Detection of expanded RNA repeats using thermostable group II intron reverse transcriptase.

Cellular accumulation of repetitive RNA occurs in several dominantly-inherited genetic disorders. Expanded CUG, CCUG or GGGGCC repeats are expressed in myotonic dystrophy type 1 (DM1), myotonic dystrophy type 2 (DM2), or familial amyotrophic lateral sclerosis, respectively. Expanded repeat RNAs (ER-RNAs) exert a toxic gain-of-function and are prime therapeutic targets in these diseases. However, efforts to quantify ER-RNA levels or monitor knockdown are confounded by stable structure and heterogeneity of the ER-RNA tract and background signal from non-expanded repeats. Here, we used a thermostable group II intron reverse transcriptase (TGIRT-III) to convert ER-RNA to cDNA, followed by quantification on slot blots. We found that TGIRT-III was capable of reverse transcription (RTn) on enzymatically synthesized ER-RNAs. By using conditions that limit cDNA synthesis from off-target sequences, we observed hybridization signals on cDNA slot blots from DM1 and DM2 muscle samples but not from healthy controls. In transgenic mouse models of DM1 the cDNA slot blots accurately reflected the differences of ER-RNA expression across different transgenic lines, and showed therapeutic reductions in skeletal and cardiac muscle, accompanied by improvements of the DM1-associated splicing defects. TGIRT-III was also active on CCCCGG- and GGGGCC-repeats, suggesting that ER-RNA analysis is feasible for several repeat expansion disorders.

Dmpk gene deletion or antisense knockdown does not compromise cardiac or skeletal muscle function in mice.

Myotonic dystrophy type 1 (DM1) is a genetic disorder in which dominant-active DM protein kinase (DMPK) transcripts accumulate in nuclear foci, leading to abnormal regulation of RNA processing. A leading approach to treat DM1 uses DMPK-targeting antisense oligonucleotides (ASOs) to reduce levels of toxic RNA. However, basal levels of DMPK protein are reduced by half in DM1 patients. This raises concern that intolerance for further DMPK loss may limit ASO therapy, especially since mice with Dmpk gene deletion reportedly show cardiac defects and skeletal myopathy. We re-examined cardiac and muscle function in mice with Dmpk gene deletion, and studied post-maturity knockdown using Dmpk-targeting ASOs in mice with heterozygous deletion. Contrary to previous reports, we found no effect of Dmpk gene deletion on cardiac or muscle function, when studied on two genetic backgrounds. In heterozygous knockouts, the administration of ASOs reduced Dmpk expression in cardiac and skeletal muscle by > 90%, yet survival, electrocardiogram intervals, cardiac ejection fraction and muscle strength remained normal. The imposition of cardiac stress by pressure overload, or muscle stress by myotonia, did not unmask a requirement for DMPK. Our results support the feasibility and safety of using ASOs for post-transcriptional silencing of DMPK in muscle and heart.

Nox4 NADPH oxidase contributes to smooth muscle cell phenotypes associated with unstable atherosclerotic plaques.

Plaque instability associated with acute coronary syndromes results in part from apoptosis and senescence of cells within the atherosclerotic (AS) lesion. Increased cellular oxidative stress has been proposed to contribute to plaque progression and changes in composition, leading to plaque instability. Our objective was to examine the role of NADPH oxidase in smooth muscle cell (SMC) phenotypes associated with an unstable plaque. Aortae were isolated from pre-lesion (8 weeks of age) and post-lesion (35 weeks of age) hypercholesterolemic mice (ApoE(-/-)/LDLR(-/-), AS), and age-matched normal C57BL/6J mice. We observed an age-dependent increase in reactive oxygen species (ROS) in aorta from AS mice, with evidence for elevated ROS prior to lesion development. Whereas macrophage infiltration was restricted to the lesion, oxidized lipids extended beyond the plaque and into the vessel wall. Consistent with these findings, we observed dynamic changes in the expression of NADPH oxidases in AS vessels. Specifically, Nox1 expression was increased early and decreased with lesion progression, while induction of Nox4 was a late event. Nox2 and p22(phox) were elevated throughout lesion development. Similar to observations in aortae, SMCs isolated from the lesion of AS aortae had decreased Nox1 and increased Nox4 levels as compared to SMCs from normal mice. AS SMCs demonstrated increased generation of ROS, cell cycle arrest, evidence of senescence, and increased susceptibility to apoptosis. Overexpression of Nox4 in normal SMCs recapitulated the phenotypes of the AS SMCs. We conclude that increased expression of Nox4 in AS may drive SMC phenotypes that lead to the plaque instability and rupture responsible for myocardial infarction and stroke.

Reducing levels of toxic RNA with small molecules.

Myotonic dystrophy (DM) is one of the most common forms of muscular dystrophy. DM is an autosomal dominant disease caused by a toxic gain of function RNA. The toxic RNA is produced from expanded noncoding CTG/CCTG repeats, and these CUG/CCUG repeats sequester the Muscleblind-like (MBNL) family of RNA binding proteins. The MBNL proteins are regulators of alternative splicing, and their sequestration has been linked with mis-splicing events in DM. A previously reported screen for small molecules found that pentamidine was able to improve splicing defects associated with DM. Biochemical experiments and cell and mouse model studies of the disease indicate that pentamidine and related compounds may work through binding the CTG*CAG repeat DNA to inhibit transcription. Analysis of a series of methylene linker analogues of pentamidine revealed that heptamidine reverses splicing defects and rescues myotonia in a DM1 mouse model.

Role for Nox1 NADPH oxidase in atherosclerosis.

OBJECTIVE: Examine the contribution of Nox1 NADPH oxidase to atherogenesis. METHODS AND RESULTS: Male apolipoprotein E deficient mice (ApoE(-/-)) and male mice deficient in both apolipoprotein E and Nox1 (ApoE(-/-) Nox1(-/y)) received an atherogenic diet for 18 weeks. Mean blood pressures, body weights, and serum cholesterol levels were similar between the two groups of mice. Deficiency of Nox1 decreased superoxide levels and reduced lesion area in the aortic arch from 43% (ApoE(-/-)) to 28% (ApoE(-/-) Nox1(-/y)). The reduction in lesion size at the level of the aortic valve in ApoE(-/-)/Nox1(-/y) was accompanied by a decrease in macrophage infiltration as compared to ApoE(-/-) mice. Carotid artery ligation in ApoE(-/-) mice induced accelerated intimal hyperplasia with decreased cellular proliferation and increased collagen content in the neointima of vessels deficient in Nox1. CONCLUSIONS: Nox1-derived ROS modify lesion composition and contribute to lesion size in a murine model of atherosclerosis.