CRISPR Corrects Duchenne Muscular Dystrophy

Summary: New studies find a drug designed to combat the gene that causes SCA2 could also be beneficial in the fight against ALS.

Source: UT Southwestern.

Using the new gene-editing enzyme CRISPR-Cpf1, researchers at UT Southwestern Medical Center have successfully corrected Duchenne muscular dystrophy in human cells and mice in the lab.

The UT Southwestern group had previously used CRISPR-Cas9, the original gene-editing system, to correct the Duchenne defect in a mouse model of the disease and in human cells. In the current work, they used a new variation of the gene-editing system to repair the defect in both a mouse model and in human cells.

“We took patient-derived cells that had the most common mutation responsible for Duchenne muscular dystrophy and we corrected them in vitro to restore production of the missing dystrophin protein in the cells. This work provides us with a promising new tool in the CRISPR toolbox,” said author Dr. Eric Olson, Chairman of Molecular Biology, Co-Director of the UT Southwestern Wellstone Muscular Dystrophy Cooperative Research Center, and Director of the Hamon Center for Regenerative Science and Medicine.

 Image shows duchenne heart muscles.

The research appears in the journal Science Advances.

CRISPR-Cpf1 differs from CRISPR-Cas9 in a number of key ways. Cpf1 is much smaller than the Cas9 enzyme, which makes it easier to package inside a virus and therefore easier to deliver to muscle cells.

It also recognizes a different sequence of DNA than Cas9 does, which provides greater flexibility in terms of use. “There will be some genes that may be difficult to edit with Cas9 but may be easier to modify with Cpf1, or vice versa. The two proteins have different biochemical properties and recognize different DNA sequences, so these properties create more options for gene-editing,” said Dr. Olson, who holds the Pogue Distinguished Chair in Research on Cardiac Birth Defects, the Robert A. Welch Distinguished Chair in Science, and the Annie and Willie Nelson Professorship in Stem Cell Research.

“By either skipping a mutation region or precisely repairing a mutation in the gene, CRISPR-Cpf1-mediated genome editing not only corrects Duchenne muscular dystrophy mutations but also improves muscle contractility and strength,” said co-author Dr. Rhonda Bassel-Duby, Professor of Molecular Biology and Associate Director of the Hamon Center for Regenerative Science and Medicine.

Duchenne muscular dystrophy is caused by a mutation to one of the longest genes in the body. When there is a DNA error in the dystrophin gene, the body doesn’t make the protein dystrophin, which serves as a sort of shock absorber for the muscle fiber. Since there are numerous places in the dystrophin gene where a mutation can occur, flexibility for gene-editing treatment is crucial.

Duchenne occurs in about 1 in every 5,000 boys, according to the Centers for Disease Control and Prevention. Duchenne muscular dystrophy is a progressive disease affecting both muscle used for movement and heart muscle, with patients typically succumbing before age 30 due to heart failure.

“CRISPR-Cpf1 gene-editing can be applied to a vast number of mutations in the dystrophin gene. Our goal is to permanently correct the underlying genetic causes of this terrible disease, and this research brings us closer to realizing that end,” Dr. Olson said.

“CRISPR-Cpf1 differs from CRISPR-Cas9 in a number of key ways, including being easier to deliver to muscle cells, said Yu Zhang, a graduate student in Dr. Olson’s lab and the first author of this study.


Other UT Southwestern researchers who contributed to this work are Dr. Hui Li, research scientist; John R. McAnally, research scientist; Dr. Kedryn Baskin, postdoctoral researcher; and John M. Shelton, senior research scientist.

Note: Dr. Rhonda Bassel-Duby and Dr. Eric Olson are consultants for Exonics Therapeutics. Drs. Olson, Bassel-Duby, Zhang, and Long are listed on the patent entitled “Prevention of muscular dystrophy by CRISPR/Cpf1-mediated gene editing” (U.S. Patent 62/426,853, 28 November 2016).

Funding: This work was supported by grants from the National Institutes of Health (NIH), a Paul D. Wellstone Muscular Dystrophy Cooperative Research Centers grant, and a Robert A. Welch Foundation grant.

Source: UT Southwestern
Image Source: images are credited to the researchers.
Original Research: Full open access research for “CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice” by Yu Zhang, Chengzu Long, Hui Li, John R. McAnally, Kedryn K. Baskin, John M. Shelton, Rhonda Bassel-Duby, and Eric N. Olson in Science Advances. Published online April 12 2017 doi:10.1126/sciadv.1602814

UT Southwestern “CRISPR Corrects Duchenne Muscular Dystrophy.” NeuroscienceNews. NeuroscienceNews, 13 April 2017.


CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice

Duchenne muscular dystrophy (DMD), caused by mutations in the X-linked dystrophin gene (DMD), is characterized by fatal degeneration of striated muscles. Dilated cardiomyopathy is one of the most common lethal features of the disease. We deployed Cpf1, a unique class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effector, to correct DMD mutations in patient-derived induced pluripotent stem cells (iPSCs) and mdx mice, an animal model of DMD. Cpf1-mediated genomic editing of human iPSCs, either by skipping of an out-of-frame DMD exon or by correcting a nonsense mutation, restored dystrophin expression after differentiation to cardiomyocytes and enhanced contractile function. Similarly, pathophysiological hallmarks of muscular dystrophy were corrected in mdx mice following Cpf1-mediated germline editing. These findings are the first to show the efficiency of Cpf1-mediated correction of genetic mutations in human cells and an animal disease model and represent a significant step toward therapeutic translation of gene editing for correction of DMD.

“CRISPR-Cpf1 correction of muscular dystrophy mutations in human cardiomyocytes and mice” by Yu Zhang, Chengzu Long, Hui Li, John R. McAnally, Kedryn K. Baskin, John M. Shelton, Rhonda Bassel-Duby, and Eric N. Olson in Science Advances. Published online April 12 2017 doi:10.1126/sciadv.1602814

Branched-chain amino acids supplementation for resistance exercise-based muscle damage

Branched-chain amino acids (BCAA) supplementation has been considered an interesting nutritional strategy to improve skeletal muscle protein turnover in several conditions. There is evidence that resistance exercise (RE)-derived biochemical markers of muscle soreness (creatine kinase (CK), aldolase, myoglobin), soreness, and functional strength may be modulated by BCAA supplementation to favor muscle adaptation. However, few studies have investigated such effects in well-controlled conditions in humans.
The study proved the potential therapeutic effects of BCAA supplementation on RE-based muscle damage in humans. The main point is that BCAA supplementation may decrease some biochemical markers related with muscle soreness but this does not necessarily reflect on muscle functionality.

About BCAAs

Branched-chain amino acids (BCAAs) are naturally occurring molecules (leucine, isoleucine, and valine) that the body uses to build proteins. The term “branched chain” refers to the molecular structure of these particular amino acids. Muscles have a particularly high content of BCAAs.
For reasons that are not entirely clear, BCAA supplements may improve appetite in cancer patients and slow the progression of amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease, a terrible condition that leads to degeneration of nerves, atrophy of the muscles, and eventual death).
BCAAs have also been proposed as a supplement to boost athletic performance.

Dietary protein usually provides all the BCAAs you need. However, physical stress and injury can increase your need for BCAAs to repair damage, so supplementation may be helpful.
BCAAs are present in all protein-containing foods, but the best sources are red meat and dairy products. Chicken, fish, and eggs are excellent sources as well. Whey protein and egg protein supplements are another way to ensure you’re getting enough BCAAs. Supplements may contain all three BCAAs together or simply individual BCAAs.

Isoleucine: lentils, chickpeas, seeds, almonds, cashews, rye, chicken, eggs, liver and soy protein

Leucine: eggs, nuts, seeds, soy, whey and whole grains

Valine: soy flour, fish and meats, grains, cottage cheese, mushrooms, vegetables and peanuts

Therapeutic Dosages
The typical dosage of BCAAs is 1 g to 5 g daily.

Therapeutic Uses
Preliminary evidence suggests that BCAAs may improve appetite in people undergoing treatment for cancer . There is also some evidence that BCAA supplements may reduce symptoms of amyotrophic lateral sclerosis (ALS, or Lou Gehrig’s disease); however, not all studies have had positive results.
Preliminary evidence from a series of small studies suggests that BCAAs might decrease symptoms of tardive dyskinesia , a movement disorder caused by long-term usage of antipsychotic drugs. BCAAs have also shown a bit of promise for enhancing recovery from traumatic brain injury.
Because of how they are metabolized in the body, BCAAs might be helpful for individuals with severe liver disease (such as cirrhosis ).
BCAAs have also been tried for aiding muscle recovery after bedrest, such as following surgery .
Although there is a little supportive evidence, on balance, current research does not indicate that BCAAs are effective as a for enhancing sports performance . mOne preliminary study hints that BCAAs might aid recovery from long distance running.
BCAAs have also as yet failed to prove effective for muscular dystrophy.

What Is the Scientific Evidence for Branched Chain Amino Acids?

Appetite in Cancer Patients
A double-blind study tested BCAAs on 28 people with cancer who had lost their appetites due to either the disease itself or its treatment. Appetite improved in 55% of those taking BCAAs (4.8 g daily) compared to only 16% of those who took placebo.

Amyotrophic Lateral Sclerosis (Lou Gehrig’s Disease)
A small double-blind study found evidence that BCAAs might help protect muscle strength in people with Lou Gehrig’s disease . Eighteen individuals were given either BCAAs (taken 4 times daily between meals) or placebo and followed for 1 year. The results showed that people taking BCAAs declined much more slowly than those receiving placebo. In the placebo group, 5 of 9 participants lost their ability to walk, 2 died, and another required a respirator. Only 1 of the 9 participants receiving BCAAs became unable to walk during the study period. This study is too small to give conclusive evidence, but it does suggest that BCAAs might be helpful for this disease.
However, other studies found no effect, and one actually found a slight increase in deaths during the study period among those treated with BCAAs compared to placebo.

Muscular Dystrophy
One double-blind, placebo-controlled study found leucine (one of the amino acids in BCAAs) ineffective at the dose of 0.2 g per kilogram body weight (for example, 15 g daily for a 75-kilogram woman) in 96 individuals with muscular dystrophy. Over the course of 1 year, no differences were seen between the effects of leucine and placebo.
Safety Issues
BCAAs are believed to be safe; when taken in excess, they are simply converted into other amino acids. However, like other amino acids, BCAAs may interfere with medications for Parkinson’s disease .

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