Myostatin propeptide gene delivery in normal dogs via AAV vector increases muscle fiber size

Myostatin has been extensively documented as a negative regulator of muscle growth. Myostatin blockades therefore offers an effective strategy for treating a number of degenerative muscle diseases, including sarcopenia and muscular dystrophy.

Explain the trial?

Dr. Quio injected a virus (AAV8- adenovirus) into one leg of dogs with Duchenne muscular dystrophy.  The virus contained the genetic information encoding myostatin propeptide.  When the virus infected the muscle cells in the dog’s leg, the viral genetic information was copied into the cellular DNA and resulted in increased muscle cellular production of myostatin propeptide.

 

The easy way to think of this is that the dog’s leg was infected with a virus carrying a myostatin propeptide gene. Then, the infected dog’s muscle cells became a factory, producing myostatin propeptide. 

 

On subsequent muscle biopsy, the muscle cells of the virus infected leg, showed increased muscle growth demonstrated by larger muscle fibers and MRI. This leg’s muscle biopsy was compared to the uninfected dog leg.  There was also a decrease in CD4 and CD8 Tcells in the infected muscle cells, demonstrating a decreased immune response.

 

What is the relevance to CMD?

Myostatin inhibition with antibodies or propeptide has not been tried in mouse models of CMD.  There is one study that looked at complete absence of myostatin in the merosin deficient mouse model.  Complete absence has been shown to increase muscle regeneration in the merosin deficient mouse model (dy/dy mouse model); but mice were not stronger and inflammation was not improved. This study was done by crossing the merosin deficient mouse model with myostatin null mice (no myostatin). The crossed mice had increased postnatal death rates, likely associated with the complete absence of myostatin.  Engvall E. et al, Elimination of myostatin does not combat muscular dystrophy in dy mice but increases postnatal lethality. Am J Pathol 2005 Feb; 166 (2):491-7.  Treatment with myostatin inhibition would not cause complete absence of myostatin; thus the postnatal lethality may not be an issue in human treatment.  The real question is whether myostatin blockade’s effect improves functional strength rather than simply increasing fiber size or muscle size.  Based on myostatin’s mechanism of action and effect on dystrophic muscle in the Duchenne mouse and dog model, myostatin may have a role in preventing progression of muscle weakness in CMD. 

 

Given the “failed” Wyeth study using MYO-029 (see below) there may be a reluctance to study myostatin inhibition in the CMD mouse models.  It might be more efficacious to proceed with studies using losartan.  Losartan may be a more powerful inhibitor of fibrosis.  Losartan increases muscle mass and blocks the effects of TGF-beta in heart and kidney.  Recent studies of losartan in mdx mice showed increased strength and less fibrosis. It might be interesting to do a head to head comparison of myostatin inhibition and losartan in two different CMD mouse models, to compare efficacy and use strength tests as the clinical outcome in addition to muscle pathology review.

 

What is myostatin’s role in the body? What role does it play in muscular dystrophy?

Myostatin has a negative effect on muscle growth. Blocking myostatin, increases muscle bulk and strength.  There are mice and cattle with mutations in the myostatin gene. These animals show increased body weight and muscle mass. Myostatin is part of the TGF-beta superfamily.  As such, myostatin blockade may play a similar role to TGF-beta blockade with losartan in decreasing fibrosis, immune response and improving muscle strength. 

 

Initial studies of myostatin blockade in Duchenne mouse model (mdx mouse) showed increase in body weight, muscle mass, muscle size and absolute muscle strength. These studies were done using intra-abdominal injections of myostatin antibodies.  There was also a decrease in muscle breakdown and a decrease in concentrations of serum (blood) creatine kinase. Creatine kinase is a protein released by muscle that is elevated in muscular dystrophy to varying degrees.  Khurana TS et al. Functional improvement of dystrophic muscle by myostatin blockade. Nature 2002 Nov 28;420 (6914):418-21.

 

Has a myostatin blockade study been done in humans?

Yes, a Phase I/II clinical trial using myostatin antibody blockade was terminated early secondary to no benefit seen. The trial was sponsored by Wyeth Pharmaceuticals. The study was done in a mixed adult patient population with muscular dystrophy. The drug, called MYO-029, was shown to be safe. Improvement in muscle function and strength was not shown; however, there was a trend towards augmented muscle mass and fiber size.  It is unclear if another trial would show a positive effect if:

1.                   increased doses were used

2.                   different clinical endpoints, aside from myostatin assay were used. Many patients’ myostatin levels were too low to be quantified accurately.

3.                   a smaller more homogeneous group of younger patients used who might have more muscle mass and accelerated muscle breakdown, leading to more positive gains in strength as an outcome measure.

 

What is myostatin propeptide?

There are several ways to block myostatin, including blocking myostatin directly with antibodies (MYO-029) or infecting a cell with a virus containing the myostatin propeptide gene.

 

Myostatin propeptide is a protein that is found naturally in the human body. It is part of the precursor form of myostatin prior to cleavage. It binds in the body to myostatin in serum (blood) regulating the effect of myostatin. Qui Y et al. The Myostatin Propeptide and the Follistatin-related Gene are Inhibitory Binding Proteins of Myostatin in the Normal Serum, J. Biol.Chem. 25 Oct 2002; 277 (43): 40735-40741. Myostatin propeptide is also made commercially in a recombinant form for therapeutic interventions.

 

There have been studies in the mouse model of Duchenne (mdx mouse) using myostatin propeptide fused to IgG-Fc.  The studies show increased mouse strength exceeding strength force seen by using myostatin antibody blockade.  This study may suggest that myostatin propeptide is a stronger inhibitor of myostatin than MYO-029.  Bogdanovich, S, et al. Myostatin propeptide-mediated amelioration of dystrophic pathophysiology, FASEB J. 2005 April; 19(6):543-9.

 

To learn more about this trial:

Duchenne muscular dystrophy (DMD), caused by mutations in the dystrophin gene, is the most common, disabling and lethal muscle disease. Myostatin has been extensively documented as a negative regulator of muscle growth. Myostatin blockades therefore offers an effective strategy for treating a number of degenerative muscle diseases, including sarcopenia and muscular dystrophy. In this study, Dr. Chunping Qiao and colleagues investigated whether gene delivery of myostatin inhibitors, specifically, the propeptide, could improve muscle growth and ameliorate the pathology in the DMD large animal model golden retriever muscular dystrophy (GRMD) dogs. They delivered the AAV8 vector encoding myostatin propeptide (AAV-MPRO) gene via hydrodynamic limb vein injection in the hindlimb of 3-month-old normal dogs, weighing 6.3 kg and 9.7 kg. The vector was delivered into one limb, the contralateral serving as a control. No vector-related adverse event was observed
during and after injection. AAV vector DNA and MPRO gene expression were detected by quantitative PCR, Western blot, and immunofluorescent staining of muscle biopsies. Over-expression of MPRO resulted in enhanced muscle growth without a CTL immune response, as evidenced by larger myofibres in multiple muscles, increased muscle volume via magnetic resonance imaging (MRI), and the lack of CD4 and CD8 T-cell infiltration in the vector-injected limbs. These preliminary data demonstrate that delivering AAV vector encoding myostatin propeptide gene into normal dogs induces muscle hypertrophy and increases their myofibre size, paving the way for future clinical studies.

Hum Gene Ther. 2008 Oct 1

 

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