Intracellular Distribution and Nuclear Activity of Antisense Oligonucleotides After Unassisted Uptake in Myoblasts and Differentiated Myotubes In Vitro.

Clinical efficacy of antisense oligonucleotides (AONs) for the treatment of neuromuscular disorders depends on efficient cellular uptake and proper intracellular routing to the target. Selection of AONs with highest in vitro efficiencies is usually based on chemical or physical methods for forced cellular delivery. Since these methods largely bypass existing natural mechanisms for membrane passage and intracellular trafficking, spontaneous uptake and distribution of AONs in cells are still poorly understood. Here, we report on the unassisted uptake of naked AONs, so-called gymnosis, in muscle cells in culture. We found that gymnosis works similarly well for proliferating myoblasts as for terminally differentiated myotubes. Cell biological analyses combined with microscopy imaging showed that a phosphorothioate backbone promotes efficient gymnosis, that uptake is clathrin mediated and mainly results in endosomal-lysosomal accumulation. Nuclear localization occurred at a low level, but the gymnotically delivered AONs effectively modulated the expression of their nuclear RNA targets. Chloroquine treatment after gymnotic delivery helped increase nuclear AON levels. In sum, we demonstrate that gymnosis is feasible in proliferating and non-proliferating muscle cells and we confirm the relevance of AON chemistry for uptake and intracellular trafficking with this method, which provides a useful means for bio-activity screening of AONs in vitro.

Cell membrane integrity in myotonic dystrophy type 1: implications for therapy.

Myotonic Dystrophy type 1 (DM1) is a multisystemic disease caused by toxic RNA from a DMPK gene carrying an expanded (CTG•CAG)n repeat. Promising strategies for treatment of DM1 patients are currently being tested. These include antisense oligonucleotides and drugs for elimination of expanded RNA or prevention of aberrant binding to RNP proteins. A significant hurdle for preclinical development along these lines is efficient systemic delivery of compounds across endothelial and target cell membranes. It has been reported that DM1 patients show elevated levels of markers of muscle damage or loss of sarcolemmal integrity in their serum and that splicing of dystrophin, an essential protein for muscle membrane structure, is abnormal. Therefore, we studied cell membrane integrity in DM1 mouse models commonly used for preclinical testing. We found that membranes in skeletal muscle, heart and brain were impermeable to Evans Blue Dye. Creatine kinase levels in serum were similar to those in wild type mice and expression of dystrophin protein was unaffected. Also in patient muscle biopsies cell surface expression of dystrophin was normal and calcium-positive fibers, indicating elevated intracellular calcium levels, were only rarely seen. Combined, our findings indicate that cells in DM1 tissues do not display compromised membrane integrity. Hence, the cell membrane is a barrier that must be overcome in future work towards effective drug delivery in DM1 therapy.

Targeting several CAG expansion diseases by a single antisense oligonucleotide.

To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUG)n triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG)(7), also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well.

Abnormal actomyosin assembly in proliferating and differentiating myoblasts upon expression of a cytosolic DMPK isoform.

DMPK, the product of the mutated gene in myotonic dystrophy type 1, belongs to the subfamily of Rho-associated serine-threonine protein kinases, whose members play a role in actin-based cell morphodynamics. Not much is known about the physiological role of differentially localized individual DMPK splice isoforms. We report here that prominent stellar-shaped stress fibers are formed during early and late steps of differentiation in DMPK-deficient myoblast-myotubes upon complementation with the short cytosolic DMPK E isoform. Expression of DMPK E led to an increased phosphorylation status of MLC2. We found no such effects with vectors that encode a mutant DMPK E which was rendered enzymatically inactive or any of the long C-terminally anchored DMPK isoforms. Presence of stellar structures appears associated with changes in cell shape and motility and a delay in myogenesis. Our data strongly suggest that cytosolic DMPK participates in remodeling of the actomyosin cytoskeleton in developing skeletal muscle cells. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.

Molecular therapy in myotonic dystrophy: focus on RNA gain-of-function.

Myotonic dystrophy (DM) is a complex, dominantly inherited, multisystem disorder and the archetypal example of an RNA gain-of-function disease. Unstable expansions of (CTG*CAG)n or (CCTG*CAGG)n repeat tracts in the DMPK and ZNF9 genes cause the two known subtypes of myotonic dystrophy, DM1 and DM2, for which no cure or effective molecular treatment exists. Focus in therapeutic development is currently on toxic, expanded (C/CUG)n RNAs. A series of recent papers provide proof of concept of promising strategies using antisense oligonucleotides or small organic compounds aimed at either complete elimination of expanded (CUG)n RNA transcripts or prevention of detrimental protein binding to thermodynamically stable (C/CUG)n hairpin structures. These developments offer new hope to patients with DM, even though several hurdles still have to be overcome before they can be introduced into clinical practice.

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.

DMPK protein isoforms are differentially expressed in myogenic and neural cell lineages.

Myotonic dystrophy type 1 (DM1) is a neuromuscular disorder caused by an unstable (CTG . CAG)n segment in the 3' untranslated region of the myotonic dystrophy protein kinase (DMPK) gene. It is commonly accepted that DMPK mRNA-based toxicity is the main contributor to DM1 manifestations; however, not much is known about the significance of the DMPK protein. To appreciate its normal and possible pathobiological role, we analyzed the patterns of DMPK splice isoform expression in mouse tissues. Long membrane-anchored DMPK dominated in heart, diaphragm, and skeletal muscle, whereas short cytosolic isoforms were highly expressed in bladder and stomach. Both isoform types were present in diverse brain regions. DMPK protein was also detectable in cultured myoblasts, myotubes, cortical astrocytes, and related cell lines of neural or muscle origin, but not in hippocampal neurons. This work identifies DMPK as a kinase with pronounced expression in diverse muscle and neural tissues that are affected in DM1.

Coiled-coil interactions modulate multimerization, mitochondrial binding and kinase activity of myotonic dystrophy protein kinase splice isoforms.

The myotonic dystrophy protein kinase polypeptide repertoire in mice and humans consists of six different splice isoforms that vary in the nature of their C-terminal tails and in the presence or absence of an internal Val-Ser-Gly-Gly-Gly motif. Here, we demonstrate that myotonic dystrophy protein kinase isoforms exist in high-molecular-weight complexes controlled by homo- and heteromultimerization. This multimerization is mediated by coiled-coil interactions in the tail-proximal domain and occurs independently of alternatively spliced protein segments or myotonic dystrophy protein kinase activity. Complex formation was impaired in myotonic dystrophy protein kinase mutants in which three leucines at positions a and d in the coiled-coil heptad repeats were mutated to glycines. These coiled-coil mutants were still capable of autophosphorylation and transphosphorylation of peptides, but the rates of their kinase activities were significantly lowered. Moreover, phosphorylation of the natural myotonic dystrophy protein kinase substrate, myosin phosphatase targeting subunit, was preserved, even though binding of the myotonic dystrophy protein kinase to the myosin phosphatase targeting subunit was strongly reduced. Furthermore, the association of myotonic dystrophy protein kinase isoform C to the mitochondrial outer membrane was weakened when the coiled-coil interaction was perturbed. Our findings indicate that the coiled-coil domain modulates myotonic dystrophy protein kinase multimerization, substrate binding, kinase activity and subcellular localization characteristics.