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Muscular dystrophy (MD) describes a hereditary disease of muscle-producing with progressive weakness and wasting. The common features of MD result from the absence or altered function of a structural component of the muscle fiber, promoting dystrophic changes characterized by cyclic rounds of necrosis, degeneration, and regeneration, resulting in remodeling of muscle tissue with fibrosis and fatty infiltration.
Duchenne MD (DMD) is the most common MD. It is caused by deletions in the gene for dystrophin. DMD is an X-linked disorder that is typically diagnosed before 5 years of age and is associated with a progressive decline in function; patients lose ambulation in their early teens, arm function in their late teens, and eventually die from cardiac or respiratory failure in early adulthood.
Gene Therapy Strategies for MD
There are several different gene therapy strategies that are being investigated for MD. One approach targets premature stop mutations, which occur when there is a substitution of one nucleotide for another, leading to a premature stop mutation in the messenger RNA (mRNA). One agent, ataluren (PCT124), reads through the stop signal and allows the ribosome to continue reading along the mRNA. Ataluren is a small molecule drug that has limited regulatory approval in some countries but is not yet approved in the United States.
Antisense oligonucleotides involve a small stretch of DNA-like material that attaches to the area of the pre-mRNA and modulates splicing, thereby affecting the transcription. This exon-skipping approach is applied to DMD via 2 strategies: (1) the 2’MOE strategy and (2) the Morpholino strategy. Both strategies increase the size of the mutation and, in DMD, allow it to bring back the reading frame such that the mRNA can read through to produce some functional protein. Eteplirsen, golodirsen, and viltolarsen are available antisense oligonucleotides that were FDA-approved in 2016, 2019, and 2020, respectively.
Gene replacement therapy uses vectors, which are viruses that shuttle the gene to the target tissue. The vectors relevant to DMD are adeno-associated virus (AAV) vectors. These vectors are employed with the goal of replacing a portion of the dystrophin gene. AVV9 and AVVrh8 are being investigated for use in DMD.
Gene editing, which is in a very early stage of preclinical development, uses CRISPR technology to edit the genome using “molecular scissors” and, in this case, an enzyme called a Cas9 nuclease, which targets a specific area in the genome using a guide RNA. This allows for insertion of a new or corrected copy of a mutated gene.
Gene Therapy Trials
There are several approaches to minidystrophin gene therapy currently in clinical trials.
SRP-9001 is being evaluated in an open-label, single-dose study in boys aged 4-7 years (N = 12) who received 2 x 1014 vg/kg. There is also an ongoing phase II study to assess the safety and efficacy of gene delivery via single IV infusion. The primary outcome measures are safety, change from baseline in minidystrophin by Western blot (WB) at 12 weeks, and secondary outcome measures are several functional measures such as the North Star Ambulatory Assessment (NSAA).
A second study is evaluating PF-06039926. It is a multicenter, open-label, single-ascending dose study in boys aged 4-12 years (N = 6). Cohort 1 received 1 x 1014 vg/kg and cohort 2 received 3 x 1014 vg/kg. The primary outcome measure is safety, with secondary outcome measures including minidystrophin expression at 2 months and 1 year.
SGT-001 is under investigation in an ongoing, open-label, single ascending-dose phase I/II study in boys aged 4-17 years. Cohort 1 received 5 x 1013 vg/kg and cohort 2 received 2 x 1014 vg/kg. The primary outcome measures are safety and microdystrophin expression.
The trial results are as follows:
For SRP-9001, they have observed no serious adverse events to date. They have seen an elevated gamma-glutamyl transferase in 3 out of 4 subjects which resolved with steroid treatment. There was transient nausea that was limited to the first week and did not correlate with liver function test elevation or any other abnormality. The immunohistochemistry (IHC) showed positive dystrophin expression in all 4 subjects, with 81.2% of fibers expressing dystrophin post treatment. Dystrophin expression by WB revealed a mean microdystrophin expression of 95.8% of normal. Creatinine kinase (CK) was found to significantly decrease over time, and the NSAA significantly increased by 6-7 points.
For PF-06939926, there was some nausea and vomiting that were treated with oral antiemetics; the vomiting fully resolved within 2-5 days and nausea within 1 week, although one patient did require hospital admission for IV antiemetics and fluids. In one subject, there was acute renal injury with complement activation. The patient was treated with hemodialysis and complement inhibitor therapy. Of note, his AAV9 neutralizing antibody titer at 2 weeks was greater than all other participants at 4 weeks. Still, outcomes of this trial are positive. The IHC in the higher-dose cohort showed 70% of fibers expressing dystrophin. NSAA data available on 2 patients form the low-dose cohort who completed 1 year of therapy showed increases from 3-6 points.
In the SGT-001 trial, a serious adverse event was observed in one subject in the first cohort (5 x 1013 vg/kg), with complement activation, thrombocytopenia, decrease in red blood cell count, and transient renal impairment. The patient was treated with a modified steroid regimen and limited course of eculizumab and made a full recovery. In the second cohort (2 x 1014 vg/kg), there was a serious adverse event in one subject with complement activation, thrombocytopenia, decrease in red blood cell count, acute kidney injury and cardio-pulmonary insufficiency. That individual also fully recovered. In the low-dose cohort, approximately 10% of fibers were positive for microdystrophin and < 5% normal dystrophin was identified by WB in one patient. There were low levels of microdystrophin by IHC and none by WB in the other 2 patients. In the high-dose cohort, 3-month biopsies showed 10% to 20% microdystrophin-positive fibers and 5% normal dystrophin by WB in the fourth patient. Fifty percent to 70% microdystrophin-positive fibers and 17.5% normal dystrophin by WB was observed in the fifth patient. Neuronal nitric oxide synthase was stabilized at the membrane and CK levels declined as well.
Gene Therapy for Limb Girdle Muscular Dystrophies (LGMD)
LGMDs are a group of disorders classified as either type 1 (of which there are 8 autosomal dominant conditions) or type 2 (of which there are 26 autosomal recessive conditions). LGMDs are associated with progressive dystrophic muscle deterioration (usually a proximal to distal gradient) that involves both skeletal and cardiac muscle.
Dysferlinopathy, which is the result of mutations in the in the dysferlin gene, is a membrane repair protein that has 2 main phenotypes: LGMD2B and Miyoshi. There have been AAV approaches to try to include the full complementary DNA in a dual-AAV delivery system. These approaches have favorable proof of concept, and they are now moving into human studies.
Sarcoglycanopathies may also be treated by gene therapy. Treatment for the gamma-sarcoglycan (SG) deficiency LGMD2C has been explored with an AAV1 intramuscular injection in a phase I trial. It was found to be safe and demonstrated positive gene expression. In the α-SG LGMD2D, there has been a phase I study with an intramuscular injection of an AAV1 vector, showing sustained expression of α-SG at 6 months. Results of a phase I/II study are pending.
In LGMD2E, the goal is replacement of β-SG gene. A phase I/II study was conducted involving single IV administration showed an 82% reduction in the CK level and improvement in motor function on the NSAA and timed motor function testing. There was also increase in β-SG expression, with a mean 51% of muscle fibers demonstrating positive expression on IHC.
Challenges to Gene Therapy in MDs
As development of gene therapy progresses, we will have to consider important issues such as the means of administration, target engagement (ie, how the vector is delivered to the muscle and tissue), potential risk of integration into the host genome, the efficiency and durability of gene expression, and potential adverse events. Immunity issues, such as the presence of preexisting neutralizing antibodies and potential immune reactions to the viral vector and new dystrophin epitopes, will also have to be considered. Approaches using ataluren and antisense oligonucleotides have been safe and relatively positive but are limited by poor generalizability, low efficiency, and potential immune responses and drug toxicities. AAV-mediated gene transfer has also been promising, with good efficacy and a favorable safety profile, but elicited immune responses continue to represent a challenge, and the clinical benefit of microdystrophin is not fully understood. There are many remaining questions with gene therapy for MD, and although significant progress has been made, there are many remaining challenges to be overcome.