Drug-induced readthrough of premature stop codons leads to the stabilization of laminin alpha2 chain mRNA in CMD myotubes.

BACKGROUND: The most common form of congenital muscular dystrophy is caused by a deficiency in the alpha2 chain of laminin-211, a protein of the extracellular matrix. A wide variety of mutations, including 20 to 30% of nonsense mutations, have been identified in the corresponding gene, LAMA2. A promising approach for the treatment of genetic disorders due to premature termination codons (PTCs) is the use of drugs to force stop codon readthrough. METHODS: Here, we analyzed the effects of two compounds on a PTC in the LAMA2 gene that targets the mRNA to nonsense-mediated RNA decay, in vitro using a dual reporter assay, as well as ex vivo in patient-derived myotubes. RESULTS: We first showed that both gentamicin and negamycin promote significant readthrough of this PTC. We then demonstrated that the mutant mRNAs were strongly stabilized in patient-derived myotubes after administration of negamycin, but not gentamicin. Nevertheless, neither treatment allowed re-expression of the laminin alpha2-chain protein, pointing to problems that may have arisen at the translational or post-translational levels. CONCLUSIONS: Taken together, our results emphasize that achievement of a clinical benefit upon treatment with novel readthrough-inducing agents would require several favourable conditions including PTC nucleotide context, intrinsic and induced stability of mRNA and correct synthesis of a full-length active protein.

The origin of tubular aggregates in human myopathies.

Tubular aggregates are morphological abnormalities characterized by the accumulation of densely packed tubules in skeletal muscle fibres. To improve knowledge of tubular aggregates, the formation and role of which are still unclear, the present study reports the electron microscopic analysis and protein characterization of tubular aggregates in six patients with 'tubular aggregate myopathy'. Three of the six patients also presented with myasthenic features. A large panel of immunochemical markers located in the sarcoplasmic reticulum, T-tubules, mitochondria, and nucleus was used. Despite differences in clinical phenotype, the composition of tubular aggregates, which contained proteins normally segregated differently along the sarcoplasmic reticulum architecture, was similar in all patients. All of these proteins, calsequestrin, RyR, triadin, SERCAs, and sarcalumenin, are involved in calcium uptake, storage, and release. The dihydropyridine receptor, DHPR, specifically located in the T-tubule, was also present in tubular aggregates in all patients. COX-2 and COX-7 mitochondrial proteins were not found in tubular aggregates, despite being observed close to them in the muscle fibre. The nuclear membrane protein emerin was found in only one case. Electron microscopy revealed vesicular budding from nuclei, and the presence of SAR-1 GTPase protein in tubular aggregates shown by immunochemistry, in all patients, suggests that tubular aggregates could arise from endoplasmic reticulum exit sites. Taken together, these results cast new light on the composition and significance of tubular aggregates.

Electrotransfer at MR imaging: tool for optimization of gene transfer protocols--feasibility study in mice.

PURPOSE: To test, by using an electrotransfer protocol for the transfection of skeletal muscle with naked plasmid complementary DNA, whether in vivo magnetic resonance (MR) imaging can help delineate either the spatial extent of the electric field when contrast agent is injected intraperitoneally or the transfection area when contrast agent is injected locally. MATERIALS AND METHODS: Three groups of five mice each were examined at 4 T. Gadopentetate dimeglumine was injected intraperitoneally before electroporation in group 1 and after electroporation in group 2. In group 3, gadopentetate dimeglumine was coinjected in situ with plasmid pCMV-beta Gal in the gastrocnemius muscle before electroporation. MR imaging and muscle preparation for histologic examination were performed 3 days later. On T1-weighted images, increase of muscle signal intensity was determined in regions of interest (ROIs) of treated legs and compared with contralateral ROIs. Comparison of signal intensity increase between groups 1 and 2 was performed with Kruskal-Wallis test. RESULTS: In groups 1 and 3, T1-weighted images of treated muscle showed zones of strongly increased signal intensity. In corresponding ROIs of groups 1, 2, and 3, the mean T1-weighted signal intensity increase at day 3 was 1.64 +/- 0.20 (SD), 1.16 +/- 0.06, and 1.58 +/- 0.17, respectively. The difference between groups 1 and 2 (ie, gadopentetate dimeglumine injected before and after electrotransfer) was significant (P <.001) both without and with correction for T2 variation (1.47 +/- 0.19 and 1.04 +/- 0.09, respectively). In group 3, after in situ coinjection of gadopentetate dimeglumine and plasmid, the area of increased signal intensity revealed at ex vivo MR imaging of the muscle showed a reasonable concordance with the transfected area revealed with beta-galactosidase on histologic sections. CONCLUSION: In vivo and ex vivo results indicate that atraumatic visualization of the permeabilized and transfected area is possible.