Thurman M. Wheeler

Reversal of RNA dominance by displacement of protein sequestered on triplet repeat RNA.

Resisting Repeats A set of diseases, including myotonic dystrophy, are caused by the expansion of a simple repeat in genomic DNA, which, when transcribed into RNA, can be toxic to other cellular processes. Ameliorating the effects of this toxic, repeat-laden RNA may also relieve the symptoms of the disease. Wheeler et al. (p. 336; see the Perspective by Cooper) developed an antisense morpholino oligonucleotide complementary to the expanded repeats found in the myotonic dystrophy protein kinase messenger RNA (mRNA). The morpholino bound the repeats in vitro and displaced the inappropriately bound and sequestered RNA splicing factor, Muscleblind-like 1. In an in vivo mouse model for myotonic dystrophy, local injection of the morpholino corrected a number of cellular defects in muscle, including the alternative mRNA splicing of several genes, among them the muscle-specific chloride channel, CIC1, leading to a marked reduction in the myotonia. An antisense oligonucleotide ameliorates the symptoms of myotonic dystrophy in transgenic mice. Genomic expansions of simple tandem repeats can give rise to toxic RNAs that contain expanded repeats. In myotonic dystrophy, the expression of expanded CUG repeats (CUGexp) causes abnormal regulation of alternative splicing and neuromuscular dysfunction. We used a transgenic mouse model to show that derangements of myotonic dystrophy are reversed by a morpholino antisense oligonucleotide, CAG25, that binds to CUGexp RNA and blocks its interaction with muscleblind-like 1 (MBNL1), a CUGexp-binding protein. CAG25 disperses nuclear foci of CUGexp RNA and reduces the overall burden of this toxic RNA. As MBNL1 is released from sequestration, the defect of alternative splicing regulation is corrected, thereby restoring ion channel function. These findings suggest an alternative use of antisense methods, to inhibit deleterious interactions of proteins with pathogenic RNAs.

Targeting nuclear RNA for in vivo correction of myotonic dystrophy

Antisense oligonucleotides (ASOs) hold promise for gene-specific knockdown in diseases that involve RNA or protein gain-of-function effects. In the hereditary degenerative disease myotonic dystrophy type 1 (DM1), transcripts from the mutant allele contain an expanded CUG repeat and are retained in the nucleus. The mutant RNA exerts a toxic gain-of-function effect, 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 messenger RNAs. Here we show that nuclear-retained transcripts containing expanded CUG (CUGexp) repeats are unusually 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 1 year after treatment was discontinued. Systemically administered ASOs were also effective for muscle knockdown of Malat1, a long non-coding RNA (lncRNA) that is retained in the nucleus. These results provide a general strategy to correct RNA gain-of-function effects and to modulate the expression of expanded repeats, lncRNAs and other transcripts with prolonged nuclear residence.

Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy

Myotonic dystrophy type 1 (DM1) is caused by toxicity of an expanded, noncoding (CUG)n tract in DM protein kinase (DMPK) transcripts. According to current evidence the long (CUG)n segment is involved in entrapment of muscleblind (Mbnl) proteins in ribonuclear aggregates and stabilized expression of CUG binding protein 1 (CUGBP1), causing aberrant premRNA splicing and associated pathogenesis in DM1 patients. Here, we report on the use of antisense oligonucleotides (AONs) in a therapeutic strategy for reversal of RNA-gain-of-function toxicity. Using a previously undescribed mouse DM1 myoblast−myotube cell model and DM1 patient cells as screening tools, we have identified a fully 2′-O-methyl-phosphorothioate-modified (CAG)7 AON that silences mutant DMPK RNA expression and reduces the number of ribonuclear aggregates in a selective and (CUG)n-length-dependent manner. Direct administration of this AON in muscle of DM1 mouse models in vivo caused a significant reduction in the level of toxic (CUG)n RNA and a normalizing effect on aberrant premRNA splicing. Our data demonstrate proof of principle for therapeutic use of simple sequence AONs in DM1 and potentially other unstable microsatellite diseases.

Myotonic dystrophy: RNA-mediated muscle disease.

Purpose of reviewThe aim of this review is to highlight recent progress in elucidating the disease mechanism in myotonic dystrophy type 1 and type 2. Recent findingsResearch on myotonic dystrophy has led to the recognition of a novel RNA-mediated disease process. In myotonic dystrophy it is the RNA rather than protein product of a disease gene that has deleterious effects on muscle cells. These unusual RNAs, which contain a long expanse of CUG or CCUG repeats, have far reaching effects on cell function by influencing the biogenesis of other cellular RNAs. One aspect of RNA metabolism that is particularly affected is the regulation of alternative splicing. By this mechanism, effects of myotonic dystrophy repeat expansions impact many different pathways, triggering a complex set of signs and symptoms. SummaryThe genetic lesion in myotonic dystrophy does not eliminate an essential muscle protein. Instead, it induces a defect of RNA processing that is potentially reversible. The nature of this disease process raises the possibility that myotonic dystrophy, among genetic disorders, may be unusually susceptible to treatment using non-gene-therapy approaches.

Splicing biomarkers of disease severity in myotonic dystrophy.

To develop RNA splicing biomarkers of disease severity and therapeutic response in myotonic dystrophy type 1 (DM1) and type 2 (DM2).

RNA interference targeting CUG repeats in a mouse model of myotonic dystrophy.

Myotonic dystrophy type 1 (DM1) is an RNA dominant disease caused by expression of DM protein kinase (DMPK) transcripts that contain an expanded CUG repeat (CUG(exp)). The toxic mRNA localizes to nuclear foci and sequesters proteins involved in the regulation of alternative splicing, such as, muscleblind-like 1 (MBNL1). Here, we used synthetic short interfering RNAs (siRNAs) to target CUG repeats and test the concept that inhibiting the expression of CUG(exp) RNA can mitigate features of DM1 in transgenic mice. Intramuscular injection and electroporation of siRNA resulted in ~70-80% downregulation of CUG(exp) transcripts. A limited survey of endogenous mouse transcripts that contain nonexpanded CUG or CAG repeats showed that most were not affected, though Txlnb containing (CUG)(9) was significantly reduced. By this strategy, the number and intensity of CUG(exp) nuclear foci were reduced and splicing of MBNL1-dependent exons was improved. These data suggest that the expanded CUG repeats are a potential target for allele-selective RNA interference.

Ribonuclear foci at the neuromuscular junction in myotonic dystrophy type 1

In myotonic dystrophy type 1 (DM1) the muscle fibers express RNA containing an expanded CUG repeat (CUG(exp)). The CUG(exp) RNA is retained in the nucleus, forming ribonuclear foci. Splicing factors in the muscleblind (MBNL) family are sequestered in ribonuclear foci, resulting in abnormal regulation of alternative splicing. In extrajunctional nuclei, these effects on splicing regulation lead to reduced chloride conductance and altered insulin receptor signaling. Here we show that CUG(exp) RNA is also expressed in subsynaptic nuclei of muscle fibers and in motor neurons in DM1, causing sequestration of MBNL1 protein in both locations. In a transgenic mouse model, expression of CUG(exp) RNA at high levels in extrajunctional nuclei replicates many features of DM1, but the toxic RNA is poorly expressed in subsynaptic nuclei and the mice fail to develop denervation-like features of DM1 myopathology. Our findings indicate that subsynaptic nuclei and motor neurons are at risk for DM1-induced spliceopathy, which may affect function or stability of the neuromuscular junction.

Identification and Characterization of Modified Antisense Oligonucleotides Targeting DMPK in Mice and Nonhuman Primates for the Treatment of Myotonic Dystrophy Type 1

Myotonic dystrophy type 1 (DM1) is the most common form of muscular dystrophy in adults. DM1 is caused by an expanded CTG repeat in the 3′-untranslated region of DMPK, the gene encoding dystrophia myotonica protein kinase (DMPK). Antisense oligonucleotides (ASOs) containing 2′,4′-constrained ethyl-modified (cEt) residues exhibit a significantly increased RNA binding affinity and in vivo potency relative to those modified with other 2′-chemistries, which we speculated could translate to enhanced activity in extrahepatic tissues, such as muscle. Here, we describe the design and characterization of a cEt gapmer DMPK ASO (ISIS 486178), with potent activity in vitro and in vivo against mouse, monkey, and human DMPK. Systemic delivery of unformulated ISIS 486718 to wild-type mice decreased DMPK mRNA levels by up to 90% in liver and skeletal muscle. Similarly, treatment of either human DMPK transgenic mice or cynomolgus monkeys with ISIS 486178 led to up to 70% inhibition of DMPK in multiple skeletal muscles and ∼50% in cardiac muscle in both species. Importantly, inhibition of DMPK was well tolerated and was not associated with any skeletal muscle or cardiac toxicity. Also interesting was the demonstration that the inhibition of DMPK mRNA levels in muscle was maintained for up to 16 and 13 weeks post-treatment in mice and monkeys, respectively. These results demonstrate that cEt-modified ASOs show potent activity in skeletal muscle, and that this attractive therapeutic approach warrants further clinical investigation to inhibit the gain-of-function toxic RNA underlying the pathogenesis of DM1.

Myotonic dystrophy: Therapeutic strategies for the future

SummaryMyotonic dystrophy (DM) is a dominantly inherited neurodegenerative disorder for which there is no cure or effective treatment. Investigation of DM pathogenesis has identified a novel disease mechanism that requires development of innovative therapeutic strategies. It is now clear that DM is not caused by expression of a mutant protein. Instead, DM is the first recognized example of an RNA-mediated disease. Expression of the mutated gene gives rise to an expanded repeat RNA that is directly toxic to cells. The mutant RNA is retained in the nucleus, forming ribonuclear inclusions in affected tissue. A primary consequence of RNA toxicity in DM is dysfunction of two classes of RNA binding proteins, which leads to abnormal regulation of alternative splicing, or spliceopathy, of select genes. Spliceopathy now is known to cause myotonia and insulin resistance in DM. As our understanding of pathogenesis continues to improve, therapy targeted directly at the RNA disease mechanism will begin to replace the supportive care currently available. New pharmacologic approaches to treat myotonia and muscle wasting in DM type 1 are already in early clinical trials, and therapies designed to reverse the RNA toxicity have shown promise in preclinical models by correcting spliceopathy and eliminating myotonia. The well-defined ribonuclear inclusions may serve as convenient therapeutic targets to identify new agents that modify RNA toxicity. Continued development of appropriate model systems will allow testing of additional therapeutic strategies as they become available. Although DM is a decidedly complex disorder, its RNA-mediated disease mechanism may prove to be highly susceptible to therapy.

Progressive myopathy in an inducible mouse model of oculopharyngeal muscular dystrophy.

The genetic basis of oculopharyngeal muscular dystrophy (OPMD) is a short expansion of a polyalanine tract (normal allele: 10 alanines, mutant allele: 11-17 alanines) in the nuclear polyadenylate binding protein PABPN1 which is essential for controlling poly(A) tail length in messenger RNA. Mutant PABPN1 forms nuclear inclusions in OPMD muscle. To investigate the pathogenic role of mutant PABPN1 in vivo, we generated a ligand-inducible transgenic mouse model by using the mifepristone-inducible gene expression system. Induction of ubiquitous expression of mutant PABPN1 resulted in skeletal and cardiac myopathy. Histological changes of degenerative myopathy were preceded by nuclear inclusions of insoluble PABPN1. Downregulation of mutant PABPN1 expression attenuated the myopathy and reduced the nuclear burden of insoluble PABPN1. These results support association between mutant PABPN1 accumulation and degenerative myopathy in mice. Resolution of myopathy in mice suggests that the disease process in OPMD patients may be treatable.