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  • Review Article
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Advances in Duchenne muscular dystrophy gene therapy

Nature Reviews Genetics volume 4, pages 774–783 (2003)Cite this article

Key Points

  • Duchenne muscular dystrophy (DMD) is characterized by a severe and progressive loss of muscle fibres, which is provoked by the dystrophin deficiency that results from frame-shifting mutations in the large dystrophin gene.

  • Conventional gene-therapy strategies (viral vectors, naked plasmids and cell transplantation) have been moderately efficient, but still have to overcome problems associated with low delivery efficiencies, immune responses, limited transgene persistence, poor cell survival and the physical barriers in muscle tissue itself.

  • Recently, relatively new and alternative strategies that are emerging from our increasing knowledge of the organization of the dystrophin gene and its role in muscle function are gaining credibility as therapeutic prospects.

  • Treatment of the mdx mouse model with DNA–RNA chimeric oligonucleotides to correct the nonsense mutation in exon 23, or with gentamicin to read through the resulting stop codon, has shown positive results. However, the efficiencies have been low and follow-up studies are required to further evaluate these methods.

  • The minimal requirements for the function of the dystrophin protein have been defined in more detail, which has allowed the construction of functional mini- and micro-dystrophins of only 6.2 kb and 3.6 kb, respectively. Now, using recombinant adeno-associated virus (rAAV) vectors, these small constructs have been efficiently delivered into the muscle of mdx mice and converted the dystrophic muscle morphology for more than six months post-injection.

  • Antisense oligonucleotides have been successfully used to modify the splicing of the dystrophin pre-mRNA so that specific exons are skipped. Despite introducing a (larger) deletion, this frame-restoring treatment induced the synthesis of BMD-like dystrophins to therapeutic levels in cultured muscle cells from a series of DMD patients with different mutations, and from the mdx mouse both in vitro and in vivo.

  • Utrophin can compensate for dystrophin deficiency and its artificial upregulation in mdx mice has reduced dystrophic pathology and improved muscle performance. Several growth and transcription factors have been identified to enhance expression from the utrophin promoter A, but the challenge remains to find an effective small-molecule drug that will be sufficiently utrophin-specific to circumvent putative adverse effects.

  • In terms of safety and efficiency, non-pathogenic rAAV and synthetic small-molecule drugs for targeted exon skipping and utrophin upregulation are the most promising techniques, and they are progressing towards clinical applications in the near future.

Abstract

Since the initial characterization of the genetic defect for Duchenne muscular dystrophy, much effort has been expended in attempts to develop a therapy for this devastating childhood disease. Gene therapy was the obvious answer but, initially, the dystrophin gene and its product seemed too large and complex for this approach. However, our increasing knowledge of the organization of the gene and the role of dystrophin in muscle function has indicated ways to manipulate them both. Gene therapy for Duchenne muscular dystrophy now seems to be in reach.

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Figure 1: Structural domains of the human full-length dystrophin, Becker mini- and micro-dystrophins, and utrophin.
Figure 2: Schematic representation of the exon-skipping strategy.
Figure 3: Therapeutic exon skipping in cultured myotubes from a DMD-patient (DL363.2) with a deletion of exons 45–54.
Figure 4: Schematic representation of the human utrophin A and B promoter regions and the factors that affect expression.

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Acknowledgements

Work in the authors' laboratories is supported by grants from the Dutch Duchenne Parent Project, the Princess Beatrix Fund (The Netherlands), the European Union, the Muscular Dystrophy Campaign (United Kingdom) and the Muscular Dystrophy Association (United States).

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Authors and Affiliations

  1. Center for Human and Clinical Genetics, Leiden University Medical Center, Wassenaarseweg 72, Leiden, 2333 AL, The Netherlands

    Judith C. T. van Deutekom & Gert-Jan B. van Ommen

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  1. Judith C. T. van Deutekom
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Correspondence to Judith C. T. van Deutekom.

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DATABASES

LocusLink

DMD

UTRN

OMIM

Becker muscular dystrophy

diabetes type 1

Duchenne muscular dystrophy

haemophilia IX

Pompe disease

FURTHER INFORMATION

Leiden muscular dystrophy pages

Glossary

EXTRACELLULAR MATRIX

In muscle, this is a thin layer (basal lamina) that contains collagen, elastin and fibronectin, which surrounds each muscle fibre. This might act as a semipermeable filter or a selective cellular barrier and is important in regeneration after damage.

TRANSDUCTION

The transfer of genetic material into a cell using a viral vector.

IMMUNOGENICITY

The properties of a virus, transgene, vector, compound or molecule that provoke an immune response.

CYTOTOXICITY

The properties of a virus, transgene, vector, compound or molecule that are toxic for cells.

ELECTROPORATION

The application of an electric current to the plasma membrane of a cell, to temporarily open pores or channels through which DNA might pass.

PRESSURIZED ISOLATED-LIMB PERFUSION

The introduction of therapeutic agents under pressure in a limb after isolation of the blood circulation by clamping.

MICROBUBBLES

Encapsulated gas microbubbles that can be used as drug or gene carriers, which are able to penetrate into the smallest membranes. When exposed to sufficiently high-amplitude ultrasound, the microbubbles rupture and release the drugs and genes that are contained in their encapsulating layer.

TRANSFECTION

The transfer of exogenous DNA into a cell.

MYOBLAST TRANSPLANTATION

The implantation of exogenous muscle-progenitor cells into muscle to generate new myofibres or to support existing myofibres.

AMINOGLYCOSIDES

A group of antibiotics (such as gentamicin) that inhibit bacterial protein synthesis and are particularly active against Gram-negative bacteria.

F-ACTIN

A protein that is involved in the contractile apparatus and the maintenance of the cytoskeleton of myofibres.

β-DYSTROGLYCAN

The α- and β-dystroglycans are the laminin-binding components of the dystrophin–glycoprotein complex, which provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix.

DYSTROBREVINS

The components of the dystrophin–glycoprotein complex that bind to syntrophin and (indirectly) to the C-terminal of dystrophin. Dystrobrevin-α recruits signalling proteins, such as neuronal nitric oxide synthase.

SYNTROPHINS

Peripheral membrane proteins that bind to the C-terminal of dystrophin, which might have a role in the process of synaptogenesis.

SPECTRIN

A large contractile submembrane protein that, similar to dystrophin, contains an actin-binding domain and a long repeat domain.

DEPENDOVIRUS

A single-stranded DNA virus from the family parvoviridae (subfamily parvovirinae), which is dependent on a co-infection with helper adenoviruses or herpes viruses for efficient replication.

EPISOMES

DNA that can replicate autonomously in the cytoplasm of host cells.

HEK-293 CELLS

Host cells that generate viral particles following transfection with the rAAV plasmid and the helper plasmid.

NEO-ANTIGEN

A foreign (transgene) product that is able to stimulate an immune response.

PRE-mRNA SPLICING

The removal of introns from the precursor mRNA molecule; the remaining exons are spliced together.

PRIMARY MUSCLE-CELL CULTURES

Cells that are taken into culture directly from a tissue biopsy. In contrast to cell lines that only contain immortalized cells, these cultures contain heterogeneous cell populations.

SPLICEOSOMAL COMPLEX

A large dynamic complex that consists of small nuclear RNA molecules and protein components. It mediates the two catalytic steps of the splicing reaction: the excision of introns from the pre-mRNA and the ligation of the two exon termini.

RNaseH

Ribonuclease H. An enzyme that cleaves RNA/DNA complexes.

PHARMACOKINETIC PROFILE

The characteristics of a drug that determine its absorption, distribution and elimination in the body.

SARCOLEMMA

The membrane that encloses a striated muscle fibre.

ACETYLCHOLINE

A neurotransmitter (C7H17NO3) that is released at autonomic synapses and neuromuscular junctions. It is active in the transmission of nerve impulses and is formed enzymatically in tissues from choline.

CpG ISLAND

Genomic regions that are rich in the CpG pattern, are resistant to methylation and are often associated with promoter activity.

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van Deutekom, J., van Ommen, GJ. Advances in Duchenne muscular dystrophy gene therapy. Nat Rev Genet 4, 774–783 (2003). https://doi.org/10.1038/nrg1180

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