Research Report on the Improvement of CMT-like Neuropathy via AAV9 Vector-based C1ORF194 Gene Therapy

Background and Research Motivation

Charcot-Marie-Tooth disease (CMT) is a rare group of genetically heterogeneous neuromuscular disorders characterized by progressive muscle weakness and atrophy, along with sensory loss. Despite extensive preclinical and clinical studies, no FDA-approved therapies currently exist for any CMT subtype. CMT’s pathogenesis involves mutations in over 100 genes, making the study of these genes’ functions and therapeutic potential critically important.

This research team previously identified C1ORF194, a novel pathogenic gene for CMT, which may function as a mitochondria-associated endoplasmic reticulum (ER) membrane (MAM) protein, playing a role in calcium homeostasis and CMT pathology. To investigate the therapeutic potential of this gene, the authors developed a C1ORF194 knockout (C1ORF194-/-) mouse model that mimics CMT pathology. The study explores using an adeno-associated virus serotype 9 (AAV9) vector to restore C1ORF194 expression for treating CMT.

Source of the Paper

The paper, authored by Zongrui Shen et al., involves researchers from Southern Medical University and the University of Texas Southwestern Medical Center. It was published on October 16, 2023, in Neurotherapeutics.

Study Design and Workflow

Experimental Model and Gene Therapy Strategy

The study used C1ORF194-/- mice, which progressively develop intermediate CMT-like phenotypes at 4 months of age, including motor and sensory defects and pathological changes in the sciatic nerve. AAV9-based gene therapy was applied, using a vector carrying a codon-optimized wild-type (WT) human C1ORF194 gene driven by a chicken β-actin promoter (CAG). The treatment involved a single intravenous injection (5 × 10¹² vg, approximately 250 μL).

Experimental Workflow

1. Gene Expression Analysis: Western blotting was performed to detect C1ORF194 protein expression in the sciatic nerve.
2. Behavioral Assessments: Motor and sensory functions were evaluated using tests such as rotarod, grip strength, and gait analysis.
3. Electrophysiological Measurements: Nerve conduction velocity (NCV) and compound muscle action potential (CMAP) of the sciatic nerve were assessed.
4. Histopathological Analyses: Included electron microscopy of the sciatic nerve, H&E staining of the gastrocnemius muscle, and Nissl staining of spinal cord motor neurons.

Research Results

Restoration of C1ORF194 Protein Expression

Untreated C1ORF194-/- mice lacked detectable C1ORF194 protein in the sciatic nerve. In contrast, AAV9-C1ORF194-treated mice showed protein expression levels close to those in WT mice.

Improvements in Motor and Sensory Function

• Motor Function: Significant improvements were observed in treated mice, including enhanced performance in rotarod and grip strength tests. Gait analysis revealed increased stride length and angle.
• Sensory Function: While the tail flick test showed some improvements in treated mice, the results were not statistically significant.

Electrophysiological and Histopathological Improvements

• Electrophysiological Metrics: Treated mice exhibited significant increases in NCV and CMAP, indicating restoration of peripheral nerve function.
• Histological Observations: Treated mice showed marked improvements in muscle atrophy, sciatic nerve myelin sheath thickness, and spinal cord motor neuron density.

Pathological Evidence

Electron microscopy revealed restoration of normal sciatic nerve myelin thickness and reduced demyelination in treated mice, suggesting that AAV9-C1ORF194 therapy alleviates peripheral nerve degeneration.

Discussion and Significance

Potential of Gene Therapy

AAV9 vectors, with their high transduction efficiency and tissue adaptability, have emerged as an ideal tool for treating monogenic neurological disorders. This study demonstrated the efficacy of AAV9-C1ORF194 in improving motor and sensory functions and reversing neuropathological changes in C1ORF194-/- mice, laying the foundation for future clinical trials.

Limitations and Optimization Directions

Despite the notable therapeutic effects, the study faces limitations, such as the restricted gene delivery efficiency due to the blood-nerve barrier in mice. Future directions include using a myelin protein zero (MPZ) promoter to enhance Schwann cell-specific expression and optimizing dosage, administration routes, and timing.

Safety Considerations

No tumorigenesis or organ pathology was observed in treated mice, providing preliminary safety data. However, immune responses and potential adverse events associated with AAV therapies warrant further investigation in larger-scale studies.

Clinical Translation Outlook

By elucidating the role of C1ORF194 in CMT and demonstrating the therapeutic potential of gene therapy, this study offers a novel approach for treating patients with C1ORF194-related CMT. Gene therapy may represent a transformative strategy for altering the disease trajectory, particularly when conventional treatments are predominantly supportive.

Conclusion

AAV9-C1ORF194 gene therapy significantly improved motor and sensory functions, as well as neuropathological outcomes in C1ORF194-/- mice, demonstrating its promise as a therapeutic strategy for CMT. Although technical challenges remain, the study provides a strong rationale for translating gene therapy into clinical settings and contributes to the expanding therapeutic arsenal for rare genetic diseases.