Duchenne Muscular Dystrophy

Function of the DMD Gene

The DMD gene is the largest gene in the human body, spanning 2.2 million base pairs and containing 79 exons. The pathogenesis of DMD primarily involves mutations in the DMD gene located on the X chromosome, which prevent muscle cells from synthesizing fully functional dystrophin protein. Dystrophin is a transmembrane cytoskeletal linking protein that plays a central role in forming a network structure beneath the sarcolemma. Its N-terminus connects to the actin cytoskeleton, while its C-terminus links to the transmembrane dystrophin-associated glycoprotein complex, thereby stably connecting intracellular contractile structures to the extracellular matrix. The absence of this protein leads to the disintegration of this connection system, resulting in loss of mechanical stability of the muscle cell membrane. Under contraction stress, the membrane becomes highly susceptible to micro-tears, leading to pathological calcium influx and calcium overload. Sustained calcium overload activates calcium-dependent proteases and phospholipases, triggering proteolysis, muscle fiber necrosis, oxidative stress, and chronic inflammation. Ultimately, the regenerative capacity of muscle satellite cells becomes exhausted, and muscle tissue is replaced by fat and fibrous tissue, leading to loss of muscle function.
 

Due to the long fragment length of the dystrophin gene, its mutation rate is relatively high, approximately 1 in 10,000, with complex and diverse mutation patterns. Deletion mutations are the most common type, accounting for 65% of all mutations, while duplication mutations account for 6-10%. The remaining 25-30% of mutations include point mutations, small deletion mutations, and insertion mutations. Deletion and duplication mutations are mainly concentrated in two hotspot regions of the gene: the 5' end (approximately 20%) and the central region (approximately 80%). Point mutations and small deletion mutations are randomly distributed without obvious mutation hotspot regions.
 

(Image placeholder: Schematic diagram of DMD and dystrophin protein - Source: Duchenne muscular dystrophy)

 

(Image placeholder: Histology of healthy muscle and DMD muscle - Source: Duchenne muscular dystrophy)

 

Gene Therapy
 

1. Stop Codon Readthrough: Approximately 15% of DMD patients have nonsense mutations caused by premature termination codons (PTCs). Aminoglycosides can bind to specific sites on the ribosome and disrupt codon-anticodon recognition at the aminoacyl-tRNA acceptor site. This mechanism can generate missense mutations that bypass PTCs, allowing normal translation to proceed and producing proteins containing full-length dystrophin.
 

2. Exon Skipping Therapy: Exon skipping therapy restores the reading frame of the DMD gene by skipping certain exons, thereby producing partially functional dystrophin. This approach is primarily achieved through antisense oligonucleotides (ASOs), which are small modified RNA fragments that specifically bind and skip specific exons during pre-mRNA splicing. Currently, researchers are exploring various strategies to improve the efficiency and delivery of ASOs, such as muscle-targeting peptide conjugation or nanoparticle delivery.
 

3. Exon Gene Excision: This method uses zinc finger nucleases to permanently remove essential splicing sequences in exon 51 of the dystrophin gene, preventing its transcription and causing exon 51 to be absent in the transcribed mRNA, thereby restoring dystrophin expression. This approach can restore the open reading frame of dystrophin in 13% of DMD patients and can coexist with existing DMD gene therapies and cell therapies.
 

4. Gene Editing Therapy: Gene editing therapy permanently corrects mutations by precisely modifying the mutation sites in the DMD gene to restore dystrophin expression. Theoretically, gene editing has the potential to permanently correct genetic defects with a single treatment, achieving normal or near-normal dystrophin expression. This method is expected to provide better functional outcomes than micro-dystrophin gene therapy because gene-edited dystrophin expression will be controlled by the endogenous dystrophin gene locus.
 

5. Micro-dystrophin Gene Therapy: Due to the enormous size of the DMD gene, developing gene transfer therapies faces challenges. However, studies have shown that delivering a smaller dystrophin gene construct can also alleviate disease phenotypes. Currently, multiple clinical trials are testing the safety and efficacy of micro-dystrophin gene therapies with different designs.
 

Mouse Models
 

1) mdx Mouse Model: The mdx mouse is the most commonly used model for exploring dystrophin gene expression and function. This model has a nonsense point mutation in exon 23 that causes premature termination codon formation, resulting in loss of full-length dystrophin expression. Although it shares the same genetic defect as DMD patients, the mdx mouse exhibits a milder muscular dystrophy phenotype, with a lifespan reduced by only 20% compared to normal mice, whereas DMD patients experience a 75% reduction in lifespan. The skeletal muscle pathology in mdx mice is relatively mild and progresses slowly with fluctuations.

2) Double Knockout Mouse Model: The most common is the double knockout mouse of the dystrophin gene and dystrophin-associated genes, specifically the mdx/utrn-/- mouse. This strain exhibits a more severe muscular dystrophy phenotype, characterized by severe muscle weakness, joint contractures, and kyphosis, with an average survival period of only 3 months.
 

MingCeler Biotech Facilitates Gene Therapy
 

Gene therapy offers hope for rare diseases, but its development and validation are inseparable from animal model support. Leveraging its self-developed TurboMice™ technology, MingCeler Biotech has successfully constructed various rare disease gene-edited mouse models, overcoming the limitations of traditional methods in terms of editing fragment length, multi-site complexity, and modeling cycles. This technology enables precise editing at virtually any target gene locus, providing the following core advantages for DMD research:
 

1. Supports Long Fragment Gene Editing: Enables knock-in, replacement, or deletion of large gene fragments, covering common deletion hotspot regions in the DMD gene (such as the central region and 5' end), and accurately constructing mouse models with mutation structures consistent with patient mutations

.2. Supports Multi-Site Point Mutation Editing: For point mutations or small fragment variations distributed in non-hotspot regions, multiple site mutations can be efficiently introduced in the same model, better simulating complex genotypes and facilitating in-depth exploration of pathogenic mechanisms.

Direct Delivery of Homozygous Mice Without Breeding Screening: Complete homozygous gene-edited mouse models can be prepared directly from embryonic stem cells in as little as 2 months.MingCeler Biotech can customize various DMD mouse models according to client needs, such as mdx mouse models, mdx/utrn-/- mice, and complex genotype models based on long fragment editing or multi-site mutations. We welcome inquiries!

 

References:

[1] Li T, Liang P. [Research progress on disease models and gene therapy of Duchenne muscular dystrophy]. Zhejiang Da Xue Xue Bao Yi Xue Ban. 2016 May 25;45(6):648-654. Chinese. doi: 10.3785/j.issn.1008-9292.2016.11.15. PMID: 28247611; PMCID: PMC10396854.

[2] Advances in Clinical Medicine, 2024, 14(4), 2420-2426.

[3] National Center for Biotechnology Information. Duchenne Muscular Dystrophy. 2021.

[4] MSD Manual Professional Edition. Duchenne Muscular Dystrophy and Becker Muscular Dystrophy. 2023.

[5] Zhao Huiwen, Shao Lijian, Kuang Bohai. Research Progress in the Treatment of Duchenne Muscular Dystrophy. Clinical Medicine Progress, 2024, 14(4): 2420-2426.

[6] Wang Xueding, Tao Yuqian, Su Qibiao, et al. Current Status of Gene Editing Research in Duchenne Muscular Dystrophy. Chinese Journal of Clinical Pharmacology, 2020, 36(04).

[7] Gene Therapy Research Progress in Duchenne Muscular Dystrophy. 2023.[8] Advances in Preclinical Models for Duchenne Muscular Dystrophy Research. 2023.

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