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Angelman syndrome is a neurodevelopmental disorder caused by disrupted function of the maternal copy of the imprinted UBE3A gene; here, targeting a long non-coding RNA that is responsible for silencing the paternal copy of UBE3A with antisense oligonucleotides is shown to partially restore UBE3A expression in the central nervous system and correct some cognitive deficits in a mouse model of the disease. Therapy for Angelman syndrome Frank Rigo and colleagues report the development of the first gene-specific therapy for Angelman syndrome, a severe neurodevelopmental disorder caused by disrupted function of the maternal copy of the imprinted gene UBE3A. The paternal copy of UBE3A is intact but silenced by a long non-coding RNA antisense transcript, UBE3A-ATS . The authors show that by reducing Ube3a-ATS with antisense oligonucleotides (ASOs), the silencing of paternal Ube3a can be reversed in cultured mouse neurons and in vivo . Some phenotypes in an Angelman syndrome mouse model, including obesity and memory impairment can also be corrected. Angelman syndrome is a single-gene disorder characterized by intellectual disability, developmental delay, behavioural uniqueness, speech impairment, seizures and ataxia 1 , 2 . It is caused by maternal deficiency of the imprinted gene UBE3A , encoding an E3 ubiquitin ligase 3 , 4 , 5 . All patients carry at least one copy of paternal UBE3A , which is intact but silenced by a nuclear-localized long non-coding RNA, UBE3A antisense transcript ( UBE3A-ATS ) 6 , 7 , 8 . Murine Ube3a-ATS reduction by either transcription termination or topoisomerase I inhibition has been shown to increase paternal Ube3a expression 9 , 10 . Despite a clear understanding of the disease-causing event in Angelman syndrome and the potential to harness the intact paternal allele to correct the disease, no gene-specific treatment exists for patients. Here we developed a potential therapeutic intervention for Angelman syndrome by reducing Ube3a-ATS with antisense oligonucleotides (ASOs). ASO treatment achieved specific reduction of Ube3a-ATS and sustained unsilencing of paternal Ube3a in neurons in vitro and in vivo . Partial restoration of UBE3A protein in an Angelman syndrome mouse model ameliorated some cognitive deficits associated with the disease. Although additional studies of phenotypic correction are needed, we have developed a sequence-specific and clinically feasible method to activate expression of the paternal Ube3a allele.

T helper 17 (T H 17) lymphocytes protect mucosal barriers from infections, but also contribute to multiple chronic inflammatory diseases. Their differentiation is controlled by RORγt, a ligand-regulated nuclear receptor. Here we identify the RNA helicase DEAD-box protein 5 (DDX5) as a RORγt partner that coordinates transcription of selective T H 17 genes, and is required for T H 17-mediated inflammatory pathologies. Surprisingly, the ability of DDX5 to interact with RORγt and coactivate its targets depends on intrinsic RNA helicase activity and binding of a conserved nuclear long noncoding RNA (lncRNA), Rmrp , which is mutated in patients with cartilage-hair hypoplasia. A targeted Rmrp gene mutation in mice, corresponding to a gene mutation in cartilage-hair hypoplasia patients, altered lncRNA chromatin occupancy, and reduced the DDX5–RORγt interaction and RORγt target gene transcription. Elucidation of the link between Rmrp and the DDX5–RORγt complex reveals a role for RNA helicases and lncRNAs in tissue-specific transcriptional regulation, and provides new opportunities for therapeutic intervention in T H 17-dependent diseases. The ability of the DEAD-box RNA helicase DDX5 to interact with master transcription factor RORγt is dependent on binding of the long noncoding RNA Rmrp ; the DDX5–RORγt complex coordinates transcription of selective T H 17 genes and is required for the pathogenicity of T H 17 cells. Modifiers of T H 17 cell pathogenicity The ability of the DEAD-box RNA helicase DDX5 to interact with master transcription factor RORγt is shown to be dependent on binding of the long noncoding RNA Rmrp . The DDX5–RORγt complex coordinates transcription of selective T-helper 17 (T H 17) genes, and is required for the pathogenicity of T H 17 lymphocytes. The discovery of this relationship between an RNA helicase and a long noncoding RNA in complex provide new insight into the role of transcriptional regulation and suggests new avenues for research into T H 17-dependent diseases.

Nusinersen is a 2′-O-methoxyethyl phosphorothioate-modified antisense drug being developed to treat spinal muscular atrophy. Nusinersen is specifically designed to alter splicing of SMN2 pre-mRNA and thus increase the amount of functional survival motor neuron (SMN) protein that is deficient in patients with spinal muscular atrophy. This open-label, phase 2, escalating dose clinical study assessed the safety and tolerability, pharmacokinetics, and clinical efficacy of multiple intrathecal doses of nusinersen (6 mg and 12 mg dose equivalents) in patients with infantile-onset spinal muscular atrophy. Eligible participants were of either gender aged between 3 weeks and 7 months old with onset of spinal muscular atrophy symptoms between 3 weeks and 6 months, who had SMN1 homozygous gene deletion or mutation. Safety assessments included adverse events, physical and neurological examinations, vital signs, clinical laboratory tests, cerebrospinal fluid laboratory tests, and electrocardiographs. Clinical efficacy assessments included event free survival, and change from baseline of two assessments of motor function: the motor milestones portion of the Hammersmith Infant Neurological Exam—Part 2 (HINE-2) and the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP-INTEND) motor function test, and compound motor action potentials. Autopsy tissue was analysed for target engagement, drug concentrations, and pharmacological activity. HINE-2, CHOP-INTEND, and compound motor action potential were compared between baseline and last visit using the Wilcoxon signed-rank test. Age at death or permanent ventilation was compared with natural history using the log-rank test. The study is registered at ClinicalTrials.gov, number NCT01839656. 20 participants were enrolled between May 3, 2013, and July 9, 2014, and assessed through to an interim analysis done on Jan 26, 2016. All participants experienced adverse events, with 77 serious adverse events reported in 16 participants, all considered by study investigators not related or unlikely related to the study drug. In the 12 mg dose group, incremental achievements of motor milestones (p<0·0001), improvements in CHOP-INTEND motor function scores (p=0·0013), and increased compound muscle action potential amplitude of the ulnar nerve (p=0·0103) and peroneal nerve (p<0·0001), compared with baseline, were observed. Median age at death or permanent ventilation was not reached and the Kaplan-Meier survival curve diverged from a published natural history case series (p=0·0014). Analysis of autopsy tissue from patients exposed to nusinersen showed drug uptake into motor neurons throughout the spinal cord and neurons and other cell types in the brainstem and other brain regions, exposure at therapeutic concentrations, and increased SMN2 mRNA exon 7 inclusion and SMN protein concentrations in the spinal cord. Administration of multiple intrathecal doses of nusinersen showed acceptable safety and tolerability, pharmacology consistent with its intended mechanism of action, and encouraging clinical efficacy. Results informed the design of an ongoing, sham-controlled, phase 3 clinical study of nusinersen in infantile-onset spinal muscular atrophy. Ionis Pharmaceuticals, Inc and Biogen.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are associated with loss of nuclear transactive response DNA-binding protein 43 (TDP-43). Here we identify that TDP-43 regulates expression of the neuronal growth-associated factor stathmin-2. Lowered TDP-43 levels, which reduce its binding to sites within the first intron of stathmin-2 pre-messenger RNA, uncover a cryptic polyadenylation site whose utilization produces a truncated, non-functional mRNA. Reduced stathmin-2 expression is found in neurons trans-differentiated from patient fibroblasts expressing an ALS-causing TDP-43 mutation, in motor cortex and spinal motor neurons from patients with sporadic ALS and familial ALS with GGGGCC repeat expansion in the C9orf72 gene, and in induced pluripotent stem cell (iPSC)-derived motor neurons depleted of TDP-43. Remarkably, while reduction in TDP-43 is shown to inhibit axonal regeneration of iPSC-derived motor neurons, rescue of stathmin-2 expression restores axonal regenerative capacity. Thus, premature polyadenylation-mediated reduction in stathmin-2 is a hallmark of ALS–FTD that functionally links reduced nuclear TDP-43 function to enhanced neuronal vulnerability.The mRNA encoding stathmin-2, a protein implicated in axonal growth, is shown to be widely suppressed by premature polyadenylation in both sporadic and C9orf72 ALS through a mechanism directly dependent on loss of nuclear TDP-43 in motor neurons.

A decrease in ataxin-2 levels leads to a reduction in the aggregation of TDP-43, markedly increased lifespan and improved motor function in a transgenic mouse model of TDP-43 proteinopathy. Neurodegeneration therapy Ataxin-2 polyglutamine expansions increase the risk for amyotrophic lateral sclerosis (ALS) and cause spinocerebellar ataxia type 2 (SCA2), two neurodegenerative diseases without a cure. A pair of papers this week report therapeutic approaches towards reducing ataxin-2. Nearly all ALS patients have toxic aggregates of the protein TDP-43 in the brain and spinal cord. Lowering ataxin-2 has been shown to suppress TDP-43 toxicity in yeast and flies, and Lindsay Becker et al . now show that lowering ataxin-2 in mice, genetically or with antisense oligonucleotides, reduces TDP-43 aggregation and toxicity, improves motor function and increases lifespan. Elsewhere in this issue, Daniel Scoles et al . test antisense oligonucleotides (ASOs) against ataxin-2 in mice models of SCA2 that recreate progressive adult-onset dysfunction and degeneration of the neuronal network. The most promising therapeutic lead is ASO7, which downregulates ATXN2 mRNA and protein and delays the onset of SCA2 phenotypes. Moreover, treatment of symptomatic mice normalizes firing of cerebellar Purkinje cells and improves motor functioning. Both papers suggest that antisense oligonucleotide-based therapeutic approaches could be used to tackle neurodegeneration. Amyotrophic lateral sclerosis (ALS) is a rapidly progressing neurodegenerative disease that is characterized by motor neuron loss and that leads to paralysis and death 2–5 years after disease onset 1 . Nearly all patients with ALS have aggregates of the RNA-binding protein TDP-43 in their brains and spinal cords 2 , and rare mutations in the gene encoding TDP-43 can cause ALS 3 . There are no effective TDP-43-directed therapies for ALS or related TDP-43 proteinopathies, such as frontotemporal dementia. Antisense oligonucleotides (ASOs) and RNA-interference approaches are emerging as attractive therapeutic strategies in neurological diseases 4 . Indeed, treatment of a rat model of inherited ALS (caused by a mutation in Sod1 ) with ASOs against Sod1 has been shown to substantially slow disease progression 5 . However, as SOD1 mutations account for only around 2–5% of ALS cases, additional therapeutic strategies are needed. Silencing TDP-43 itself is probably not appropriate, given its critical cellular functions 1 , 6 . Here we present a promising alternative therapeutic strategy for ALS that involves targeting ataxin-2. A decrease in ataxin-2 suppresses TDP-43 toxicity in yeast and flies 7 , and intermediate-length polyglutamine expansions in the ataxin-2 gene increase risk of ALS 7 , 8 . We used two independent approaches to test whether decreasing ataxin-2 levels could mitigate disease in a mouse model of TDP-43 proteinopathy 9 . First, we crossed ataxin-2 knockout mice with TDP-43 (also known as TARDBP ) transgenic mice. The decrease in ataxin-2 reduced aggregation of TDP-43, markedly increased survival and improved motor function. Second, in a more therapeutically applicable approach, we administered ASOs targeting ataxin-2 to the central nervous system of TDP-43 transgenic mice. This single treatment markedly extended survival. Because TDP-43 aggregation is a component of nearly all cases of ALS 6 , targeting ataxin-2 could represent a broadly effective therapeutic strategy.

Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5′ end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood–brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration. Genes in the rodent brain are knocked down by DNA/RNA heteroduplexes injected intravenously.

Talking antisense: rescue of SMN2 in motor neurone disease Spinal muscular atrophy (SMA) is a motor neurone disease caused by a mutation in a gene called SMN1 that is necessary for the survival of motor neurons. Humans have a duplicate gene, SMN2 , but that is barely expressed. One promising form of therapy involves increasing SMN2 expression. It has been assumed that it would be necessary to increase the expression of SMN2 in spinal cord motor neurons to achieve a therapeutic effect. Not so. In a mouse model of SMA, subcutaneous, peripheral administration of an antisense oligonucleotide that corrects a splicing defect in SMN2 is shown to provide a much more powerful therapy than direct delivery to the brain. Surprisingly, peripheral rescue is found to be essential for long-term rescue of SMA, and biomarkers suggest that the liver has an important role of the liver in SMA pathogenesis. Spinal muscular atrophy (SMA) is a motor neuron disease and the leading genetic cause of infant mortality; it results from loss-of-function mutations in the survival motor neuron 1 ( SMN1 ) gene 1 . Humans have a paralogue, SMN2 , whose exon 7 is predominantly skipped 2 , but the limited amount of functional, full-length SMN protein expressed from SMN2 cannot fully compensate for a lack of SMN1 . SMN is important for the biogenesis of spliceosomal small nuclear ribonucleoprotein particles 3 , but downstream splicing targets involved in pathogenesis remain elusive. There is no effective SMA treatment, but SMN restoration in spinal cord motor neurons is thought to be necessary and sufficient 4 . Non-central nervous system (CNS) pathologies, including cardiovascular defects, were recently reported in severe SMA mouse models and patients 5 , 6 , 7 , 8 , reflecting autonomic dysfunction or direct effects in cardiac tissues. Here we compared systemic versus CNS restoration of SMN in a severe mouse model 9 , 10 . We used an antisense oligonucleotide (ASO), ASO-10-27, that effectively corrects SMN2 splicing and restores SMN expression in motor neurons after intracerebroventricular injection 11 , 12 . Systemic administration of ASO-10-27 to neonates robustly rescued severe SMA mice, much more effectively than intracerebroventricular administration; subcutaneous injections extended the median lifespan by 25 fold. Furthermore, neonatal SMA mice had decreased hepatic Igfals expression, leading to a pronounced reduction in circulating insulin-like growth factor 1 (IGF1), and ASO-10-27 treatment restored IGF1 to normal levels. These results suggest that the liver is important in SMA pathogenesis, underscoring the importance of SMN in peripheral tissues, and demonstrate the efficacy of a promising drug candidate.

Hutchinson–Gilford progeria syndrome (HGPS) is a rare, invariably fatal childhood premature aging disorder caused by a pre-messenger RNA (mRNA) splicing defect in the LMNA gene. We used combined in vitro screening and in vivo validation to systematically explore the effects of target sequence, backbone chemistry and mechanism of action to identify optimized antisense oligonucleotides (ASOs) for therapeutic use in HGPS. In a library of 198 ASOs, the most potent ASOs targeted the LMNA exon 12 junction and acted via non-RNase H-mediated mechanisms. Treatment with an optimized lead candidate resulted in extension of lifespan in a mouse model of HGPS. Progerin mRNA levels were robustly reduced in vivo, but the extent of progerin protein reduction differed between tissues, suggesting a long half-life and tissue-specific turnover of progerin in vivo. These results identify a novel therapeutic agent for HGPS and provide insight into the HGPS disease mechanism. Optimized antisense oligonucleotides that can reduce progerin tissue expression in vivo pave the way for development of a novel therapeutic approach for Hutchinson–Gilford progeria syndrome.

Misregulation of long non-coding RNA (lncRNA) genes has been linked to a wide variety of cancer types. Here we report on Mammary Tumor Associated RNA 25 ( MaTAR25 ), a nuclear enriched and chromatin associated lncRNA that plays a role in mammary tumor cell proliferation, migration, and invasion, both in vitro and in vivo. MaTAR25 functions by interacting with purine rich element binding protein B (PURB), and associating with a major downstream target gene Tensin1 ( Tns1 ) to regulate its expression in trans . The Tns1 protein product is a critical component of focal adhesions linking signaling between the extracellular matrix and the actin cytoskeleton. Knockout of MaTAR25 results in down-regulation of Tns1 leading to a reorganization of the actin cytoskeleton, and a reduction of focal adhesions and microvilli. We identify LINC01271 as the human ortholog of MaTAR25 , and importantly, increased expression of LINC01271 is associated with poor patient prognosis and metastasis. Our findings demonstrate that LINC01271 represents a potential therapeutic target to alter breast cancer progression. A group of long non-coding RNAs (lncRNAs), Mammary Tumor Associated RNA s   1-30 ( MaTARs   1-30 ), are differentially expressed between mammary tumor cells and normal mammary epithelial cells. Here the authors report that MaTAR25 plays a role in breast cancer cell proliferation, migration and invasion by regulating the expression of the Tensin1 gene in trans .

Genetic correction of MeCP2 levels largely reversed the behavioural, molecular and physiological deficits associated with MECP2 duplication syndrome in a transgenic mouse model; similarly, reduction of MeCP2 levels using an antisense oligonucleotide strategy resulted in phenotypic rescue in adult transgenic mice, and dose-dependently corrected MeCP2 levels in cells from patients with MECP2 duplication. Potential reversal of a developmental disorder MECP2 duplication syndrome is a childhood disorder caused by duplication of the MECP2 gene and, consequently, increased MECP2 protein levels. Huda Zoghbi and colleagues report that genetic correction of MECP2 levels largely reverses the behavioural, molecular and physiological deficits in a transgenic mouse model. Reducing MECP2 levels using an antisense oligonucleotide (ASO) strategy—which has greater potential for therapeutic application—similarly resulted in phenotypic rescue in adult transgenic mice and dose-dependently corrected MECP2 levels in cells from patients with MECP2 duplication. These findings suggest that a disorder caused by copy number variation can be reversed after symptoms have emerged. Copy number variations have been frequently associated with developmental delay, intellectual disability and autism spectrum disorders 1 . MECP2 duplication syndrome is one of the most common genomic rearrangements in males 2 and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections and early death 3 , 4 , 5 . The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical-pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question that we addressed was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders 6 , 7 , 8 , including loss of MeCP2 in Rett syndrome 9 , indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we propose that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. By generating and characterizing a conditional Mecp2- overexpressing mouse model, here we show that correction of MeCP2 levels largely reverses the behavioural, molecular and electrophysiological deficits. We also reduced MeCP2 using an antisense oligonucleotide strategy, which has greater translational potential. Antisense oligonucleotides are small, modified nucleic acids that can selectively hybridize with messenger RNA transcribed from a target gene and silence it 10 , 11 , and have been successfully used to correct deficits in different mouse models 12 , 13 , 14 , 15 , 16 , 17 , 18 . We find that antisense oligonucleotide treatment induces a broad phenotypic rescue in adult symptomatic transgenic MECP2 duplication mice ( MECP2 -TG) 19 , 20 , and corrected MECP2 levels in lymphoblastoid cells from MECP2 duplication patients in a dose-dependent manner.