Research Summary
This summary is intended to help the Duchenne community understand the current research approaches being pursued to find treatments for Duchenne. Due to the complex pathophysiology of the disease, many approaches are related and interactive. The organization of the research approaches used in this summary is just one way to categorize them.
Many sources were used to compile the information, and only public information was listed. Due to the dynamic and ever-changing world of research, at times this summary may be out of date. Our hope is to keep it as current as possible. This summary is designed to complement the CureDuchenne: Research Index, which provides a comprehensive list of nearly every strategy being pursued. Readers are encouraged to cross-reference them.
Research projects funded in some amount by CureDuchenne have been identified below. If you have any comments, corrections or suggestions, please email
1. Research Overview
Since the cloning of the dystrophin gene almost 25 years ago, Duchenne muscular dystrophy has gone from a disorder viewed often as incurable or hopeless to one with numerous potential treatment options. From a genetic standpoint, methods to increase dystrophin or utrophin production are showing great promise. Gene-based therapies that have recently emerged spotlight conventional gene replacement strategies, RNA-based technology, and pharmacological approaches, according to Aure'lie Goyenvalle1, Jane T. Seto2, Kay E. Davies, and Jeffrey Chamberlain in "Therapeutic approaches to muscular dystrophy" in Human Molecular Genetics (2011).
While the proof of principle has been demonstrated in animal models, several clinical trials have recently been undertaken to investigate the feasibility of these strategies in patients. In particular, antisense-mediated exon skipping has shown encouraging results for the treatment of dystrophic muscle. Below is a summary of recent progress made in all therapeutic approaches to Duchenne Muscular Dystrophy. The amount of research in DMD is vast and in no way does this summary capture every detail. However, it does give a snapshot overview of all therapeutic strategies that are being pursued. All strategies aim to either replace or compensate for the lack of dystrophin which characterizes Duchenne, or lessen the severity of the disease. These strategies, which will be discussed in more detail below, include:
Gene delivery therapy aimed at reintroducing a functional recombinant version of the dystrophin gene
Tricking the gene into producing dystrophin, such as modification of the dystrophin pre-mRNA, commonly referred to as exon skipping
Read-through strategies for nonsense mutations
Cell-based therapies
Approaches which compensate for the lack of dystrophin such as utrophin upregulation, myostation inhibition/HDAC inhibition, and IGF-1.
Pharmacologic solutions using FDA approved products, either alone or in combination.
All strategies face major challenges imposed by the nature of DMD:
Need to target different muscles in the body, including respiratory and cardiac muscles,
Need for long-term effect,
Potential immune response and the problem of fibrosis,
Requirement for various versions of a drug to address different mutations.
Considering disease frequency and severity, drug development programs in muscular dystrophy are under-represented. Investment in the DMD space has seen some growth recently, as big pharma searches for new opportunities and companies such as Shire and Genzyme have proven the high price, small market- size model can work. As noted below, an example is the recent partnership between Prosensa, a biotech in the DMD space developing an exon skipping therapeutic, and GSK. At the same time, patient advocacy groups for rare and orphan diseases have become more effective and visible, bringing to the surface more research opportunities in DMD. This all bodes well for boys who have Duchenne and, hopefully, there is a therapeutic on the horizon that will ameliorate this disease.
2. Viral Vector Mediated Gene Therapy
The prospect of using gene replacement is a conceptually simple approach to therapy in DMD. The aim is to provide an alternative copy of the functional dystrophin gene for patients rather than repair the locus within the patient's genome. An advantage of gene replacement for DMD is that it is not dependent on any particular mutation in a patient's gene.
However, identifying safe and efficient methods to deliver a replacement dystrophin or dystrophin-like gene to muscles body-wide is a daunting challenge. Much of the work in the field is currently aimed at optimizing delivery methods for targeted and long-term expression of dystrophin in muscles throughout the body. Results from recent studies in larger animal models and in early human trials highlight immunological complications associated with viral vector-mediated gene transfer as the major barrier to clinical success.
The development of all these vectors for dystrophin gene transfer, or any type of gene transfer for that matter, has proceeded slowly, following the death of a patient in the early'90s who was receiving a gene therapy to treat partial ornithine transcarbamylase deficiency. This event impacted the entire of field of gene therapy development. It has taken until now, 20 years later, for gene therapy to again be considered a viable option for DMD and other therapies and renewed development efforts are underway.
Three types of viral vectors have primarily been used by researchers studying the muscular dystrophies. They are:
Adeno-associated viral (AAV)
All three vectors have shown some success in transduction and stable expression of striated muscles; however, only the second, adeno-associated virus (AAV) vectors, have progressed to being used in clinical trials. Adeno viruses are more efficient in immature or regenerating muscle due to down-regulation of the primary coxsackie and adenovirus receptor during muscle maturation. In adult mdx mice and in the dog model immunosuppression was required for long-term transduction. For this reason, these viruses are not being pursued in DMD gene therapy. Lentiviruses are a class of retroviral vectors that stably integrate transgenes into the genomes of quiescent and non-quiescent cells. Integration into the host genome, however, can cause insertional mutagenesis, leading to potential activation of proto-oncogenes -- although this occurs at a low frequency. Lentiviral vectors have low inherent immunogenicity and a relatively large carrying capacity, but they have not been shown to achieve widespread transduction of tissues in vivo and therapeutic effect would be hard to achieve. Consequently, the utility of this vector system for the muscular dystrophies appears primarily for stable transduction of myogenic stem cells or stem cell progenitors and mesoangioblasts with mini-dystrophin expression cassettes.
AAVs are single-stranded DNA parvoviruses that require a helper virus for replication and assembly. However, the recombinant form (rAAV), despite carrying no viral genes, can be produced at high titers in the absence of a helper virus and infect both dividing and non-dividing cells. Stable gene expression following intramuscular rAAV injection has been reported for up to two years in mice and more than seven years in dogs and rhesus monkeys. To date, more than nine primate serotypes (AAV1-9) have been described, with the different serotypes all displaying different tissue tropisms. In striated muscles, high transduction efficiencies have been achieved with rAAV1, 6, 7, 8 and 9. Of all the serotypes tested, rAAV6 also showed the best capability of achieving high transduction in the heart of mice.
Recombinant AAV vectors have attracted widespread interest for the development of muscular dystrophy therapies due to the ability of several serotypes to be efficiently extravasated from capillaries and to subsequently transduce underlying muscle tissues. This ability has been exploited to develop systemic gene delivery techniques for body-wide delivery of dystrophin to striated muscles. Unlike adenovirus, rAAV vectors appeared to exhibit low immunogenicity. A phase I trial for hemophilia b using rAAV2 reported no adverse events in patients receiving intramuscular injections. Further studies in dogs and humans now suggest that these vectors have the potential for a cellular immune response that could greatly influence the nature of clinical application.
The limited carrying capacity of the rAAV vectors, compared with the sheer size of the dystrophin gene, which encodes 14 kb mRNA and spans 2.2 Mb, has meant that truncated versions of dystrophin must be used. The construction of these 'mini'- or 'micro' - dystrophins follows the observation that some very mildly affected Becker muscular dystrophy (BMD) patients can carry large genomic deletions of dystrophin. Work from several labs has shown that two large regions of dystrophin - most most of the rod domain (which normally contains 24 spectrin-like repeats) and the C-terminal domain - can be truncated with minimal functional impact. In mdx mice, treatment with "mini-dystrophin" using rAAV vectors has been shown to reverse most morphological abnormalities in young and old mdx mice and the more severe dystrophin:utrophin double-knockout mouse mode. These studies have prompted researchers to take rAAVs with mini dystrophin into the clinical phase.
While AAV vectors do not elicit a cellular immune response in mice, multiple labs have now observed that AAVs 1, 2, 6, and 8 can elicit an immune response in the dog model for DMD, in monkeys, and in humans. However, studies in dystrophic dogs also showed that this T-cell response could be blocked with transient immuno-suppression. Transient immune suppression has also been applied with success in non-human primates.
There is also some concern that dystrophin itself may be immunogenic in dystrophin-deficient patients. In the only human trial, Dystrophin Immunity in Duchenne's Muscular Dystrophy - NEJM of dystrophin gene transfer, done by Nationwide Children's Hospital and Asklepios Biopharmaceutical, intramuscular injections of rAAV2-minidystrophin into the biceps of six patients showed robust mini-dystrophin-specific T-cell activity from as early as day 15 post-vector treatment in one patient.Another patient showed a T-cell response before vector treatment, and this response was intermittently positive over two years follow-up. No dystrophin-specific antibodies were detected in the serum of any study patients. However, none of the six patients displayed any detectable exogenous dystrophin, regardless of the presence or absence of an immune response against dystrophin, and all patients appeared to express 'revertant' dystrophin-positive myofibers. The authors proposed dystrophin epitopes from revertant dystrophin fibers expressing functional truncated dystrophin after spontaneous in-frame splicing in some patients could prime the T-cell response. However, other investigators postulate that the immune system tends to tolerize patients against revertant fibers - and this could explain the lack of immune response to the transfer of mouse dystrophin into mdx mice. Capsid-specific T-cell responses are also sometimes primed by the delivery of the rAAV vector to muscle, but this does not necessarily cause loss of transgene expression.
Dr. Jeffrey Chamberlain of the Muscular Dystrophy Cooperative Research Center - DNA Plasmids at the University of Washington is also working to deliver mini dystrophin via a rAAV and is in preclinical development and anticipates filing and IND in 2012. CureDuchenne has invested in his research.
It has been shown that an inflammatory immune response is often observed following delivery of rAAV vectors that express immunogenic proteins such as bacterial proteins under the control of a ubiquitously active promoter, like cytomegalovirus. The use of a muscle-specific promoter such as creatine kinase has been shown to effectively block this response. Thorough purification of the viral vectors from contaminants that could potentially be immunogenic, such as products of residual viral genes in vectors or serum products, can also help reduce the immune response.
Strategies to reduce the vector genome dosage would also help prevent elicitation of an immune response and improve expression levels following systemic administration. This could be achieved by codon optimization of the microdystrophin transgenes, or by the use of vascular ligatures or compression bandages to increase the dwell time of the vector in order to increase the chance of transduction.
Other strategies that have been employed to ensure body-wide dissemination of the vector include the use of high-volume or high-pressure injections, or simultaneous perfusion with agents that promote endothelial permeability, such as histamine. Finally, delivery of micro-utrophin in place of micro-dystrophin could circumvent any innate immune response against dystrophin in DMD patients. Recent work has shown that micro-utrophin performed favorably relative to micro-dystrophin in dystrophin:utrophin double-knockout mice.
Another strategy that is being pursued by Nationwide Children's Hospital is to inject a rAAV carrying the gene for follistatin, a muscle growth stimulant into the quadriceps muscles of BMD boys. The work is being carried out by Center for Gene Therapy :: Clinical Trials :: The Research Institute at Nationwide Children's Hospital. This first study will be used to verify that the procedure is safe to and to observe any effects in quadriceps muscle size. The investigators submitted the IND in October of 2011 and plan on enrolling patients shortly. The study will have a low dose and a high dose arm, and safety monitoring as well as evaluation of muscle size will be included.
The second way of dealing with the large size of the dystrophin expression plasmids is to dispense with vectors and rely on the curiously high efficiency of direct transfection into muscle of naked plasmids delivered under hydrostatic pressure, a strategy that has produced promising results in primates and dogs. There is a planned trial of this approach under the auspices of the Genethon in France.
3. Antisense - Mediated Exon Skipping
The majority of DMD mutations disrupt the open reading frame, resulting in the absence of a functional dystrophin protein at the sarcolemma of muscle fibers. The related allelic disorder, BeckerMD, is caused by mutations that create shortened but in-frame transcripts with production of partially functional dystrophin, leading to variable symptoms that range from borderline Duchenne to virtually asymptomatic. Antisense-induced exon-skipping strategies, which aim to remove or "skip over" the mutated or additional exon(s) to restore the reading frame, have been shown to induce the expression of these 'Becker-like' shortened forms of dystrophin protein, retaining crucial functions. Antisense oligonucleotide (AO)-mediated exon skipping uses different oligonucleotide chemistries to target specific exon(s) and "skips" over them to restore the reading frame. The dystrophin gene has been mapped in a way that shows which exon(s) need to be skipped to restore the reading frame for a particular mutation. (click here to see map that shows how to find which exon(s) to skip).
The principle of the exon-skipping therapy for Duchenne was first demonstrated in 1998 in cultured mouse cells in vitro. Since then, numerous in vivo studies have provided pre-clinical evidence for the therapeutic potential of an antisense strategy for Duchenne in several animal models. In particular, the mdx mouse model, which harbors a nonsense mutation in exon 23, has been used extensively to test efficacy of the AO approach using various oligonucleotide chemistries, such as2-'O-methyl antisense oligonucleotides, phosphorodiamidate morpholino oligomers (PMOs), locked nucleic acid oligonucleotides and peptide nucleic acid oligonucleotides. The 2-'O-methyl and PMO backbone chemistries have been taken forward into clinical trials by three different companies, and a summary of their progress is below. Both development programs are focusing on exon 51 for their first target, as skipping over this exon will impact the largest number of boys. Work on subsequent exons is underway, primarily being driven by Prosensa's strategy to advance more than one exon at a time, versus AVI's strategy of proving proof of principle in one exon and then moving into others.
Both the 2'O-methyls and PMOs are small molecules and if you inject them intravenously or subcutaneously, they go into the blood stream and then will be eliminated by the kidneys. The advantage of the 2'O-methyls is their phosphorothioate backbone binds to serum proteins, causing them to act like a carrier. The unbound AO is cleared by the kidneys, but the AO bound to the serum is protected and, therefore, the serum half-life of 2'O-methyl AOs is much higher(by several weeks) than the serum half-life of the PMO's. By contrast, the morpholinos are unable to bind to serum proteins and are thus filtered out more quickly. The serum half-life of the morpholinos is between only two and four hours. Thus the morpholinos have a very brief window of opportunity; they either go to the muscle and are very effective there, or they are filtered out by the kidney before they have a chance to do anything. At this time, morpholinos are more expensive to make than the 2'O-methyls, and higher doses are needed, making them a more costly therapeutic option.
The 2'O-methyl chemistry is being advanced by Prosensa of the Netherlands and GSK. In 2009, Prosensa formed a partnership with GSK whereby GSK got the development program rights to exon 51 and options to other exons if certain milestones were met. (See Prosensa and GSK sign Duchenne Muscular Dystrophy partnership). In September of 2011, Prosensa announced it was advancing the development of exons 44, 45 and 53 into the next stage (See Prosensa Advances Three Exon Skipping Candidates for Duchenne Muscular Dystrophy into the Next Development Stage - Prosensa to receive up to £27M in development and milestone payments from GSK - Prosensa).
In a Phase 1 study completed in 2007, Results of Prosensa's Extended Phase I/II Exon-skipping Trial in Duchenne Muscular Dystrophy Published in the New England Journal of Medicine , Prosensa reported that its product PRO150 (now known as GSK 2402968)was found to be safe and well tolerated in four DMD patients, from 10 - 13 years old and selected on the basis of mutational status, muscle condition and positive response to exon skipping 51 in vitro. The boys received a single injection of PRO150 in a small area of muscle in the lower leg. In a biopsy taken four weeks later, dystrophin expression was seen in most of the muscle fibers at levels that are expected to be clinically relevant. Currently, GSK is concluding a second, phase I/II systemic trial involving 15 DMD patients, receiving five weekly injections. Results showed dystrophin in greater than 95% of muscle fibers in at least some subjects at every dose. All participants then entered a phase I/IIa extension study in which each boy received the highest dose of the drug by subcutaneous injection (under the skin) once a week. At the end of 12 weeks of the extension study, the distance the participants could walk in six minutes had increased by a mean of 30 meters. No serious adverse events were observed, and the majority of adverse events were due to local injection site reactions. Some protenuria was present but resolved upon drug termination and did not present a danger. GSK is planning a safety and tolerability study for non-ambulatory DMD patients and a Phase II double blind study for ambulatory patients.(see Phase II Double blind Exploratory Study of GSK2402968 in Ambulant Subjects With Duchenne Muscular Dystrophy - Full Text View -
AVI Biopharma of Bothell, Wash. is currently developing the PMO chemistry in DMD and has completed Phase 1 studies in Europe. In the Phase I study, AVI-4658 (also known as eteplirsen) was given to seven DMD patients, 12 - 18 years old, administered as an IM injection in the extensor digitorum brevis. Two boys received a low dose of 0.09 mg in 900 ul and 7 boys received 0.9 mg in 900 ul. Each boy received a saline injection in the contralateral EDB. Muscle biopsies were taken before treatment and at three or four weeks and examined for dystrophin production. Results showed that AVI-4658 was safe and well tolerated and induced dystrophin production in a dose responsive manner. A phase 1b/2 clinical study of AVI-4658, in which the drug was infused in boys with Duchenne by IV once a week for 12 weeks, was completed in the United Kingdom in early 2010. Results demonstrated variable dystrophin expression from one participant to the next, in some cases as high as 55% of muscle fibers. Safety issues were mild to moderate and not considered likely to be related to the experimental drug.(see AVI BioPharma - News Release). AVI received regulatory clearance from the FDA in June 2011 to initiate clinical trials in the US and initiated dosing in August of 2011. The placebo-controlled study of 12 patients, which will be conducted at Nationwide Children's Hospital in Columbus, Ohio, is designed to evaluate the efficacy and safety of eteplirsen in DMD patients over 24 weeks of dosing. Patients enrolled in the study will receive once weekly intravenous infusions of either 50mg/kg of eteplirsen, 30mg/kg of eteplirsen or placebo, and will be evaluated on a number of safety and efficacy endpoints. The efficacy endpoints will include biochemical markers in muscle biopsies, such as the production of the dystrophin protein and markers of immune-inflammatory response, as well as clinical outcomes to measure muscle strength, function and degree of ambulation (AVI BioPharma - News Release).
AVI is taking a different approach from Prosensa/GSK in that they are not advancing another exon development program in parallel with their exon 51 program, most likely as the result of a business decision. They do have another related compound, a peptide-conjugated PMO (PPMO), which was studied in non-primates where some toxicity was seen. At this point, AVI seems to be focusing solely on its PMO program.
What is now being developed is single-exon skipping, which will affect most types of deletions on the dystrophin gene. But there are also some deletions that need two exons to be skipped to create an in-frame reading frame. We know from experiments in cultured cells and animal models that skipping two exons is possible. The biggest hurdle to advancing multiple exon skipping is the regulatory pathway. More than likely, the proof of principle for exon skipping will be proven first in single exon skipping and then a regulatory pathway for multiple exon skipping will be defined.
From an exon skipping perspective, duplications are very difficult. If an AO were administered that targeted the duplicated exon, the AO would recognize both exons and skip both. For single-exon duplications, there might be a solution as a third exon before or after the duplication could be skipped as a way to restore the reading frame. But for larger duplications, it becomes very complex and thus very challenging.
An additional challenge is currently there is no animal model with a Duchenne duplication. Utilizing the highly experienced Transgenic Mouse Core at Nationwide Children's Hospital, Kevin Flanigan is currently creating a mouse that carries a duplication of exon 2 in the DMD gene which represents the most commonly duplicated exon among DMD patients. This animal model will be highly valuable for testing the efficacy of single duplicated exon skipping, and is expected to be a very useful reagent for carrying out studies necessary for demonstrating efficacy to regulatory agencies.
Three parties, Genethon, Amsterdam Molecular Therapeutics and Kay Davies and Luis Garcia are working on an approach where exon skipping is achieved through inserting the code for exon skipping via a rAAV. The advantage to this approach is that you only have to transfer the gene once and then the therapy is there forever. However, the problem researchers are trying to address is t it is not very efficient. It is quite difficult to treat an entire limb in a human because you have to inject the virus vectors into an artery under pressure. Once it is injected, it is irreversible. This poses risks in that if a toxicity issue were found, it would be challenging to deal with.
Three parties,Genethon, Amsterdam Molecular Therapeutics and Kay Davies and Luis Garcia are working on an approach where exon skipping is achieved through inserting the code for exon skipping via a rAAV. The advantage to this approach is that you only have to transfer the gene once and then the therapy is there forever. However, the problem researchers are trying to address is t it is not very efficient. It is quite difficult to treat an entire limb in a human because you have to inject the virus vectors into an artery under pressure. Once it is injected, it is irreversible. This poses risks in that if a toxicity issue were found, it would be challenging to deal with.
Read through Strategy for Nonsense Mutations: Ataluren (PTC124®)*
4. Read through Strategy for Nonsense Mutations: Ataluren (PTC124®)
In about 10 - 15% of Duchenne boys, the disease is caused by a point mutation that causes a change in a triplet codon, so that it no longer codes for an amino acid, but instead codes for a 'stop' signal ('nonsense' codons UAA, UAG or UGA). Translation of the dystrophin protein is prematurely stopped, and the short fragment is non-functional and/or degraded. This type of Duchenne is commonly referred to as nmDMD. A promising therapy for nmDMD is ataluren, an orally delivered small molecule designed to selectively induce ribosomal read through of premature stop codons but not normal termination codons. Ataluren was developed after gentamicin, an aminoglycoside, was shown to promote read through in mammalian models and in the mdx mouse model but presented lack of potency, potential toxicity issues and administration issues. These proof-of-concept experiments led researchers to use high throughput screening methods to identify compounds that suppressed the early termination but not normal termination codons, and did not present the potency, toxicity and administration issues associated with gentamicin. In mdx mice and muscle cell cultures from patients, ataluren, a non-aminoglycoside, was found to promote dystrophin production in primary muscle cells in humans and in mdx mice expressing dystrophin nonsense alleles. Additionally, ataluren was found to restore striated muscle function in mdx mice within 2two- to eight weeks of drug exposure.
PTC Therapeutics, of South Plainfield, New Jersey has completed phase I and IIa and b clinical trials with ataluren. In Phase I, ataluren, delivered as a single or multiple dose, was found to be safe and well tolerated and supported the initiation of Phase II trials. Sixty two healthy adult male and female volunteers were administered the drug as part of Phase 1. In Phase 2, 38 nmDMD patients were given ataluren at one of three dose levels for 28 days. The drug was found to be safe and well tolerated with infrequent adverse events. Plasma concentrations correlating to activity in preclinical models were found at the middle and high dose. Additionally, patients receiving ataluren showed qualitative increases in muscle dystrophin expression and reductions in serum creatinine kinase levels. These patients were followed in an open label, long-term safety study. In April of 2008, a Phase 2b study was initiated and by February of 2009 was fully enrolled at 173 nm DMD patients, ahead of schedule by two months. In the Spring of 2010, despite there being no safety events in either the IIa or b studies, it was determined that the IIb study failed to meet all trial endpoints, including the primary outcome the six- minute walk test. (See WMS Report on Phase 2b). All further studies were halted. After extensive analysis, PTC Therapeutics found that if the highest dose cohort was excluded, a clinically meaningful delay in loss of ambulation was demonstrated. The company may pursue this with the FDA and some compassionate use of the low dose may be initiated.
5. Stem Cell Transplantation
Even before the discovery of the dystrophin gene, the idea of transplanting immature muscle cells from healthy donors into dystrophic muscle was mooted as a potential therapy. However, it was shown that myoblasts from normal mice could repair the spontaneously degenerating muscle fibers in the mdx mouse, the murine homologue of Duchenne, and generate near normal levels of dystrophin protein within them . It was also clear, however, that the efficiency, particularly that of distribution of donor cells from the site of injection, was too low to be of practical use as a whole body therapy. Nonetheless, a number of human phase I trials were conducted with mixed results. One research group, the lab of Dr. Jacques P Tremblay of Laval University in Quebec, has persisted in a systematic exploration of ways of improving this technology in a series of human trial, using immunosuppression and other techniques. However, the efficacy of distribution, 1mm or less from the injection tracks, remains too low to be applicable to more than a few selected muscles.
Cell therapy has been resuscitated in recent years by a series of demonstrations that cells other than those conventionally thought of as myogenic are able to give rise to skeletal muscle as well as a number of other cell types. What excites interest from a therapeutic viewpoint is the finding, in some cases, of widespread delivery via the blood. A number of demonstrations (Ferrari) that cells derived from bone marrow grafts could make a small but detectable contribution to regenerating muscle of the host, led to the idea that the bone marrow might act as a pool for distribution of stem or precursor cells to the various solid tissue of the body (Blau). Such a mechanism would overcome the main problem of myoblast transplantation, namely the poor dispersion of cells from the graft site but, up to now, it has been too ineffectual to be of physiological value (Ferrari).
Efficient delivery via the vasculature has, however, been demonstrated for rare clones derived apparently from endothelial cells called 'mesoangioblasts' and for bone marrow mesenchymal cells converted to a myogenic precursor phenotype by exposure to a cocktail of growth factors and transfected with a construct encoding the intracellular domain of 'Notch'. Of these, the former has been developed for application to a human trial by Dr. Giulio Cossu and a small-scale trial in Duchenne has been initiated.* A good summary of the current status of his work is captured on the CureDuchenne Founder’s Blog» Blog Archive » Dr. Giulio Cossu Explains Duchenne Stem Cell Research for Cure Duchenne.
6. Compensatory Proteins
A promising pharmacological treatment for DMD aims to increase levels of utrophin, a homologue of dystrophin, in muscle fibers of affected patients to compensate for the absence of dystrophin. Proof of principle studies in mdx mice have established that elevation of utrophin levels in dystrophic muscle fibers can restore sarcolemnal expression of the dystrophin associated protein complex (DAPC) members and alleviate the dystrophic pathology. The mechanisms of transcriptional control of utrophin provide multiple targets for pharmacological interventions.
In particular, knowledge of the utrophin-A promoter has initiated the search for small molecules that can stimulate the transcription of utrophin. Such utrophin-based drug therapy for Duchenne presents many advantages, as it should be effective for all Duchenne patients, regardless of the specific gene defect. Additionally, it could be administered to patients systemically because utrophin overexpression in tissues other than muscles does not seem to cause detrimental effect.
High-throughput screenings, which enable the rapid assessment of thousands of potentially beneficial compounds from chemical libraries, have allowed the identification of a small molecule (BMN195) that has recently been tested in a phase I clinical trial by BioMarin Pharmaceuticals, who licensed the molecule from Summit plc. While BMN195 showed promising utrophin up-regulation potential in pre-clinical models, it could not reach efficient plasma level concentration due to poor bioavailability during the clinical study. Since there were no safety issues with BMN195, Summit plc has taken back development of BMN195, is investigating the possibility of better formulations of the drug and is committed to moving the Duchenne program forward.
Several other groups have reported approaches for increasing the levels of utrophin through other targets such as RhoA, heregulin, L-arginine, and calpain inhibition, although none of them has been shown to increase the level of utrophin substantially. In a study published in 2010, administration of L-arginine concurrently with prednisone to dystrophic mouse produced a modest effect in terms of reducing fibrosis and increasing muscle strength but chronic administration induced some toxicity. In a study published in 2010 in the dystrophic mouse, increased expression of integrins and ADAM12 have been shown to improve pathology, in addition to increasing the levels of utrophin. Agents such as biglycan that stabilize the DAPC also hold therapeutic promise. Custom-designed proteins with zinc finger motifs have been shown to act as strong transcriptional activators of the utrophin A promoter. Ervasti and colleagues are also attempting to deliver utrophin protein directly through the TAT-Utrophin technology, described below.
Currently there are four ongoing development programs advancing utrophin up-regulation. All are in the pre-clinical stage. The first, mentioned above, is SMT C1100, Summit Pharmaceuticals (see FAQ's on Summit 1100). Although Biomarin has discontinued the program, Summit is committed to carrying forward the program and is looking for development partners.
The second is a recombinant human biglycan being developed by Tivorsan . The company is currently going through the pre-clinical development steps necessary to file an IND.
The third program is being developed by PTC Therapeutics, the same company that developed Ataluren, through its Project Catalyst. No lead candidate has been decided on, but several compounds look promising.*
A fourth approach to up-regulating utrophin is being researched by Dr. Ervasti of the University of Minnesota, Minneapolis, Minn. and developed by Retrophin, llc, an emerging biotech committed to rare and life threatening diseases. Dr. Ervasti has come up with a way to transport utrophin, acting as a substitute for dystrophin, to the muscle cells. This approach requires that utrophin is attached to another protein called TAT. This new fused protein (or chimera) is then transported into the cell. Dr. Ervasti has promising preliminary results that demonstrate improvement in a mouse model treated with this therapy. Currently, Dr. Ervasti is investigating the fused proteins' optimal dosage, frequency of administration, and mode of delivery. In addition, Dr. Ervasti is investigating ways to scale production of TAT-utrophin in preparation for preclinical safety and toxicology studies in animals.
Myostatin (GDF8) is an inactive protein produced in the muscle cells and found in circulation in the bloodstream. Myostatin is activated when its propeptide is degraded and it binds to activin type II in muscle cell membrane. This leads to a cascade of events which eventually reach the muscle cell nucleus and block muscle forming genes. Mutations in myostatin cause marked increases in muscle mass, suggesting that this transforming growth factor-β (TGF-β) superfamily member negatively regulates muscle growth and its blockade could lead to increase muscle building and help mitigate the muscle wasting in Duchenne patients. Myostatin inhibitors include myostatin antibodies, myostatin propeptide, follistatin and follistatin-related protein. Researchers are focusing on determining which, if any, would be effective in promoting muscle growth in Duchenne.
The first approach to myostatin inhibition is via a humanized antibody targeted against myostatin. In a Phase 1/11 trial done by Kathryn Wagner of the Wellstone Dystrophy Center at Johns Hopkins University in collaboration with Wyeth Laboratories of Collegeville, Penn., MYO-029, a humanized antibody against myostatin, was found to be safe at the doses studied but resulted in no improvement in muscle function. Although Wyeth has decided not to pursue this antibody for this indication, other antibodies are being prepared for further research.
Acceleron Pharmaceuticals of Cambridge Mass. had an antibody, ACE-031, that acts as a decoy for myostatin and binds it before it binds with ACTRIIB and inhibits muscle growth. The company completed its Phase I studies with good results. The drug was found to be safe, there were positive signs the drug caused significant increases in lean muscle mass and there was a trend in improved grip strength. In early 2010, Phase II studies were initiated in five sites in Canada. Due to some safety concerns, the FDA decided these studies be terminated, despite the fact these safety concerns resolve upon discontinuation of treatment. In September 2010, Acceleron announced a partnership with Shire to continue to develop ACE-03 and both companies remain committed to advancing the program.
PTC Therapeutics, through research project called Project Catalyst, also has a program in myostatin inhibition at the very early stages of development. No lead candidate has been identified as of yet.
Another approach to myostatin inhibition is through the use of histone deacetylase (HDAC) inhibitors to indirectly target myostatin by influencing follistatin expression. Lorenzo Puri and colleagues are studying HDAC inhibition in the mdx mouse model and are showing positive effects. There are many HDAC inhibitors in clinical trials for other disease states. Should HDAC inhibition move forward into clinical trials, the fact that the concept is already in humans should be advantageous in many respects.
Insulin-like growth factor (IGF-1) is a target in treating Duchenne as it plays an important role in the regeneration of damaged muscles by activating muscle cell proliferation and fusion. It is thought that increasing levels of IGF-1 in Duchenne patients may help repair damaged muscles and preserve muscle function. PTC Therapeutics, via its Project Catalyst program is exploring IGF-1's muscle regenerative role and is evaluating it as a target for Duchenne. No lead candidate has been chosen.
A clinical trial studying Increlex, the first rhIGF-1 (recombinant human insulin-like growth factor-1)therapy approved in the United States for long-term treatment of growth failure in children with Severe Primary Insulin -like growth factor deficiency (IGFD), is being studied in Duchenne patients in a clinical trial at Cincinnati Children's Hospital. Results have not been announced.
7. Pre-Approved Pharmacologics Being Repurposed Either Singly or in Combination
Increasingly, researchers are looking for new uses for already FDA-approved products. This strategy offers advantages in terms of time it takes a therapy to get to patients, research and development costs, and a decreased risk of safety concerns once a product is on the market. In recent years in Duchenne research, there has been a push to explore repurposing existing products. Promising candidates our summarized below.
Another target that is being studied in Duchenne is the nNOS pathway. In Duchenne, the lack of dystrophin causes a lack of nitric oxide, which in healthy muscle cells stimulates cGMP, a molecule that is needed for healthy muscle function. This lack of nitric oxide can be compensated for by inhibiting an enzyme called PDE-5. Blocking PDE-5 directly allows for an increase in cGMP, increased vasodilation and promotes healthy cell activity.
Two products, already approved for use in penile erectile dysfunction, are being studied in Duchenne for their PDE-5 inhibition effect. Revatio® (sildenafil) also known as Viagra® is in Phase II at the Kennedy Krieger Research Institute.* The trial is looking at Revatio's ability to improve cardiac function in participants with Duchenne who are 15 to 50 years old. The primary outcome is a change in left ventricular end-systolic volume, a measurement of cardiac function. Cialis (tadalafil) is being studied at Cedars Sinai Medical Center in Los Angeles in male participants who are 18 to 50 years old and either have a diagnosis of Becker or who are healthy volunteers. In the first part of the Tadalafil in Becker Muscular Dystrophy study, baseline measurements, including bloodflow before and after exercise, will be established in participants with Becker verses healthy controls. In the second part of the study, participants with Becker will be administered tadalafil for two days and the measurements will be repeated. This study, ongoing at Cedar Sinai, has been extended to 1) determine if tadalafil or sildenafil can improve muscle blood flow during exercise in boys with Duchenne; and 2) to inform the design of a subsequent, randomized, multi-center trial with clinical endpoints.
Isosorbide dinitrate, a vasodilator used for angina pectoris, can increase nitric oxide (NO) production in cells, which is needed for healthy muscle functioning. Ibuprofen is an NSAID that has well-known anti-inflammatory properties A recent study, Nitric oxide release combined with nonsteroidal anti inflammatory activity prevents muscular dystrophy pathology and enhances stem cell therapy from the University of Milan suggests that a drug that combines both NO-stimulating properties and anti-inflammatory properties in mice that lack dystrophin leads to improved muscle cell structure and function. Professor Clementi from the University of Milan is taking this combination forward in humans.
There is some evidence to suggest that approved drugs for the management of hypertension could be used preventatively in Duchenne boys to prevent cardiomyopathy. Several strategies, using already drugs, in combination or alone, are being evaluated for safety and efficacy in Duchenne boys. ACE inhibitors are almost considered standard of care in Duchenne. Researchers are looking for more effective compounds to manage cardiomyopathies and also compounds that have an additive effect to make existing therapies more effective.
Losartan, (brand name Cozaar) an ACEII inhibitor that is used to lower blood pressure works by blocking the production of TGFβ. As noted below, TGFβ has a deleterious effect on muscle repair and researchers are looking for compounds that inhibit it to decrease the muscle damage due to the lack of dystrophin. Researchers have shown that mdx mice treated with Losartan had decreased cardiac and skeletal muscle fibrosis and improved cardiac systolic function. The Treat NMD TACT committee reviewed the pre-clinical data for Losartan and a potential trial design in January of 2010. Researchers are working to carry this study forward. Jerry Mendell, of Nationwide Children's in Columbus, has funding from the MDA to study Lisinopril versus Losartan in a multi-year study "Treatment of the Dystrophin Deficient Cardiomyopathy".
One study, Ramipril versus Carvedilol is comparing the efficacy of an ACE inhibitor and a beta-blocker in combination on myocardial tissue properties and heart function through MRI and myocardial tissue ultrasound.
Lisinopril is also being studied in combination with CoQ10, an anti-oxidant , to measure its ability to ameliorate the decline in cardiac muscle function that occurs in muscular dystrophies. The Cooperative International Neuromuscular Research Group is conducting the study and is currently enrolling patients.
Spironolactone, a diuretic, in combination with lisinopril, an ace inhibitor, when administered early showed preventative cardiac effects in mdx mice in a recent study done by Jill Rafael-Fortney from Ohio State University. These promising results are leading to clinical trials to test spironolactone and lisinopril in humans and also to evaluate the effects of spironolactone with other compounds (losartan and prednisone). More can be learned by listening to a recent podcast where Jill discusses the results of her animal work and her next steps.
8. Anti-Inflammatories/Anti-Oxidants
TGFβ is thought to interfere with muscle repair by stimulating the deposition of connective tissue proteins (collagen) in regenerating muscle in a process known as fibrosis. Thus blocking it can enhance muscle repair. Researchers at the University of Pittsburgh have shown that decorin, a proteoglycan normally found in the human body, can deactivate TGFβ. Furthermore, decorin was shown to prevent fibrosis of the muscle in mice suggesting that TGFβ inhibition may prove effective in treating debilitating muscle diseases such as Duchenne.
Duchenne is associated with chronic inflammation that results in muscle cell death. NF-kB is a transcriptional factor that induces the production of many inflammatory proteins responsible for the pathogenesis of inflammatory diseases. Thus, targeting NF-kB may reduce inflammation, and improve muscle function. Researchers at Ohio State University demonstrated that targeting NK-kB with a chemical inhibitor led to improved muscle function in mice suggesting that NF-kB inhibition may become an effective option in treating individuals with Duchenne.
Flavocoxid has been shown to be a potent anti inflammatory via NF-kB inhibition and is in Phase I safety study.
TNF-alpha is pro-inflammatory and pro-fibrotic and these cytokines are responsible for the pathogenesis of inflammatory diseases. Targeting TNF-alpha has proven effective at reducing inflammation and improving quality of life in patients with rheumatoid arthritis and inflammatory bowel disease. TNF-alpha is found to be elevated in Duchenne and in mdx muscles. Therefore, TNF-alpha has been identified as a target in Duchenne. In fact, a 2004 study (Grounds et al.) demonstrated that TNF-alpha blockade reduced dystrophic muscle breakdown in mice. Four compounds (BKT-104, cV1q, LMP420, etanercept) are being studied in animal models and results look promising.
Increasing α7-integrin promotes muscle cell proliferation, adhesion, and resistance to apoptosis. In the mdx mouse model of Duchenne it has been found that laminin-111 upregulates α7-integrin, thus promoting muscle cell adhesion and reducing muscle disease. Human Laminin-111 is a protein delivered via IV or IM injection and is anticipated to be used in conjunction with other therapies that are being developed. Prothelia, a biotech company is conducting the development of this approach and is currently in the pre-clinical stage.
Corticosteroids are useful palliative treatments and considered the standard of care for patients with Duchenne. However, long term use is limited by side effects such as bone loss, muscle loss, and cardiomyopathies. These side effects are associated with binding of the activated glucocorticoid receptor to promoter regions of genes and subsequent transcription in a process called transactivation. The beneficial anti-inflammatory properties are believed to be attributed to inhibition of pro-inflammatory molecules such as NF-kB. VBP15, being developed by Reveragen Biopharma,* is a steroidal analogue that does not appear to possess trans-activation properties yet still retains anti-inflammatory capacity (NF-kB inhibition). Furthermore, VBP15 has shown efficacy in the mdx mouse model for Duchenne. In fact, after four months of treatment in the MDX model, VBP15 was shown to be as effective as prednisone but exhibit less side effects. Thus, VBP15 may represent an effective, yet safer alternative to traditional glucocorticoids in the treatment of Duchenne. The company is finalizing its development plan and is planning on submitting an IND in 2013.
It has been suggested that anti-oxidants, which target reactive oxygen species, could be of benefit in Duchenne by acting to minimize the oxidative stress and boost mitochondrial function, which has been shown to be reduced in Duchenne muscle cells. In dystrophic mice, anti-oxidants have been shown to reduce muscle necrosis and protect against reactive oxygen species.
There are many types of anti-oxidants that are being researched in DMD and the CureDuchenne: Research Index has a substantial list. Those in clinical trial are summarized below.
Catena®, (idebenone), an antioxidant, is in clinical trials for Duchenne and has been approved in Canada for use in Friedrich's Ataxia. Currently Santhera, the company developing Catena, is recruiting patients for the DELOS phase III trial. In summer 2011, Santhera received a US patent for the use of Catena in Duchenne.
Another anti-oxidant compound being studied, currently in Phase 11/111, is Sunphenon Epigallocatechin-Gallate (EGCg) the major extract of green tea. Phase II/III study is a double blind, placebo controlled, randomized study of 40 patients over 18 months looking at safety and tolerability. A secondary efficacy outcome (Six- Minute Walk Test) will be evaluated over 36 months.
9. Supplements
Nutritional support, although often overlooked, may be an important component of Duchenne treatment. A list of the most promising nutritional supplements that are available is listed below.
Glutamine, a potent anti-oxidant has been studied in Duchenne patients. The Cooperative International Neuromuscular Research Group found that although there were no statistically significant results, a disease-modifying effect of glutamine in younger Duchenne patients cannot be ruled out.
Calcium and Vitamin D are two compounds that are critical for healthy bone mineralization and density. Duchenne boys, especially those on corticosteroids, can develop bone weakening osteoporosis. In A review of nutrition in Duchenne muscular dystrophy, it was suggested that calcium and vitamin supplements would be beneficial. Although no formal clinical trials have been conducted on providing supplemental vitamin D and calcium to MD patients, the practice has been recommended by at least one MD researcher (Leighton S 2003).
Alpha Lipoic Acid, a potent anti-oxidant and Hydroxy-Beta-Methylbutyrate (HBM) a metabolite of the essential amino acid leucine are two compounds that seem to prevent certain kinds of cell damage in the body and also play a role in muscle growth and regeneration. In a 2006 study titled Nutritional therapy improves function and complements corticosteroid therapy in mdx, it was found that alpha lipoic acid and HBM, along with two other compounds, showed a therapeutic effect when administered as a four-compound therapy with or without corticosteroids. No formal testing has been done in humans.
Other supplements that are suggested to have a positive effect in Duchenne are Creatine, Green Tea Extract, Co Q10 and Arginine. These have been discussed previously in different sections in this summary.