Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat1–3 and are retained in the nucleus4, 5. The mutant RNA exerts a toxic gain-of-function6, making it an appropriate target for therapeutic ASOs. However, despite improvements in ASO chemistry and design, systemic use of ASOs is limited because uptake in many tissues, including skeletal and cardiac muscle, is not sufficient to silence target mRNAs7, 8. Here we show that nuclear-retained transcripts containing expanded CUG (CUGexp) repeats are extraordinarily sensitive to antisense silencing. In a transgenic mouse model of DM1, systemic administration of ASOs caused a rapid knockdown of CUGexp RNA in skeletal muscle, correcting the physiological, histopathologic, and transcriptomic features of the disease. The effect was sustained for up to one year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat-1, a long noncoding RNA (lncRNA) that is retained in the nucleus9. These results provide a general strategy to correct RNA gain-of-function and modulate the expression of expanded repeats, lncRNAs, and other transcripts with prolonged nuclear residence.

Objective Studies of mice with mild Marfan syndrome (MFS) have correlated the development of thoracic aortic aneurysm (TAA) with improper stimulation of non-canonical (Erk-mediated) TGFβ signaling by the angiotensin type I receptor (AT1r). This correlation was largely based on comparable TAA modifications by either systemic TGFβ neutralization or AT1r antagonism. However, subsequent investigations have called into question some key aspects of this mechanism of arterial disease in MFS. To resolve these controversial points, here we made a head-to-head comparison of the therapeutic benefits of TGFβ neutralization and AT1r antagonism in mice with progressively severe MFS (Fbn1mgR/mgR mice). Approach and Results Aneurysm growth, media degeneration, aortic levels of phosphorylated Erk and Smad proteins and the average survival of Fbn1mgR/mgR mice were compared after a ∼3 month long treatment with placebo and either the AT1r antagonist losartan or the TGFβ neutralizing antibody 1D11. In contrast to the beneficial effect of losartan, TGFβ neutralization either exacerbated or mitigated TAA formation depending on whether treatment was initiated before (post-natal day 16; P16) or after (P45) aneurysm formation, respectively. Biochemical evidence related aneurysm growth with Erk-mediated AT1r signaling, and medial degeneration with TGFβ hyperactivity that was in part AT1r-dependent. Importantly, P16-initiated treatment with losartan combined with P45-initiated administration of 1D11 prevented death of Fbn1mgR/mgR mice from ruptured TAA. Conclusions By demonstrating that promiscuous AT1r and TGFβ drive partially overlapping processes of arterial disease in MFS mice, our study argues for a therapeutic strategy against TAA that targets both signaling pathways while sparing the early protective role of TGFβ.

The muscle-specifIc enhancer element located downstream of the myosin light chain (MLC) locus encoding MLC1 and MLC3 contains three binding sites (A, B, and C) for the myogenic determination factor MyoD. A 173-base-pair region of the MLC gene enhancer, including these three sites, retains full enhancer function when transfected into muscle cells. Whereas mutation of either upstream MyoD binding site (A or B) has a mild effect on muscle-specific enhancer activity, mutation of the third MyoD binding site (C) substantially weakens the enhancer, both in muscle cells or in nonmusde cells cotransfected with a MyoD, myogenin, or myf5 expression vector. Site C is necessary but insuicient, since double mutation of two MyoD binding sites (A plus B) abrogates enhancer activity. Thus, site C requires either site A or B for enhancer function. This study shows a hierarchy of function among the three MyoD binding sites in the MLC enhancer. We propose that a protein-DNA complex Is formed with at least two ofthese sites (A and C or B and C) to effect activation of the locus encoding MLC1/3 during myogenesis.The activation of the myogenic program in muscle precursor cells involves a hierarchy of regulatory factors, which coordinate the expression of multiple contractile proteins. Among the structural genes that are induced during muscle differentiation, the myosin alkali light chain proteins MLC1 and MLC3 are generated from a single genetic locus by transcription from two different promoters and alternate splicing ofthe pre-mRNAs (1-3). The muscle-specific regulation of both rat and human gene expression of MLC1/3 depends upon a strong enhancer element, located =24 kilobases (kb) downstream of the MLC1 gene promoter in both species (4, 5). (The human locus has been assigned the symbol MYLI.) This element causes a dramatic increase in the transcription of linked reporter genes exclusively in myotubes, independent of its distance, position, or orientation relative to the promoter (4, 5). In addition, transgenic mice carrying multiple copies of a MLC1 promoter-chloramphenicol acetyltransferase (CAT) gene transcription unit linked to the MLC enhancer activate the transgene exclusively in skeletal muscles, concurrently with the onset of endogenous MLC1 transcription (6). These studies have identified the transcriptional control elements necessary to activate the locus encoding MLC1/3 at the appropriate fetal stage and indicated that in rodents, the MLC gene enhancer is sufficient to induce developmentally regulated expression from the MLC1 promoter exclusively in skeletal muscle cells.The sequence conservation between the rat and human MLC enhancers (5) suggests that they represent an original component of the ancestral mammalian MLC1/3 locus and are targets for factors involved in the developmental and tissue-specific regulation of muscle gene transcription. Potential candidates for these regulatory proteins include a related group of myogenic factors-MyoD (7), myogenin (8, 9), myf5 (10), and MRF4 (11)-some of which autoregul...

This study provides Class II evidence of the muscle cell penetration, exon skipping, and induction of novel dystrophin expression by eteplirsen, as confirmed by 4 assays.

Two skeletal myosin light chains, MLC 1 and MLC3, are generated from a single gene by transcription from two different promoters and alternate splicing of the pre-mRNAs. To define DNA sequences involved in MLC transcriptional control, we constructed a series of plasmid vectors in which segments of the rat MLC locus were linked to a CAT gene and assayed for expression in muscle and nonmuscle cells. Whereas sequences proximal to the two MLC promoters do not appear to contain tissue-specific regulatory elements, a 0.9-kb DNA segment, located >24 kb downstream of the MLC~ promoter, dramatically increases CAT gene expression in differentiated myotubes but not in undifferentiated myoblasts or nonmuscle cells. The ability of this segment to activate gene expression to high levels, in a distance-, promoter-, position-, and orientation-independent way, defines it as a strong muscle-specific enhancer element.[Key Words: Rat myosin light chain 1/3; CAT assay; enhancer; muscle-specific expression]