Intraperitoneal administration of AAV9-shRNA inhibits target gene expression in the dorsal root ganglia of neonatal mice.

BACKGROUND: There is considerable interest in inducing RNA interference (RNAi) in neurons to study gene function and identify new targets for disease intervention. Although short interfering RNAs (siRNAs) have been used to silence genes in neurons, in vivo delivery of RNAi remains a major challenge, especially by systemic administration. We have developed a highly efficient method for in vivo gene silencing in dorsal root ganglia (DRG) by using short hairpin RNA-expressing single-stranded adeno-associated virus 9 (ssAAV9-shRNA). RESULTS: Intraperitoneal administration of ssAAV9-shRNA to neonatal mice resulted in highly effective and specific silencing of a target gene in DRG. We observed an approximately 80% reduction in target mRNA in the DRG, and 74.7% suppression of the protein was confirmed by Western blot analysis. There were no major side effects, and the suppression effect lasted for more than three months after the injection of ssAAV9-shRNA. CONCLUSIONS: Although we previously showed substantial inhibition of target gene expression in DRG via intrathecal ssAAV9-shRNA administration, here we succeeded in inhibiting target gene expression in DRG neurons via intraperitoneal injection of ssAAV9-shRNA. AAV9-mediated delivery of shRNA will pave the way for creating animal models for investigating the molecular biology of the mechanisms of pain and sensory ganglionopathies.

Non-human primate model of amyotrophic lateral sclerosis with cytoplasmic mislocalization of TDP-43.

Amyotrophic lateral sclerosis is a fatal neurodegenerative disease characterized by progressive motoneuron loss. Redistribution of transactive response deoxyribonucleic acid-binding protein 43 from the nucleus to the cytoplasm and the presence of cystatin C-positive Bunina bodies are considered pathological hallmarks of amyotrophic lateral sclerosis, but their significance has not been fully elucidated. Since all reported rodent transgenic models using wild-type transactive response deoxyribonucleic acid-binding protein 43 failed to recapitulate these features, we expected a species difference and aimed to make a non-human primate model of amyotrophic lateral sclerosis. We overexpressed wild-type human transactive response deoxyribonucleic acid-binding protein 43 in spinal cords of cynomolgus monkeys and rats by injecting adeno-associated virus vector into the cervical cord, and examined the phenotype using behavioural, electrophysiological, neuropathological and biochemical analyses. These monkeys developed progressive motor weakness and muscle atrophy with fasciculation in distal hand muscles first. They also showed regional cytoplasmic transactive response deoxyribonucleic acid-binding protein 43 mislocalization with loss of nuclear transactive response deoxyribonucleic acid-binding protein 43 staining in the lateral nuclear group of spinal cord innervating distal hand muscles and cystatin C-positive cytoplasmic aggregates, reminiscent of the spinal cord pathology of patients with amyotrophic lateral sclerosis. Transactive response deoxyribonucleic acid-binding protein 43 mislocalization was an early or presymptomatic event and was later associated with neuron loss. These findings suggest that the transactive response deoxyribonucleic acid-binding protein 43 mislocalization leads to α-motoneuron degeneration. Furthermore, truncation of transactive response deoxyribonucleic acid-binding protein 43 was not a prerequisite for motoneuronal degeneration, and phosphorylation of transactive response deoxyribonucleic acid-binding protein 43 occurred after degeneration had begun. In contrast, similarly prepared rat models expressed transactive response deoxyribonucleic acid-binding protein 43 only in the nucleus of motoneurons. There is thus a species difference in transactive response deoxyribonucleic acid-binding protein 43 pathology, and our monkey model recapitulates amyotrophic lateral sclerosis pathology to a greater extent than rodent models, providing a valuable tool for studying the pathogenesis of sporadic amyotrophic lateral sclerosis.

Intraperitoneal AAV9-shRNA inhibits target expression in neonatal skeletal and cardiac muscles.

Systemic injections of AAV vectors generally transduce to the liver more effectively than to cardiac and skeletal muscles. The short hairpin RNA (shRNA)-expressing AAV9 (shRNA-AAV9) can also reduce target gene expression in the liver, but not enough in cardiac or skeletal muscles. Higher doses of shRNA-AAV9 required for inhibiting target genes in cardiac and skeletal muscles often results in shRNA-related toxicity including microRNA oversaturation that can induce fetal liver failure. In this study, we injected high-dose shRNA-AAV9 to neonates and efficiently silenced genes in cardiac and skeletal muscles without inducing liver toxicity. This is because AAV is most likely diluted or degraded in the liver than in cardiac or skeletal muscle during cell division after birth. We report that this systemically injected shRNA-AAV method does not induce any major side effects, such as liver dysfunction, and the dose of shRNA-AAV is sufficient for gene silencing in skeletal and cardiac muscle tissues. This novel method may be useful for generating gene knockdown in skeletal and cardiac mouse tissues, thus providing mouse models useful for analyzing diseases caused by loss-of-function of target genes.

Efficient In Vivo Delivery of siRNA to the Liver by Conjugation of α-Tocopherol.

RNA interference is a powerful tool for target-specific knockdown of gene expression. However, efficient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ, which is essential for therapeutic applications, has not been established. In this study we used α-tocopherol (vitamin E), which has its own physiological transport pathway to most of the organs, as a carrier molecule of siRNA in vivo. The α-tocopherol was covalently bound to the antisense strand of 27/29-mer siRNA at the 5'-end (Toc-siRNA). The 27/29-mer Toc-siRNA was designed to be cleaved by Dicer, producing a mature form of 21/21-mer siRNA after releasing α-tocopherol. The C6 hydroxyl group of α-tocopherol, associated with antioxidant activity, was abolished. Using this new vector, intravenous injection of 2 mg/kg of Toc-siRNA, targeting apolipoprotein B (apoB), achieved efficient reduction of endogenous apoB messenger RNA (mRNA) in the liver. The downregulation of apoB mRNA was confirmed by the accumulation of lipid droplets in the liver as a phenotype. Neither induction of interferons (IFNs) nor other overt side effects were revealed by biochemical and pathological analyses. These findings indicate that Toc-siRNA is effective and safe for RNA interference-mediated gene silencing in vivo.

Efficient in vivo delivery of siRNA to the liver by conjugation of alpha-tocopherol.

RNA interference is a powerful tool for target-specific knockdown of gene expression. However, efficient and safe in vivo delivery of short interfering RNA (siRNA) to the target organ, which is essential for therapeutic applications, has not been established. In this study we used alpha-tocopherol (vitamin E), which has its own physiological transport pathway to most of the organs, as a carrier molecule of siRNA in vivo. The alpha-tocopherol was covalently bound to the antisense strand of 27/29-mer siRNA at the 5'-end (Toc-siRNA). The 27/29-mer Toc-siRNA was designed to be cleaved by Dicer, producing a mature form of 21/21-mer siRNA after releasing alpha-tocopherol. The C6 hydroxyl group of alpha-tocopherol, associated with antioxidant activity, was abolished. Using this new vector, intravenous injection of 2 mg/kg of Toc-siRNA, targeting apolipoprotein B (apoB), achieved efficient reduction of endogenous apoB messenger RNA (mRNA) in the liver. The downregulation of apoB mRNA was confirmed by the accumulation of lipid droplets in the liver as a phenotype. Neither induction of interferons (IFNs) nor other overt side effects were revealed by biochemical and pathological analyses. These findings indicate that Toc-siRNA is effective and safe for RNA interference-mediated gene silencing in vivo.

Posterior Reversible Encephalopathy Syndrome and Reversible Cerebral Vasoconstriction Syndrome after Rapid Blood Transfusion.

We herein report a case of posterior reversible encephalopathy syndrome (PRES) and reversible cerebral vasoconstriction syndrome (RCVS) that occurred immediately after blood transfusion. A 64-year-old Japanese woman was diagnosed with liver cirrhosis due to hepatitis B 2 years ago. She was admitted to our hospital with hemorrhagic shock due to esophageal variceal rupture. She was hospitalized with rapid blood pumping transfusion, after which consciousness disorder appeared, and her blood pressure suddenly increased. Magnetic resonance imaging revealed PRES and RCVS. We speculated that hypoalbuminemia and blood transfusion might have been involved in the development of PRES and RCVS.

[Subcortical calcification on CT in Borden type III intracranial dural arteriovenous fistula].

We herein report a 67-year-old female who presented with progressive dementia and disturbance of consciousness. Brain CT showed multiple subcortical calcifications with edema. Enhanced CT showed multiple abnormal vessels in the left hemisphere. Electroencephalography indicated diffuse spike and slow wave complex, so non-convulsive status epilepticus was diagnosed. Cerebral angiography revealed several feeder arteries with retrograde leptomeningeal venous drainage. We diagnosed her with Borden type III cerebral dural arteriovenous fistulas. Trans-arterial embolization with n-butyl-2-cyanoacrylate was performed, and she has experienced no epileptic attacks for at least ten months. Calcification changes are sometimes seen in Borden type II dural arteriovenous fistulas but not in aggressive types, such as Borden type III. It is important to suspect dural arteriovenous fistulas when we encounter patients with progressive dementia or/and epilepsy with cerebral calcification lesions, as this may be a treatable disease condition.

Prevalence and clinicoradiological features of spinocerebellar ataxia type 34 in a Japanese ataxia cohort.

INTRODUCTION: Spinocerebellar ataxia (SCA) type 34, a form of autosomal dominantly inherited ataxia, has recently been associated with mutations in the ELOVL4 gene. However, a genetic study of the prevalence of SCA34 in an ataxia cohort has never been reported. METHODS: We performed a mutation screening of ELOVL4 in a cohort of 153 undiagnosed index ataxia patients, selected after excluding for common SCA types, in a series of 506 Japanese index ataxia patients. RESULTS: Heterozygous mutation c.698C > T (p.T233M) was detected in an index patient with multisystem neurodegeneration including ataxia and erythrokeratodermia skin lesions, an archetypal skin phenotype in SCA34. The patient's father also presented with ataxia but not skin lesions. Although this mutation has been recently reported in a single English-Canadian patient, the present study confirms its cosegregation with the ataxia phenotype in the Japanese kindred. Brain magnetic resonance imaging (MRI) of the patient and his father revealed marked pontine and cerebellar atrophy as well as the hot cross bun sign, that is common in cerebellar type of multiple system atrophy and was also described in SCA34 patients harboring two other mutations: p.L168F and p.W246G. CONCLUSION: This represents the first genetic study of the prevalence of SCA34 in an ataxia cohort and demonstrates its low prevalence (0.2%) in ataxia patients. The broad SCA34 clinical spectrum suggests variable multisystem neurodegeneration. Clinicians should be aware of this rare disease entity, particularly if erythrokeratodermia or the hot cross bun sign in MRI are present in undiagnosed degenerative ataxia patients.

New RNAi strategy for selective suppression of a mutant allele in polyglutamine disease.

In gene therapy of dominantly inherited diseases with small interfering RNA (siRNA), mutant allele specific suppression may be necessary for diseases in which the defective gene normally has an important role. It is difficult, however, to design a mutant allele-specific siRNA for trinucleotide repeat diseases in which the difference of sequences is only repeat length. To overcome this problem, we use a new RNA interference (RNAi) strategy for selective suppression of mutant alleles. Both mutant and wild-type alleles are inhibited by the most effective siRNA, and wild-type protein is restored using the wild-type mRNA modified to be resistant to the siRNA. Here, we applied this method to spinocerebellar ataxia type 6 (SCA6). We discuss its feasibility and problems for future gene therapy.

Proteolytic cleavage and cellular toxicity of the human alpha1A calcium channel in spinocerebellar ataxia type 6.

Spinocerebellar ataxia type 6 (SCA6) is a neurodegenerative disease caused by small CAG repeat expansion in the alpha1A calcium channel gene. We found that the human alpha1A calcium channel protein expressed in human embryonic kidney 293T cells produces a 75 kDa C-terminal fragment. This fragment is more toxic to cells than the full-length alpha1A calcium channel, regardless of polyglutamine tract length. In cells stably transfected with plasmids of full-length alpha1A calcium channel cDNAs, the C-terminal fragment protein is present in the mutant transformant but not in the wild-type one, indicative that this C-terminal fragment with the expanded polyglutamine tract is more resistant to proteolysis than that with the normal sized polyglutamine tract. We speculate that the toxic C-terminal fragment, in which resistance to proteolysis is rendered by the expanded polyglutamine, has a key role in the pathological mechanism of SCA6.

[Relapsing polychondritis with an intracranial granuloma: a case report].

We report a case of relapsing polychondritis (RP) with an intracranial granuloma. A 67-year-old man developed progressive disorientation during the course of RP with left auricular chondritis and episcleritis. He had history of sinusitis and rupture of an aneurysm in middle cerebral artery. Laboratory examinations revealed high erythrocyte sedimentation rate and positive C-reactive protein. Head CT and MRI with contrast enhancement showed a mass adjacent to the falx cerebri and lesions in the frontal skull base. The mass was surrounded by extensive perifocal edema that spread mainly into the frontal white matter on both sides. Histologically, the mass displayed an inflammatory granuloma. By removal of the mass, edema decreased around the granuloma, and his disorientation improved markedly. Surgical findings revealed the granuloma was separated from sinusitis. There are a few reports on RP with an intracranial granuloma.

FALS with Gly72Ser mutation in SOD1 gene: report of a family including the first autopsy case.

Clinical information on familial amyotrophic lateral sclerosis (FALS) with Gly72Ser mutation in the Cu/Zn superoxide dismutase-1 (SOD1) gene has been limited and autopsy findings remain to be clarified. We describe one Japanese family with ALS carrying Gly72Ser mutation in the SOD1 gene, in which autopsy was performed on one affected member. The autopsied female patient developed muscle weakness of the left thigh at age 66 and showed transient upper motor neuron signs. She died of respiratory failure 13 months after onset without artificial respiratory support. There were no symptoms suggesting bladder or rectal dysfunction throughout the clinical course. Her brother with ALS was shown to have Gly72Ser mutation in the SOD1 gene. Histopathologically, motor neurons were markedly decreased throughout the whole spinal cord, whereas corticospinal tract involvement was very mild and was demonstrated only by CD68 immunohistochemistry. Degeneration was evident in the posterior funiculus, Clarke's nucleus, posterior cerebellar tract, and Onuf's nucleus. Neuronal hyaline inclusions were rarely observed in the neurons of the spinal cord anterior horn including Onuf's nucleus, and were immunoreactive for SOD1. To date, neuron loss in Onuf's nucleus has hardly been seen in ALS, except in the patients showing prolonged disease duration with artificial respiratory support. Involvement of Onuf's nucleus may be a characteristic pathological feature in FALS with Gly72Ser mutation in the SOD1 gene.

In vivo application of an RNAi strategy for the selective suppression of a mutant allele.

Gene therapy for dominantly inherited diseases with small interfering RNA (siRNA) requires mutant allele-specific suppression when genes in which mutation causes disease normally have an important role. We previously proposed a strategy for selective suppression of mutant alleles; both mutant and wild-type alleles are inhibited by most effective siRNA, and wild-type protein is restored using mRNA mutated to be resistant to the siRNA. Here, to prove the principle of this strategy in vivo, we applied it to our previously reported anti-copper/zinc superoxide dismutase (SOD1) short hairpin RNA (shRNA) transgenic (Tg) mice, in which the expression of the endogenous wild-type SOD1 gene was inhibited by more than 80%. These shRNA Tg mice showed hepatic lipid accumulation with mild liver dysfunction due to downregulation of endogenous wild-type SOD1. To rescue this side effect, we generated siRNA-resistant SOD1 Tg mice and crossed them with anti-SOD1 shRNA Tg mice, resulting in the disappearance of lipid accumulation in the liver. Furthermore, we also succeeded in mutant SOD1-specific gene suppression in the liver of SOD1(G93A) Tg mice, a model for amyotrophic lateral sclerosis, using intravenously administered viral vectors. Our method may prove useful for siRNA-based gene therapy for dominantly inherited diseases.

Silencing efficiency differs among tissues and endogenous microRNA pathway is preserved in short hairpin RNA transgenic mice.

In short hairpin RNA (shRNA) transgenic mice, the tissue difference in gene silencing efficiency and oversaturation of microRNA (miRNA) pathway have not been well assessed. We studied these problems in our previously-reported anti-copper/zinc superoxide dismutase (SOD1) shRNA transgenic mice. Although there was a tissue difference (liver and skeletal muscle, >95%; central nervous system and lung, approximately 80%), the target gene silencing was systemic and our anti-SOD1 shRNA transgenic mice recapitulated the SOD1-null mice. Neither endogenous miRNAs nor their target gene levels were altered, indicating the preservation of endogenous miRNA pathways. We think that the shRNA transgenic mice can be utilized for gene analysis.

Efficient regulation of viral replication by siRNA in a non-human primate surrogate model for hepatitis C.

RNA interference (RNAi) represents a new technology which could offer potential applications for the therapeutics of human diseases. RNAi-mediated therapy has recently been shown to be effective toward infectious diseases in in vitro and rodent models, however, it remains unclear whether RNAi therapy with systemic application could be effective in primates. In this study, we examined if RNAi therapy could be effective toward infectious diseases by using a non-human primate surrogate model for hepatitis C. Administration into marmosets of cationic liposome-encapsulated siRNA (CL-siRNA) for GB virus B (GBV-B), which is most closely related to hepatitis C virus, repressed GBV-B replication in a dose-dependent manner. Especially, 5 mg/kg of the CL-siRNA completely inhibited the viral replication. Since the serum interferons (IFNs) were induced by CL-siRNA in vivo, inhibition of viral regulation by anti-GBV-B CL-siRNA may include an antiviral effect of IFN. However, contribution of induced IFN may be partial, since the control CL-siRNA which induced a stronger IFN response than GBV-B CL-siRNA could only delay the viral replication. Our results suggest the feasibility of systemic administration of CL-siRNA as an antiviral strategy.