Search

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.

Cell-to-cell communications are critical determinants of pathophysiological phenotypes, but methodologies for their systematic elucidation are lacking. Herein, we propose an approach for the Systematic Elucidation and Assessment of Regulatory Cell-to-cell Interaction Networks (SEARCHIN) to identify ligand-mediated interactions between distinct cellular compartments. To test this approach, we selected a model of amyotrophic lateral sclerosis (ALS), in which astrocytes expressing mutant superoxide dismutase-1 (mutSOD1) kill wild-type motor neurons (MNs) by an unknown mechanism. Our integrative analysis that combines proteomics and regulatory network analysis infers the interaction between astrocyte-released amyloid precursor protein (APP) and death receptor-6 (DR6) on MNs as the top predicted ligand-receptor pair. The inferred deleterious role of APP and DR6 is confirmed in vitro in models of ALS. Moreover, the DR6 knockdown in MNs of transgenic mutSOD1 mice attenuates the ALS-like phenotype. Our results support the usefulness of integrative, systems biology approach to gain insights into complex neurobiological disease processes as in ALS and posit that the proposed methodology is not restricted to this biological context and could be used in a variety of other non-cell-autonomous communication mechanisms., Neuron-astrocyte communication plays a key role in pathophysiology, however systematic approaches to unveil it are limited. Here, the authors propose SEARCHIN, a multi-modal integrated workflow, as a tool to identify cross-compartment ligand-receptor interactions, applied to ALS models.

Purpose Pathological stimuli or injury to the peripheral nervous system can trigger neuropathic pain with common clinical features such as allodynia and hypersensitivity. Although various studies have identified molecules or genes related to neuropathic pain, the essential components are still unclear. Therefore, in this study, we investigated the molecular and genetic factors related to neuropathic pain. Methods We extracted candidate genes in the dorsal root ganglion (DRG) from three nerve injury mouse models and a sham-operated model (sciatic nerve ligation and resection, sural nerve resection, spared nerve injury [SNI], and sham) using DNA microarray to elucidate the genes responsible for the neuropathic pain mechanism in the SNI model, which exhibits hypersensitivity in the hindpaw of the preserved sural nerve area. We eliminated as many biases as possible. We then focused on an upregulated endogenous vasopressin receptor and clarified whether it is closely associated with traumatic neuropathic pain using a knockout mouse and drug-mediated suppression of the gene. Results Algorithm analysis of DNA microarray results identified 50 genes significantly upregulated in the DRG of the SNI model. Two independent genes-cyclin-dependent kinase-1 (CDK-1) and arginine vasopressin receptor 1A (V1a)-were subsequently identified as candidate SNI-specific genes in the DRG by quantitative PCR analysis. Administration of V1a agonist to wild-type SNI mice significantly alleviated neuropathic pain. However, V1a knockout mice did not exhibit higher hypersensitivity to mechanical stimulation than wild-type mice. In addition, V1a knockout mice showed similar pain behaviors after SNI to wild-type mice. Conclusion Through the DNA microarray analysis of several neuropathic models, we detected specific genes related to chronic pain. In particular, our results suggest that V1a in the DRG may partially contribute to the mechanism of neuropathic pain.

Parkinson’s disease (PD) is a neurodegenerative disease caused by the loss of dopaminergic neurons in the substantia nigra. A characteristic pathological feature of PD is cytoplasmic accumulation of α-synuclein (SNCA) protein. Multiplication of the SNCA gene in familial PD and pathological accumulation of SNCA protein during progression of sporadic PD suggest that increased SNCA protein levels increase the risk of PD. Thus, reducing SNCA expression levels could delay PD onset or modify the disease course. For efficient knock down, we designed and synthesized an amido-bridged nucleic acids (AmNA)-modified antisense oligonucleotide (ASO) that targeted SNCA with improved stability and cellular uptake in vivo. AmNA-ASO efficiently downregulated SNCA at both the mRNA and protein level in vitro and in vivo. Notably, AmNA-ASO was efficiently delivered into the mouse brain by intracerebroventricular injection without the aid of additional chemicals. Furthermore, administration of AmNA-ASO ameliorated neurological defects in PD model mice expressing human wild type SNCA. Taken together, these findings suggest that AmNA-ASO is a promising therapeutic strategy for SNCA-associated pathology in PD.

Duchenne muscular dystrophy (DMD) is caused by mutations in DMD, which codes for dystrophin. Because the progressive and irreversible degeneration of muscle occurs from childhood, earlier therapy is required to prevent dystrophic progression. Exon skipping by antisense oligonucleotides called phosphorodiamidate morpholino oligomers (PMOs), which restores the DMD reading frame and dystrophin expression, is a promising candidate for use in neonatal patients, yet the potential remains unclear. Here, we investigate the systemic efficacy and safety of early exon skipping in dystrophic dog neonates. Intravenous treatment of canine X-linked muscular dystrophy in Japan dogs with a 4-PMO cocktail resulted in ∼3%-27% in-frame exon 6-9 skipping and dystrophin restoration across skeletal muscles up to 14% of healthy levels. Histopathology was ameliorated with the reduction of fibrosis and/or necrosis area and centrally nucleated fibers, significantly in the diaphragm. Treatment induced cardiac multi-exon skipping, though dystrophin rescue was not detected. Functionally, treatment led to significant improvement in the standing test. Toxicity was not observed from blood tests. This is the first study to demonstrate successful multi-exon skipping treatment and significant functional improvement in dystrophic dogs. Early treatment was most beneficial for respiratory muscles, with implications for addressing pulmonary malfunction in patients.

Duchenne muscular dystrophy (DMD), the most common lethal heritable childhood disease, is caused by mutations in the DMD gene that result in the absence of functional dystrophin protein. Exon skipping mediated by antisense oligonucleotides has recently emerged as an effective approach for the restoration of dystrophin, and skipping of exon 51 of DMD has received accelerated approval. Identifying antisense sequences that can provide the highest possible skipping efficiency is crucial for future clinical applications. Herein, we systematically tested two-step antisense oligonucleotide walks along human DMD exon 53 in order to define sequence-dependent effects of antisense oligonucleotide binding sites in human rhabdomyosarcoma cell lines. The first rough whole-exon 53 walk enabled the identification of a target region, and a second walk of this region was used to determine an optimal antisense oligonucleotide sequence (NS-065/NCNP-01) for exon 53 skipping. This oligonucleotide strongly promoted exon 53 skipping in a dose-dependent manner during pre-mRNA splicing in rhabdomyosarcoma and DMD patient-derived cells, and it restored dystrophin protein levels in patient-derived cells. NS-065/NCNP-01, a phosphorodiamidate morpholino oligomer, appears to be a promising candidate for treating exon 53 skipping, and it is potentially applicable to 10.1% of patients with DMD. Keywords: Duchenne muscular dystrophy, dystrophin, exon 53, exon skipping, antisense therapeutics, morpholino

Brain endothelial cells (BECs) are involved in the pathogenesis of ischemic stroke. Recently, several microRNAs (miRNAs) in BECs were reported to regulate the endothelial function in ischemic brain. Therefore, modulation of miRNAs in BECs by a therapeutic oligonucleotide to inhibit miRNA (antimiR) could be a useful strategy for treating ischemic stroke. However, few attempts have been made to achieve this strategy via systemic route due to lack of efficient delivery-method toward BECs. Here, we have developed a new technology for delivering an antimiR into BECs and silencing miRNAs in BECs, using a mouse ischemic stroke model. We designed a heteroduplex oligonucleotide, comprising an antimiR against miRNA-126 (miR-126) known as the endothelial-specific miRNA and its complementary RNA, conjugated to α-tocopherol as a delivery ligand (Toc-HDO targeting miR-126). Intravenous administration of Toc-HDO targeting miR-126 remarkably suppressed miR-126 expression in ischemic brain of the model mice. In addition, we showed that Toc-HDO targeting miR-126 was delivered into BECs more efficiently than the parent antimiR in ischemic brain, and that it was delivered more effectively in ischemic brain than non-ischemic brain of this model mice. Our study highlights the potential of this technology as a new clinical therapeutic option for ischemic stroke.

Dystrophin, encoded by the DMD gene on the X chromosome, stabilizes the sarcolemma by linking the actin cytoskeleton with the dystrophin-glycoprotein complex (DGC). In-frame mutations in DMD cause a milder form of X-linked muscular dystrophy, called Becker muscular dystrophy (BMD), characterized by the reduced expression of truncated dystrophin. So far, no animal model with in-frame mutations in Dmd has been established. As a result, the effect of in-frame mutations on the dystrophin expression profile and disease progression of BMD remains unclear. In this study, we established a novel rat model carrying in-frame Dmd gene mutations (IF rats) and evaluated the pathology. We found that IF rats exhibited reduced expression of truncated dystrophin in a proteasome-independent manner. This abnormal dystrophin expression caused dystrophic changes in muscle tissues but did not lead to functional deficiency. We also found that the expression of additional dystrophin named dpX, which forms the DGC in the sarcolemma, was associated with the appearance of truncated dystrophin. In conclusion, the outcomes of this study contribute to the further understanding of BMD pathology and help elucidate the efficiency of dystrophin recovery treatments in Duchenne muscular dystrophy, a more severe form of X-linked muscular dystrophy., Editor's choice: The newly established rat model carrying in-frame mutations in the Dmd gene exhibits the dystrophic phenotype and abnormal dystrophin expression profile, similar to patients with Becker muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a fatal X-linked disorder caused by nonsense or frameshift mutations in the DMD gene. Among various treatments available for DMD, antisense oligonucleotides (ASOs) mediated exon skipping is a promising therapeutic approach. For successful treatments, however, it is requisite to rigorously optimise oligonucleotide chemistries as well as chemical modifications of ASOs. To achieve this, here, we aim to develop a novel enhanced green fluorescence protein (EGFP)-based reporter assay system that allows us to perform efficient and high-throughput screenings for ASOs. We design a new expression vector with a CAG promoter to detect the EGFP fluorescence only when skipping of mdx-type exon 23 is induced by ASOs. Then, an accurate screening was successfully conducted in C57BL/6 primary myotubes using phosphorodiamidate morpholino oligomer or locked nucleic acids (LNA)/2′-OMe mixmers with different extent of LNA inclusion. We accordingly generated a novel transgenic mouse model with this EGFP expression vector (EGFP-mdx23 Tg). Finally, we confirmed that the EGFP-mdx23 Tg provided a highly sensitive platform to check the effectiveness as well as the biodistribution of ASOs for exon skipping therapy. Thus, the assay system provides a simple yet highly sensitive platform to optimise oligonucleotide chemistries as well as chemical modifications of ASOs.

Duchenne muscular dystrophy (DMD) is a lethal genetic disorder caused by an absence of the dystrophin protein in bodywide muscles, including the heart. Cardiomyopathy is a leading cause of death in DMD. Exon skipping via synthetic phosphorodiamidate morpholino oligomers (PMOs) represents one of the most promising therapeutic options, yet PMOs have shown very little efficacy in cardiac muscle. To increase therapeutic potency in cardiac muscle, we tested a next-generation morpholino: arginine-rich, cell-penetrating peptide-conjugated PMOs (PPMOs) in the canine X-linked muscular dystrophy in Japan (CXMDJ) dog model of DMD. A PPMO cocktail designed to skip dystrophin exons 6 and 8 was injected intramuscularly, intracoronarily, or intravenously into CXMDJ dogs. Intravenous injections with PPMOs restored dystrophin expression in the myocardium and cardiac Purkinje fibers, as well as skeletal muscles. Vacuole degeneration of cardiac Purkinje fibers, as seen in DMD patients, was ameliorated in PPMO-treated dogs. Although symptoms and functions in skeletal muscle were not ameliorated by i.v. treatment, electrocardiogram abnormalities (increased Q-amplitude and Q/R ratio) were improved in CXMDJ dogs after intracoronary or i.v. administration. No obvious evidence of toxicity was found in blood tests throughout the monitoring period of one or four systemic treatments with the PPMO cocktail (12 mg/kg/injection). The present study reports the rescue of dystrophin expression and recovery of the conduction system in the heart of dystrophic dogs by PPMO-mediated multiexon skipping. We demonstrate that rescued dystrophin expression in the Purkinje fibers leads to the improvement/prevention of cardiac conduction abnormalities in the dystrophic heart.

Gapmer-type antisense oligonucleotides have not yet been approved for the treatment of central nervous system diseases, whereas steric-blocking-type antisense oligonucleotides have been well-developed for clinical use. We here characterize a new type of double-stranded oligonucleotides, overhanging-duplex oligonucleotides, which are composed of the parent gapmer and its extended complementary RNA. By intracerebroventricular injection, overhanging oligonucleotides show greater silencing potency with more efficient delivery into mouse brains than the parent single-stranded gapmer. Structure-activity relationship analyses reveal that the potency enhancement requires 13-mer or more overhanging oligonucleotides with a phosphorothioate backbone. Overhanging oligonucleotides provide a new platform of therapeutic oligonucleotides for gene modulation in the central nervous system.

Duchenne muscular dystrophy (DMD) and the less severe Becker muscular dystrophy (BMD) are due to mutations in the DMD gene. Previous reports show that in-frame deletion of exons 45–55 produces an internally shorted, but functional, dystrophin protein resulting in a very mild BMD phenotype. In order to elucidate the molecular mechanism leading to this phenotype, we generated exon 45–55 deleted dystrophin transgenic/mdx (Tg/mdx) mice. Muscular function of Tg/mdx mice was restored close to that of wild type (WT) mice but the localization of the neuronal type of nitric oxide synthase was changed from the sarcolemma to the cytosol. This led to hyper-nitrosylation of the ryanodine receptor 1 causing increased Ca2+ release from the sarcoplasmic reticulum. On the other hand, Ca2+ reuptake by the sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) was restored to the level of WT mice, suggesting that the Ca2+ dysregulation had been compensated by SERCA activation. In line with this, expression of sarcolipin (SLN), a SERCA-inhibitory peptide, was upregulated in mdx mice, but strongly reduced in Tg/mdx mice. Furthermore, knockdown of SLN ameliorated the cytosolic Ca2+ homeostasis and the dystrophic phenotype in mdx mice. These findings suggest that SLN may be a novel target for DMD therapy.

The blood-brain barrier (BBB) is increasingly regarded as a dynamic interface that adapts to the needs of the brain, responds to physiological changes, and gets affected by and can even promote diseases. Modulation of BBB function at the molecular level in vivo is beneficial for a variety of basic and clinical studies. Here we show that our heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide and its complementary RNA, conjugated to α-tocopherol as a delivery ligand, efficiently reduced the expression of organic anion transporter 3 (OAT3) gene in brain microvascular endothelial cells in mice. This proof-of-concept study demonstrates that intravenous administration of chemically synthesized HDO can remarkably silence OAT3 at the mRNA and protein levels. We also demonstrated modulation of the efflux transport function of OAT3 at the BBB in vivo. HDO will serve as a novel platform technology to advance the biology and pathophysiology of the BBB in vivo, and will also open a new therapeutic field of gene silencing at the BBB for the treatment of various intractable neurological disorders.

Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches available for the treatment of several neuromuscular disorders, including Duchenne muscular dystrophy. The main weakness of this treatment arises from the low efficiency and sporadic nature of delivery of the neutrally charged PMO into muscle fibers, the mechanism of which is unknown.Recently, using wild-type and dystrophic mdx52 mice, we showed that muscle fibers took up PMO more efficiently during myotube formation. Interestingly, through in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. Next, we tested the therapeutic potential of PMO in laminin-alpha2 (laminin-α2) chain-null dy 3K/dy 3K mice, a model of merosin-deficient congenital muscular dystrophy 1A (MDC1A) with active muscle regeneration. We confirmed the recovery of the laminin-α2 chain following skipping of the mutated exon 4 in dy 3K/dy 3K mice, which prolonged the life span of the animals slightly. These findings support the theory that PMO entry into fibers is dependent on the developmental stage in myogenesis rather than on dystrophinless muscle membranes, and provide a platform for the future development of PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration. Herein, we describe the methods for PMO transfection/injection and evaluation of the efficacy of exon skipping in the laminin-α2-deficient dy 3K/dy 3K mouse model both in vitro and in vivo.

Emerging concerns regarding heart failure, arrhythmia, and sudden death in patients with muscular dystrophy are of significant clinical importance. On the other hand, little attention has been paid to renal dysfunction because these patients have low serum creatinine levels. Serum cystatin C, unaffected by muscle quantity, is a potentially superior marker for estimating renal function. Here, we present cases with muscular dystrophy in which estimated glomerular filtration rate (GFR) by cystatin C (eGFRcys) provided good agreement with simultaneously measured GFR by inulin renal clearance (differences less than 20%). Sudden death with acute heart failure occurred in a patient with underlying renal dysfunction and elevated BNP. Neurologists and cardiologists should evaluate renal function using GFR with cystatin C in patients with muscular dystrophy.

Within the field of RNA therapeutics, antisense oligonucleotide-based therapeutics are a potentially powerful means of treating intractable diseases. However, if these therapeutics are used for the treatment of neurological disorders, safe yet efficient methods of delivering antisense oligonucleotides across the blood-brain barrier to the central nervous system must be developed. Here, we examined the use of angubindin-1, a binder to the tricellular tight junction, to modulate paracellular transport between brain microvascular endothelial cells in the blood-brain barrier for the delivery of antisense oligonucleotides to the central nervous system. This proof-of-concept study demonstrated that intravenously injected angubindin-1 increased the permeability of the blood-brain barrier and enabled transient delivery of subsequently administered antisense oligonucleotides into the mouse brain and spinal cord, leading to silencing of a target RNA without any overt adverse effects. We also found that two bicellular tight junction modulators did not produce such a silencing effect, suggesting that the tricellular tight junction is likely a better target for the delivery of antisense oligonucleotides than the bicellular tight junction. Our delivery strategy of modulating the tricellular tight junction in the blood-brain barrier via angubindin-1 provides a novel avenue of research for the development of antisense oligonucleotide-based therapeutics for the treatment of neurological disorders.

Duchenne muscular dystrophy (DMD) is a lethal hereditary muscle disease caused by mutations in the gene encoding the muscle protein dystrophin. These mutations result in a shift in the open reading frame leading to loss of the dystrophin protein. Antisense oligonucleotides (ASOs) that induce exon skipping correct this frame shift during pre-mRNA splicing and partially restore dystrophin expression in mouse and dog models. We conducted a phase 1, open-label, dose-escalation clinical trial to determine the safety, pharmacokinetics, and activity of NS-065/NCNP-01, a morpholino ASO that enables skipping of exon 53. Ten patients with DMD (6 to 16 years old), carrying mutations in the dystrophin gene whose reading frame would be restored by exon 53 skipping, were administered NS-065/NCNP-01 at doses of 1.25, 5, or 20 mg/kg weekly for 12 weeks. The primary endpoint was safety; the secondary endpoints were pharmacokinetics and successful exon skipping. No severe adverse drug reactions were observed, and no treatment discontinuation occurred. Muscle biopsy samples were taken before and after treatment and compared by reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence, and Western blotting to assess the amount of exon 53 skipping and dystrophin expression. NS-065/NCNP-01 induced exon 53 skipping in dystrophin-encoding mRNA in a dose-dependent manner and increased the dystrophin/spectrin ratio in 7 of 10 patients. Furthermore, the amount of exon skipping correlated with the maximum drug concentration in plasma (Cmax) and the area under the concentration-time curve in plasma (AUC0-t ). These results indicate that NS-065/NCNP-01 has a favorable safety profile and promising pharmacokinetics warranting further study in a phase 2 clinical trial.

Incorporation of 2′-O-methyl-2-thioribothymidine (s2Tm) into antisense oligoribonucleotides significantly enhanced the exon skipping activity in Duchenne muscular dystrophy model mice.

The reported prevalence of left ventricular noncompaction (LVNC) varies widely and its prognostic impact remains controversial. We sought to clarify the prevalence and prognostic impact of LVNC in patients with Duchenne/Becker muscular dystrophy (DMD/BMD).We evaluated the presence of LNVC in patients with DMD/BMD aged 4-64 years old at the study entry (from July 2007 to December 2008) and prospectively followed-up their subsequent courses (n=186). The study endpoint was all-cause death and the presence of LVNC was blinded until the end of the study (median follow-up: 46 months; interquartile range: 41-48 months).There were no significant differences in baseline characteristics between patients with LVNC (n=35) and control patients without LVNC (n=151), with the exception of LV function. Patients with LVNC showed, in comparison with patients without LVNC, a significant negative correlation between age and LVEF (R=-0.7 vs. R=-0.4) at baseline; and showed a significantly greater decrease in absolute LVEF (-8.6 ± 4.6 vs. -4.3 ± 4.5, p0.001) during the follow-up. A worse prognosis was observed in patients with LVNC (13/35 died) than in patients without LVNC (22/151 died, Log-rank p0.001). Multivariate Cox analysis revealed that LVNC is an independent prognostic factor (relative hazard 2.67 [95% CI: 1.19-5.96]).LVNC was prevalent in patients with DMD/BMD. The presence of LVNC is significantly associated with a rapid deterioration in LV function and higher mortality. Neurologists and cardiologists should pay more careful attention to the presence of LVNC.

Phosphorodiamidate morpholino oligomer (PMO)-mediated exon skipping is among the more promising approaches to the treatment of several neuromuscular disorders including Duchenne muscular dystrophy. The main weakness of this approach arises from the low efficiency and sporadic nature of the delivery of charge-neutral PMO into muscle fibers, the mechanism of which is unknown. In this study, to test our hypothesis that muscle fibers take up PMO more efficiently during myotube formation, we induced synchronous muscle regeneration by injection of cardiotoxin into the tibialis anterior muscle of Dmd exon 52-deficient mdx52 and wild-type mice. Interestingly, by in situ hybridization, we detected PMO mainly in embryonic myosin heavy chain-positive regenerating fibers. In addition, we showed that PMO or 2′-O-methyl phosphorothioate is taken up efficiently into C2C12 myotubes when transfected 24–72 h after the induction of differentiation but is poorly taken up into undifferentiated C2C12 myoblasts suggesting efficient uptake of PMO in the early stages of C2C12 myotube formation. Next, we tested the therapeutic potential of PMO for laminin-α2 chain-null dy3K/dy3K mice: a model of merosin-deficient congenital muscular dystrophy (MDC1A) with active muscle regeneration. We confirmed the recovery of laminin-α2 chain and slightly prolonged life span following skipping of the mutated exon 4 in dy3K/dy3K mice. These findings support the idea that PMO entry into fibers is dependent on a developmental stage in myogenesis rather than on dystrophinless muscle membranes and provide a platform for developing PMO-mediated therapies for a variety of muscular disorders, such as MDC1A, that involve active muscle regeneration.

Currently, antisense oligonucleotides (AONs)-mediated exon skipping therapy is the most promising therapy for Duchenne muscular dystrophy. We performed preclinical studies using dystrophic mdx52 mice and CXMD J dystrophic dogs to obtain proof of concept of AONs-mediated exon skipping therapy. Then, Japan participated in the worldwide clinical trials of drisapersen (Phase III). Finally, we developed a new AON targeting exon 53 in collaboration with Nippon Shinyaku Co., Ltd. in Japan and started an investigator-initiated clinical study at the National Center Hospital of NCNP (Phase I). This early phase exploratory study is the first clinical trial of an AON developed in Japan and a good example of translational research for treatment of DMD in Japan. In this chapter, we summarize the long development of the drug and discuss the future direction of splicing therapy for DMD.

A promising therapeutic approach for Duchenne muscular dystrophy (DMD) is exon skipping using antisense oligonucleotides (AOs). In-frame deletions of the hinge 3 region of the dystrophin protein, which is encoded by exons 50 and 51, are predicted to cause a variety of phenotypes. Here, we performed functional analyses of muscle in the exon 52-deleted mdx (mdx52) mouse, to predict the function of in-frame dystrophin following exon 51-skipping, which leads to a protein lacking most of hinge 3. A series of AOs based on phosphorodiamidate morpholino oligomers was screened by intramuscular injection into mdx52 mice. The highest splicing efficiency was generated by a two-oligonucleotide cocktail targeting both the 5' and 3' splice sites of exon 51. After a dose-escalation study, we systemically delivered this cocktail into mdx52 mice seven times at weekly intervals. This induced 20-30% of wild-type (WT) dystrophin expression levels in all muscles, and was accompanied by amelioration of the dystrophic pathology and improvement of skeletal muscle function. Because the structure of the restored in-frame dystrophin resembles human dystrophin following exon 51-skipping, our results are encouraging for the ongoing clinical trials for DMD. Moreover, the therapeutic dose required can provide a suggestion of the theoretical equivalent dose for humans.

DNA damage is a proposed pathogenic factor in neurodegenerative disorders such as Parkinson disease. To probe the underpinning mechanism of such neuronal perturbation, we sought to produce an experimental model of DNA damage. We thus first assessed DNA damage by in situ nick translation and emulsion autoradiography in the mouse brain after administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 4 x 20 mg/kg, ip, every 2 h), a neurotoxin known to produce a model of Parkinson disease. Here we show that DNA strand breaks occur in vivo in this mouse model of Parkinson disease with kinetics and a topography that parallel the degeneration of substantia nigra neurons, as assessed by FluoroJade labeling. Previously, nitric oxide synthase and cyclooxygenase-2 (Cox-2) were found to modulate MPTP-induced dopaminergic neuronal death. We thus assessed the contribution of these enzymes to DNA damage in mice lacking neuronal nitric oxide synthase (nNOS), inducible nitric oxide synthase (iNOS), or Cox-2. We found that the lack of Cox-2 and nNOS activities but not of iNOS activity attenuated MPTP-related DNA damage. We also found that not only nuclear, but also mitochondrial, DNA is a target for the MPTP insult. These results suggest that the loss of genomic integrity can be triggered by the concerted actions of nNOS and Cox-2 and provide further support to the view that DNA damage may contribute to the neurodegenerative process in Parkinson disease.

When fused with the protein transduction domain (PTD) derived from the human immunodeficiency virus TAT protein, proteins can cross the blood-brain barrier and cell membrane and transfer into several tissues, including the brain, making protein therapy feasible for various neurological disorders. We have constructed a powerful antiapoptotic modified Bcl-X(L) protein (originally constructed from Bcl-X(L)) fused with PTD derived from TAT (TAT-modified Bcl-X(L)), and, to examine its clinical effectiveness in a mouse model of familial amyotrophic lateral sclerosis (ALS), transgenic mice expressing human Cu/Zn superoxide dismutase (SOD1) bearing a G93A mutation were treated by intrathecal infusion of TAT-modified Bcl-X(L). We demonstrate that intrathecally infused TAT-fused protein was effectively transferred into spinal cord neurons, including motor neurons, and that intrathecal infusion of TAT-modified Bcl-X(L) delayed disease onset, prolonged survival, and improved motor performance. Histological studies show an attenuation of motor neuron loss and a decrease in the number of cleaved caspase 9-, cleaved caspase 3-, and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive cells in the lumbar cords of TAT-modified Bcl-X(L)-treated G93A mice. Our results indicate that intrathecal protein therapy using a TAT-fused protein is an effective clinical tool for the treatment of ALS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease with progressive cell death of upper and lower motor neurons. In this study, we measured monocyte chemotactic protein-1 (MCP-1) and vascular endothelial growth factor (VEGF) levels in cerebrospinal fluid (CSF) and serum by enzyme-linked immunosorbent assay (ELISA) in 42 ALS patients, and compared these levels with those of control subjects with other neurodegenerative disorders or with those of normal controls. MCP-1 levels in CSF were significantly higher in ALS patients than in the control group. VEGF levels in CSF tended to be lower in ALS patients than in the control group, but not significantly. A positive correlation was found between MCP-1 levels in CSF of ALS patients and the total Norris scale. The elevation of MCP-1/VEGF ratio in CSF was more specific to ALS patients compared to other neurological diseases such as Parkinson's disease (PD) and spinocerebellar ataxia (SCA) and to controls. Our data suggested that both MCP-1 levels and MCP-1/VEGF ratio in CSF may be useful markers for the clinical diagnosis of ALS.

Mutations in superoxide dismutase-1 (SOD1) cause a form of the fatal paralytic disorder amyotrophic lateral sclerosis (ALS), presumably by a combination of cell-autonomous and non–cell-autonomous processes. Here, we show that expression of mutated human SOD1 in primary mouse spinal motor neurons does not provoke motor neuron degeneration. Conversely, rodent astrocytes expressing mutated SOD1 kill spinal primary and embryonic mouse stem cell–derived motor neurons. This is triggered by soluble toxic factor(s) through a Bax-dependent mechanism. However, mutant astrocytes do not cause the death of spinal GABAergic or dorsal root ganglion neurons or of embryonic stem cell–derived interneurons. In contrast to astrocytes, fibroblasts, microglia, cortical neurons and myocytes expressing mutated SOD1 do not cause overt neurotoxicity. These findings indicate that astrocytes may play a role in the specific degeneration of spinal motor neurons in ALS. Identification of the astrocyte-derived soluble factor(s) may have far-reaching implications for ALS from both a pathogenic and therapeutic standpoint.

Objective Early-onset ataxia with ocular motor apraxia and hypoalbuminemia (EAOH)/ataxia with oculomotor apraxia type 1 (AOA1) is an autosomal recessive form of cerebellar ataxia. The causative protein for EAOH/AOA1, aprataxin (APTX), interacts with X-ray repair cross-complementing 1 (XRCC1), a scaffold DNA repair protein for single-strand breaks (SSBs). The goal of this study was to prove the functional involvement of APTX in SSB repair (SSBR). Methods We visualized the SSBR process with a recently developed laser irradiation system that allows real-time observation of SSBR proteins and with a local ultraviolet-irradiation system using a XPA-UVDE cell line that repairs DNA lesions exclusively via SSBR. APTX was knocked down using small interference RNA in the cells. Oxidative stress-induced DNA damage and cell death were assessed in EAOH fibroblasts and cerebellum. Results Our systems showed the XRCC1-dependent recruitment of APTX to SSBs. SSBR was impaired in APTX-knocked-down cells. Oxidative stress in EAOH fibroblasts readily induced SSBs and cell death, which were blocked by antioxidants. Accumulated oxidative DNA damage was confirmed in EAOH cerebellum. Interpretation This study provides the first direct evidence for the functional involvement of APTX in SSBR and in vivo DNA damage in EAOH/AOA1, and suggests a benefit of antioxidant treatment.

Duchenne muscular dystrophy is a lethal X-linked muscle disorder. We have already reported that osteopontin (OPN), an inflammatory cytokine and myogenic factor, is expressed in the early dystrophic phase in canine X-linked muscular dystrophy in Japan, a dystrophic dog model. To further explore the possibility of OPN as a new biomarker for disease activity in Duchenne muscular dystrophy, we monitored serum OPN levels in dystrophic and wild-type dogs at different ages and compared the levels to other serum markers, such as serum creatine kinase, matrix metalloproteinase-9, and tissue inhibitor of metalloproteinase-1. Serum OPN levels in the dystrophic dogs were significantly elevated compared with those in wild-type dogs before and 1 hour after a cesarean section birth and at the age of 3 months. The serum OPN level was significantly correlated with the phenotypic severity of dystrophic dogs at the period corresponding to the onset of muscle weakness, whereas other serum markers including creatine kinase were not. Immunohistologically, OPN was up-regulated in infiltrating macrophages and developmental myosin heavy chain-positive regenerating muscle fibers in the dystrophic dogs, whereas serum OPN was highly elevated. OPN expression was also observed during the synergic muscle regeneration process induced by cardiotoxin injection. In conclusion, OPN is a promising biomarker for muscle regeneration in dystrophic dogs and can be applicable to boys with Duchenne muscular dystrophy.

The expression profile of the protease-activated receptor-2 (PAR-2) and effects of PAR-2 gene knockout (PAR-2 KO) on the infarct size were investigated after 60 minutes of transient middle cerebral artery occlusion (tMCAO) in mice in relation to phosphorylated extracellular signal-regulated kinase (p-ERK) and astrocyte activation. PAR-2 was normally distributed mainly in neurons of the central nervous system (CNS), and strongly upregulated at 8–24 hours after tMCAO. Deficiency of PAR-2 gene significantly increased the infarct volume and the number of TUNEL-positive cells at 24 hours of reperfusion. The strong neuronal expression of p-ERK was induced at 5 minutes as a peak after reperfusion in wild-type mice, but the signal change was significantly reduced in PAR-2 KO mice. Astroglial activation was also greatly inhibited at 24 hours after tMCAO in PAR-2 KO mice. These results show that the deficiency of PAR-2 gene increases the acute ischemic cerebral injury associating with suppression of neuronal ERK activation and reactive astroglial activation.

Background: Distal myopathy with rimmed vacuoles (DMRV) is an autosomal-recessive disorder with preferential involvement of the tibialis anterior muscle that starts in young adulthood and spares quadriceps muscles. The disease locus has been mapped to chromosome 9p1-q1, the same region as the hereditary inclusion body myopathy (HIBM) locus. HIBM was originally described as rimmed vacuole myopathy sparing the quadriceps; therefore, the two diseases have been suspected to be allelic. Recently, HIBM was shown to be associated with the mutations in the gene encoding the bifunctional enzyme, UDP- N -acetylglucosamine 2-epimerase/ N -acetylmannosamine kinase ( GNE ). Objective: To determine whether DMRV and HIBM are allelic. Methods: The GNE gene was sequenced in 34 patients with DMRV. The epimerase activity in lymphocytes from eight DMRV patients was also measured. Results: The authors identified 27 unrelated DMRV patients with homozygous or compound-heterozygous mutations in the GNE gene. DMRV patients had markedly decreased epimerase activity. Conclusions: DMRV is allelic to HIBM. Various mutations are associated with DMRV in Japan. The loss-of-function mutations in the GNE gene appear to cause DMRV/HIBM.

The newly recognized ataxia-ocular apraxia 1 (AOA1; MIM 208920) is the most frequent cause of autosomal recessive ataxia in Japan and is second only to Friedreich ataxia in Portugal. It shares several neurological features with ataxia-telangiectasia, including early onset ataxia, oculomotor apraxia and cerebellar atrophy, but does not share its extraneurological features (immune deficiency, chromosomal instability and hypersensitivity to X-rays). AOA1 is also characterized by axonal motor neuropathy and the later decrease of serum albumin levels and elevation of total cholesterol. We have identified the gene causing AOA1 and the major Portuguese and Japanese mutations. This gene encodes a new, ubiquitously expressed protein that we named aprataxin. This protein is composed of three domains that share distant homology with the amino-terminal domain of polynucleotide kinase 3'- phosphatase (PNKP), with histidine-triad (HIT) proteins and with DNA-binding C2H2 zinc-finger proteins, respectively. PNKP is involved in DNA single-strand break repair (SSBR) following exposure to ionizing radiation and reactive oxygen species. Fragile-HIT proteins (FHIT) cleave diadenosine tetraphosphate, which is potentially produced during activation of the SSBR complex. The results suggest that aprataxin is a nuclear protein with a role in DNA repair reminiscent of the function of the protein defective in ataxia-telangiectasia, but that would cause a phenotype restricted to neurological signs when mutant.

The thymus is a critical organ for the elimination of autoreactive T cells by apoptosis. We studied the expression of apoptosis-associated genes, bcl-xL, bad, caspase-3, and c-myc family genes in myasthenia gravis (MG) thymuses. We observed that the mRNA levels of myc family genes, c-myc and max, were markedly reduced in MG thymuses. These results indicate that c-myc-mediated signaling is abnormal in MG thymuses. The levels of molecules whose expressions are associated with myc, such as STAM, prothymosin-alpha, and NFkappaB, were also analyzed.

Objectives - To examine the expression of OX40, an activated memory T-cell marker, and its ligand (OX40L), a set of molecules for T-cell-B-cell interaction, and other lymphocyte activation markers in the thymuses of myasthenia gravis (MG) and controls. Material and methods - We studied the expression of OX40, OX40L, IL-2Ra and HLA-DR in the thymic tissues of MG and controls using immunocytochemistry and flowcytometry. Results - In both hyperplastic thymus of MG and control thymus, OX40 + cells were scattered mainly in the medulla with much fewer OX40L + cells being distributed in the corticomedullary junctions. IL-2Ra and HLA-DR were expressed in the medulla at higher frequencies as compared with OX40 in controls as well as MG. In contrast, the numbers of OX40 + cells around the germinal centers (GC) were significantly greater than those of control thymuses, and some mononuclear cells in GC were OX40L + . A considerable number of OX40 + cells were seen in the thymic tissues adjacent to thymomas. OX40 + cells were CD4 + CD8 or CD4 + CD8 + and were mostly HLA-DR . (The coexpression of OX40 and IL-2Ra on activated CD4 + T cells was previously reported.) Conclusion - OX40, expressed in a fraction of activated CD4 + T cells, may be upregulated in thymic tissues adjacent to GC and thymoma in MG, and OX40 may interact with OX40L in GC to enhance anti-acetylcholine receptor antibody production in MG.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system and one of the earliest changes in inflammatory focus involves the activation of vascular endothelial cells. We determined the plasma level of plasminogen activator inhibitor-1 (PAI-1), a key regulator of fibrinolysis and cell migration, in patients with MS. The level of plasma PAI-1 was significantly higher in active MS cases when compared to stable MS and controls. Plasma concentrations of tissue plasminogen activator, transforming growth factor beta-1, and lipoprotein-a remained normal in spite of disease activity. These results suggested that PAI-1 plasma levels are associated with MS disease activity and is a good marker for MS relapse.

Currently, phase 2/3 clinical trials of exon 51 skipping for Duchenne Muscular Dystrophy (DMD) are being conducted and have been shown promising results for NDA. Next to the patients treatable by exon 51 skipping, the patients amenable to exon 53 skipping are the second largest population; therefore a development of exon 53 skipping drug has high priority. National Center of Neurology and Psychiatry, with Kyoto-based pharmaceutical company Nippon Shinyaku, had jointly developed the exon 53 skipping drug since 2009 and started an investigator-initiated clinical trial from June 2013 (UMIN Clinical Trial ID: UMIN000010964, ClinicalTrial.gov: NCT02081625). The NS-065/NCNP-01 is a morpholino based antisense oligonucleotide that has been developed to skip the exon 53 of the dystrophin gene and to treat DMD patient amenable to the exon 53 skipping. It has been confirmed potent efficacy and high safety in pre-clinical studies. This clinical trial is an exploratory phase 1, single-site, first-in-human study. The primary endpoints are the safety and tolerability and the secondary endpoints are the pharmacokinetics and efficacies (dystrophin recovery). Three doses cohort design (1.25 mg/kg, 5 mg/kg and 20 mg/kg) was adopted and all subjects were dosed weekly intravenous infusion for 12 weeks. Mainly nonambulant subjects were recruited because the dose and duration in this trial was not enough to expect functional improvement. Of total 10 subjects, each three subjects were randomly assigned to 1.25 mg/kg or 5 mg/kg cohorts, and four subjects were assigned to 20 mg/kg cohort. An in vitro confirmation of dystrophin recovery and exon 53 skipping in subject-derived cells was one of inclusion criteria. Subject's mutations were classified into any of exons 45-52, 48-52 or 49-52 deletion; all NS-065/NCNP-01 treated cells satisfied the criteria. One week after an initial dosing for the first subject in each cohort, next subjects were dosed. Safety review committee advised the principal investigator whether or not to proceed to next cohort. At the end of 2014, dosing to all subjects had completed; no serious adverse events were observed. Dystrophin expression is to be evaluated by Western blotting and immunofluorescent staining. The analysis is still ongoing, but an immunofluorescent image analysis will be performed as objective and quantitative as possible by automated measurements. We would report progress of the first-in-human study of NS-065/NCNP-01; the morpholino based exon 53 skipping drug for DMD.

We report two brothers from a Japanese family with a late-onset distal myopathy characterized by rimmed vacuoles and dysferlin deficiency with no inflammatory infiltration and dystrophic changes in muscle biopsy. Mutations in the GNE, dysferlin, caveolin 3, emerin, and lamin A/C genes were excluded. We speculate that dysferlin is involved in the pathogenesis of the myopathy in these patients, which may represent a new disease entity presenting as a distal myopathy.

Duchenne muscular dystrophy (DMD), one of the most common and lethal genetic disorders, and the mdx mouse myopathies are caused by a lack of dystrophin protein. These dystrophic muscles contain sporadic clusters of dystrophin-expressing revertant fibers (RFs), as detected by immunohistochemistry. RFs are known to arise from muscle precursor cells with spontaneous exon skipping (alternative splicing) and clonally expand in size with increasing age through the process of muscle degeneration/regeneration. The expansion of revertant clusters is thought to represent the cumulative history of muscle regeneration and proliferation of such precursor cells. However, the precise mechanisms by which RFs arise and expand are poorly understood. Here, to test the effects of mutation types and aging on RF expansion and muscle regeneration, we examined the number of RFs in mdx mice (containing a nonsense mutation in exon 23) and mdx52 mice (containing deletion mutation of exon 52) with the same C57BL/6 background at 2, 6, 12, and 18months of age. Mdx mice displayed a significantly higher number of RFs compared to mdx52 mice in all age groups, suggesting that revertant fiber expansion largely depends on the type of mutation and/or location in the gene. A significant increase in the expression and clustering levels of RFs was found beginning at 6months of age in mdx mice compared with mdx52 mice. In contrast to the significant expansion of RFs with increasing age, the number of centrally nucleated fibers and embryonic myosin heavy chain-positive fibers (indicative of cumulative and current muscle regeneration, respectively) decreased with age in both mouse strains. These results suggest that mutation types and aging differently affect revertant fiber expansion in mdx and mdx52 mice.

We present an analysis of 1- to 5-μm, near-infrared broadband images of Comet P/Halley 1986 III covering a 10 4 -km square region of the inner coma obtained on three consecutive nights in 1986 March during post-perihelion passage. In all images, the coma is extended in the sunward direction and appears distinctly non-spherical, similar to morphology in the 10-μm made by Hayward et al. (1987) 3.0 days later during the Giotto encounter. Marked variation in the coma's overall structure and brightness also was apparent, including the presence of a jet feature which we associate with a short-term outburst of material from the comet nucleus. The observed coma surface brightness dependence upon nucleocentric distance ( r ) at all wavelengths in both the comet dust tail and in the jet deviates from the dependence predicted by the “steady state” model for comet nucleus ablation, with the radial decrease in surface brightness being slower than r −1 on the jet side of the nucleus and faster that r −1 on the tail side. The near-infrared colors of the coma are not constant as a function of nucleocentric distance, suggesting that the grain properties are not uniform across the coma. Based on an elementary dynamical analysis of the trajectories of dust particles ablated from the nucleus we argue that these observations may be consistent with the hypothesis that particles emitted in jets fragments in the outflow on time scales of a few hours.

We describe a Japanese family with hereditary spinocerebellar ataxia characterized by initial emaciation and myoclonus. The proband first noted truncal ataxia, myoclonus in the shoulder and general emaciation at age 24. The other affected members of the family also had such emaciation in the early stage of the disease. The DNA analyses of the family revealed that the patients of the family are associated with the expansions of CAG repeats for Machado-Joseph disease (MJD) on the long arm of chromosome 14. Although the clinical features of MJD are very variable, general emaciation in an early stage of the disease and systemic myoclonus have not been documented. Because it is sometimes difficult to distinguish among hereditary spinocerebellar ataxias such as spinocerebellar ataxia type 1 (SCA1) or dentatorubropallidoluysian atrophy (DRPLA) by clinical features, a genetic examination provides better understanding of such a rare and ambiguous type of hereditary spinocerebellar ataxia.

Duchenne muscular dystrophy (DMD) is a severe muscular disorder. It was reported that multiple exon skipping (MES), targeting exon 45–55 of the DMD gene, might improve patients’ symptoms because patients who have a genomic deletion of all these exons showed very mild symptoms. Thus, exon 45–55 skipping treatments for DMD have been proposed as a potential clinical cure. Herein, we detected the expression of endogenous exons 44–56 connected mRNA transcript of the DMD using total RNAs derived from human normal skeletal muscle by reverse transcription polymerase chain reaction (RT-PCR), and identified a total of eight types of MES products around the hotspot. Surprisingly, the 5′ splice sites of recently reported post-transcriptional introns (remaining introns after co-transcriptional splicing) act as splicing donor sites for MESs. We also tested exon combinations to generate DMD circular RNAs (circRNAs) and determined the preferential splice sites of back-splicing, which are involved not only in circRNA generation, but also in MESs. Our results fit the current circRNA-generation model, suggesting that upstream post-transcriptional introns trigger MES and generate circRNA because its existence is critical for the intra-intronic interaction or for extremely distal splicing.