); (2) Animals received intraperitoneal anti-CD20 (10 mg/kg, clone 18B12) every 3 weeks before AAV administration, combined with oral rapamycin (4 mg/kg) given three times weekly for 10 weeks (IMS) + AAV9-Des-UFµDys2; (3) IMS + AAV9-LSP-UFµDys2 (liver-directed tolerogenic vector, 2×10¹³ vg/kg) followed by AAV9-Des-UFµDys2; and (4) vehicle control. Immune profiling was performed by flow cytometry on splenocytes. Anti-µDys antibodies were assessed by western blot, and anti-AAV9 capsid antibodies by ELISA. µDys expression in cardiac muscle, skeletal muscle, and brain was quantified by western blot and qPCR.

Results:
Liver-directed µDys delivery combined with IMS (Group 3) resulted in a significant expansion of splenic Tregs (p<0.0001) and depletion of effector T cells compared to muscle-directed µDys alone (Group 1). Group 3 exhibited an 83% reduction in anti-µDys antibodies compared to Group 1 (p<0.0001). Correspondingly, µDys expression was significantly increased in cardiac muscle (p<0.0001), skeletal muscle (p<0.0001), and brain (p=0.0015) in Group 3 versus Group 1, despite comparable vector genome copy numbers, indicating immune-mediated enhancement rather than increased transduction efficiency.

Conclusion:
Liver-directed µDys expression combined with transient IMS induces antigen-specific immune tolerance and significantly enhances therapeutic microdystrophin expression across multiple tissues. This clinically translatable tolerogenic strategy represents a promising approach to improve the safety and efficacy of AAV gene therapy for DMD.