Small molecules to rescue folding-defective sarcoglycans: in vivo assessment of novel therapeutic strategies

LGMD2C-F, or sarcoglycanopathies, are rare genetic diseases that, disrupting the sarcoglycan (SG) complex, affect the sarcolemma stability during muscle contraction. Most of the reported cases are due to missense mutations originating a folding-defective sarcoglycan that is eliminated, although potentially functional, by the quality control of the cell. To prevent mutant degradation and sarcoglycan complex disruption, we have designed and proven in vitro two small molecule-based pharmacological approaches. The first one utilizes inhibitors of the E3 ubiquitin ligase HRD1 to reduce the degradation of alpha-SG mutants (protein rescue), whereas the second approach, CFTR correctors to foster the folding process of the mutants (protein repair). Our successful in vitro strategy needs now to be confirmed in vivo. However, the animal models of sarcoglycanopathy, at present available, are unsuitable to our purposes as we need animals expressing a folding-defective sarcoglycan. Therefore, also considering the large number of reported sarcoglycan missense mutants, the first aim of our project is the generation and characterization of novel LGMD2D mouse models by the transduction of sgca null mice with rAAVs (recombinant adeno associated viruses) expressing different missense mutants of the human alpha-SG. To this intent, during the first year, we cloned the human alpha-SG cDNA, either wild type or carrying missense mutations, under the control of the truncated muscle creatine kinase promoter/enhancer to assure muscle specific expression of the transgene. The cassette was then utilized to engineer AAVs of the serotype 2/1, especially targeting skeletal muscle tissue. For the generation of the animal models, AAVs are intraperitoneally injected in 1-2 day old pups of the sgca null mouse to achieve whole body-muscle transduction. No major effects on survival, body weight and general behavior have been observed after AAV-injection and during animal growth. At 6, 10 and 16 weeks post transduction, the expression of the human transgene is at first quantified by qRT-PCR and western blot analysis of different muscles of the legs, diaphragm, as well as heart. The development of dystrophic phenotype is evaluated in muscle by histological analysis, whereas SG localization is monitored by immunofluorescence analysis. Evaluation of muscle performance of these “humanized” mice (muscle strength and fatigability) will be estimated in vivo, and also ex vivo. We are confident that, once fully characterized, these animals will become suitable sarcoglycanopathy models to test in vivo our therapeutic strategy. Important aim of the project is also the assessment of efficacy of selected small molecules in human pathological samples. The SG-complex has been successfully rescued in primary myotubes from a patient carrying the R34H and V247M amino acid substitution on alpha-SG, whereas experiments with myogenic cells from a homozygote R77C-alpha-SG patient are ongoing.