iPS Cells Used to Correct Genetic Mutations that Cause Muscular Dystrophy

iPS Cells Used to Correct Genetic Mutations that Cause Muscular Dystrophy

Thursday, 27 November 2014

This image shows immunofluorescence staining

of skeletal cells differentiated from DMD-iPS

cells. Untreated DMD skeletal cells do not

express dystrophin (green) due to the deletion of

exon 44. However, after any of the three

correction strategies are applied to iPS cells,

differentiation into skeletal cells results in normal

dystrophin expression. Scale bar, 50 μm.

Credit: Dr. Akitsu Hotta, Kyoto University.

Researchers at the Center for iPS Cell Research and Application (CiRA), Kyoto University, show that induced pluripotent stem (iPS) cells can be used to correct genetic mutations that cause Duchenne muscular dystrophy (DMD). The research, published in Stem Cell Reports, demonstrates how engineered nucleases, such as TALEN and CRISPR, can be used to edit the genome of iPS cells generated from the skin cells of a DMD patient. The cells were then differentiated into skeletal muscles, in which the mutation responsible for DMD had disappeared.

DMD is a severe muscular degenerative disease caused by a loss-of-function mutation in the dystrophin gene. It inflicts 1 in 3500 boys and normally leads to death by early adulthood. Currently, very little is available in terms of treatment for patients outside palliative care. One option gaining interest is genomic editing by TALEN and CRISPR, which have quickly become invaluable tools in molecular biology. These enzymes allow scientists to cleave genes at specific locations and then modify the remnants to produce a genomic sequence to their liking. However, programmable nucleases are not pristine and often mistakenly edit similar sequences that vary a few base pairs from the target sequence, making them unreliable for clinical use because of the potential for undesired mutations.

For this reason, induced pluripotent stem cells (iPS cells) are ideal models, because they provide researchers an abundance of patient cells on which to test the programmable nucleases and find optimal conditions that minimize off-target modifications. CiRA scientists took advantage of this feature by generating iPS cells from a DMD patient. They used several different TALEN and CRISPR to modify the genome of the iPS cells, which were then differentiated into skeletal muscle cells. In all cases, dystrophin protein expression was convalesced, and in some cases, the dystrophin gene was fully corrected.

One key to the success was the development of a computational protocol that minimized the risk of off-target editing. The team built a database that all possible permutations of sequences up to 16 base pairs long. Among these, they extracted those that only appear once in the human genome, i.e. unique sequences. DMD can be caused by several different mutations; in the case of the patient used in this study, it was the result of the deletion of exon 44. The researchers therefore built a histogram of unique sequences that appeared in a genomic region that contained this exon. They found a stack of unique sequences in exon 45.

to Akitsu Hotta, who headed the project and holds joint positions at CiRA and the Institute for Integrated Cell-Materials Sciences at Kyoto University:

"Nearly half the human genome consists of repeated sequences. So even if we found one unique sequence, a change of one or two base pairs may result in these other repeated sequences, which risks the TALEN or CRISPR editing an incorrect region. To avoid this problem, we sought a region that hit high in the histogram".

With this target, the team considered three strategies to modify the frame-shift mutation of the dystrophin gene: exon skipping by connecting exons 43 and 46 to restore the reading frame, frame shifting by incorporating insertion or deletion (indel) mutations, and exon knock-in by inserting exon 44 before exon 45. All three strategies effectively increased dystrophin synthesis in differentiated skeletal cells, but only the exon knock-in approach recovered the gene to its natural state. Importantly, editing showed very high specificity, suggesting that their computational approach can be used to minimize off-target editing by the programming nucleases.

Moreover, the paper provides a proof-of-principle for using iPS cell technology to treat DMD in combination with TALEN or CRISPR. The group now aims to expand this protocol to other diseases.

First author Lisa Li explains: "We show that TALEN and CRISPR can be used to correct the mutation of the DMD gene. I want to apply the nucleases to correct mutations for other genetic-based diseases like point mutations".

Source: Center for iPS Cell Research and Application - Kyoto University

Contact: Akemi Nakamura

Reference:

Precise correction of the DYSTROPHIN gene in Duchenne Muscular Dystrophy patient-derived iPS cells by TALEN and CRISPR-Cas9

Hongmei Lisa Li, Naoko Fujimoto, Noriko Sasakawa, Saya Shirai, Tokiko Ohkame, tetsushi Sakuma, Michihiro tanaka, Naoki Amano, Akira Watanabe, Hidetoshi Sakurai, Takashi Yamamoto, Shinya Yamanaka, and Akitsu Hotta

Stem Cell Reports, November 26, 2014, DOI: http://dx.doi.org/10.1016/j.stemcr.2014.10.013

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Cardiac Stem Cell Therapy may Heal Heart Damage Caused by Duchenne Muscular Dystrophy

Late-breaking basic science research presented at American Heart Association Scientific Sessions shows stem cell treatment restores heart function damaged by muscular disease

Tuesday, 18 November 2014

Researchers at the Cedars-Sinai Heart Institute have found that injections of cardiac stem cells might help reverse heart damage caused by Duchenne muscular dystrophy, potentially resulting in a longer life expectancy for patients with the chronic muscle-wasting disease.

The study results were presented today at a Breaking Basic Science presentation during the American Heart Association Scientific Sessions in Chicago. After laboratory mice with Duchenne muscular dystrophy were infused with cardiac stem cells, the mice showed steady, marked improvement in heart function and increased exercise capacity.

Duchenne muscular dystrophy, which affects 1 in 3,600 boys, is a neuromuscular disease caused by a shortage of a protein called dystrophin, leading to progressive muscle weakness. Most Duchenne patients lose their ability to walk by age 12. Average life expectancy is about 25. The cause of death often is heart failure because the dystrophin deficiency leads to cardiomyopathy, a weakness of the heart muscle that makes the heart less able to pump blood and maintain a regular rhythm.

"Most research into treatments for Duchenne muscular dystrophy patients has focused on the skeletal muscle aspects of the disease, but more often than not, the cause of death has been the heart failure that affects Duchenne patients," said Eduardo Marbán, MD, PhD, director of the Cedars-Sinai Heart Institute and study leader.

"Currently, there is no treatment to address the loss of functional heart muscle in these patients."

During the past five years, the Cedars-Sinai Heart Institute has become a world leader in studying the use of stem cells to regenerate heart muscle in patients who have had heart attacks. In 2009, Marbán and his team completed the world's first procedure in which a patient's own heart tissue was used to grow specialized heart stem cells. The specialized cells were then injected back into the patient's heart in an effort to repair and regrow healthy muscle in a heart that had been injured by a heart attack. Results, published in The Lancet in 2012, showed that one year after receiving the experimental stem cell treatment, heart attack patients demonstrated a significant reduction in the size of the scar left on the heart muscle.

Earlier this year, Heart Institute researchers began a new study, called ALLSTAR, in which heart attack patients are being infused with allogeneic stem cells, which are derived from donor-quality hearts.

Recently, the Heart Institute opened the nation's first Regenerative Medicine Clinic, designed to match heart and vascular disease patients with appropriate stem cell clinical trials being conducted at Cedars-Sinai and other institutions.

"We are committed to thoroughly investigating whether stem cells could repair heart damage caused by Duchenne muscular dystrophy," Marbán said.

In the study, 78 lab mice were injected with cardiac stem cells. Over the next three months, the lab mice demonstrated improved pumping ability and exercise capacity in addition to a reduction in heart inflammation. The researchers also discovered that the stem cells work indirectly, by secreting tiny fat droplets called exosomes. The exosomes, when purified and administered alone, reproduce the key benefits of the cardiac stem cells.

Marbán said the procedure could be ready for testing in human clinical studies as soon as next year. The process to grow cardiac-derived stem cells was developed by Marbán when he was on the faculty of Johns Hopkins University. Johns Hopkins has filed for a patent on that intellectual property and has licensed it to Capricor, a company in which Cedars-Sinai and Marbán have a financial interest. Capricor is providing funds for the ALLSTAR clinical trial at Cedars-Sinai.

The Cedars-Sinai Heart Institute has been at the forefront of developing investigational stem cell treatments for heart attack patients.

Source: Cedars-Sinai Medical Center

Contact: Sally Stewart

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Researchers Develop New Muscular Dystrophy Treatment Approach Using Human Stem Cells

Researchers Develop New Muscular Dystrophy Treatment Approach Using Human Stem Cells

Saturday, 05 May 2012

Researchers from the University of Minnesota's Lillehei Heart Institute have effectively treated muscular dystrophy in mice using human stem cells derived from a new process that – for the first time – makes the production of human muscle cells from stem cells efficient and effective.

The research, published today in Cell Stem Cell, outlines the strategy for the development of a rapidly dividing population of skeletal myogenic progenitor cells (muscle-forming cells) derived from induced pluripotent (iPS) cells. iPS cells have all of the potential of embryonic stem (ES) cells, but are derived by reprogramming skin cells. They can be patient-specific, which renders them unlikely to be rejected, and do not involve the destruction of embryos.

This is the first time that human stem cells have been shown to be effective in the treatment of muscular dystrophy.

According to U of M researchers – who were also the first to use ES cells from mice to treat muscular dystrophy – there has been a significant lag in translating studies using mouse stem cells into therapeutically relevant studies involving human stem cells. This lag has dramatically limited the development of cell therapies or clinical trials for human patients.

The latest research from the U of M provides the proof-of-principle for treating muscular dystrophy with human iPS cells, setting the stage for future human clinical trials.

"One of the biggest barriers to the development of cell-based therapies for neuromuscular disorders like muscular dystrophy has been obtaining sufficient muscle progenitor cells to produce a therapeutically effective response," said principal investigator Rita Perlingeiro, Ph.D., associate professor of medicine in the Medical School's Division of Cardiology.

"Up until now, deriving engraftable skeletal muscle stem cells from human pluripotent stem cells hasn't been possible. Our results demonstrate that it is indeed possible and sets the stage for the development of a clinically meaningful treatment approach."

Upon transplantation into mice suffering from muscular dystrophy, human skeletal myogenic progenitor cells provided both extensive and long-term muscle regeneration which resulted in improved muscle function.

To achieve their results, U of M researchers genetically modified two well-characterized human iPS cell lines and an existing human ES cell line with the PAX7 gene. This allowed them to regulate levels of the Pax7 protein, which is essential for the regeneration of skeletal muscle tissue after damage. The researchers found this regulation could prompt naïve ES and iPS cells to differentiate into muscle-forming cells.

Up until this point, researchers had struggled to make muscle efficiently from ES and iPS cells. PAX7 – induced at exactly the right time – helped determine the fate of human ES and iPS cells, pushing them into becoming human muscle progenitor cells.

Once Dr. Perlingeiro's team was able to pinpoint the optimal timing of differentiation, the cells were well suited to the regrowth needed to treat conditions such as muscular dystrophy. In fact, Pax7-induced muscle progenitors were far more effective than human myoblasts at improving muscle function. Myoblasts, which are cell cultures derived from adult muscle biopsies, had previously been tested in clinical trials for muscular dystrophy, however the myoblasts did not persist after transplantation.

"Seeing long-term maintenance of these cells without major adverse side effects is exciting," said Perlingeiro.

"Our research proves that these differentiated stem cells have real staying power in the fight against muscular dystrophy."

According to John Wagner, M.D., scientific director of clinical research at the University's Stem Cell Institute and renowned blood and marrow transplant expert,

"This research is a phenomenal breakthrough. Dr. Perlingeiro and her collaborators have overcome one of the most significant obstacles to moving stem cell therapies into the treatment of children with devastating and life threatening muscular dystrophies."

The U of M researchers say alternative methods of Pax7 induction will need to be investigated before this study can be turned into a human clinical trial. Their method of delivering the Pax7 protein involved genetic modification of cells with viruses and because viruses sometimes cause mutations, they add risk to a clinical trial. But the U of M researchers are committed to developing a safe and effective clinical protocol, and are actively testing alternate methods of delivering Pax7.

Source: University of Minnesota Academic Health Center 

Contact: Caroline Marin

Reference:

Human ES- and iPS-Derived Myogenic Progenitors Restore DYSTROPHIN and Improve Contractility upon Transplantation in Dystrophic Mice

Radbod Darabi, Robert W. Arpke, Stefan Irion, John T. Dimos, Marica Grskovic, Michael Kyba, Rita C.R. Perlingeiro

Cell Stem Cell, Volume 10, Issue 5, 610-619, 4 May 2012, 10.1016/j.stem.2012.02.015

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Stem Cells That Repair Injured Muscles

Research effort may have important implications for muscular dystrophy Saturday, 07 March 2009 A University of Colorado at Boulder research team has identified a type of skeletal muscle stem cell that contributes to the repair of damaged muscles in mice, which could have important implications in the treatment of injured, diseased or aging muscle tissue in humans, including the ravages of muscular dystrophy. The newly identified stem cells are found within populations of satellite cells located between muscle fibres and the surrounding connective tissue that are responsible for the repair and maintenance of skeletal muscles, said Professor Bradley Olwin of CU-Boulder's molecular, cellular and developmental biology department. When muscle fibres are stressed or traumatized, satellite cells divide to make more specialized muscle cells and repair the muscle, said Olwin. The stem cell population identified by the CU team within the satellite cells – dubbed "satellite-SP" cells – were shown to renew the satellite cell population after injection into injured muscle cells, contributing to recovery of muscle tissue in the laboratory mice. "This research shows how satellite cells can maintain their populations within injured tissues," said Olwin. "The hope is this new method will allow us to repair damaged or diseased skeletal muscle tissue." A paper on the subject was published in the March 5 issue of the journal Cell Stem Cell. Co-authors on the study included the MCD biology department's Kathleen Tanaka, John Hall and Andrew Troy, as well as Dawn Cornelison from the University of Missouri and Susan Majka from the University of Colorado Denver. Stem cells are distinguished by their ability to renew themselves through cell division and differentiate into specialized cell types. In healthy skeletal muscle tissue, the population of satellite cells is constantly maintained, leading the CU-Boulder team to believe that at least some of the satellite cell population in the mouse study included stem cells. For the study, the researchers injected 2,500 satellite-SP cells into a population of satellite cells within injured mouse muscle tissue. They found that 75 percent of the satellite cells that reproduced were derived from the previous satellite-SP cells injected into the tissue. The results demonstrated the injected satellite-SP cells were renewing the satellite cell pool, Olwin said. "The key point here is we are not just repairing the tissue," said Olwin. "We injected a permanent, self-renewing population of stem cells. One advantage of using this technology is that we can use a relatively small number of stem cells and do the job with a small number of injections – in this case, only one." The research has implications for a number of human diseases, he said. In muscular dystrophy, the loss of a protein called dystrophin causes the muscle to literally tear itself apart, a process that cannot be repaired without cell-based intervention. Although injected cells will repair the muscle fibres, maintaining the muscle fibres requires additional cell injections. Reference: Syndecan-4-Expressing Muscle Progenitor Cells in the SP Engraft as Satellite Cells during Muscle Regeneration Kathleen Kelly Tanaka, John K. Hall, Andrew A. Troy, D.D.W. Cornelison, Susan M. MajkaandBradley B. Olwin Cell Stem Cell, Volume 4, Issue 3, 217-225, 6 March 2009, doi:10.1016/j.stem.2009.01.016 ......... ZenMaster

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Stem Cells Restore Muscle in Muscular Dystrophy

Muscle stem cell transplant boosts diseased muscle function and replenishes stem cell pool Friday, 11 July 2008 Researchers at the Joslin Diabetes Center have demonstrated for the first time that transplanted muscle stem cells can both improve muscle function in animals with a form of muscular dystrophy and replenish the stem cell population for use in the repair of future muscle injuries. By injecting purified stem cells isolated from adult skeletal muscle, researchers have shown they can restore healthy muscle and improve muscle function in mice with a form of muscular dystrophy. Those muscle-building stem cells were derived from a larger pool of so-called satellite cells that normally associate with mature muscle fibers and play a role in muscle growth and repair. In addition to their contributions to mature muscle, the injected cells also replenished the pool of regenerative cells normally found in muscle. Those stem cells allowed the treated muscle to undergo subsequent rounds of injury repair, they found. "I'm very excited about this," said lead author Amy J. Wagers, Ph.D., Principal Investigator in the Joslin Section on Developmental and Stem Cell Biology, principal faculty member at the Harvard Stem Cell Institute and Assistant Professor of Stem Cell and Regenerative Biology at Harvard University. "This study indicates the presence of renewing muscle stem cells in adult skeletal muscle and demonstrates the potential benefit of stem cell therapy for the treatment of muscle degenerative diseases such as muscular dystrophy." "Our work shows proof-of-concept that purified muscle stem cells can be used in therapy," said Wagers, noting that in some cases the stem cells replaced more than 90 percent of the muscle fibers. Such an advance would require isolation of stem cells equivalent to those in the mouse from human muscle, something Wagers said her team is now working on. The study was designed to test the concept that skeletal muscle precursor cells could function as adult stem cells and that transplantation of these cells could both repair muscle tissue and regenerate the stem cell pool in a model of Duchenne muscular dystrophy, she said. Duchenne muscular dystrophy is the most common form of the disease and is characterized by rapidly progressing muscle degeneration. The disease is caused by a genetic mutation and there is currently no cure. The data from this new study demonstrate that regenerative muscle stem cells can be distinguished from other cells in the muscle by unique protein markers present on their surfaces. The authors used these markers to select stem cells from normal adult muscle and transferred the cells to diseased muscle of mice carrying a mutation in the same gene affected in human Duchenne muscular dystrophy. Satellite cells were first described decades ago and have since generally been considered as a homogeneous group, Wagers said. While anatomically they look similar under a microscope, they nonetheless show considerable variation in their physiology and function. In a previous study, Wagers' identified a set of five markers that characterize the only subset of satellite cells responsible for forming muscle, which they also refer to as skeletal muscle precursors or SMPs. In the new study, the researchers analyzed the stem cell and regenerative properties of those SMPs. When engrafted into muscle of mice lacking dystrophin, purified SMPs contributed to up to 94 percent of muscle fibers, restoring dystrophin expression and significantly improving muscle structure and contractile function, they report. (The dystrophin gene encodes a protein important for muscle integrity. Mice lacking dystrophin, also known as mdx mice, are a model for Duchenne muscular dystrophy, the most prevalent form of muscular dystrophy.) "Importantly, high-level engraftment of transplanted SMPs in mdx animals shows therapeutic value — restoring defective dystrophin gene expression, improving muscle histology, and rescuing physiological muscle function," the researchers said. "Moreover, in addition to generating mature muscle fibers, transplanted SMPs also re-seed the satellite cell niche and are maintained there such that they can be recruited to participate in future rounds of muscle regeneration.” "Taken together, these data indicate that SMPs act as renewable, transplantable stem cells for adult skeletal muscle. The level of myofiber reconstitution achieved by these myogenic stem cells exceeds that reported for most other myogenic cell populations and leads to a striking improvement of muscle contraction function in SMP-treated muscles. These data thus provide direct evidence that prospectively isolatable, lineage-specific skeletal muscle stem cells provide a robust source of muscle replacement cells and a viable therapeutic option for the treatment of muscle degenerative disorders." Wagers noted however that there may be complications in the delivery of cell therapy in humans, particularly for those with conditions influencing skeletal muscle throughout the body. Even so, the new findings present an "opportunity to understand what happens [to these regenerative cells] in disease and identify factors and pathways that may boost their activity," she said. "We may get a handle on drugs that could target muscle impairment" not only in those with muscular dystrophies, but also in elderly people suffering from the muscle wasting that comes with age. "Once the healthy stem cells were transplanted into the muscles of the mice with muscular dystrophy, they generated cells that incorporated into the diseased muscle and substantially improved the ability of the treated muscles to contract," said Wagers. "At the same time, the transplantation of the healthy stem cells replenished the formerly diseased stem cell pool, providing a reservoir of healthy stem cells that could be re-activated to repair the muscle again during a second injury." According to the paper, these cells provide an effective source of immediately available muscle regenerative cells as well as a reserve pool that can maintain muscle regenerative activity in response to future challenges. "This work demonstrates, in concept, that stem cell therapy could be beneficial for degenerative muscle diseases," Wagers said. Wagers also said the study will lead to other studies in the near-term that will identify pathways that regulate these muscle stem cells in order to figure out ways to boost the normal regenerative potential of these cells. These could include drug therapies or genomic approaches, she said. In the long-term, the idea will be to replicate these findings in humans. "This is still very basic science, but I think we're going to be able to move forward in a lot of directions. It opens up many exciting avenues," she said. The Wagers Lab at Joslin studies both hematopoietic stem cells, which constantly maintain and can fully regenerate the entire blood system, as well as skeletal muscle stem cells, involved in skeletal muscle growth and repair. The work is aimed particularly at defining novel mechanisms that regulate the migration, expansion, and regenerative potential of these two distinct adult stem cells. About Joslin Diabetes Center Joslin Diabetes Center is the world's largest diabetes clinic, diabetes research center and provider of diabetes education. Joslin is dedicated to ensuring people with diabetes live long, healthy lives and offers real hope and progress toward diabetes prevention and a cure for the disease. Founded in 1898 by Elliott P. Joslin, M.D., Joslin is an independent non-profit institution affiliated with Harvard Medical School. Reference: Highly Efficient, Functional Engraftment of Skeletal Muscle Stem Cells in Dystrophic Muscles Massimiliano Cerletti, Sara Jurga, Carol A. Witczak, Michael F. Hirshman, Jennifer L. Shadrach, Laurie J. Goodyear, and Amy J. Wagers Cell, Vol 134, 37-47, 11 July 2008 ......... ZenMaster

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Embryonic Stem Cell Transplantation Improves Muscular Dystrophy in Mice

Embryonic Stem Cell Transplantation Improves Muscular Dystrophy in Mice Sunday, 20 January 2008 Using embryonic stem cells from mice, UT Southwestern Medical Center researchers have prompted the growth of healthy – and more importantly, functioning – muscle cells in mice afflicted with a human model of Duchenne muscular dystrophy. The study represents the first time transplanted embryonic stem cells have been shown to restore function to defective muscles in a model of muscular dystrophy. The researchers’ newly developed technique, which involves stringent sorting to preserve all stem cells destined to become muscle, avoids the risk of tumour formation while improving the overall muscle strength and coordination of the mice, the researchers found. The mice used in the study lacked dystrophin, the same protein that humans with the fatal wasting disease also are missing. The study, headed by Dr. Rita Perlingeiro, assistant professor of developmental biology and molecular biology, is available online today and in the February issue of Nature Medicine. “We envision eventually developing a stem-cell therapy for humans with muscular dystrophy, if we are able to successfully combine this approach with the technology now available to make human embryonic stem cells from reprogrammed skin cells,” Dr. Perlingeiro said. “These cells can be transplanted into the muscle, and they cause muscle regeneration resulting in stronger contractility.” The study represents a major step in the field, she said, because the researchers were able to tease out exactly the cells they wanted. “The problem had been that embryonic stem cells make everything,” Dr. Perlingeiro said. “They make a great variety of cells. The trick is to pull out only the one type you want.” The UT Southwestern researchers focused on manipulating genes that are active in the very early stages as embryonic stem cells start to develop into more specialized cells. At first, they activated a gene called Pax3, which is involved in creating muscle cells, and then injected those cells into the animals’ muscles. Those cells caused tumours containing many different types of cells, indicating that there were still residual undifferentiated embryonic stem cells in the cultures at the time of implantation. “Even if there are 10 undesirable cells, that’s too many,” Dr. Perlingeiro said. The researchers then began using fluorescent dyes to sort cells depending on whether some surface markers were turned on while others were turned off. By analogy, it was as if they were dealing with a crowd of people and wanted to pull out only those with red hair, green scarves and blue coats, while those with red hair, green scarves and no coats would be disqualified. The final selection of cells, containing only one type, was again injected into the animals’ hind-limb muscles. After a month, the fluorescent dyes showed that the cells had deeply penetrated the muscle, an indication that they were growing and reproducing as desired, and many of the muscle fibres also contained dystrophin, the key protein lacking in muscular dystrophy. After three months, the mice also showed no signs of tumours. Tests of isolated muscles showed that the treated muscles were significantly stronger than untreated mice lacking dystrophin, although not quite as strong as those of normal mice. The treated mice also were tested for coordination. Again, their performance was better than that of untreated mice, but not as good as that of normal mice. “The improved coordination is significant because it shows the embryonic stem cells have benefited the animal’s quality of life, not simply caused an isolated growth with no overall improvement,” Dr. Perlingeiro said. The researchers will next investigate whether these transplanted cells can make “muscle stem cells,” which are partially developed cells in muscle tissue that serve as a reserve to replenish muscles. They also are testing their implantation approach in animal models of other types of muscular dystrophy. ......... ZenMaster

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microRNA scan uncovers reasons behind muscle dystrophies

Massive microRNA scan uncovers leads to treating muscle degeneration Thursday, 18 October 2007 Researchers have discovered the first microRNAs – tiny bits of code that regulate gene activity – linked to each of 10 major degenerative muscular disorders, opening doors to new treatments and a better biological understanding of these debilitating, poorly understood, often untreatable diseases. The study, to be published online this week by the Proceedings of the National Academy of Sciences, was led by Iris Eisenberg, PhD, of the Program in Genomics at Children’s Hospital Boston. Louis Kunkel, PhD, director of the Program in Genomics and an investigator with the Howard Hughes Medical Institute, was senior investigator. The disorders include the muscular dystrophies (Duchenne muscular dystrophy, Becker muscular dystrophy, limb girdle muscular dystrophies, Miyoshi myopathy, and fascioscapulohumeral muscular dystrophy); the congenital myopathies (nemaline myopathy); and the inflammatory myopathies (polymyositis, dermatomyositis, and inclusion body myositis). While past studies have linked them with an increasing number of genes, it's still largely unknown how these genes cause muscle weakness and wasting, and, more importantly, how to translate the discoveries into treatments. For instance, most muscular dystrophies begin with a known mutation in a “master gene”, leading to damaged or absent proteins in muscle cells. In Duchenne and Becker muscular dystrophies, the absent protein is dystrophin, as Kunkel himself discovered in 1987. Its absence causes muscle tissue to weaken and rupture, and the tissue becomes progressively non-functional through inflammatory attacks and other damaging events that aren’t fully understood. “The initial mutations do not explain why patients are losing their muscle so fast,” says Eisenberg. “There are still many unknown genes involved in these processes, as well as in the inflammatory processes taking place in the damaged muscle tissue.” She and Kunkel believe microRNAs may help provide the missing genetic links. Their team analyzed muscle tissue from patients with each of the ten muscular disorders, discovering that 185 microRNAs are either too abundant or too scarce in wasting muscle, compared with healthy muscle. Discovered in humans only in the past decade, microRNAs are already known to regulate major processes in the body. Therefore, Eisenberg believes microRNAs may be involved in orchestrating the tissue death, inflammatory response and other major degenerative processes in the affected muscle tissue. The researchers used bioinformatics to uncover a list of genes the microRNAs may act on, and now plan to find which microRNAs and genes actually underlie these processes. The findings raise the possibility of slowing muscle loss by targeting the microRNAs that control these “cascades” of damaging events. This approach is more efficient than targeting individual genes. The team also defined the abnormal microRNA “signatures” that correspond to each of the ten wasting diseases. They hope these will shed light on the genes and disease mechanisms involved in the most poorly understood and least treatable of the degenerative disorders, such as inclusion body myositis. “At this point, it’s very theoretical, but it’s possible,” says Eisenberg. Article: Distinctive patterns of microRNA expression in primary muscular disorders Iris Eisenberg et al. Proc. Natl. Acad. Sci. USA, 10.1073/pnas.0708115104 ......... ZenMaster

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