Inhibition of myostatin with emphasis on follistatin as a therapy for muscle disease

Abstract

In most cases, pharmacologic strategies to treat genetic muscle disorders and certain acquired disorders, such as sporadic inclusion body myositis, have produced modest clinical benefits. In these conditions, inhibition of the myostatin pathway represents an alternative strategy to improve functional outcomes. Preclinical data that support this approach clearly demonstrate the potential for blocking the myostatin pathway. Follistatin has emerged as a powerful antagonist of myostatin that can increase muscle mass and strength. Follistatin was first isolated from the ovary and is known to suppress follicle-stimulating hormone. This raises concerns for potential adverse effects on the hypothalamic-pituitary-gonadal axis and possible reproductive capabilities. In this review we demonstrate a strategy to bypass off-target effects using an alternatively spliced cDNA of follistatin (FS344) delivered by adeno-associated virus (AAV) to muscle. The transgene product is a peptide of 315 amino acids that is secreted from the muscle and circulates in the serum, thus avoiding cell-surface binding sites. Using this approach our translational studies show increased muscle size and strength in species ranging from mice to monkeys. Adverse effects are avoided, and no organ system pathology or change in reproductive capabilities has been seen. These findings provide the impetus to move toward gene therapy clinical trials with delivery of AAV-FS344 to increase size and function of muscle in patients with neuromuscular disease.

Figures

FIGURE 1

Myostatin null animals exhibit increased muscle mass. Adult myostatin null mice demonstrating increased size (right) as compared to wildtype (left) animals. Reprinted with permission from Lee SJ, McPherron AC. Curr Opin Genet Dev 1999:5:604−607.

FIGURE 2

Blocking the myostatin pathway. Myostatin (M) activation requires stepwise proteolytic cleavages of the precursor protein. After the signal peptide (SP) is removed, a second cleavage event leaves two fragments: an N-terminal propeptide domain of ≈28 kD and C-terminal domain of 12.5 kD destined to become the active myostatin protein. Parallel fragments of the myostatin C-terminal are linked through a disulfide bond, referred to as the myostatin C-terminal dimer that remains noncovalently complexed to the N-terminal propeptide. This noncovalent complex circulates in the blood maintaining the myostatin C-terminal dimer in a latent, inactive state. A third cleavage at amino acid 76 affects the ability of the propeptide to bind the active C terminal domain. Myostatin can be found in the serum or locally in an inactive state when bound to follistatin (FS), follistatin-related gene (FLRG), and growth and differentiation factor-associated serum protein-1 (GASP-1). These peptides block the activation of the myostatin pathway. If the pathway is not inhibited, the active myostatin dimer binds to the activin receptor type IIB (ActRIIB), which then recruits and activates by transphosphorylation the type I receptor (ALK4 or ALK5). Smad2 and Smad3 are subsequently activated and form aggregates with Smad4 and then are translocated to the nucleus, activating target gene transcription.

FIGURE 3

The follistatin gene consists of six exons. Alternative splicing generates two isoforms, FS317 and FS344. Alternative splicing occurs at the 3′ end of the gene between exon 5 and exon 6. Splicing out of intron 5 generates a stop codon immediately following the last amino acid of exon 5, and leads to the termination of the coding sequence for FS317. An alternative splice site results in the inclusion of exon 6 and generates FS344. After translation and prior to activation, follistatin undergoes further posttranslational modification by cleavage of the 29 amino acid signal peptide. This results in polypeptides FS315 (long-isoform from FS344) and FS288 (short-isoform from FS317).

FIGURE 4

Myostatin inhibitor proteins increase muscle mass and strength in wildtype C57Bl/6 mice. (a) Gross hindlimb muscle mass is increased in all myostatin-inhibitor-protein-treated mice at 725 days of age compared with AAV1-GFP-injected controls. (b) Total body mass is significantly increased in AAV1-FS344-injected (**P < 0.01) and AAV1-GASP-1-injected (*P < 0.05) mice compared with AAV1-GFP controls at 725 days of age (n = 10). (c) The mass of individual hindlimb and forelimb muscles is increased in mice injected with AAV expressing myostatin inhibitory proteins (n = 10). *P < 0.05. (d) Hindlimb grip strength improves >2 years in all treated mice with the greatest differences in AAV1-FS344 treated animals compared with AAV1-GFP controls (n = 10). Error bars represent standard error.

FIGURE 5

Single injection of AAV1.FS-344 increases muscle mass and strength in young mdx mice. (a) Gross hindlimb muscle mass is increased in AAV1.FS-344-injected mdx animals at 180 days of age compared with AAV1.GFP-injected controls. (b) The mass of individual hindlimb and forelimb muscles is increased at 180 days of age in mice injected at 3 weeks of age with AAV1.FS-344 paired with AAV1.GFP controls (n = 15). *P ≤ 0.05. (c) Grip strength is improved in a dose-dependent manner in young mdx mice injected at 3 weeks of age with AAV1-FS-344 followed for 180 days (n = 15). Red, high-dose AAV1.FS; blue, low-dose AAV1.FS-344; green, AAV1.GFP controls. Error bars represent standard errors.

FIGURE 6

Effects of AAV1.FS344 on muscle enzymes, strength, and morphology in older mice. (a) Serum creatine kinase levels (units/liter) are decreased at 3 months after injection with AAV1.FS-344 compared with AAV1.GFP-injected controls. (*P < 0.05; n = 10.) Error bars represent standard errors. Aged mdx mice treated with AAV1.FS-344. (b) Hindlimb grip strength is significantly increased (P < 0.05) at 275 days and beyond in aged mdx mice treated with AAV1.FS-344 at 210 days of age (n = 15). Red, high-dose AAV1.FS; green, AAV1.GFP controls. (c) Hematoxylin and eosin (H&E) stain of aged gastrocnemius (pre-treatment 180 days) demonstrates reduced pathology at 560 days.