What is Duchenne Muscular Dystrophy?
 

Duchenne Muscular Dystrophy (DMD) is a rare X-linked recessive genetic disorder primarily caused by mutations in the DMD gene, leading to the absence of dystrophin protein. This protein is crucial for maintaining the stability of muscle cell membranes.

The incidence of DMD is approximately 1 in 3,500 to 5,000 male births, with an estimated 450,000 to 600,000 patients worldwide. In China, the prevalence is about 8.5 per 100,000 males, with approximately 60,000 cumulative patients.

DMD primarily affects skeletal and cardiac muscles. Patients typically present with a waddling gait, toe walking, and lumbar lordosis. As the disease progresses, patients lose ambulatory ability around age 12. Cardiac involvement can lead to dilated cardiomyopathy, conduction abnormalities, and arrhythmias. Ultimately, the average life expectancy is reduced to approximately 26 years.

 

Pathogenesis and Treatment Approaches
 

The DMD gene is the largest gene in the human genome, spanning 2.2 million base pairs and containing 79 exons. DMD is primarily caused by mutations in the dystrophin gene located on the X chromosome, leading to the absence or dysfunction of dystrophin protein. This results in loss of muscle cell membrane stability and progressive muscle cell destruction. The encoded dystrophin protein is primarily distributed in skeletal muscle, cardiac muscle, and the brain, where it stabilizes the cytoskeleton and protects muscle cells.

The large size of the dystrophin gene makes it prone to mutations (mutation rate approximately 1/10,000), with complex and diverse mutation patterns. Deletion mutations account for 65% of all mutations, duplication mutations for 6-10%, and the remaining 25-30% include point mutations, small deletions, and insertions. Deletion and duplication mutations primarily occur in two hotspot regions: the 5' end (approximately 20%) and the central region (approximately 80%). Point mutations and small deletions are randomly distributed without distinct hotspot regions.

(Image: Schematic diagram of DMD and dystrophin protein)
 

(Image: Histology of healthy muscle and DMD muscle)

 

Currently, DMD remains an incurable disease. However, with in-depth research into its pathogenesis and the continuous development of emerging technologies such as gene therapy and stem cell therapy, treatment options are increasing.

(I) Glucocorticoid Therapy

Glucocorticoids are currently the only drugs proven to improve muscle strength and delay disease progression in DMD patients. Prednisone is commonly used to enhance muscle strength and delay loss of ambulation. Its mechanism involves reducing inflammatory damage to muscles to slow disease progression. However, glucocorticoids do not address the root cause of DMD, and their therapeutic effects are limited.

(II) Stem Cell Therapy

Stem cell therapy involves transplanting autologous or allogeneic stem cells into DMD patients to differentiate into muscle cells and express dystrophin. Current research indicates that the ability of stem cells to differentiate into muscle cells is limited, with only a small number of bone marrow mesenchymal stem cells (BM-MSCs) capable of promoting muscle fiber formation.

(III) Gene Therapy

Currently, there are five main approaches to DMD gene therapy:

1. Stop Codon Readthrough

Approximately 15% of DMD patients have nonsense mutations causing premature termination codons (PTCs). Aminoglycosides can bind to specific sites on the ribosomal RNA and disrupt codon-anticodon recognition at the aminoacyl-tRNA acceptor site. This principle can generate missense mutations that bypass PTCs, allowing normal translation to proceed and producing full-length dystrophin protein.

2. Exon Skipping Therapy

Exon skipping therapy restores the reading frame of the DMD gene by skipping certain exons, producing partially functional dystrophin. This approach primarily uses antisense oligonucleotides (ASOs), which are small modified RNA fragments that specifically bind and skip specific exons during pre-mRNA splicing. Researchers are exploring various strategies to improve ASO efficiency and delivery, such as muscle-targeting peptides 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. This results in the absence of exon 51 in the transcribed mRNA, restoring dystrophin expression. This approach can restore the open reading frame in 13% of DMD patients and can coexist with existing DMD gene and cell therapies.

4. Gene Editing Therapy

Gene editing therapy permanently corrects mutations by precisely modifying the mutation site in the DMD gene, restoring dystrophin expression. Theoretically, gene editing has the potential to permanently correct the genetic defect with a single treatment, achieving normal or near-normal dystrophin expression. This approach may provide better functional outcomes than micro-dystrophin gene therapy, as gene-edited dystrophin expression would be controlled by the endogenous dystrophin gene locus.

5. Micro-dystrophin Gene Therapy

The large size of the DMD gene presents challenges for developing gene transfer therapies. However, studies show that delivering a smaller dystrophin gene construct can also ameliorate the disease phenotype. Currently, multiple clinical trials are testing the safety and efficacy of micro-dystrophin gene therapies with different designs.
 

Gene-Edited 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, leading to premature termination codon and loss of full-length dystrophin expression. Although sharing the same genetic defect as DMD patients, mdx mice exhibit a milder muscular dystrophy phenotype, with a lifespan reduced by only 20% compared to normal mice, whereas DMD patients experience a 75% reduction. 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 dystrophin gene and dystrophin-associated gene double knockout mouse, specifically mdx/utrn-/- mice. This strain exhibits a more severe muscular dystrophy phenotype, characterized by severe muscle weakness, joint contractures, and kyphosis, with an average survival of only 3 months. This supports the notion that dystrophin-associated proteins have compensatory effects in dystrophin deficiency.

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 developed multiple rare disease mouse models. The TurboMice™ technology overcomes the challenges of long modeling cycles and low success rates for complex models. It enables editing at virtually any target gene locus and can generate complete homozygous gene-edited mouse models 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 mice and mdx/utrn-/- mice. 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. 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.

[4] Merck Manual Professional Edition. Duchenne Muscular Dystrophy and Becker Muscular Dystrophy. Pediatrics: Inherited Muscular Disorders.

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

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

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