Nonsense mutations promote premature translational termination and cause anywhere from 5-70% of the individual cases of most inherited diseases. Studies on nonsense-mediated cystic fibrosis have indicated that boosting specific protein synthesis from <1% to as little as 5% of normal levels may greatly reduce the severity or eliminate the principal manifestations of disease. To address the need for a drug capable of suppressing premature termination, we identified PTC124-a new chemical entity that selectively induces ribosomal readthrough of premature but not normal termination codons. PTC124 activity, optimized using nonsense-containing reporters, promoted dystrophin production in primary muscle cells from humans and mdx mice expressing dystrophin nonsense alleles, and rescued striated muscle function in mdx mice within 2-8 weeks of drug exposure. PTC124 was well tolerated in animals at plasma exposures substantially in excess of those required for nonsense suppression. The selectivity of PTC124 for premature termination codons, its well characterized activity profile, oral bioavailability and pharmacological properties indicate that this drug may have broad clinical potential for the treatment of a large group of genetic disorders with limited or no therapeutic options.
Mutations in a number of cardiac sarcomeric protein genes cause hypertrophic cardiomyopathy (HCM). Previous findings indicate that HCM-causing mutations associated with a truncated cardiac troponin T (TnT) and missense mutations in the -myosin heavy chain share abnormalities in common, acting as dominant negative alleles that impair contractile performance. In contrast, Lin et al.[Lin, D., Bobkova, A., Homsher, E. & Tobacman, L. S. (1996) J. Clin. Invest. 97, 2842-2848] characterized a TnT point mutation (Ile79Asn) and concluded that it might lead to hypercontractility and, thus, potentially a different mechanism for HCM pathogenesis. In this study, three HCMcausing cardiac TnT mutations (Ile79Asn, Arg92Gln, and ⌬Glu160) were studied in a myotube expression system. Functional studies of wild-type and mutant transfected myotubes revealed that all three mutants decreased the calcium sensitivity of force production and that the two missense mutations (Ile79Asn and Arg92Gln) increased the unloaded shortening velocity nearly 2-fold. The data demonstrate that TnT can alter the rate of myosin cross-bridge detachment, and thus the troponin complex plays a greater role in modulating muscle contractile performance than was recognized previously. Furthermore, these data suggest that these TnT mutations may cause disease via an increased energetic load on the heart. This would represent a second paradigm for HCM pathogenesis.
Mutations in the  -myosin heavy chain gene are believed to cause hypertrophic cardiomyopathy (HCM) by acting as dominant negative alleles. In contrast, a truncated cardiac troponin T (TnT) that causes HCM implies that altered stoichiometry of contractile proteins may also cause cardiac hypertrophy. Wild-type and HCM-mutant (truncated) TnT were studied in a novel quail myotube expression system. Unexpectedly, antibody staining demonstrated incorporation of both forms of human cardiac TnT into the sarcomeres of quail myotubes. Functional studies of wild type and mutant transfected myotubes of normal appearance revealed that calcium-activated force of contraction was normal upon incorporation of wild type TnT, but greatly diminished for the mutant TnT. These findings indicate that HCM-causing mutations in TnT and  -myosin heavy chain share abnormalities in common, acting as dominant negative alleles that impair contractile performance. This diminished force output is the likely stimulus for hypertrophy in the human heart. ( J. Clin. Invest. 1996. 98:2456-2461.)
In this study the ability of magnetodendrimers to efficiently label cultured muscle stem cells and allow for subsequent in vivo cell detection was determined. Magnetodendrimer-labeled cells exhibited normal growth rates in culture, and retained their capacity to undergo proliferation and form normal myotubes. Labeled stem cells possessed high in vivo proton relaxivities that enhanced MRI contrast properties and enabled us to noninvasively monitor the stem cells' incorporation into dystrophic muscle. Well defined regions of decreased signal intensity were observed in both T 2 -and T 1 -weighted image sequences. MRI was used to longitudinally follow stem cell dynamics in dystrophic muscle with in-plane resolutions on the order of a single muscle fiber (22 ؋ 43 m 2 ). Regions of decreased signal intensity were well correlated with iron accumulation and other histochemical markers of stem cell incorporation. The monitoring of stem cell transfer efficacy and incorporation (1) in a clinical setting currently necessitates the use of invasive techniques (2,3). This can be problematic in subjects with extensive muscle damage and necrosis, such as children with Duchenne's muscular dystrophy. Although it has been shown that both direct intramuscular (4,5) and intravenous injection of normal muscle stem cells (3,5-7) can lead to the reexpression of dystrophin and the rescue of dystrophic muscle (8), the transfer efficacy is limited and stem cell incorporation may be transient because of the host immune response. Techniques to noninvasively monitor such transplants are essential to accelerate the development of cell therapies by reducing the labor associated with conventional cell-detection strategies, as well as to minimize the risk associated with invasive muscle biopsies. We therefore sought to develop high-resolution MRI techniques capable of detecting and following the fate of exogenous muscle stem cell transplants using magnetodendrimers.Magnetodendrimers are a novel class of MR contrast reagents that are effective for nonspecific in vitro cellular labeling. Magnetodendrimers consist of an iron oxide core coated with carboxylated poly(amidoamine) generation 4.5 dendrimers. Dendrimers function as a "synthetically controllable mesh" or matrix that can be used to encapsulate iron particles (9,10), particularly superparamagnetic iron oxide (SPIO). Dendrimers have been demonstrated to have a high nonspecific affinity for cellular membranes, and thus are an efficient vehicle for facilitating the intracellular accumulation of SPIO by a non-receptor-mediated process. The use of magnetodendrimers eliminates the need for complex cellular labeling strategies that rely on the overexpression of iron-binding receptors or the conjugation of iron oxides to specific ligands. SPIOs have net magnetic moments that are at least 4 orders of magnitude higher than the same number of paramagnetic spins, resulting in large local field gradients that dephase nearby protons and dramatically reduce proton relaxation times. When cells are labeled w...
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