Myostatin Knockout Achieved in Dogs via CRISPR

In mammals, reduced levels of myostatin or increased levels of follistatin, which acts to inhibit myostatin production, can be achieved by a variety of methods ranging from gene therapy to RNA interference, the standard panoply of technologies used to adjust the amounts of a particular protein in animal studies. Suitably altered levels of myostatin or follistatin result in greatly increased muscle growth, lower amounts of body fat, and in mice at least a possible but disputed modest life extension to go along with it. The most direct methodology is knockout of the myostatin gene, and this is the path chosen of late by Chinese researchers in their work on dogs, using CRISPR, one of the latest advances in genetic editing technology.

The creation of genetically altered, heavily muscled dogs is not an isolated line of research. There is at least one dog breed in which a myostatin mutation has occurred naturally, and the same goes for cows, another species heavily influenced by centuries of quite sophisticated human breeding strategies. There are even a few human natural myostatin mutants presently alive, as well-muscled as their animal peers. Gene therapies have been used for nearly a decade to create "mighty mouse" myostatin and follistatin mutants. Early this year scientists demonstrated myostatin knockout pigs using TALENs, another modern improvement on older methods of gene therapy.

Obviously this is a road that at some point branches away from the production of improved animal lineages towards the production of enhancements and therapies for humans. The conventional view is that enhanced muscle production is a viable therapy for the collection of wasting diseases known as myopathies and sarcopenia, the characteristic progressive loss of muscle mass and strength that occurs with aging. This would be a compensatory approach, a way to improve quality of life by overriding some of the results of damage or disease on the natural balance of muscle tissue repair and regeneration, but without actually fixing the damage itself.

I have in the past argued that myostatin or follistatin gene therapies look very much like an all upside treatment, something that everyone should undergo in an ideal world, not just older or sicker people, after it has been developed for use in humans. On the other hand SENS Research Foundation staffer Michael Rae has suggested more caution; if you look back at some of the archived posts on animal studies of myostatin knockout you'll see some of the data to back up that point of view. A decade of animal studies, naturally occurring mutant lineages, numerous mammalian examples of successful gene therapy, and early human trials of the same for myopathies is enough for some people, however. It is interesting to note that BioViva CEO Liz Parrish recently became the first publicly acknowledged healthy recipient of follistatin gene therapy, carried out as a first step towards spurring greater progress towards human clinical trials and treatments aimed at slowing or reversing the effects of aging.

All in all, if you were going to pick one gene therapy to move on with today, myostatin or follistatin would be near the top of the list given the present state of the art and the level of experience present in the scientific community. This is no doubt why the researchers here chose it as a first step in their program of producing genetically altered dog lineages. Their ultimate goal is the production of new models for disease research, not enhancement or treatments per se, but work on myostatin is sufficiently well advanced that it makes a good test case for the newer technologies and methodologies used along the way.

First Gene-Edited Dogs Reported in China

Scientists in China say they are the first to use gene editing to produce customized dogs. They created a beagle with double the amount of muscle mass by deleting a gene called myostatin. The dogs have "more muscles and are expected to have stronger running ability, which is good for hunting, police (military) applications. The goal of the research is to explore an approach to the generation of new disease dog models for biomedical research. Dogs are very close to humans in terms of metabolic, physiological, and anatomical characteristics."

Genome editing refers to newly developed techniques that let scientists easily disable genes or rearrange their DNA letters. The method used to change the beagles, known as CRISPR-Cas9, is particularly inexpensive and precise. Last month, the work was highlighted as part of a large Chinese effort to modify animals using CRISPR. The list of animals already engineered using gene editing in China includes goats, rabbits, rats, and monkeys. The efforts were described as a national scientific priority and part of China's effort to establish world-class research.

The dog researchers took much the same approach, directly introducing the gene-editing chemicals - a DNA snipping enzyme, Cas9, and a guide molecule that zeroes in to a particular stretch of DNA - into more than 60 dog embryos. Their objective was to damage, or knock out, both copies of the myostatin gene so that the beagles' bodies would not produce any of the muscle-inhibiting protein that the gene manufactures. In the end, of 65 embryos they edited, 27 puppies were born, but only two, a female and a male, had disruptions in both copies of the myostatin gene. They named the female Tiangou, after the "heaven dog" in Chinese myth. They named the male Hercules. In Hercules the gene editing was incomplete, and that a percentage of the dog's muscle cells were still producing myostatin. But in Tiangou, the disruption of myostatin was complete and the beagle "displayed obvious muscular phenotype," or characteristics.

Comments

Hello Reason!
When do you think this will be available to humans?? 5 years? 10? 20?
Which is your best bet?
Thanks

Posted by: Josep at October 20th, 2015 10:23 AM

By the way Reason (or anybody) do you know a book, explainig the history and perspectives of rejuvenation biotechnology, suitable for politicians and law makers? I want to give it as a gift to the president of my country. I guess Aubrey's one is a little bit old and too technical, and it doesn't explain the field as a whole (for example Human Longevity Inc., Calico, Palo Alto Prize etc).
Thanks!

Posted by: Josep at October 20th, 2015 10:29 AM

@Josep: Technically it is already available if you have a hundred thousand dollars, know the right people, and can talk them into it. The quality is an open question though, in the sense that the coverage of tissues in humans from gene therapies like this is still hit and miss.

Since there are early trials in humans for muscular dystrophy, 10 years seems reasonable for passage through the official system of regulation, after which it will be available to a small number of patients.

By that time I'd expect there to be a small but thriving medical tourism industry that can offer gene therapies. The yardstick here is how long it took stem cell therapy medical tourism to establish itself solidly, with reputable clinics, and a body of practice large enough so that experienced clinicians are reasonably easy to find and train. I think that gene therapies post-CRISPR should go much the same way, and in much the same time frame.

On books, 100 Plus (2011) is aimed at the layperson and a gentle introduction, but might be more general than you want, covering much more than SENS-style research:

https://www.fightaging.org/archives/2011/08/changing-the-mythology-thoughts-on-sonia-arrisons-100-plus.php

Posted by: Reason at October 20th, 2015 10:42 AM

10 years... Well, a bit too long :-(
On books, I already had thought about 100 plus. I will buy it and see how it is.
Thanks!

Posted by: Josep at October 20th, 2015 11:13 AM

@Reason the Follistatin therapy is considerably cheaper than TERT from what I understand.

Posted by: Steve H at October 20th, 2015 11:34 AM

This could be a bridge to the general public for rejuvenation treatment even if it is in itself a poor treatment. Big muscles win a lot of attention.
Lets hope Brad Pitt or some other famous person does the treatment in Mexico and makes it look good for a lot of people.
Also I wonder if athletes could do this and still compete, are the rules for doping updated to adress gene-edits?

Posted by: Arren at October 20th, 2015 10:40 PM

I still wondering if Liz Parish of BioViva will end up looking like a female bodybuilder who has taken anabolic steroids?

Posted by: Jim at October 21st, 2015 3:31 AM

@Arren: As Michael Rae says in the linked comments, the muscles are bigger but weak.

Posted by: Antonio at October 21st, 2015 3:53 AM

There are also other possible benefits to Follistatin depending on the type used, some animal data and anecdotal human data indicating it may help with atherosclerosis. We will no doubt find out in due course, data is king lets find out!

Posted by: Steve H at October 21st, 2015 8:35 AM

"As Michael Rae says in the linked comments, the muscles are bigger but weak"

That is his opinion, but the real studies shows something else.

Please read/refer to real studies/data vs. comments/opinions.

See here:

"The therapy, developed at Nationwide Children’s Hospital by Dr. Mendell and Dr. Brian Kaspar, is based on adeno-associated virus delivery of follistatin 344 to increase muscle strength and prevent muscle wasting and fibrosis. Because follistatin’s mechanism of action is not mutation specific, it could potentially help other forms of muscular dystrophy. Another clinical trial in patients with inclusion body myositis is also being conducted at Nationwide Children’s Hospital."

http://milobiotechnology.com/2015/04/09/first-duchenne-muscular-dystrophy-patient-treated-in-follistatin-gene-therapy-trial/

http://www.prnewswire.com/news-releases/first-duchenne-muscular-dystrophy-patient-treated-in-follistatin-gene-therapy-trial-300064098.html

here are the studies under Clinical Trials:

https://clinicaltrials.gov/ct2/show/NCT01519349?term=Follistatin+Gene+Therapy&rank=2

https://clinicaltrials.gov/ct2/show/NCT01519349?term=Follistatin+Gene+Therapy&rank=2

We need to look at studies' results (in humans) as opposed to theories, that are nice until proved (or disproved).

Posted by: alc at October 21st, 2015 8:41 AM

@Antonio, why the muscles are weaker?

Posted by: Josep at October 21st, 2015 8:49 AM

Here is another new development by a different team.

It reinforce the findings that the therapy delivered via AAV bring the muscles back to normal.

"The dogs are now six to seven months old and continue to develop normally."

... caveat: the studies were done in mice and dogs, with planned for humans ...

One interesting thing mentioned by researchers is "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."

here are the links:

http://www.medicalnewstoday.com/releases/301465.php

http://www.ncbi.nlm.nih.gov/pubmed/26264580

Posted by: alc at October 23rd, 2015 10:15 AM

Posted by: Antonio at October 21st, 2015 3:53 AM: As Michael Rae says in the linked comments, the muscles are bigger but weak

Posted by: alc at October 21st, 2015 8:41 AM: That is his opinion, but the real studies shows something else.

Please read/refer to real studies/data vs. comments/opinions.

I will first note, without I hope too much chiding, that your citations are to comments/opinions in press accounts, not to real studies/data. My statement was not based on my own opinion or anyone else's, but on the findings of studies of myostatin knockout or inhibition via follistatin (eg. (1,2)) which report increased muscle strength (compared to untreated animals with muscular dystrophy or other problems) and less (not the absence of) muscle wasting and fibrosis (compared to untreated animals with muscular dystrophy or other problems), but is not at all the same as bringing the muscles back to normal. The gains in strength come from an increase in muscle tissue mass: the quality of that muscle is still compromised relative to normal animals, leading to reduced force generation per unit mass.

This is a marvelous improvement relative to the baseline scenario, but not something that an otherwise-healthy but aging person should embrace as a way to rejuvenate aging muscle, particularly since degradation in muscle structural quality, and not just quantity of muscle tissue, is critical to to the loss of muscle strength with age.

The dogs are now six to seven months old and continue to develop normally.

This means only that they don't develop any develop abnormalities resulting from the gene therapy itself, not that they are the same as normal, wild-type dogs. Indeed, they haven't yet reported anything about the quantity, strength, or quality of these dogs' muscles: just reported (semi-formally, in a letter to the editor) the generation of such animals.

References
1: Amthor H, Macharia R, Navarrete R, Schuelke M, Brown SC, Otto A, Voit T, Muntoni F, Vrbóva G, Partridge T, Zammit P, Bunger L, Patel K. Lack of myostatin results in excessive muscle growth but impaired force generation. Proc Natl Acad Sci U S A. 2007 Feb 6;104(6):1835-40. Epub 2007 Jan 31. Erratum in: Proc Natl Acad Sci U S A. 2007 Mar 6;104(10):4240. PubMed PMID: 17267614; PubMed Central PMCID: PMC1794294.

2: Mendias CL, Bakhurin KI, Faulkner JA. Tendons of myostatin-deficient mice are small, brittle, and hypocellular. Proc Natl Acad Sci U S A. 2008 Jan 8;105(1):388-93. Epub 2007 Dec 27. PubMed PMID: 18162552; PubMed Central PMCID: PMC2224222.

Posted by: Michael at July 22nd, 2016 12:56 PM

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