Research Strategies Targeting Dystrophin Production

Though not yet approved by the FDA, several experimental therapies are in various stages of clinical development.

One category of experimental therapies aims to address the cause of DMD, namely, the lack of the dystrophin protein, by restoring production of a functioning form of dystrophin.

Investigational Gene Transfer Therapy

What Gene Therapy is Designed to Do?

Many neuromuscular diseases are monogenic, meaning that they are caused by mutations in a single gene that lead to muscle or motor neuron cells producing little to no working protein.1 Gene transfer therapy is an experimental treatment that is being studied in clinical trials for monogenic neuromuscular diseases like DMD.2

The goal of gene transfer therapy is to slow or stabilize disease by introducing a potentially functioning version of the gene into disease-affected cells.1,3



Components of Gene Transfer Therapy

Gene transfer therapies consist of 3 core components—the vector, promoter, and transgene—with each playing an essential role.2-4



Designing a gene transfer therapy involves selecting appropriate components. The vector is a vehicle that delivers the promoter and the transgene into disease-affected cells. The experimental vector is selected based on two main qualities: 1) its ability to target the disease-affected cells (skeletal muscle [including diaphragm] and cardiac muscle cells) in DMD, and 2) a low potential to stimulate an immune response.2-4

One of the most common vectors used in investigational gene transfer therapy is the adeno-associated virus (AAV) vector.4,5 There are many different types of AAV or serotypes, which vary in the types of tissues that they target and the immune responses they elicit.3 If a patient has been exposed to a specific AAV serotype in the past, they may have pre-existing immunity (antibodies) to that AAV serotype. Because pre-existing immunity is one of many factors that can influence the efficacy and toxicity of gene transfer therapy, it also impacts patient eligibility to receive gene transfer therapy in a clinical trial.4,5 Learn more about antibody testing.



Another component, the promoter, is a segment of DNA that works like an “on/off switch” for protein expression in the target cells.3,6,7 Different promoters have diverse patterns and strengths of expression in target cells. In designing a gene transfer therapy, researchers select a promoter that ensures protein expression in target cells. For example, to slow or stabilize disease in DMD, researchers select a promoter that turns on robust and uniform protein expression across skeletal muscle cells (including those of the diaphragm) and cardiac muscle cells.


The transgene is engineered with the goal of creating a functioning version of the affected gene. Once the transgene is delivered into target cells, it is designed to direct protein expression in the cells. In DMD, the full-length dystrophin gene is too large to fit in the vector. Instead, researchers have engineered a transgene that is a truncated version of the dystrophin gene. This transgene aims to direct protein expression of a truncated but functional form of dystrophin in the disease-affected cells.3

Gene Editing

Gene editing is an experimental technique in the very early stages of development.5 The goal of gene editing is to change specific building blocks in the dystrophin gene, like changing letters in an instruction manual, so that cells can produce dystrophin.5 These strategies use a technique called CRISPR/Cas (also called CRISPR/Cas9 or simply CRISPR) gene editing.5

One way that researchers are using CRISPR is to cut out “errors” in the dystrophin gene in heart and muscle cells so that the cells can now read the gene and make dystrophin protein.8 Another potential strategy uses CRISPR to replace the errors in the dystrophin gene with parts from a healthy gene, with the goal of restoring the cell’s ability to produce dystrophin.5

CRISPR gene editing is also being explored in muscle stem cells in Duchenne. Researchers are editing the dystrophin gene in muscle stem cells and investigating whether they can develop into new muscle cells that produce dystrophin.5 The ultimate goal is to regenerate healthy muscle to replace the weakened muscle in Duchenne.

Emerging Exon-skipping Approaches


Current exon-skipping approaches are based on the chemistry of phosphorodiamidate morpholino oligomers, or PMOs.9 PMOs are synthetic molecules that are structurally similar to RNA. PMOs have the same nucleic acid bases found in RNA, and are designed to be both more stable and also customizable. 9,11 This customizable quality allows PMOs to target and bind to specific pre-messenger RNA sequences—including the mutated sequences responsible for causing Duchenne.9,10,12 In this way, it is hoped that additional exon deletions will ultimately be amenable to exon-skipping therapies using this approach.13


Preclinical research has shown that the ability of PMOs to effectively target and enter muscle cells can be enhanced by conjugating a PMO with a cell-penetrating peptide, or CPP. The resulting conjugate is called a peptide phosphorodiamidate morpholino oligomer, or PPMO. PPMOS in development are being evaluated to determine if they enhance tissue penetration.9,12,14 More research is needed to determine the efficacy, safety, and dosing frequency of PPMOs for DMD patients, and potentially, to expand the range of diseases amenable to treatment.9,12,14

1. Gardlík R, Pálffy R, Hodosy J, Lukács J, Turna J, Celec P. Vectors and delivery systems in gene therapy. Med Sci Monit. 2005;11:RA110-RA112. 2. Min YL, Bassel-Duby R, Olson EN. CRISPR Correction of Duchenne Muscular Dystrophy. Annu Rev Med. 2019;70:239-255. 3. Asher DR, Thapa K, Dharia SD, et al. Clinical development on the frontier: gene therapy for Duchenne muscular dystrophy. Expert Opin Biol Ther. 2020;20:263-274. 4. Rabinowitz J, Chan Y, Samulski R. Adeno‐associated virus (AAV) versus immune response. Viruses. 2019;11(102):1‐11. 5. Meliani A, Leborgne C, Triffault S, Jeanson-Leh L, Veron P, Mingozzi F. Determination of anti-adeno-associated virus vector neutralizing antibody titer with an in vitro reporter system. Hum Gene Ther Methods. 2015;26:45-53. 6. Naso MF, Tomkowicz B, Perry WL III, Strohl WR. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs. 2017;31:317-334. 7. Zheng C, Baum BJ. Evaluation of promoters for use in tissue-specific gene delivery. Method Mol Biol. 2008;434:205-219. 8. Wang JZ, Wu P, Shi ZM, Xu YL, Liu ZJ. The AAV-mediated and RNA-guided CRISPR/Cas9 system for gene therapy of DMD and BMD. Brain Dev. 2017;39(7):547-556. 9. Tsoumpra MK, Fukumoto S, Matsumoto T, Takeda S, Wood MJA, Aoki Y. Peptide-conjugate antisense based splice-correction for Duchenne muscular dystrophy and other neuromuscular diseases. EBioMed. 2019;45:630-645. 10. Summerton JE. Morpholino, siRNA, and S-DNA compared: impact of structure and mechanism of action on off-target effects and sequence specificity. Curr Top Med Chem. 2007; 7(7):651-660. 11. Moulton JD. Guide for Morpholino Users: Toward Therapeutics. J Drug Des Devel Ther. 2016;3(2):1023-1035. 12. Moulton HM, Moulton JD. Morpholinos and their conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy. Biochim Biophys Acta. 2010;1798(12):2296-2303. 13. Anwar S, He M, Lim KRQ, Maruyama R, Yokota T. A Genotype-Phenotype Correlation Study of Exon Skip-Equivalent In-Frame Deletions and Exon Skip-Amenable Out-of-Frame Deletions across the DMD Gene to Simulate the Effects of Exon-Skipping Therapies: A Meta-Analysis. J Pers Med. 2021;11(1):46. 14. Wu B, Moulton HM, Iversen PL, et al. Effective rescue of dystrophin improves cardiac function in dystrophin-deficient mice by a modified morpholino oligomer. Proc Natl Acad Sci USA. 2008;105(39):14814-14819.