Muscular Dystrophy is a Stem Cell-Based Disease


Michael Rudnicki, who has done pioneering work in muscle stem cell biology and muscle regeneration, and whose work has been featured several times on this blog, has struck again. Rudnicki, who serves as director of the Regenerative Medicine Program at The Ottawa Hospital and a professor at the University of Ottawa and holds the prestigious Canada Research Chair in Molecular Genetics, teamed up with workers from the Sprott Centre for Stem Cell Research and the Sinclair Centre for Regenerative Medicine to investigate the role of muscle-specific stem cells in patients who suffer from Duchenne muscular dystrophy. This new earth-shaking study, which was published in the journal Nature Medicine (November 16, 2015), has changed the way we think about muscular dystrophy and will almost certainly force people to rethink the treatments and cures for this dreadful disease.

According to this new study, Duchenne muscular dystrophy directly affects muscle stem cells, and is, largely a disease of muscle stem cells.

Rudicki said: “For nearly 20 years, we’ve thought that the muscle weakness observed in patients with Duchenne muscular dystrophy is primarily due to problems in their muscle fibers, but our research shows that it is also due to intrinsic defects in the function of their muscle stem cells. This completely changes our understanding of Duchenne muscular dystrophy and could eventually lead to far more effective treatments.”

Muscular dystrophy comes in several different forms, but the predominant sign of muscular dystrophy is progressive muscle weakness. Altogether, muscular dystrophy refers to a group of more than 30 genetic diseases, all of which cause progressive weakness and degeneration of skeletal muscles used during voluntary movement. Approximately half of all who suffer from muscular dystrophy have Duchenne muscular dystrophy (DMD). Because muscular dystrophy results from mutations in the dystrophin gene, which is on the X chromosome, the vast majority of muscular dystrophy patients are male. Girls can be carriers of muscular dystrophy and can be mildly affected.

Interestingly, somewhere around one-third of boys who suffer from DMD have no family history of the disease. Because the dystrophin gene is so large, spontaneous mutations in it are probably relatively common.

The signs and symptoms typically appear between the ages of 2 and 3, and may include frequent falls, difficulty getting up from a lying or sitting position, trouble running and jumping, a strange, shuffling way of walking or having a tendency to walk on their toes, calf muscles that are abnormally large, muscle pain and stiffness, and some learning disabilities.

Becker muscular dystrophy (BMD) has signs and symptoms that are largely similar to those of DMD, but BMD tends to be a milder form of the disease that progresses more slowly. Symptoms typically begin in the teens but, some patients may not experience symptoms until their mid-20s and some may not experience symptoms until later.

There are also several different types of muscular dystrophy-type diseases. Steinert’s disease or myotonic muscular dystrophy, which is characterized by an inability to relax muscles at after contractions, is the most common form of adult-onset muscular dystrophy. The first muscles to be affected are the muscles of the face and neck. Facioscapulohumeral muscular dystrophy affects the muscles of the face and shoulders, where symptoms first begin. When patients with facioscapulohumeral raise their arms, their shoulder blades noticeably protrude. This disease may first manifest itself in children, teenagers as late as age 40. This disease tends to affect one side more than the other.

Limb-girdle muscular dystrophy affects the muscles of the shoulders and hips. There are over 20 inherited forms of this disease, and because this condition is not due to mutations in dystrophin, but to mutations in genes that encode proteins that interact with dystrophin, the inheritance of limb-girdle muscular dystrophy is not sex-linked. Some forms of this disease are recessive and some are dominant. Patients with this type of muscular dystrophy usually trip more often because they have trouble raising the front part of their feet. Some autosomal recessive forms of the disorder are now known to be due to a deficits in proteins called sarcoglycans or dystroglycan.

Congenital muscular dystrophy is extremely varible and is probably a cluster of several different diseases caused by mutations in different genes. Some of types of congenital muscular dystrophy show sex-linked inheritance while others do not. Most cases of congenital muscular dystrophy result from the absence of a muscle protein called merosin, which is found in the connective tissue that surrounds muscle fibers. Other types of congenital muscular dystrophy have normal merosin and still others result from abnormal motor neuron migration. Clinically, this disease is also extremely variable and can manifest itself at birth or before age 2, progress slowly or rapidly, and cause mild disability or severe impairment.

Muscular dystrophy affects all ethnic groups and occurs globally. It affects around 1 in every 3,500 to 6,000 male births each year in the United States.  DMD affects approximately one in 3,600 boys.

Because DMD results from mutations in the dystrophin gene, the vast majority of muscular dystrophy research was based on a simple model in which the Dystrophin protein played a structural role in the structural integrity of muscle fibers. Abnormal versions of the Dystrophin protein caused the muscle fibers to become damaged and die as a result of contraction.  Dystrophin anchors the cytoskeleton of the muscle fibers, which are essential for muscle contraction, to the muscle cell membrane, and then to the extracellular matrix outside the cell that serves as a foundation upon which the muscle cells are built.

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However in this current study, Rudnicki and his team discovered that muscle stem cells also express the dystrophin protein. This is a revelation because Dystrophin was thought to be protein that ONLY appeared in mature muscle. However, in this study, it became exceedingly clear that in the absence of Dystrophin, muscle stem cells generated ten-fold fewer muscle precursor cells, and, consequently, far fewer functional muscle fibers. Dystrophin is also a component of a signal transduction pathway that allows muscle stem cells to properly ascertain if they need to replace dead or dying muscle.  Muscle stem cells repair the muscle in response to injury or exercise by dividing to generate precursor cells that differentiate into muscle fibers.

“Muscle stem cells that lack dystrophin cannot tell which way is up and which way is down,” said Dr. Rudnicki. “This is crucial because muscle stem cells need to sense their environment to decide whether to produce more stem cells or to form new muscle fibres. Without this information, muscle stem cells cannot divide properly and cannot properly repair damaged muscle.”

Even though Rudnicki used mice as a model system in these experiments, the Dystrophin protein is highly conserved in most vertebrate animals. Therefore, it is highly likely that these results will also apply to human muscle stem cells.

Treatment for DMD patients is limited to steroids to decrease muscle inflammation and muscle cell death, and physical therapy to increase muscle use and prevent muscle atrophy. These approaches only delay the progression of the disease and alleviate symptoms. Gene therapy experiments and trials are in progress and even show some promise, but Rudnicki’s work tell us that gene therapy approaches must target muscle stem cells as well as muscle fibers if they are to work properly.

“We’re already looking at approaches to correct this problem in muscle stem cells,” said Dr. Rudnicki. “I’m not sure if we will ever cure Duchenne muscular dystrophy, but I’m very hopeful that someday in the future, we will have new therapies that correct the ability of muscle stem cells to repair the muscles of afflicted patients and turn this devastating, lethal disease into a chronic but manageable condition.”

This paper has received high praise from the likes of Ronald Worton, who was one of the co-discovers of the dystrophin gene with Louis Kunkel in 1987.  Worton later served as Vice-President of Research at The Ottawa Hospital from 1996 to 2007.

“When we discovered the gene for Duchenne muscular dystrophy, there was great hope that we would be able to develop a new treatment fairly quickly,” said Dr. Worton, who is now retired. “This has been much more difficult than we initially thought, but Dr. Rudnicki’s research is a major breakthrough that should renew hope for researchers, patients and families.”

New Gene Therapy Effectively Treats All Muscles in Dogs With Muscular Dystrophy


The X-linked genetic disease, muscular dystrophy, affects the structure and function of skeletal muscles. Muscular dystrophy patients harbor mutations in a gene that encodes a protein known as dystrophin. Dystrophin attaches the internal skeleton of skeletal muscle cells to the cell membrane. In turn, proteins in the skeletal muscle membrane attach to the intracellular matrix that acts as the foundational material upon which muscle cells (and other cells) sit. Therefore, the dystrophin protein serves to attach skeletal muscle cells to the extracellular matrix. The loss of dystrophin causes muscles to separate from the cell matrix and detach from each other. The lack of attachment of muscles to each other causes them to degenerate and die.

Dystrophin

The death of skeletal muscles in muscular dystrophy patients leads to the replacement of what was once skeletal muscle with scar tissue, fatty tissue, or even bone. Because muscular dystrophy is caused by mutations in an X-linked gene, the majority of muscular dystrophy patients are boys. The losses of muscle structure, function, and mass cause patients to lose their ability to walk and eventually breath (since the diaphragm is a skeletal muscle) as they age. Thus muscular dystrophy tends to put patients in wheelchairs and condemn them to respirators.

The most common form of muscular dystrophy is called Duchenne Muscular Dystrophy or DMD. Close to 250,000 people in the United States suffer from muscular dystrophy. Treatment options are very limited and usually palliative. However, a research team from the University of Missouri has successfully treated dogs that suffer from DMD. They are optimistic that human clinical trials can be planned in the next few years.

This is a remarkable finding, especially, when you consider that the dystrophin gene is extremely large. In fact, the dystrophin gene is the largest gene in the human genome. This makes gene therapy treatments for DMD problematic.

Dongsheng Duan, who serves as the lead scientist in this study, and is the Margaret Proctor Mulligan Professor in Medical Research at the MU School of Medicine “This is the most common muscle disease in boys, and there is currently no effective therapy. This discovery took our research team more than 10 years, but we believe we are on the cusp of having a treatment for the disease.

Duan continued: “Due to its size, it is impossible to deliver the entire gene with a gene therapy vector, which is the vehicle that carries the therapeutic gene to the correct site in the body,” Duan said. “Through previous research, we were able to develop a miniature version of this gene called a microgene. This minimized dystrophin protected all muscles in the body of diseased mice.”

Duan and his colleagues worked for almost ten years to develop a viable strategy that can safely transfer the micro-dystrophin gene to every muscle in a the body of dogs that have a canine form of DMD. Dogs are an excellent model system for human medicine, since dogs are about the same size as a human boy. Successful treatment of DMA dogs can provide the foundation for human clinical trials.

In this new study, Duan and his team demonstrated that by using a common virus to deliver the micro-dystrophin gene to all the muscles in the body of a diseased dog. Duan and others injected DMA dogs with this genetically engineered virus when they were two-three months old. For dogs, this is about the time when they begin to show some of the DMD-associated signs and symptoms. Now, these dogs are six-seven months old and they are experiencing normal development and muscular activity.

“The virus we are using is one of the most common viruses; it is also a virus that produces no symptoms in the human body, making this a safe way to spread the dystrophin gene throughout the body,” Duan said. “These dogs develop DMD naturally in a similar manner as humans. It’s important to treat DMD early before the disease does a lot of damage as this therapy has the greatest impact at the early stages in life.”

Gene Editing in iPS Cells Corrects Genetic Mutations That Cause Muscular Dystrophy


Induced pluripotent stem cells or iPSCs have many of the same characteristics as embryonic stem cells. One such feature is the ability to be grown in culture and manipulated like genuine tissue culture cells.

To that end a research group at the Center for iPS Cell Research and Application (CiRA) have used iPSCs made from the cells of patients with Duchenne muscular dystrophy (DMD) to show that such mutations can be efficiently fixed.

This research, which was published in Stem Cell Reports, demonstrates how a new group of engineered nucleases, such as TALEN and CRISPR, can edit the genome of iPS cells generated from skin cells isolated from a DMD patient. After being genetically fixed, these iPSCs were differentiated into skeletal muscles, and it was clear that the mutation responsible for DMD had disappeared.

DMD is a severe muscular degenerative disease caused by loss-of-function mutations in the dystrophin gene. DMD affects 1 in 3500 boys and normally leads to death by early adulthood. The treatments for this disease are largely palliative.

However, the capability to edit the genomes of mutant cells is a formerly unknown option that was once only for the realms of science fiction. Two nucleated called TALEN and CRISPR have quickly become invaluable tools in molecular biology. These enzymes allow scientists to cleave genes at specific locations and then modify the cut ends to generate a specifically chosen genomic sequence. However, these programmable nucleases are not perfect and often mistakenly edit similar sequences that vary a few base pairs from the target sequence. This makes them unreliable for clinical use because of the potential for creating new, undesired mutations.

For precisely this reason, iPSCs are ideal model systems because they provide researchers an abundance of patient cells on which to test the programmable nucleated, and determine the optimal conditions that minimize off-target modifications. CiRA scientists used this very feature to generating iPS cells from a DMD patient. Then they utilized several different TALENs and CRISPRs to modify the genome of the iPS cells, which were then differentiated into skeletal muscle cells. In all cases, dystrophin protein expression was restored, and in some cases, the dystrophin gene was fully corrected.

One of the reasons for the success in this project was the development of a computational protocol that minimized the risk of off-target editing. The CiRA team built a database that contained all possible combination of sequences up to 16 base pairs long. Among these, they isolated those sequences that only appear once in the human genome. DMD can be caused by several different mutations. For example, in the case of the patient used in this study, it was the result of the deletion of exon 44. After building a histogram of unique sequences that appeared in a genomic region that contained this exon, the CiRA group found a cluster of unique sequences in exon 45.

The head researcher for this project, Akitsu Hotta, who headed the project and holds joint positions at CiRA and the Institute for Integrated Cell-Materials Sciences at Kyoto University, said:  “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.”

This 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”.

Growing Skeletal Muscle in the Laboratory


Skeletal muscle – that type of voluntary muscle that allows movement – has proven difficult to grow in the laboratory. While particular cells can be differentiated into skeletal muscle cells, forming a coherent, structurally sound skeletal muscle is a tough nut to crack from a research perspective.

Another problem dogging muscle research is the difficulty growing new muscle in patients with muscle diseases such as muscular dystrophy or other types of disorders that weaken and degrade skeletal muscle.

Now research groups at the Boston Children’s Hospital Stem Cell Program have reported that they can boost the muscle mass and even reverse the disease of mice that suffer from a type of murine muscular dystrophy. To do this, this group use a combination of three different compounds that were identified in a rapid culture system.

This ingenious rapid culture system uses the cells of zebrafish (Danio rerio) embryos to screen for these muscle-inducing compounds. These single cells are placed into the well of a 96-well plate, and then treated with various compounds to determine if those chemical induce the muscle formation. To facilitate this process, the zebrafish embryo cells express a very special marker that consists of the myosin light polypeptide 2 gene fused to a red-colored protein called “cherry.” When cells become muscle, they express the myosin light polypeptide 2 gene at high levels. Therefore, any embryo cell that differentiates into muscle should glow a red color.

(A) myf5-GFP;mylz2-mCherry double-transgenic expression recapitulates expression of the endogenous genes. myf5-GFP is first detected at the 11-somite stage. mylz2-mCherry expression is not observed until 32 hpf. Scale bars represent 200 mm. (B) myf5-GFP;mylz2-mCherry embryos were dissociated at the oblong stage and cultured in zESC medium. Images were taken 48 hr after plating. Scale bars represent 250 mm.
(A) myf5-GFP;mylz2-mCherry double-transgenic expression recapitulates expression of the endogenous genes. myf5-GFP is first detected at the 11-somite
stage. mylz2-mCherry expression is not observed until 32 hpf. Scale bars represent 200 mm.
(B) myf5-GFP;mylz2-mCherry embryos were dissociated at the oblong stage and cultured in zESC medium. Images were taken 48 hr after plating. Scale bars
represent 250 mm.

Once a cocktail of muscle-inducing chemicals were identified in this assay, those same chemicals were used to treat induced pluripotent stem cells made from cells taken from patients with muscular dystrophy.  Those iPSCs were treated with the combination of chemicals identified in the zebrafish embryo screen as muscle inducing agents.

Zebrafish embryo culture system

The results were outstanding.  Leonard Zon from the Division of Hematology/Oncology, Children’s Hospital Boston and Dana-Farber Cancer Institute and his colleagues showed that a combination of basic Fibroblast Growth Factor, an  adenylyl cyclase activator called forskolin, and the GSK3β inhibitor BIO induced skeletal muscle differentiation in human induced pluripotent stem cells (iPSCs).  Furthermore, these muscle cells produced engraftable myogenic progenitors that contributed to muscle repair when implanted into mice with a rodent form of muscular dystrophy.

Representative hematoxylin and eosin staining (H&E) images and immunostaining on TA sections of preinjured NSG mice injected with 1 3 105 iPSCs at day 14 of differentiation. Muscles injected with BJ, 00409, or 05400 iPSC-derived cells stain positively for human d-Sarcoglycan protein (red). Fibers were counterstained with Laminin (green). No staining is observed in PBS-injected mice or when 00409 fibroblast cells were transplanted. Because the area of human cell engraftment could not be specifically distinguished on H&E stained sections, which must be processed differently from sections for immunostaining, the H&E images shown do not represent the same muscle region as that shown in immunofluorescence images. Scale bars represent 100 mm, n = 3 per sample.
Representative hematoxylin and eosin staining
(H&E) images and immunostaining on TA sections
of preinjured NSG mice injected with 1 3 105
iPSCs at day 14 of differentiation. Muscles injected
with BJ, 00409, or 05400 iPSC-derived cells
stain positively for human d-Sarcoglycan protein
(red). Fibers were counterstained with Laminin
(green). No staining is observed in PBS-injected
mice or when 00409 fibroblast cells were transplanted.
Because the area of human cell engraftment
could not be specifically distinguished on
H&E stained sections, which must be processed
differently from sections for immunostaining, the
H&E images shown do not represent the same
muscle region as that shown in immunofluorescence
images. Scale bars represent 100 mm, n = 3
per sample.

Zon hopes that clinical trials can being soon in order to translate these remarkable results into patients with muscle loss within the next several years.  Zon and his co-workers are also screening compounds to address other types of disorders beyond muscular dystrophy.

This paper represents the application of shear and utter genius.  However, there is one caveat.  The mice into which the muscles were injected were immunodeficient mice whose immune systems are unable to reject transplanted tissues.  In human patients with muscular dystrophy, an immune response against dystrophin, the defective protein, has been an enduring problem (for a review of this, see T. Okada and S. Takeda, Pharmaceuticals (Basel). 2013 Jun 27;6(7):813-836).  While there have been some technological developments that might circumvent this problem, transplanting large quantities of muscle cells might be beyond the pale.  Muscular dystrophy results from disruption of an important junction between the muscle and substratum to which the muscle is secured.  This connection is mediated by the “dystrophin-glycoprotein complex.”  Structural disruptions of this complex (shown below) lead to unanchored muscle that cannot contract properly, and eventually atrophies and degrades.

Dystrophin-glycoprotein complex. Molecular structure of the dystrophin-glycoprotein complex and related proteins superimposed on the sarcolemma and subsarcolemmal actin network (redrawn from Yoshida et al. [5], with modifications). cc, coiled-coil motif on dystrophin (Dys) and dystrobrevin (DB); SGC, sarcoglycan complex;SSPN, sarcospan; Syn, syntrophin; Cav3, caveolin-3; N and C, the N and C termini, respectively; G, G-domain of laminin; asterisk indicates the actin-binding site on the dystrophin rod domain; WW, WW domain.
Dystrophin-glycoprotein complex. Molecular structure of the dystrophin-glycoprotein complex and related proteins superimposed on the sarcolemma and subsarcolemmal actin network (redrawn from Yoshida et al. [5], with modifications). cc, coiled-coil motif on dystrophin (Dys) and dystrobrevin (DB); SGC, sarcoglycan complex;SSPN, sarcospan; Syn, syntrophin; Cav3, caveolin-3; N and C, the N and C termini, respectively; G, G-domain of laminin; asterisk indicates the actin-binding site on the dystrophin rod domain; WW, WW domain.
This is a remarkable advance, but until the host immune response issue is satisfactorily addressed, it will remain a problem.

Researchers from the University of Minnesota Use Genetically Corrected Stem Cells To Repair Muscles


University of Minnesota researchers from the Lillehei Heart Institute have combined genetic engineering techniques to repair mutations in abnormal muscle cells with cellular reprogramming to generate stem cells that can repair and regenerate muscle regeneration in a mouse model for Duchenne Muscular Dystrophy (DMD). This research is a proof-of-principle experiment that determines the feasibility of combining induced pluripotent stem cell technology and genetic engineering techniques that correct mutations to treat muscular dystrophy. Experimental strategies such as these could represent a major step forward in autologous cell-based therapies for DMD. Furthermore, it might pave the way for clinical trials to test this approach in reprogrammed human pluripotent cells from muscular dystrophy patients.

University of Minnesota researchers combined three groundbreaking technologies to achieve effective muscular dystrophy therapy in a mouse model of DMD. First, researchers reprogrammed skin cells into induced pluripotent stem cells (iPSCs). iPSCs are capable of differentiating into any of the mature cell types within an adult organism. In this case, the University of Minnesota researchers generated pluripotent cells from the skin of mice that carry mutations in two genes; the dystrophin and utrophin genes. Mice with mutations in both the dystrophin and utrophin genes develop a severe case of muscular dystrophy that resembles the type of disease observed in human DMD patients. This provided a model system platform that successfully mimicked what would theoretically occur in humans.

The second technology employed is a genetic correction tool developed at the University of Minnesota. In this case, they used a transposon, which is a segment of DNA that can jump from one location to another within the genome. The specific transposon used is the “Sleeping Beauty Transposon.” The use of this transposon allowed them to transport genes into cells in a convenient manner. The Lillehei Heart Institute researchers used the Sleeping Beauty transposon to deliver a gene called “micro-utrophin” into the iPSCs made from the DMD mice.

Sleeping Beauty Transposon

Human micro-utrophin can support muscle fiber strength and prevent muscle fiber injury throughout the body. However, there is one essential difference micro-utrophin and dystrophin: dystrophin is absent in muscular dystrophy patients, but if it is introduced into the bodies of DMD patients, their immune system will initiate a devastating immune response against it. However, in those same patients, utrophin is active and functional, which makes it essentially “invisible” to the immune system. This invisibility allows the micro-utrophin to replace dystrophin build and repair muscle fibers within the body.

Utrophin

The third technology utilized is a method to produce skeletal muscle stem cells from pluripotent cells. This procedure was developed in the laboratory of Rita Perlingeiro, who is also the principal investigator of this latest study.

Rita Perlingeiro Ph.D.
Rita Perlingeiro Ph.D.

Perlingeiro’s technology gives pluripotent cells a short pulse of a muscle stem cell protein called Pax3, which nudges the pluripotent cells to become skeletal muscle stem cells, which can then be exponentially expanded in culture. These Pax3-induced muscle stem cells were then transplanted back into the same strain of DMD mice from which the pluripotent stem cells were originally derived.

Pax3 and 7

When combined, these platforms created muscle-generating stem cells that would not be rejected by the body’s immune system. According to Perlingeiro, the transplanted cells performed very well in the dystrophic mice, and they generated functional muscle and responded to muscle fiber injury.

“We were pleased to find the newly formed myofibers expressed the markers of the correction, including utrophin,” said Perlingeiro, a Lillehei endowed scholar within the Lillehei Heart Institute and an associate professor in the University of Minnesota Medical School. “However, a very important question following transplantation is if these corrected cells would self-renew, and produce new muscle stem cells in addition to the new muscle fibers.”

By injuring the transplanted muscle and watching it repair itself, the researchers demonstrated that the transplanted muscle stem cells endowed the recipient mice with fully functional muscle cells. This latest project provides the proof-of-principle for the feasibility of combining induced pluripotent stem cell technology and genetic correction to treat muscular dystrophy.

“Utilizing corrected induced pluripotent stem cells to target this specific genetic disease proved effective in restoring function,” said Antonio Filareto, Ph.D., a postdoctoral fellow in Perlingeiro’s laboratory and the lead author on the study. “These are very exciting times for research on muscular dystrophy therapies.”

These studies pave the way for testing this approach in a clinical trial that would use reprogrammed human pluripotent cells from muscular dystrophy patients.

According to Perlingeiro, “Developing methods to genetically repair muscular dystrophy in human cells, and demonstrating efficacy of muscle derived from these cells are critical near-term milestones, both for the field and for our laboratory. Testing in animal models is essential to developing effective technologies, but we remained focused on bringing these technologies into use in human cells and setting the stage for trials in human patients.”

This research was published in Nature Communications.