α-Synuclein antisense oligonucleotides as a disease-modifying therapy for Parkinson's disease.

Parkinson's disease (PD) is a prevalent neurodegenerative disease with no approved disease-modifying therapies. Multiplications, mutations, and single nucleotide polymorphisms in the SNCA gene, encoding α-synuclein (aSyn) protein, either cause or increase risk for PD. Intracellular accumulations of aSyn are pathological hallmarks of PD. Taken together, reduction of aSyn production may provide a disease-modifying therapy for PD. We show that antisense oligonucleotides (ASOs) reduce production of aSyn in rodent preformed fibril (PFF) models of PD. Reduced aSyn production leads to prevention and removal of established aSyn pathology and prevents dopaminergic cell dysfunction. In addition, we address the translational potential of the approach through characterization of human SNCA-targeting ASOs that efficiently suppress the human SNCA transcript in vivo. We demonstrate broad activity and distribution of the human SNCA ASOs throughout the nonhuman primate brain and a corresponding decrease in aSyn cerebral spinal fluid (CSF) levels. Taken together, these data suggest that, by inhibiting production of aSyn, it may be possible to reverse established pathology; thus, these data support the development of SNCA ASOs as a potential disease-modifying therapy for PD and related synucleinopathies.

PMP22 antisense oligonucleotides reverse Charcot-Marie-Tooth disease type 1A features in rodent models.

Charcot-Marie-Tooth disease type 1A (CMT1A) is caused by duplication of peripheral myelin protein 22 (PMP22) and is the most common hereditary peripheral neuropathy. CMT1A is characterized by demyelination and axonal loss, which underlie slowed motor nerve conduction velocity (MNCV) and reduced compound muscle action potentials (CMAP) in patients. There is currently no known treatment for this disease. Here, we show that antisense oligonucleotides (ASOs) effectively suppress PMP22 mRNA in affected nerves in 2 murine CMT1A models. Notably, initiation of ASO treatment after disease onset restored myelination, MNCV, and CMAP almost to levels seen in WT animals. In addition to disease-associated gene expression networks that were restored with ASO treatment, we also identified potential disease biomarkers through transcriptomic profiling. Furthermore, we demonstrated that reduction of PMP22 mRNA in skin biopsies from ASO-treated rats is a suitable biomarker for evaluating target engagement in response to ASO therapy. These results support the use of ASOs as a potential treatment for CMT1A and elucidate potential disease and target engagement biomarkers for use in future clinical trials.

Intrathecal administration of antisense oligonucleotide against p38α but not p38ß MAP kinase isoform reduces neuropathic and postoperative pain and TLR4-induced pain in male mice.

p38 mitogen-activated protein kinase (MAPK) consists of two major isoforms: p38α and p38ß; however, it remains unclear which isoform is more important for chronic pain development. Recently, we developed potent, long-lasting, and p38 MAPK subtype-specific antisense oligonucleotides (ASOs). We examined the therapeutic effects of isoform-specific ASOs in several chronic pain models following single intrathecal injection (300 µg/10 µl) in CD1 mice. In the chronic constriction injury (CCI) model, p38α MAPK ASO, given on post-operative day 5, reduced CCI-induced mechanical allodynia in male but not female mice. In contrast, mechanical allodynia after CCI in both sexes was not affected by p38ß MAPK ASO. Intrathecal injection of p38α or p38ß ASO resulted in a partial reduction (≈ 50%) of spinal p38α or p38ß mRNA level, respectively, in both sexes at two weeks. In contrast, intrathecal injection of the ASOs did not affect p38α and p38ß MAPK mRNA levels in dorsal root ganglia. Intrathecal p38α ASO also reduced postoperative pain (mechanical and cold allodynia) in male mice after tibia fracture. However, intrathecal p38α ASO had no effect on mechanical allodynia in male mice after paclitaxel treatment. Intrathecal p38α MAPK ASO pre-treatment also prevented TLR4-mediated mechanical allodynia and downregulated levels of p38α MAPK and phosphorylated p38 MAPK following intrathecal treatment of lipopolysaccharide. In summary, our findings suggest that p38α MAPK is the major p38 MAPK isoform in the spinal cord and regulates chronic pain in a sex and model-dependent manner. Intrathecal p38α MAPK ASO may offer a new treatment for some chronic pain conditions.

Antisense oligonucleotides selectively suppress target RNA in nociceptive neurons of the pain system and can ameliorate mechanical pain.

There is an urgent need for better treatments for chronic pain, which affects more than 1 billion people worldwide. Antisense oligonucleotides (ASOs) have proven successful in treating children with spinal muscular atrophy, a severe infantile neurological disorder, and several ASOs are currently being tested in clinical trials for various neurological disorders. Here, we characterize the pharmacodynamic activity of ASOs in spinal cord and dorsal root ganglia (DRG), key tissues for pain signaling. We demonstrate that activity of ASOs lasts up to 2 months after a single intrathecal bolus dose. Interestingly, comparison of subcutaneous, intracerebroventricular, and intrathecal administration shows that DRGs are targetable by systemic and central delivery of ASOs, while target reduction in the spinal cord is achieved only after direct central delivery. Upon detailed characterization of ASO activity in individual cell populations in DRG, we observe robust target suppression in all neuronal populations, thereby establishing that ASOs are effective in the cell populations involved in pain propagation. Furthermore, we confirm that ASOs are selective and do not modulate basal pain sensation. We also demonstrate that ASOs targeting the sodium channel Nav1.7 induce sustained analgesia up to 4 weeks. Taken together, our findings support the idea that ASOs possess the required pharmacodynamic properties, along with a long duration of action beneficial for treating pain.

MBNL1-mediated regulation of differentiation RNAs promotes myofibroblast transformation and the fibrotic response.

The differentiation of fibroblasts into myofibroblasts mediates tissue wound healing and fibrotic remodelling, although the molecular programme underlying this process remains poorly understood. Here we perform a genome-wide screen for genes that control myofibroblast transformation, and identify the RNA-binding protein muscleblind-like1 (MBNL1). MBNL1 overexpression promotes transformation of fibroblasts into myofibroblasts, whereas loss of Mbnl1 abrogates transformation and impairs the fibrotic phase of wound healing in mouse models of myocardial infarction and dermal injury. Mechanistically, MBNL1 directly binds to and regulates a network of differentiation-specific and cytoskeletal/matrix-assembly transcripts to promote myofibroblast differentiation. One of these transcripts is the nodal transcriptional regulator serum response factor (SRF), whereas another is calcineurin Aß. CRISPR-Cas9-mediated gene-editing of the MBNL1-binding site within the Srf 3'UTR impairs myofibroblast differentiation, whereas in vivo deletion of Srf in fibroblasts impairs wound healing and fibrosis. These data establish a new RNA-dependent paradigm for myofibroblast formation through MBNL1.

MBNL Sequestration by Toxic RNAs and RNA Misprocessing in the Myotonic Dystrophy Brain.

For some neurological disorders, disease is primarily RNA mediated due to expression of non-coding microsatellite expansion RNAs (RNA(exp)). Toxicity is thought to result from enhanced binding of proteins to these expansions and depletion from their normal cellular targets. However, experimental evidence for this sequestration model is lacking. Here, we use HITS-CLIP and pre-mRNA processing analysis of human control versus myotonic dystrophy (DM) brains to provide compelling evidence for this RNA toxicity model. MBNL2 binds directly to DM repeat expansions in the brain, resulting in depletion from its normal RNA targets with downstream effects on alternative splicing and polyadenylation. Similar RNA processing defects were detected in Mbnl compound-knockout mice, highlighted by dysregulation of Mapt splicing and fetal tau isoform expression in adults. These results demonstrate that MBNL proteins are directly sequestered by RNA(exp) in the DM brain and introduce a powerful experimental tool to evaluate RNA-mediated toxicity in other expansion diseases.

Loss of MBNL leads to disruption of developmentally regulated alternative polyadenylation in RNA-mediated disease.

Inhibition of muscleblind-like (MBNL) activity due to sequestration by microsatellite expansion RNAs is a major pathogenic event in the RNA-mediated disease myotonic dystrophy (DM). Although MBNL1 and MBNL2 bind to nascent transcripts to regulate alternative splicing during muscle and brain development, another major binding site for the MBNL protein family is the 3' untranslated region of target RNAs. Here, we report that depletion of Mbnl proteins in mouse embryo fibroblasts leads to misregulation of thousands of alternative polyadenylation events. HITS-CLIP and minigene reporter analyses indicate that these polyadenylation switches are a direct consequence of MBNL binding to target RNAs. Misregulated alternative polyadenylation also occurs in skeletal muscle in a mouse polyCUG model and human DM, resulting in the persistence of neonatal polyadenylation patterns. These findings reveal an additional developmental function for MBNL proteins and demonstrate that DM is characterized by misregulation of pre-mRNA processing at multiple levels.

RNA-protein interactions in unstable microsatellite diseases.

A novel RNA-mediated disease mechanism has emerged from studies on dominantly inherited neurological disorders caused by unstable microsatellite expansions in non-coding regions of the genome. These non-coding tandem repeat expansions trigger the production of unusual RNAs that gain a toxic function, which involves the formation of RNA repeat structures that interact with, and alter the activities of, various factors required for normal RNA processing as well as additional cellular functions. In this review, we explore the deleterious effects of toxic RNA expression and discuss the various model systems currently available for studying RNA gain-of-function in neurologic diseases. Common themes, including bidirectional transcription and repeat-associated non-ATG (RAN) translation, have recently emerged from expansion disease studies. These and other discoveries have highlighted the need for further investigations designed to provide the additional mechanistic insights essential for future therapeutic development.

Compound loss of muscleblind-like function in myotonic dystrophy.

Myotonic dystrophy (DM) is a multi-systemic disease that impacts cardiac and skeletal muscle as well as the central nervous system (CNS). DM is unusual because it is an RNA-mediated disorder due to the expression of toxic microsatellite expansion RNAs that alter the activities of RNA processing factors, including the muscleblind-like (MBNL) proteins. While these mutant RNAs inhibit MBNL1 splicing activity in heart and skeletal muscles, Mbnl1 knockout mice fail to recapitulate the full-range of DM symptoms in these tissues. Here, we generate mouse Mbnl compound knockouts to test the hypothesis that Mbnl2 functionally compensates for Mbnl1 loss. Although Mbnl1(-/-) ; Mbnl2(-/-) double knockouts (DKOs) are embryonic lethal, Mbnl1(-/-) ; Mbnl2(+/-) mice are viable but develop cardinal features of DM muscle disease including reduced lifespan, heart conduction block, severe myotonia and progressive skeletal muscle weakness. Mbnl2 protein levels are elevated in Mbnl1(-/-) knockouts where Mbnl2 targets Mbnl1-regulated exons. These findings support the hypothesis that compound loss of MBNL function is a critical event in DM pathogenesis and provide novel mouse models to investigate additional pathways disrupted in this RNA-mediated disease.