M. Elbaz

Losartan, a therapeutic candidate in congenital muscular dystrophy: Studies in the dy2J/dy2J Mouse

Lamininα2‐deficient congenital muscular dystrophy type 1A (MDC1A) is a cureless disease associated with severe disability and shortened lifespan. Previous studies have shown reduced fibrosis and restored skeletal muscle remodeling following treatment with losartan, an angiotensin II type I receptor blocker. We therefore evaluated the effect of losartan treatment in the dy2J/dy2J mouse model of MDC1A.

The Ras Antagonist, Farnesylthiosalicylic Acid (FTS), Decreases Fibrosis and Improves Muscle Strength in dy2J/dy2J Mouse Model of Muscular Dystrophy

The Ras superfamily of guanosine-triphosphate (GTP)-binding proteins regulates a diverse spectrum of intracellular processes involved in inflammation and fibrosis. Farnesythiosalicylic acid (FTS) is a unique and potent Ras inhibitor which decreased inflammation and fibrosis in experimentally induced liver cirrhosis and ameliorated inflammatory processes in systemic lupus erythematosus, neuritis and nephritis animal models. FTS effect on Ras expression and activity, muscle strength and fibrosis was evaluated in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy. The dy2J/dy2J mice had significantly increased RAS expression and activity compared with the wild type mice. FTS treatment significantly decreased RAS expression and activity. In addition, phosphorylation of ERK, a Ras downstream protein, was significantly decreased following FTS treatment in the dy2J/dy2J mice. Clinically, FTS treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter. Significant reduction of fibrosis was demonstrated in the treated group by quantitative Sirius Red staining and lower muscle collagen content. FTS effect was associated with significantly inhibition of both MMP-2 and MMP-9 activities. We conclude that active RAS inhibition by FTS was associated with attenuated fibrosis and improved muscle strength in the dy2J/dy2J mouse model of congenital muscular dystrophy.

Improved muscle strength and mobility in the dy2J/dy2J mouse with merosin deficient congenital muscular dystrophy treated with Glatiramer acetate

The therapeutic effect of Glatiramer acetate, an immune modulating agent, was evaluated in the dy(2J)/dy(2J) mouse with merosin deficient congenital muscular dystrophy, which is a milder variant of the dy/dy mouse. The treated mice showed significant improvement in hind limb muscle strength measured by electronic grip strength meter and in motor performance quantified by video detection software. Glatiramer acetate treatment was associated with significantly increased expression of regeneration transcription factors MyoD and myogenin, and attenuation of the fibrosis markers vimentin and fibronectin. No effective treatment is currently available in congenital muscular dystrophy and Glatiramer acetate may present a new potential treatment for this disorder.

Sustained expression and safety of human GNE in normal mice after gene transfer based on AAV8 systemic delivery

GNE myopathy is an autosomal recessive adult onset disorder caused by mutations in the GNE gene. GNE encodes the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetyl mannosamine kinase, the key enzyme in the biosynthesis pathway of sialic acid. Additional functions for GNE have been described recently, but the mechanism leading from GNE mutation to this myopathy is unclear. Therefore a gene therapy approach could address all potential defects caused by GNE mutations in muscle. We show that AAV8 viral vectors carrying wild type human GNE cDNA are able to transduce murine muscle cells and human GNE myopathy-derived muscle cells in culture and to express the transgene in these cells. Furthermore, the intravenous administration of this viral vector to healthy mice allows expression of the GNE transgene mRNA and of the coexpressed luciferase protein, for at least 6months in skeletal muscles, with no clinical or pathological signs of focal or general toxicity, neither from the virus particles nor from the wild type human GNE overexpression. Our results support the future use of an AAV8 based vector platform for a safe and efficient therapy of muscle in GNE myopathy.

G.P.211

Merosin deficient congenital muscular dystrophy type 1A (MDC1A) is a devastating incurable disorder of childhood. We previously studied the effect of Glatiramer acetate (GA), an anti-inflammatory agent and S- farnesylthiosalicilate acid (FTS), an anti-fibrotic agent, as a single agent in the dy2J/dy2J mouse model of MDC1A. Each of the two medications showed a different pattern of disease on three primary outcome measures: strength, fibrosis and animal mobility. GA monotheraphy resulted in significant improvement in hind limb muscle strength and movement parameters, but without a decrease in fibrosis measurement. FTS monotheraphy was associated with increased hind limb muscle strength, decreased fibrosis, but with no improvement in movement parameters. In the present study, the combination of GA and FTS was found to increase both fore (32%) and hind limb (75.8%) muscle strength, more than treatment with either drug alone (fore limb: no significant change for GA and for FTS; hind limb: GA 52.7% vs. FTS 42%). In addition, GA-FTS treatment resulted in significant reduction in muscle fibrosis (35.4%) compared to untreated dy2J/dy2J mice. However, no improvement in animal mobility was shown. According to the results, combined GA and FTS increased both fore and hind limb muscle strength more than either drug by itself and significantly ameliorated muscle fibrosis. Thus, GA-FTS treatment might be considered as a potential candidate for future clinical trials in children with CMD.

P.8.10 Losartan up-regulates NFκB signaling pathway and favors survival versus apoptosis in the dy2J/dy2J mouse model of Congenital Muscular Dystrophy

Congenital Muscular Dystrophy (CMD) is a group of genetic disorders characterized by progressive loss of muscle strength and integrity. Merosin deficient congenital muscular dystrophy type 1A (MDC1A) is a common form of this disorder. Children affected with MDC1A suffer from early onset severe hypotonia and weakness with significant motor milestone delay. Often they do not achieve independent ambulation and die in the second or third decade. Despite extensive advances in diagnosis, cellular and molecular understanding, MDC1A remains a disease without a cure or any proven therapeutic option to relieve or slow disease progression. Our experimental data suggest that treatment with Losartan, an Angiotensin II type I receptor antagonist, results in significant clinical improvements and amelioration of fibrosis in the dy2J/dy2J mouse model of CMD through inhibition of TGFβ and MAPK signaling. We further examined Losartan’s effect on the cellular network, focusing on NFκB signaling. Previous studies suggested a role of NFκB in promoting muscle inflammation, necrosis and degeneration in Duchenne muscular dystrophy patients and animal models. Contrary to this, here we show that Losartan’s beneficial effect in the dy2J/dy2J of CMD is associated with NFκB signaling up-regulation manifested by enhanced serum TNFα level, decreased IκB-β protein level (NFκB inhibitor) and P65 accumulation in gastrocnemius nuclei of the dy2J/dy2J mice. A more in-depth investigation revealed that Losartan induced a modification in the NFκB gene expression towards pro-survival profile as cIAP2, TRAF2 and FTH mRNA levels were markedly increased following treatment. Losartan also induced the expression of anti-apoptotic BCl2 protein and down-regulated the expression of pro-apoptotic caspase 3 protein. Our study indicates that in the dy2J/dy2J mice of CMD, Losartan treatment resulted in NFκB activation with shifting from apoptosis/damage targeting pathway to a profile favoring cell survival.

P3.48 Losartan improves muscle strength and ameliorates fibrosis in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy

Losartan improves muscle strength and ameliorates fibrosis in the dy2J/dy2J mouse model of merosin deficient congenital muscular dystrophy M. Elbaz , S. Aga-Mizrachi , N. Yanay , O. Dadush , K. Ettinger , Z. Brunschwig , M. Rabie , V. Barak , Y. Nevo 1 1 Hadassah, Hebrew University Hospital, Pediatric Neurology Unit, Jerusalem, Israel, 2 Hadassah, Hebrew University Hospital, Immunology Laboratory for Tumor Diagnosis, Jerusalem, Israel