OTC:ICOTF | TSX:MSCL.V
Satellos Bioscience Inc. (OTC:MSCL) is a Canadian biotechnology company dedicated to developing therapies for the treatment of life-threatening, degenerative muscle disorders based on relevant discoveries by the company’s scientific co-founder. Satellos is currently focused on the development of a new class of compounds that could help to restore muscle regeneration in those suffering from Duchenne muscular dystrophy (DMD), the most common genetic disorder diagnosed in childhood, with additional applications possible in other degenerative disorders. A number of therapies have recently been approved for DMD, however none is curative and whether they impact the natural progression of the disease is still being tested, thus additional therapies are still needed.
DMD results from a lack of dystrophin protein in muscle cells (discussed later in the article), thus many current treatment options are directed toward increasing the amount of dystrophin in a patients’ cells. Satellos is taking a different approach that is not predicated on the presence of functional dystrophin. Through studies on muscle stem cells, researchers at Satellos have identified abnormal cellular division in DMD patients as a contributing factor to the disease along with a signaling pathway that could be exploited to correct this issue. The company is diligently working to identify compounds that could activate this pathway, with preliminary results showing modulation of this pathway leads to increased muscle strength and performance in a mouse model of DMD. Satellos is the only company we are aware of that is targeting muscle stem cells as a therapeutic approach to DMD, which could ultimately change the entire treatment paradigm for the disease.
Muscle Stem Cells
Muscle stem cells (or satellite cells) are required for the normal growth, maintenance, and repair of skeletal muscle. While normally inactive, the cells can be induced to divide following injury. Satellite cells are a heterogeneous population of cells that include both a population of cells capable of self-renewal and a population of progenitor cells capable of muscle repair (Kuang et al., 2007). Satellite cells are located in a niche between the basal lamina and the myofibers. Their polarity leads to differentiated expression of certain molecules on the basal and apical surface of the cell; integrin-A7 and -B1, and dystroglycan are expressed on the basal surface (Blanco-Bose et al., 2001) while M-cadherin and NCAM are expressed on the apical surface (Chargé et al., 2004).
As shown in the following image, satellite cells can divide via two mechanisms: symmetric division (self-renewal) in which both daughter cells are identical satellite cells, and asymmetric division in which one satellite cell and one progenitor cell are produced (Chang et al., 2016). In addition, once produced, progenitor cells can go through a finite number of divisions to repair muscle.
Interestingly, it was reported that Dmd, the gene that encodes dysrophin, is expressed in satellite cells, which was seen using both RNA-Seq to identify the Dmd transcript and immunofluorescence to identify the dystrophin protein (Dumont et al., 2015). Dystrophin is known to associate with the cell polarity regulating protein MARK2 (Yamashita et al., 2010) and cell polarity proteins are implicated in asymmetric cell division of satellite cells (Troy et al., 2012). In the mdx mouse model, satellite cells lack dystrophin and have decreased expression of MARK2, which leads to the loss of polarization and a significant reduction in asymmetric cell division. It is this lack of asymmetric cell division that leads to a gradual loss of myogenic progenitor cells and impaired muscle regeneration, which is shown in the following image. These results indicate that DMD may be due at least in part to a deficiency in muscle stem cell division and suggests that targeting stem cells may prove to be beneficial for DMD patients.
Targeting Muscle Stem Cells for Treating DMD
Dystrophin deficiency in skeletal muscle makes them susceptible to damage in DMD patients (Cohn et al., 2000), however a deficit of dystrophin in muscle stem cells is also a part of the mechanism by which DMD patients experience progressive skeletal muscle wasting (Dumont et al., 2015). The loss of dystrophin in satellite cells results in loss of polarity in those cells and ultimately a decrease in asymmetric cell divisions, myogenic progenitors, and ultimately the degeneration of myofibers and replacement with adipose and fibrotic tissue.
The current approaches to treating DMD patients are all less than ideal, thus a novel mechanism of action to target is desperately needed, particularly one that would be applicable to all DMD patients regardless of mutational status. Recently, Satellos scientists identified novel signaling pathways that are implicated in asymmetric cellular division that could be therapeutically exploited.
A small molecule screen was performed with 640 well characterized pharmacological compounds to determine if any stimulated satellite stem cell expansion using myofibers isolated from flexor digitorum brevis muscles, with Wnt7a utilized as a positive control (Wang et al., 2019). Forty-three candidate compounds were identified that induced satellite stem cell expansion, as shown in the following image. While Wnt7a was utilized as a positive control, p38MAPK signaling is known to drive satellite cell commitment (Bernet et al., 2014), thus the identification of compounds that inhibit the p38MAPK pathway was further validation that the assay was capable of identifying compounds that drive satellite stem cell expansion. Lapatinib, an FDA approved EGFR-Erbb2 inhibitor, was identified as a lead compound along with ZM 449829, another inhibitor of EGFR (Brown et al., 2000). Microarray data showed that Egfr, Aurka, and Aurkb are all expressed in satellite cells and are the likely targets of the inhibitors in the screen.
To confirm the activity of the lead compounds and show it is not specific to FDB muscle satellite cells, myofibers from extensor digitorum longus (EDL) muscles were cultured. Treatment of these cells with lapatinib resulted in a shift toward symmetric stem cell division, which included an 83% decrease in the number of asymmetric divisions observed and a 71% increase in the number of satellite stem cells, as shown in the two figures below. There was no change in the total number of satellite cells due to EGFR inhibition, thus indicating it does not alter proliferation.
While EGFR inhibition resulted in a decrease in asymmetric cell division and an increase in symmetric division, addition of EGF to growth medium resulted in an increase in the number of asymmetric divisions compared to vehicle, as shown in the following image.
How EGFR signaling functions in satellite stem cells without dystrophin was examined in EDL myofibers isolated from mdx mice. Following one hour of EGF stimulation, the cells were stained for phosphorylated EGFR (a sign of activation). The following graph on the left shows that EGFR was stimulated (with a significant increase in the percentage of polarized cells), thus indicating it retains normal activity even if dystrophin is not present. The following graph on the right shows that EGF is able to restore the number of asymmetric divisions in mdx mice to levels identical to the untreated wildtype mice, thus showing the potential for augmenting the EGFR pathway to restore normal muscle stem cell function.
Since DMD is a long-term, progressive muscle disease, the long-term effect of EGF treatment on mdx cells was studied through electroporation of TA muscles in mdx mice with an EGF expression vector. At both 30- and 150-days post-intervention (dpi), muscles electroporated with the EGF expression vector showed an increase in muscle mass, as shown in the following image. In addition, there was an increase in the total number of myofibers and an increase in force in the EGF expression vector electroporated muscles.
These results indicate that stimulating the EGFR signaling pathway results in the long-term augmentation of muscle strength in mdx mice through increased asymmetric muscle stem cell division, an improvement in regeneration potential in muscle stem cells, and decreased disease progression in the mdx mouse model of DMD. Importantly, these effects are seen even in the absence of dystrophin. Thus, the EGFR signaling pathway could represent a novel target for pharmaceutical intervention in the treatment of DMD patients.
Case Reports of Milder Symptoms in DMD Patients
Targeting muscle stem cells will not change the fact that DMD patients lack muscle dystrophin, which is thought to be the root cause of the disease. However, there are case reports in the medical literature of DMD patients with a very mild form of the disease even though they have mutations that result in little to no dystrophin expression. These reports support the notion that a patient can have functional muscles even with little to no dystrophin.
Zatz et al. describe two case studies of patients with DMD and very mild phenotypes (Zatz et al., 2014). The first case study involved two half-brothers. One brother was diagnosed with DMD at age 7 and was wheel-chair bound at age 9. The other brother had some muscle weakness at age 13, however as of age 15 (when the report came out) was only mildly affected with some difficulty running but normal walking. Dystrophin levels in both brothers was <5% of normal and despite both of them having a similar lack of dystrophin they had very different phenotypes.
The second case study involved a 16-year-old who has an out-of-frame deletion in the dystrophin gene and was negative for dystrophin by immunostaining. In spite of this, the patient is doing well with discrete calf hypertrophy and a normal stride.
Mild DMD phenotypes are also seen in animal models of the disease. The model with the most similarity to the human condition is the golden retriever muscular dystrophy (GRMD) dog (Cooper et al., 1988). These dogs have progressive muscle degeneration, atrophy, fibrosis, and elevated serum creatine kinase (CK), with death typically occurring at around 1-2 years of age. In a Brazilian research center, two dogs were reported on that had a very mild phenotype despite the absence of muscle dystrophin (Zucconi et al., 2010). The dogs remained fully ambulatory and had normal lifespans in spite of the fact they had no muscle dystrophin, elevated serum CK, and no sign of utrophin upregulation. Further analysis of these dogs led to the identification of candidate genomic region that associated with the mild phenotype. This genomic region contained a muscle-specific transcription factor binding site that drives the overexpression of Jagged1 (Vieira et al. 2015). The dogs with increased Jagged1 expression had a greater proliferative capacity of their myoblasts, thus suggesting Jagged1 could be involved in muscle cell proliferation and repair.
The important point from these case studies is that they show it is possible to have functional muscle activity with little to no muscle dystrophin and that signaling pathways exist that can affect muscle cell proliferation and repair in the absence of dystrophin. Thus, this supports the therapeutic strategy focused on restoring proper muscle stem cell division rather than restoring the expression of dystrophin.
Background on Duchenne Muscular Dystrophy
DMD is an X-linked recessive disorder that is the most common muscular dystrophy, which are a group of diseases that result in progressive weakness and loss of muscle mass. DMD occurs in approximately 1 in 5000 boys and the average age of diagnosis is five years of age (Moat et al., 2013).
Patients typically present with gross motor delay, gait abnormalities, and frequent falls. Most boys with DMD will gain strength until approximately six years of age, however after this progressive deterioration in strength will occur and without treatment most will need a wheelchair around age 10 (Falzarano et al., 2015). Cardiomyopathy presents in all patients by the age of 18, however most are asymptomatic due to limited physical activity (Nigro et al., 1990). Respiratory complications are found in all patients and respiratory failure is the leading cause of death for DMD patients (Phillips et al., 2001).
DMD is caused by a mutation in the DMD gene, which encodes the dystrophin protein (Hoffman et al., 1987). The DMD gene contains more than 2.5 million base pairs of the X chromosome (approximately 1.5% of the X chromosome). The coding sequence is 86 exons, which leads to a 14,000 base pair messenger RNA that is predominantly expressed in skeletal and cardiac muscle, with lesser amounts expressed in the brain (Muntoni et al., 2003). Three different promoters give rise to different full-length isoforms, with additional isoforms derived from alternative splicing events (Sadoulet-Puccio et al., 1996).
The dystrophin protein is 427 kDa and comprises four domains. It is associated with the plasma membrane of cardiac and skeletal muscle (sarcolemma) where it interacts with various integral membrane proteins that together form the dystrophin-glycoprotein complex (DGC). The main role of the DGC is to stabilize the muscle fibers during contractions as well as propagate cell survival and cellular defense signaling pathways (Rando 2001).
Mutations in the DMD gene include intragenic deletions (60-65%), duplications (5-15%), and various combinations of point mutations, intronic deletions, and exonic insertions. While deletions can happen anywhere in the gene, two “hotspots” exist within exons 2-19 toward the 5′ end of the gene and exons 45-55 in the middle of the gene (Muntoni et al., 2003). There is no direct correlation between the size of the deletion in the DMD gene and disease severity, with the phenotype for DMD patients being dependent upon whether the mutation disrupts the reading frame.
The most widely used animal model of DMD is the mdx mouse, which has a point mutation in exon 23 of the mdx gene that prevents the production of the dystrophin protein (Sicinski et al., 1989). These animals exhibit a number of similar phenotypes as DMD patients, including elevated CK levels, irregular muscle histology and necrosis, decreased respiratory function with age, and multiple cardiac phenotypes, including myocardial fibrosis by 6 months of age (Stuckey et al., 2012).
Therapeutic Options for DMD
There is currently no cure for DMD, and all therapeutic approaches are designed to manage symptoms, treat expected complications, and improve a patient’s quality of life as much as possible. Multiple advances over the past 30 years has resulted in an increase in expected survival time, with standards of care for DMD patients published in 2010 (Bushby et al., 2010).
Standard of care for DMD patients begins with corticosteroids. Multiple short-term and long-term studies have shown that their use leads to muscle strength improvement across a number of parameters (Matthews et al., 2016). In addition, corticosteroid therapy has been shown to increase ambulation by approximately three years (Biggar et al., 2006). Emflaza® (deflazacort) was approved by the FDA for the treatment of DMD in 2017 and is currently approved to treat DMD patients 2 years of age and older, regardless of mutation status. In 2020, Emflaza generated revenues of $139 million for PTC Therapeutics (EvaluatePharam).
While offering a number of positive benefits to patients, corticosteroid therapy results in a number of potentially serious side effects that must be effectively managed. These side effects include excessive weight gain, gastric complications, cataracts, hypertension, behavioral changes, bone fracture, and growth suppression (Bushby et al., 2010).
The adverse effects of corticosteroids are hypothesized to be caused by transactivation of downstream signaling pathways following binding to the glucocorticoid receptor. Vamorolone is a compound that binds to the same receptors as corticosteroids but without the transactivation effects. It is currently in a Phase 2b trial following positive results from a Phase 2a study in which vamorolone exhibited an improved side effect profile compared to glucocorticoids (Conklin et al., 2018).
Newer therapies developed for DMD include gene addition, exon skipping, stop codon readthrough, and genome editing techniques, which are all designed to try to restore some level of dystrophin expression.
• Gene Addition: The idea behind gene therapy is that if a functional copy of the DMD gene can be inserted into patients’ cells it may help to restore muscle function. Adeno-associated virus (AAV) is the only viral vector that will infect muscle, however its genome is relatively small (~4.5 kb) and the full-length DMD mRNA is very large (14 kb). As a workaround, researchers discovered that as long as two domains of dystrophin are present, the actin-binding domain at the N-terminus and the dystroglycan binding domain at the C-terminus, the protein retains at least partial activity. Thus, multiple ‘microdystrophins’ have been designed that can fit inside the AAV vector.
SRP-9001: This microdystrophin is being developed by Sarepta Therapeutics. In January 2021, the company reported results from a Phase 2 clinical trial in which there was no statistically significant difference in functional motor ability between patients treated with SRP-9001 and placebo after 48 weeks. The company is still planning to move the drug into a Phase 3 trial.
PF-06939926: This microdystrophin is being developed by Pfizer. It is currently being tested in a Phase 3 clinical trial following positive results reported in a Phase 1b trial.
While the idea of adding a functional microdystrophin through gene delivery is an intriguing one, the strategy does have a few drawbacks. First, the human body has over 500 muscles, thus it is very difficult to get viral infection of all them. Second, since AAV is episomal, normal muscle cell turnover will result in loss of the transgene over time. Third, an immune response can be developed to AAV, the microdystrophin, or both. Fourth, the manufacturing of the viral vector is quite time consuming and improvements in large scale manufacturing of AAV will be necessary in order to treat a large number of patients.
Lastly, the FDA recently held a workshop to discuss safety concerns surrounding the use of AAV, as the agency noted that approximately one-third of recent AAV gene therapy trials had a treatment-emergent serious adverse event. For example:
• A clinical trial in 2020 of an Audentes Therapeutics gene therapy lead to severe liver-related side effects and the deaths of three patients.
• Multiple patients in Solid Biosciences’ Phase 1/2 clinical trial of its DMD therapy experienced immune-related side effects, including acute kidney injury and complement activation.
• Pfizer recently limited enrollment in a clinical trial of its DMD gene therapy following three cases of muscle weakness, two of which involved myocarditis.
A DMD treatment that can avoid the use of AAV would be advantageous, as it would prevent any of the above-mentioned complications.
• Exon Skipping: In patients that harbor a mutation in the DMD gene that disrupts the reading frame of the dystrophin mRNA, exon skipping is utilized in an effort to prevent the exon carrying the mutation from being included in the final mRNA. Four antisense oligonucleotides (ASO) have been approved that target mutations in three different exons.
Exondys 51 (eteplirsen): Approved in 2016, this treatment targets mutations in exon 51, which covers approximately 14% of DMD patients. It generated $415 million in revenues for Sarepta Therapeutics in 2020 (EvaluatePharma).
Vyondys 53 (golodirsen): Approved in 2019 and targets mutations in exon 53, which covers approximately 8% of DMD patients. It generated $41 million in revenues for Sarepta Therapeutics in 2020 and is estimated to generate $212 million in revenues in 2026 (EvaluatePharam).
Viltepso (viltolarsen): Approved in 2020 and targets mutations in exon 53. It generated $23 million in revenues for NS Pharma in 2020 and is estimated to generate $417 million in revenues in 2026 (EvaluatePharma).
Amondys 45 (casimersen): Approved in 2021 and targets mutations in exon 45, which covers approximately 8% of DMD patients. It is estimated to generate $216 million in revenues in 2026 (EvaluatePharma).
Exon skipping can result in expression of a truncated dystrophin, however given the turnover of the ASO it is necessary to continually dose patients. In addition, it is very difficult to achieve expression of the truncated dystrophin in all muscle cells in the body. Given the specificity of each compound, only a certain percentage of DMD patients can be treated effectively by each drug.
• Stop Codon Readthrough: In patients with a mutation in the DMD gene that results in a premature stop codon (nonsense mutations), stop codon readthrough is an option to bypass the stop codon and produce a functional dystrophin. The field began in 1979 where stop codon readthrough was reported for aminoglycosides (Palmer et al., 1979). The aminoglycoside antibiotic gentamicin was tested in DMD patients and was shown to significantly increase the level of dystrophin to 15% of normal (Malik et al., 2010).
Translarna (ataluren): This compound was approved for the treatment of DMD by the E.U. in 2014. It generated $192 million in revenues for PTC Therapeutics in 2020 and is forecast to generate $261 million in 2026 (EvaluatePharma).
• Genome Editing Techniques: CRISPR-Cas9 technology can be utilized to incur double stranded DNA breaks at specific places in the genome. DNA repair can initiate homologous recombination (in dividing cells) or non-homologous end joining (in non-dividing cells). For non-dividing cells such as muscle, non-homologous end joining can be used to delete exons with mutations to restore the reading frame of the DMD gene.
Conclusion
DMD is a devastating disease for which new therapeutic approaches are desperately needed. While new treatments can delay the progression of the disease, none of them are curative and all are either limited to only a subset of patients, have limited efficacy, or have severe side effects. The data discussed above points to a potential new approach to treating the disease by targeting a signaling pathway that could normalize muscle stem cell division. This approach would likely be available for all DMD patients regardless of mutation status, can likely be modulated with a small molecule compound, and could be used in conjunction with currently approved therapies. Satellos scientists are currently following up on these results and we look forward to updates from the company as it works to find an effective therapy for DMD patients.
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