This company is working on a potential approach to knock down myotonic dystrophy, Here is their letter to teh community of DMD and DM1
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Audentes Therapeutes Letter to Myotonic Dystrophy Community
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A better Myotonic Dystrophy Mouse
There is more going on in myotonic dystrophy type 1 than just alternative splicing
by Ana María Rodríguez, Ph.d., Baylor College of Medicine
Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy that affects multiple organ systems. People with this condition develop progressive muscle wasting and weakness in their lower legs, hands, neck and face. Their muscles feel stiff and tight, causing them to be slow to relax certain muscles and therefore have difficulty releasing the hand from a handshake or a doorknob. In addition, people with this condition may have fatigue, muscle pain, difficulty swallowing, cataracts, irregularities in their heartbeat and respiratory complications. In his laboratory at Baylor College of Medicine, Dr. Thomas A. Cooper is leading the way to better understand this rare but devastating condition.
“Muscle wasting in this disease, which happens over decades, is responsible for the death of 60 percent of the patients,” said Cooper, who is professor of pathology and immunology, of molecular and cellular biology and of molecular physiology and biophysics at Baylor College of Medicine. “In this study we wanted to develop a novel model of the disease that would allow us to study muscle wasting in more detail.”
DM1 is caused by a striking expansion of three-letter repeats (CTG) in the DMPK gene. While the unaffected population carries 5 to 37 repeats, people with the condition have 50 to 3000 repeats. The RNA transcripts containing the CTG repeat expansion accumulate in the cell nucleus. This disturbs the normal cellular processing and distribution of molecules, such as muscleblind-like (MBNL) proteins, and induces up-regulation of others, such as the CELF1 protein. These alterations result in abnormal alternative splicing, which is thought to play a central role in the development of DM1. However, how these changes triggered by the expansion of the CTG repeat lead to muscle wasting still is not completely understood.
“We think that the current animal models of DM1 do not provide researchers with a complete and practical tool to investigate the mechanisms involved in muscle loss,” said Dr. Ginny Morriss, postdoctoral associate in the Cooper lab and the first author of this work. “This disease has many different components. Current animal models have some of the molecular components, but the physiological components, what’s happening to the tissue, are mostly missing. We wanted to develop a mouse modelof DM1 that clearly showed muscle loss and to implement a strategy that would allow us to study the pathways involved in muscle wasting.”
A mouse model of reversible DM1
The researchers genetically engineered a skeletal muscle-specific mouse model of DM1 that allowed them to induce the development of the disease at will. When induced, the mice expressed 960 CUG repeats of a particular region of the human DMPK gene and the RNA transcripts containing the CUG repeat expansion accumulated inside the cell nucleus triggering the chain of events that resulted in progressive muscle wasting. When the researchers ‘turned off’ the expression of the 960 CUG repeats, RNA accumulation and muscle loss progressively reverted.
In this model, the researchers saw alternative splicing that was consistent with findings in previous studies that correlated it with muscle weakness. They also validated signaling pathway changes that had been previously found by others. Importantly, they saw signaling pathway changes that had not been described before. These new changes stratified with how severe muscle wasting was in the mice, showing a clear association between specific signaling pathways and muscle loss.
“We validated the upregulation of the activity of protein AMPK-alpha, which had been shown previously by another group in another model. AMPK-alpha regulates the way the muscles metabolize and function,” Morriss said. “One of the new changes we discovered in our model was the dramatic reduction of signaling activity mediated by PDGFR-beta, which is involved in energy metabolism pathways.”
In addition, Cooper, Morriss and their colleagues found a connection with the human condition. They analyzed human tissue samples from patients and unaffected individuals and found in the patients the same signaling pathway changes they had found in their mouse model.
“The field has been focusing on alternative splicing. But, one of the things our findings tell us is that, although many of the characteristics of the disease result from alternative splicing defects, in addition there are other mechanisms at play and therefore other potential targets to treat this disease. There is more going on here than just alternative splicing,” said Cooper, who also is the S. Donald Greenberg and R. Clarence and Irene H. Fulbright Professor and a member of the Dan L Duncan Comprehensive Cancer Center at Baylor.
“Now we have a mouse model in which we can test mechanisms involved in the disease. Because we made our model reversible, we can use it to test hypotheses about how the repeats cause the characteristics of the disease. We can systematically test each one of those hypothesis independently in our model blocking each signaling event specifically and determining how much that affects the disease. We can in this way determine how much each of the disease components, signaling pathways and alternative splicing, contribute to the disease,” Cooper said.
Explore furtherResearchers reveal abnormal myokine signaling in congenital myotonic dystrophy
New Paradigm in Reversing RNA defects in Myotonic Dystrophy
In an elaborate and well documented speech on Tuesday Sept the 5th 2017 Dr. Yeo of University of California in San Diego revealed a new way of reversing the defects caused by RNA in myotonic dystrophy. Both in Laboratory test and in animal models this new way of reversing the defect is now well documented.
The key finds were that this new approach can highly target the repeated CTG repeats and eliminate the foci, not only in DM1 but in other triple expansion repeat diseases including Huntington’s and DM2. The approach uses a molecules called RCas9 through a virus that enable this to travel into the cells. Its a highly innovative and welcome addition the to the arsenal of potential therapies that are coming to help patients with myotonic dystrophy.
Special Focus on CRISPR: CRISPR on the moveSeptember 2017by Jeffrey Bouley | Email the authorEDIT CONNECTSHARING OPTIONS:
Special Focus: CRISPR Gene EditingCRISPR on the moveGene-editing technology continues to evolve In the relatively short time since gene editing involving clustered regularly interspaced short palindromic repeats (CRISPR) arrived on the life-sciences scene—most particularly in the handful of years since we got CRISPR/Cas9 and a much more simplified editing process—the technology has seen its ups and downs with regard to how safe, specific, efficient and reliable it is. But there is no doubt the technology continues to advance and almost certainly will hold a key position in the genomics arena for a long time to come. Germany’s Merck KGaA (not to be confused with U.S.-based Merck & Co.), for example, recently developed an alternative CRISPR genome editing method that it says makes CRISPR “more efficient, flexible and specific, giving researchers more experimental options and faster results that can accelerate drug development and access to new therapies.” Merck KGaA calls the new technique proxy-CRISPR and maintains that it provides access to previously unreachable areas of the genome. Most natural CRISPR systems, found in bacteria, cannot work in human cells without significant re-engineering, the company notes; however, proxy-CRISPR is said to provide a simpler and quicker method to increase their usability without the need to re-engineer native CRISPR proteins. “With more flexible and easy-to-use genome-editing technologies, there is greater potential in research, bioprocessing and novel treatment modalities,” said Udit Batra, a member of the company’s executive board and CEO of its Life Science unit. “As a leader in genome editing, Merck’s new technology is just one example of our commitment to solving challenges in the genome editing field, and we will continue to make CRISPR research a priority.” The company has filed several patent applications on the proxy-CRISPR technology, just one of several CRISPR patent application filings made by the company since 2012. Merck’s research on proxy-CRISPR, “Targeted Activation of Diverse CRISPR-Cas Systems for Mammalian Genome Editing via Proximal CRISPR Targeting,” was published in the April 7, 2017, edition of Nature Communications. The new technology is a follow-on to Merck’s existing CRISPR applications, and the company’s next suite of genome-editing tools for the research community—planned for launch later this year—is expected to include novel and modified versions of Cas and Cas-like proteins. More progress on the RNA front Researchers in the medical school at the University of California, San Diego (UC San Diego) in a 2016 study repurposed the CRISPR/Cas9 technique to track RNA in live cells in a method called RNA-targeting Cas9 (RCas9). In a new study, published Aug. 10 in Cell, the team took RCas9 a step further, using the technique to correct molecular mistakes that lead to microsatellite repeat expansion diseases, which include myotonic dystrophy types 1 and 2, the most common form of hereditary amyotrophic lateral sclerosis and Huntington’s disease. “This is exciting because we’re not only targeting the root cause of diseases for which there are no current therapies to delay progression, but we’ve re-engineered the CRISPR/Cas9 system in a way that’s feasible to deliver it to specific tissues via a viral vector,” said senior author Dr. Gene Yeo, professor of cellular and molecular medicine at UC San Diego School of Medicine. Microsatellite repeat expansion diseases arise because there are errant repeats in RNA sequences that are toxic to the cell, in part because they prevent production of crucial proteins. These repetitive RNAs accumulate in the nucleus or cytoplasm of cells, forming dense knots, called foci. In this proof-of-concept study, Yeo’s team used RCas9 to eliminate the problem-causing RNAs associated with microsatellite repeat expansion diseases in patient-derived cells and cellular models of the diseases in the laboratory. There is still a ways to go before RCas9 could be tested in patients, though, Yeo acknowledged. One bottleneck is efficient delivery of RCas9 to patient cells, as the non-infectious adeno-associated viruses that are commonly used in gene therapy are typically too small to hold Cas9 to target DNA. Yeo’s team made a smaller version of Cas9 by deleting regions of the protein that were necessary for DNA cleavage, but dispensable for binding RNA. “The main thing we don’t know yet is whether or not the viral vectors that deliver RCas9 to cells would illicit an immune response,” he said. “Before this could be tested in humans, we would need to test it in animal models, determine potential toxicities and evaluate long-term exposure.”
Bone Problems in children with myotonic dystrophy
Editors Note: Clubfoot is a well know manifestation of congenital myotonic dystrophy. Other orthopedic issues are discussed in this article as well.
Orthopaedic Manifestations of Congenital Myotonic Dystrophy During Childhood and Adolescence
Journal of Pediatric Orthopaedics: March 2009 – Volume 29 – Issue 2 – p 208-213
doi: 10.1097/BPO.0b013e3181982bf6
Selected Topics
BUY
Congenital myotonic dystrophy (CMD) is a dominantly inherited disorder manifested in childhood by muscle weakness which can be profound at birth, but which progressively improves over the first few years. Congenital myotonic dystrophy represents the severe end of the spectrum of myotonic dystrophy, which in milder cases may not be diagnosed until adulthood. The goal of the study was to identify and quantitate the musculoskeletal deformities which may significantly affect the function of children with CMD.
Methods: A retrospective chart and radiograph review was performed after Institutional Review Board approval for all cases of myotonic dystrophy from 1987 to 2004 followed at a children‘s specialty orthopaedic hospital. Inclusion criteria were either a conclusive testing for CMD by gene testing, electromyography, and/or muscle biopsy in the child or parent and the presence of a typical clinical picture. Skeletal manifestations were classified by body segment (upper extremity, hand, spine, hip, lower extremity, foot) and by the type of deformity. Surgical procedures and outcomes were also documented.
Results: Thirty children and adolescents met the inclusion criteria. The male/female ratio was 1 (15 boys and 15 girls). In 27 cases, the mother transmitted the disease, and in 2 cases, the father transmitted the disease; in one case, it was impossible to reconstitute the family history of the child who was adopted. The mean age at onset of gait was 29 months. Twenty-two (73%) out of 30 children underwent surgery for lower extremity-, foot-, or spinal-related deformities. The mean follow-up was 11.4 years (range, 3-20 years).
No contractures or deformities were observed in the upper extremities. Spinal deformities affected 9 patients (30%), and 3 of these required surgery. These spinal deformities when present usually had an early onset and included thoracolumbar scoliosis as well as kyphoscoliosis. Problems at the level of the hips and knee were infrequent and included only 2 patients who had unilateral hip abduction contracture and 1 patient who had significant fixed knee flexion contracture. Congenital clubfoot occurred in 5 patients (17%) and generally responded well after posteromedial release and recurrence occurred in only one case. Developmental equinusand equinovarus exclusive of clubfoot affected 7 patients (23%), 70% of whom required surgery. Outcome after Achilles tendon lengthening was positive, and many of the children began walking soon after the Achilles lengthening, and recurrence did not occur.
Conclusions: Child with CMD are at high risk for musculoskeletal deformities of the spine and lower extremities. In our experience, correction and improved function were likely after surgery.
Level of Evidence: Retrospective study; level IV
NIH sponsored research shows promise
April 9, 2019
Small molecule targets cause of adult onset muscular dystrophy
At a Glance
- Researchers developed a small molecule that, in mice, blocks the mutated RNA responsible for adult onset muscular dystrophy.
- The findings suggest a new avenue to develop therapeutics for this condition.
Muscular dystrophy includes over 30 inheritable diseases. These are characterized by progressive weakness and degeneration of the muscles. Some types of muscular dystrophy appear in childhood, while others may not appear until adulthood.
Myotonic dystrophy is the most common form of adult onset muscular dystrophy. People with this disorder experience a delay in relaxing their muscles after using them. Type 1 usually affects the lower legs, hands, neck, and face; whereas, type 2 typically affects the neck, shoulders, elbows, and hips. They are caused by mutations in different genes.
Type 1 is caused by a mutation in the dystrophia myotonica protein kinase (DMPK) gene. This mutation causes three nucleotides, CTG, to repeat multiple times in the gene’s DNA. Most people have between 5 to 34 CTG repeats in this gene; however, people with type 1 myotonic dystrophy have from 50 to 5,000.
These extra repeats result in a toxic messenger RNA (mRNA) that traps proteins and forms clumps within cells. This interferes with many important proteins that regulate muscle gene products, leading to serious defects in the muscle cells.
To test whether a small molecule could target the altered RNA and block it from trapping proteins, a team led by Dr. Matthew Disney at Scripps Research Institute carried out experiments in muscle cells taken from patients with myotonic dystrophy type 1 and a mouse model of the disease. The research was supported in part by NIH’s National Institute of Neurological Disorders and Stroke (NINDS) and an NIH Director’s Pioneer Award. Results were published online on March 29, 2019, in the Proceedings of the National Academies.
The researchers designed a small molecule to specifically target the altered RNA’s 3D structure, which folds into a hairpin shape. They first tested its activity in cells taken from patients with myotonic dystrophy. The molecule, called Cugamycin, cleaved 40% of the DMPKmRNA in cells taken from patients, but not in healthy control cells. It also reduced the mRNA’s binding to MBNL1, an important protein it commonly traps, by about 30%.
The team then tested the molecule in a mouse model of myotonic dystrophy type 1 that has 250 CTG repeats. Mice were treated with Cugamycin every other day for one week. Treated mice showed 40% less of the mutated mRNA in their lower leg muscles than untreated mice. They also showed partial improvement in their ability to relax their lower leg muscles.
The toxic mRNA from the mutated DMPK gene alters the expression of 326 genes in this mouse model. Cugamycin treatment restored the normal expression of 177 of these. In an analysis of more than 15,000 other genes, the researchers found no off-target effects.
“The results suggest that our technology can be used to treat myotonic dystrophy type 1 and similar categories of inherited diseases, and without unintended, off-target effects,” Disney says.
These findings demonstrate that small molecules can be designed to selectively target and destroy mutated RNA molecules that cause human disease. However, more studies are needed before this molecule could be tested in people.
—by Tianna Hicklin, Ph.D.