Promising Treatments: Gene Therapies

            DMD is universally fatal, and there is no known cure for the disease at present. For the most part, treatment is limited to symptom alleviation and quality of life improvement, with an early focus on prolonging a patient’s ability to walk on their own for as long as possible. Standard treatment options involve light physical therapy and prescription of corticosteroids such as prednisone and deflazacort.1,2,3,4 The mechanism of these corticosteroids has not been fully outlined, however various pathways have been proposed, including the inhibition of degenerative proteolytic pathways7, greater ability for muscle regeneration due to increased myogenic stem cells8,9, and attenuation of cellular Ca2+ influx10. Although corticosteroids are the only pharmaceutical option with a proven beneficial clinical effect, the extent of their ability to obstruct the progression of the disease is impermanent, with the only objective being to delay and minimize symptoms for as long as possible. As such, there is still a significant unmet need for a lasting effective treatment for DMD.

            Although current widely available treatment options are relatively limited, current studies have implicated genetic techniques with extraordinary potential for significantly more effective treatment options. Researchers are currently exploring a variety of promising novel therapies for muscular dystrophies; however this page will focus primarily on treatment at the cellular and genetic levels, aimed at restoring dystrophin itself.

            One of the most widely looked at treatment methods currently being explored is a technique called exon skipping. As discussed in previous pages, Duchenne-type muscular dystrophy results from a disruption of the reading frame of the dystrophin gene, leaving no functional dystrophin expressed. In Becker Muscular Dystrophy, the mutation does not affect the reading frame, leading to the expression of a shorter, but often still semi functional dystrophin protein, usually resulting in significantly milder symptoms than DMD. The goal of exon skipping is to delete the damaged exon, as well as any necessary adjoining exons, such that the reading frame is restored and the muscle cells are able to produce a Becker-like protein that is truncated, but still functional.

Diagram of exon-skipping. Left: disruption of the reading frame due to a mutation of exon 50 in DMD. Right: restoration of the reading frame by AO induced suppression of exon 51, restoring the translational reading frame and allowing for production of a slightly shorter, functional dystrophin protein. (Ref 21)

            Exon skipping was first considered after it was observed that in many DMD patients, a few individual muscle fibers were occasionally found to contain functional dystrophin. It was determined that these rare, dystrophin-positive revertant fibers regained their expression of dystrophin due to a second mutation at the original site that restored the reading frame.5,6 In 2001, Mann et al. demonstrated therapeutic effects of this technique of the dystrophin gene in the mdx mouse, a very commonly used mouse model of human muscular dystrophies. Using 2′-O-methyl antisense oligoribonucleotides (AOs) complementary to specific sites on pre-mRNA, researchers were able to disrupt splicing of exon 23, the location of the malign mutation in mdx mice, and exclude it from the final mRNA product. The removal of exon 23 restored the reading frame of the mdx dystrophin gene, with subsequent analysis revealing correct expression and localization of dystrophin and the DAPC molecule gamma-sarcoglycan in treated cells.11 Many other skipped exons have been identified that correct the reading frame in a number of different DMD causing mutation sites, including the excision of exon 46 for an exon 45 deletion and exon 51 for an exons 48-50 deletion.12,13 By 2006, over 100 exon skip inducing AOs had been identified.14 The first clinical human DMD drug making use of exon skipping, Exondys 51 (eteplirsen), was approved in 2016. In spite of the enormous potential of this technique, dystrophin restoration by most drugs remains at less than ideal levels.15 Current research is thus focused on identifying drugs that increase efficacy of AOs when cotreated.15

            An even more novel, but perhaps even more promising method of treating DMD is gene therapy via direct genome editing. An emerging method of genome editing, CRISPR/Cas9, has proven to be faster and less expensive than other methods, in addition to being highly accurate. CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats) are a group of DNA sequences found in prokaryotes which act as a defense mechanism against viruses, or phages. These sequences are derived from bacteria that have previously been infected by a specific phage, the destroyed DNA of which is integrated into the bacteria’s own genome. Should the bacteria be infected with the same phage, the matching of the viral and CRISPR DNA allows the bacteria to recognize the invader. It then produces a guide RNA (sgRNA) from the CRISPR DNA, which binds to the foreign DNA. The guide RNA recruits Cas9, a CRISPR associated endonuclease, to cleave viral DNA at specific sites, destroying it.16

            This CRISPR/Cas9 targeted DNA cleavage method can be utilized on the eukaryotic genome to alter a gene, by cutting DNA at specific sites using sgRNA and Cas9, allowing researchers to add or remove genetic information.17 The applications of such a technology are vast, with the potential to treat a large myriad of ailments, including DMD. The potential of CRISPR/Cas9 to treat DMD was first demonstrated when researchers used this technique in a 2014 study to treat mdx mice. The result was mice that were genetically mosaic, with cells exhibiting a range of between 2% and 100% dystrophin recovery.18 In another study, researchers were able to use CRISPR/Cas9 to delete the entire region between exons 45 and 55, the primary hotspot for DMD causing mutations, in human induced pluripotent stem cells (hiPSC).19 Unlike exon skipping which acts by preventing the splicing of an exon, this treatment acts on the DNA itself, which gives it the ability to correct mutations on introns as well. It also has an advantage over exon skipping in that it permanently restores dystrophin expression permanently19 and due to the ability to make insertions with CRISPR/Cas9, may potentially allow for the full restoration of dystrophin, rather than merely a truncated form.

            The most common means of delivery of the Cas9 enzyme and sgRNA is an adeno-associated virus (AAV) vector. This creates a limit on this method, however. Because of the size of the muscular system in the body, a large dose of AAV is necessary for a desirable effect; high doses of AAV vector are known to cause liver damage and risk activating an immune response, however. In a recent study, researchers were able to create double-stranded, self-complementary AAV (scAAV) that showed functional recovery of dystrophin in heart and skeletal muscles with a 20 times lower dosage than the amount of standard single stranded AAV required to achieve the same effect.20 Continuous research into improving the delivery and stability of CRISPR/Cas9 vectors is needed, but the ability of CRISPR/Cas9 editing to restore dystrophin expression, especially if used at a young age before the onset of necrosis and fibrosis, holds great promise in its potential to be the ultimate cure for this devastating disease.


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