Antisense oligonucleotides (AONs) hold promise for therapeutic correction of many genetic diseases via exon skipping, and the first AON-based drugs have entered clinical trials for neuromuscular disorders. However, despite advances in AON chemistry and design, systemic use of AONs is limited because of poor tissue uptake, and recent clinical reports confirm that sufficient therapeutic efficacy has not yet been achieved. Here we present a new class of AONs made of tricyclo-DNA (tcDNA), which displays unique pharmacological properties and unprecedented uptake by many tissues after systemic administration. We demonstrate these properties in two mouse models of Duchenne muscular dystrophy (DMD), a neurogenetic disease typically caused by frame-shifting deletions or nonsense mutations in the gene encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, respiratory failure and neurocognitive impairment. Although current naked AONs do not enter the heart or cross the blood-brain barrier to any substantial extent, we show that systemic delivery of tcDNA-AONs promotes a high degree of rescue of dystrophin expression in skeletal muscles, the heart and, to a lesser extent, the brain. Our results demonstrate for the first time a physiological improvement of cardio-respiratory functions and a correction of behavioral features in DMD model mice. This makes tcDNA-AON chemistry particularly attractive as a potential future therapy for patients with DMD and other neuromuscular disorders or with other diseases that are eligible for exon-skipping approaches requiring whole-body treatment.

The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic ␤-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.diabetes ͉ mutagenesis ͉ soluble N-ethylmaleimide-sensitive factor attachment protein receptor ͉ schizophrenia ͉ exocytosis I ntracellular membrane fusion events within eukaryotic cells are mediated by proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family. The prototypic SNARE-mediated fusion event is synaptic vesicle exocytosis, in which the membrane-associated SNAP-25 and syntaxin-1A interact with the vesicle-associated membrane protein (VAMP) to create a stable ternary core complex with 1:1:1 stoichiometry (1). This interaction has been extensively studied in various systems including neurons, neuroendocrine chromaffin cells, and pancreatic ␤-islet cells, all of which use the neuronal core complex proteins to regulate secretion (2, 3). In vivo knockout and mutagenesis studies in invertebrate systems have allowed SNARE function to be analyzed at the level of the synapse (4), but their scope has been limited by the failure of these organisms to model higher neurological functions in which SNARE function is strongly implicated. A major goal, therefore, has been the generation of a mammalian model of SNARE dysfunction in which the effects on complex neurological and behavioral phenotypes can be assessed.Both Vamp2 and Snap25 knockouts are perinatally lethal in homozygous mice, and heterozygotes show no apparent phenotype, so detailed analysis of viable affected individuals has not been possible (5, 6). The spontaneous mouse mutant coloboma carries a heterozygous deletion spanning four genes including Snap25 (7), but its locomotor hyperactivity phenotype is likely to depend on the additional loss of one or more of the other genes within the deleted region. Most recently, a viable syntaxin-1a knockout mouse has been described with subtle defects in long-term potentiation and fear-memory behavior, although normal synaptic transmission is unaffected likely because of functional compensation by syntaxin-1b (8). Existing mammalian SNARE mutations have therefore offered only very limited insights into the consequences of SNARE dysfunction in an adult system and have yielded no informat...

Duchenne muscular dystrophy (DMD) is an X-linked recessive disease caused, in most cases, by the complete absence of the 427 kDa cytoskeletal protein, dystrophin. There is no effective treatment, and affected individuals die from respiratory failure and cardiomyopathy by age 30. Here, we investigated whether cardiomyopathy could be prevented in animal models of DMD by increasing diaphragm utrophin or dystrophin expression and thereby restoring diaphragm function. In a transgenic mdx mouse, where utrophin was over expressed in the skeletal muscle and the diaphragm, but not in the heart, we found cardiac function, specifically right and left ventricular ejection fraction as measured using in vivo magnetic resonance imaging, was restored to wild-type levels. In mdx mice treated with a peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO) that resulted in high levels of dystrophin restoration in the skeletal muscle and the diaphragm only, cardiac function was also restored to wild-type levels. In dystrophin/utrophin-deficient double-knockout (dKO) mice, a more severely affected animal model of DMD, treatment with a PPMO again produced high levels of dystrophin only in the skeletal muscle and the diaphragm, and once more restored cardiac function to wild-type levels. In the dKO mouse, there was no difference in heart function between treatment of the diaphragm plus the heart and treatment of the diaphragm alone. Restoration of diaphragm and other respiratory muscle function, irrespective of the method used, was sufficient to prevent cardiomyopathy in dystrophic mice. This novel mechanism of treating respiratory muscles to prevent cardiomyopathy in dystrophic mice warrants further investigation for its implications on the need to directly treat the heart in DMD.

Duchenne muscular dystrophy (DMD) is a severe neuromuscular disorder caused by mutations in the dystrophin gene that result in the absence of functional protein. Antisense-mediated exon skipping is one of the most promising approaches for the treatment of DMD and recent clinical trials have demonstrated encouraging results. However, antisense oligonucleotide-mediated exon skipping for DMD still faces major hurdles such as extremely low efficacy in the cardiac muscle, poor cellular uptake and relatively rapid clearance from circulation, which means that repeated administrations are required to achieve some therapeutic efficacy. To overcome these limitations, we previously proposed the use of small nuclear RNAs (snRNAs), especially U7snRNA to shuttle the antisense sequences after vectorization into adeno-associated virus (AAV) vectors. In this study, we report for the first time the efficiency of the AAV-mediated exon skipping approach in the utrophin/dystrophin double-knockout (dKO) mouse which is a very severe and progressive mouse model of DMD. Following a single intravenous injection of scAAV9-U7ex23 in dKO mice, near-normal levels of dystrophin expression were restored in all muscles examined, including the heart. This resulted in a considerable improvement of their muscle function and dystrophic pathology as well as a remarkable extension of the dKO mice lifespan. These findings suggest great potential for AAV-U7 in systemic treatment of the DMD phenotype.