Follistatin augments the power of myostatin inhibition discovered in mutant models J.D. Lowderman The Professional Program in Biotechnology, Texas A&M University, College Station 77802

Introduction Follistatin augments the power of myostatin inhibition discovered in mutant models Myostatin has 2 potent inhibitory roles: One is to decrease the number of myofibers formed prenatally, and the other is to inhibit the growth of muscles postnatally and on into adulthood Myostatin was first identified through degenerate PCR Knowledge of myostatin was gained gradually through the study of mutant models from 1997-2007 Through myostatin exploration, scientists discovered that the myostatin inhibitor follistatin is an extremely potent muscle enhancer Using AAV gene therapy, inserting the follistatin gene as a treatment for muscle wasting disorders has been very successful in both mice and primate studies

Discovery of Myostatin Initial goal: identify new members of the transforming growth factor-β superfamily Used degenerate PCR Determined myostatin is produced predominately by skeletal muscle Determined its function by creating MSTN knockout mice Adapted from McPherron, A. et al. Nature 387 (1997): 83-90.

Adapted from McPherron, A. et al. Nature 387 (1997): 83-90.

Are the drawbacks caused by MSTN mutation? The Belgian Blue and Piedmontese cattle were the first MSTN mutants discovered Produce normal meat in higher quantities and have higher feed efficiency Drawbacks: smaller internal organs, delayed sexual maturation, reduced female fertility, and lower viability of offspring Are the drawbacks caused by MSTN mutation? http://www.freerepublic.com/focus/f-news/1865890/posts

The next homozygous mutant discovered was a 4.5 yr old German boy His evaluation suggests that some of the drawbacks observed in the Piedmontese cattle are not due to MSTN mutation He has normal internal organs and he had a normal birth (birth weight in 75th percentile) Muscles greater than 2 times the mass of controls Adapted from J. Lee. N Engl J Med 350 (2004): 2682 -2688.

Whippet Study Discovery that some whippet dogs were also MSTN mutants provided more insight Only “side effects” of homozygote: 2 muscles susceptible to cramping, 50% have overbite Most desired phenotype is the heterozygote because they are the fastest sprinters Showed that a heterozygote can have significantly more muscle Compare to previous studies

http://www.freerepublic.com/focus/f-news/1865890/posts Adapted from Quignon, C. D. et al. PLoS Genet 3 (2007): e79.

http://www.freerepublic.com/focus/ f-news/1865890/posts

New Goal Inhibit myostatin in adult animals to serve as a therapy for muscle wasting disorders, to increase feed efficiency in farm animals, and even to serve as a muscle enhancement for athletes and the average muscle enthusiast The first studies used a myostatin antibody These were successful in animal models

The Next Strategy Use AAV gene therapy because of its safety and affinity for muscle fibers Goal was to insert a gene that produces a protein that inhibits myostatin Several were explored, and they all inhibited myostatin effectively However, follistatin not only strongly inhibited myostatin but apparently follistatin also inhibits additional factors that negatively regulate muscle 2 alternatively spliced isoforms: FS315 and FS288

Adapted from Rodino-Klapac et al., Muscle Nerve 39 (2009): 283-296.

Adapted from Lee, S. J. PLoS One 2 (2007): e789.

Follistatin transduction is an effective treatment in mdx mice Follistatin not only enhances muscle mass in mice, but also ameliorates the symptoms of muscular dystrophy Anabolic steroids (the only available treatment at the moment) have numerous side effects and are not effective Not only was follistatin transduction successful at increasing muscle mass, but it also reduced the symptoms of muscular dystrophy Adapted from Rodino-Klapac et al., Muscle Nerve 39 (2009): 283-296

Will the success in mice translate to human trials? Since the myostatin antibody studies did not translate to human therapies, will the FS315 success in mice lead to a therapy in humans? It depends on how similar humans are to macaques http://www2.gsu.edu/~wwwvir/VirusInfo/pages/longtailed1_jpg.htm

Conclusion Follistatin augments the power of myostatin inhibition discovered in mutant models Although myostatin has a potent negative effect on musculature, FS315 cancels this effect as well as other factors which reduce musculature The alternatively spliced FS315 is specific to muscles, and this is enhanced by AAV gene therapy which has a natural affinity for muscle fibers The mouse studies led to primate studies, which should in the near future lead to human studies One flaw was the use of immunosuppression