Adeno-associated viruses illustration
An illustration of adeno-associated viruses. Credit: Getty Images

Duchenne muscular dystrophy (DMD) is caused by loss-of-function variants in the dystrophin gene. Therefore, genetic approaches that aim to target the affected muscle cells and restore the disrupted reading frame of the dystrophin gene have curative potential.

So, we know the defective gene that underlies DMD pathology and we live in an era of great developments for gene therapy. It seems like we have it all to conquer this battle. Even though, DMD is still a lethal X-linked recessive disorder. The question is, “Why?”

A Limiting Delivery System

Viral vectors are widely used as delivery systems. In general, safety profiles of adeno-associated viruses (AAVs) are better than that of adenoviruses and retroviruses. However, many subtypes of AAVs are available, and they differ in their biodistribution and interaction with the host’s immune response.

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DMD affects muscle globally, including the myocardium and diaphragm. Hence, when searching for a potential curative approach it is essential to ensure it is sufficiently disseminated throughout all muscles without adverse effects.

Read more about DMD Treatment

To have high tropism for skeletal, diaphragm, and cardiac muscles, AAV-based vectors have to be administered systemically, frequently requiring high titers. This poses a severe risk of liver toxicity, since the AAV serotypes currently used in clinics present significant biodistribution in the liver.

In addition, the presence of the viral capsid may generate a dose-dependent innate immune response against the vector, thereby compromising the therapeutics. In some cases, this is accompanied by an increase in liver enzymes, which can be managed with glucocorticoids under careful monitoring.

“The vector itself is a limiting step for many of these novel genetic approaches; there has to be a revolution in the efficiency, tropism and yield of viral vector production in order for the therapies to become commercially and clinically viable,” Himič and Davies wrote.

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Researchers have been working on these issues. For instance, “It is now possible to decrease the dose of the vector to mitigate these limitations; the use of a self-complementary AAV to deliver gRNA achieves sufficient CRISPR-Cas9 editing efficiency with a 20-fold lower dose than with the previous single-stranded AAV,” Himič and Davies added.

Also, the use of tissue-specific promoters would allow enhancement of the expression of dystrophin gene at high levels in demanding tissues, such as skeletal and cardiac muscles, while ensuring minimal effects in off-target organs.

The Dystrophin Gene

“A major limitation for the use of gene therapy for the treatment of DMD is the size of the dystrophin gene which exceeds by far the packaging capacity of AAV-based vectors,” Kirschner wrote in the Journal of Perinatal Medicine.

AAVs are relatively small and can only pack about 5 kb of DNA. In contrast, the dystrophin gene is one of the largest in the human genome; its cDNA has approximately 14 kb.

“Thought must be put into the design of the dystrophin transgene,” Asher et al wrote. “In addition to fitting within AAV, the transgene must encode a protein that is stable, functional, properly localized to the sarcolemma, successful in restoration of the [dystrophin-associated protein complex], effective in preserving cell integrity, and able to ultimately improve clinical function.”

Other Complicating Factors

In fact, curing DMD may not be as simple as it sounds.

Preexisting immunity to AAV renders some patients ineligible for treatment. Therefore, alternative methods to circumvent AAV antibodies are of utmost importance to allow the administration of gene therapy to these patients. “This is an area of active investigation, with the application of methods such as plasmapheresis or T-cell suppression using medications like rituximab and rapamycin,” Asher et al said.

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Additional reasons could be related to muscle cells, as mentioned by Himič and Davies in the European Journal of Human Genetics. “Muscle is a post-mitotic tissue, and therefore simply halting dystrophin loss will not replace muscle that has already been lost.”

Ultimately, the scenario worsens due to challenges in designing clinical trials for gene therapy in rare diseases.

“For example, development of antibodies to the vector upon gene therapy administration serves as a barrier to receiving future gene therapies derived from that vector and may also exclude them from participation in other clinical trials,” Asher et al explained.

This is a complicated issue and a major burden on preclinical studies because participants receiving suboptimal doses may still develop immunity that, consequently, will compromise future dose escalation studies. For that reason, the establishment of an optimal dose range for safety and efficacy is critical in DMD gene therapy trials.


Himič V, Davies KE. Evaluating the potential of novel genetic approaches for the treatment of Duchenne muscular dystrophy. Eur J Hum Genet. 2021;29(9):1369-1376. doi:10.1038/s41431-021-00811-2

Asher DR, Thapa K, Dharia SD, et al. Clinical development on the frontier: gene therapy for Duchenne muscular dystrophy. Expert Opin Biol Ther. 2020;20(3):263-274. doi:10.1080/14712598.2020.1725469

Kirschner J. Postnatal gene therapy for neuromuscular diseases – opportunities and limitations. J Perinat Med. 2021;49(8):1011-1015. doi:10.1515/jpm-2021-0435