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A4 - AAV Vectors - Preclinical and Proof-of-Concept In-Vivo Studies (Excluding Non-Human Primates)

18: Breaking Ground in CMT1B Treatment: AAV9-Mediated Dual RNAi and Gene Replacement Therapy Targeting Schwann Cells Improves Myelination and Peripheral Nerve Function in Mice

Type: Oral Abstract Session

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Session Title: AAV Vectors - Preclinical and Proof-of-Concept: Therapy Focus

Charcot-Marie-Tooth (CMT) encompasses a diverse group of hereditary sensory and motor neuropathies resulting in progressive disability and pain. Lacking a cure for these debilitating diseases, current patient care strategies involve symptom management, surgery, rehabilitation, and occupational therapy. CMT1B, a demyelinating subtype, is associated with over 200 different mutations in Myelin Protein Zero (MPZ) gene. Most mutations in MPZ gene are autosomal dominant and exhibit toxic gain-of-function effects, resulting in the aggregation of mutant MPZ protein within Schwann cells (SCs) followed by endoplasmic reticulum (ER) stress, defective myelination, reduced nerve conduction, failure of peripheral nerve and subsequent muscle wasting and weakness. Creating a single, comprehensive therapy for CMT1B is challenging due to the diverse mutations throughout the MPZ gene. In response, we developed a novel, universal AAV gene therapy approach termed AAV9-KDAR (KnockDown And Replacement). This strategy involves knocking down mutated MPZ using RNAi to reduce the aggregate burden in SCs, and concurrently replacing it with a functional RNAi-resistant MPZ (rMPZ) gene provided by the same vector to promote re-myelination. Given that MPZ constitutes over 50% of proteins in the myelin sheath, its replacement is essential for enhancing myelination. Twelve micro-RNAs targeting MPZ gene (miMPZ) were designed and cloned. To identify lead miRNAs capable of targeting both mutant and wild-type MPZ (mut/wtMPZ) mRNA without affecting rMPZ expression, we co-transfected HEK293 cells with mut/wtMPZ plasmids, rMPZ, and each miMPZ, followed by luciferase assays, western blot, and qRT-PCR analyses. Then, the effects of AAV-based MPZ knockdown, rMPZ replacement, and MPZ-KDAR were investigated separately in an ex-vivo model of severe early-onset CMT1B-R98C’s DRG/ SCs co-cultures. Reduction of mutant MPZ, decreased ER stress, and increased myelination were assessed using IF staining, western blot, and qRT-PCR. Off-target effects of miMPZ were investigated by RNA-seq analysis in human Schwann cells. Next, we tested the in vivo efficacy of AAV9-KDAR therapy at two-time points: pre and early onset of CMT1B disease. We administered 2.5E+11 or 5E+11 vg of AAV9-miMPZ-rMPZ via intracerebroventricular or intrathecal injections on neonatal and adult CMT1B-R98C mice, respectively. Molecular, behavioral, and electrophysiological assays were performed at 6 months post-injection. Lead miMPZs successfully reduced mut/wtMPZ levels by > 85-fold in both ex vivo and in vivo models without affecting rMPZ expression. Interestingly, silencing MPZ alone worsened demyelination symptoms, and replacement of MPZ alone was insufficient to reverse ER stress and restore myelination. In contrast, CMT1B-DRG/SCs co-cultures treated with KDAR approach exhibited enhanced myelination, increased numbers of MBP + internodes, and reduced ER stress biomarkers. Similarly, we detected a significant reduction in mut/wtMPZ and a 60-fold increase in rMPZ expression in treated sciatic nerves accompanied by improvement in treadmill and rotarod motor performance by 50% , as well as CMAP and NCV electrophysiological outcomes in treated pre- and post-onset CMT1B-R98C mice compared to untreated controls. Furthermore, no significant off-target effects were detected by RNA-seq analysis on lead miRNA-treated human SCs. This study marks a groundbreaking development in gene therapy for CMT1B, providing proof of concept for treating CMT1B through AAV9-KDAR approach. Nonetheless, additional research is imperative to optimize AAV doses, enhance efficacy, and assess in vivo safety thoroughly.

Daniella Munezero1, Jingting Zhu1, Arun Paripati1, Russell Chuah1, Lauren Taylor1, Afrooz Rashnonejad1,2

1Center for Gene Therapy, Abigail Wexner Research Institute, Nationwide Children's Hospital, Columbus, OH,2Department of Pediatrics, The Ohio State University, Columbus, OH"

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