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Targeting skeletal muscle pathology associated with Duchenne muscular dystrophy with novel valproic acid-related compounds

Project Description

Introduction: Duchenne muscular dystrophy (DMD), a severe muscle wasting disease, is due to mutations in the DMD gene resulting in lack of dystrophin protein in skeletal and cardiac muscle1, contraction-induced injury2, muscle fibre necrosis3. chronic inflammation4 and fibrosis5. It is apparent from recent clinical trials of genetic medicines to restore dystrophin protein expression that skeletal muscle fibrosis compromises their therapeutic benefit. Valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, has been shown to reduce fibrosis in a variety of tissues6 through the inhibition of collagen production7-10 by a number of mechanisms including selective regulation of Smads11, activation of P13K/Akt/mTOR pathway7, and epigenetic implications of HDAC inhibition12. VPA has been reported to ameliorate disease pathology in a mouse model of DMD through enhancement of α7 integrin levels, induction of muscle hypertrophy, inhibition of apoptosis and reduction of muscle fibrosis7. No study has yet been performed to examine the effect of the combined use of VPA and dystrophin restoration. Since VPA is associated with some side effects13, it is imperative to explore safer or more efficacious variants to develop a potential therapy for DMD that has translational potential.

Hypothesis: With the construction of a novel chemical library of compounds related to VPA by Prof Williams, it is proposed here to test the efficacy of these novel compounds as new therapeutic treatments against the fibrotic pathology associated with DMD. These compounds have already been demonstrated to have efficacy against the distinct cellular targets/mechanisms of VPA in regulating cellular functions (Williams, unpublished data). This novel chemical library may provide a significant advantage in the development of new treatments for dystrophic skeletal muscle pathology particularly when combined with a dystrophin replacement therapy. The overall aim of the work is to show that an effective anti-fibrotic treatment when combined with a treatment that restores dystrophin protein expression has a therapeutically beneficial effect compared to dystrophin restorative treatment alone. Comparison will be made to VPA throughout to establish relative efficacy and safety profiles.

Specific aim 1: In vitro determination of anti-fibrotic efficacy.

Efficacy of the novel chemical library to reverse skeletal muscle pathology associated with DMD will initially be examined in DMD patient muscle cells and mouse muscle cells derived from an established small animal model of the disease, the mdx mouse. These cells will be grown in culture and differentiated using established protocols within the Popplewell laboratory. The effect of daily chemical treatment on the expression of α7 integrin and fibrotic markers, myotube hypertrophy, and level of apoptosis will be assessed using published techniques7. The signalling pathway used by the novel compounds in muscle cells will be examined through established protocols within the Williams laboratory.

Specific aim 2: In vivo determination of anti-fibrotic efficacy.

The novel chemical library will also be tested in vivo in the mdx mouse using twice daily intraperitoneal (IP) injections from 4 weeks of age until 24 weeks of age. Anti-fibrotic effect of the chemical treatment will be assessed by immunohistochemical (IHC) staining of sectioned bodywide muscle, and by western blot and real-time (RT)-PCR. The effect of treatment on myofibre integrity, inflammation and Akt signalling will also be examined.

Specific aim 3: In vivo assessment of combination of treatment with dystrophin restoration.

The overall aim of the work is to show that an effective anti-fibrotic treatment when combined with a treatment that restores dystrophin protein expression has a therapeutically beneficial effect compared to dystrophin restorative treatment alone. Four week old mdx mice will be injected systemically with microdystrophin AAV9 vector followed by twice daily IP injections of VPA-related chemicals for 22 weeks. The combined effect of dystrophin protein expression and chemical treatment on locomotor phenotype will be examined at four weekly intervals using rod grip strength measurements and activity cage evaluation; electrophysiological analysis of muscle will be made at conclusion of treatment. Body-wide muscles will be harvested and dystrophin expression, restoration of the dystrophin-associated protein complex and anti-fibrosis efficacy assessed using IHC staining, western blot analysis and real-time (RT)-PCR. Serum samples will be collected and assayed for creatine kinase, and for liver and kidney markers of toxicity.

Funding Notes

Applicants should already have or be expected to obtain a First or upper Second Class degree in a relevant discipline. This studentship is fully funded for three years. It covers tuition fees at the UK/EU rate and includes a stipend at the standard Research Council rate (currently £16,296 per annum). Funding is available for UK and EU students.


1. Hoffman EP, Brown RH, Kunkel LM. (1987). Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell. 51(6):919-28.

2. Consolino CM, Brooks SV. (2004). Susceptibility to sarcomere injury induced by single stretches of maximally activated muscles of mdx mice. J Appl Physiol. 96(2):633-8.

3. Vallejo-Illarramendi A, Toral-Ojeda I, Aldanondo G, et al. (2014). Dysregulation of calcium homeostasis in muscular dystrophies. Expert Rev Mol Med. 16:e16.

4. Chen YW, Nagaraju K, Bakay M, et al. (2005). Early onset of inflammation and later involvement of TGFbeta in Duchenne muscular dystrophy. Neurology. 65(6):826-34.

5. Serrano AL, Muñoz-Cánoves P. (2016). Fibrosis development in early-onset muscular dystrophies: Mechanisms and translational implications. Semin Cell Dev Biol. pii: S1084-9521(16)30304-4.

6. Khan S, Ahirwar K, Jena G (2016). Anti-fibrotic effects of valproic acid: role of HDAC inhibition and associated mechanisms. Epigenomics 8(8):1087-101.

7. Gurpur PB, Liu J, Burkin DJ, Kaufman SJ. (2009). Valproic acid activates the PI3K/Akt/mTOR pathway in muscle and ameliorates pathology in a mouse model of Duchenne muscular dystrophy. Am J Pathol. 174(3):999-1008.

8. Marumo T, Hishikawa K, Yoshikawa M, et al. (2010). Histone deacetylase modulates the proinflammatory and -fibrotic changes in tubulointerstitial injury. Am J Physiol Renal Physiol. 298(1):F133-41.

9. Pang M, Zhuang S. (2010). Histone deacetylase: a potential therapeutic target for fibrotic disorders. J Pharmacol Exp Ther. 335(2):266-72.

10. Van Beneden K, Geers C, Pauwels M, et al. (2013). Comparison of trichostatin A and valproic acid treatment regimens in a mouse model of kidney fibrosis. Toxicol Appl Pharmacol. 271(2):276-84.

11. Seet LF, Toh LZ, Finger SN, et al. (2016). Valproic acid suppresses collagen by selective regulation of Smads in conjunctival fibrosis. J Mol Med. 94(3):321-34.

12. Consalvi S, Saccone V, Giordani L, et al. (2011). Histone deacetylase inhibitors in the treatment of muscular dystrophies: epigenetic drugs for genetic diseases. Mol Med. 17(5-6):457-65.

13. Star K, Edwards IR, Choonara I (2014). Valproic acid and fatalities in children: a review of individual case safety reports in VigiBase. PLoS One. 9(10):e108970.

How good is research at Royal Holloway, University of London in Biological Sciences?

FTE Category A staff submitted: 24.00

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