# Proteomics-based evaluation of AAV dystrophin gene therapy outcomes in mdx skeletal muscle

## TL;DR

This study uses proteomics to assess how well different AAV-based dystrophin therapies work in a mouse model of Duchenne muscular dystrophy.

## Contribution

A novel proteomics-based method is introduced to evaluate dystrophin replacement therapies in dystrophic mice.

## Key findings

- Proteomics reveals changes in protein expression profiles in dystrophic and treated mouse muscles.
- Successful dystrophin expression restores muscle homeostasis to varying degrees.
- Potential biomarkers for evaluating dystrophin therapy effectiveness are identified.

## Abstract

Duchenne muscular dystrophy (DMD) is a fatal genetic muscle-wasting disease characterized by loss of dystrophin protein. Therapeutic attempts to restore a functional copy of dystrophin to striated muscle are under active development, and many utilize adeno-associated viral (AAV) vectors. However, the limited cargo capacity of AAVs precludes delivery of full-length dystrophin, a 427 kDa protein, to target tissues. Recently, we developed a method to express large dystrophin constructs using the protein trans-splicing mechanism mediated by split inteins and myotropic AAV vectors. The efficacy of this approach to restore muscle function in mdx4cv mice was previously assessed using histology, dystrophin immunolabeling, and Western blotting. Here, we expand our molecular characterization of dystrophin constructs with variable lengths using a mass spectrometry–based proteomics approach, providing insight into unique protein expression profiles in skeletal muscles of wild-type, dystrophic mdx4cv, and AAV-treated mdx4cv mice. Our data reveal several affected cellular processes in mdx4cv skeletal muscles with changes in the expression profiles of key proteins to muscle homeostasis, whereas successful expression of dystrophin constructs results in an intermediate to complete restoration. This study highlights several biomarkers that could be used in future preclinical or clinical studies to evaluate the effectiveness of therapeutic strategies.

This study describes a new method using proteomics to evaluate the efficacy of three different dystrophin replacement approaches using AAV vectors.