Inhibition of CASK expression by adeno associated virus-mediated RNA interference in medial prefrontal cortex affects social behavior in the adult mouse （アデノ随伴ウィルスを用いたRNA干渉による内側前頭前皮質でのCASKの発現抑制で成体マウスの社会行動が阻害される。）

信州大学(Shinshu university)博士（医学）雑誌に発表。 / 信州医学雑誌 69(1):1-45 (2021); doi:10.11441/shinshumedj.69.45. ©　信州医学会Thesis曹 雪杉. Inhibition of CASK expression by adeno associated virus-mediated RNA interference in medial prefrontal cortex affects social behavior in the adult mouse （アデノ随伴ウィルスを用いたRNA干渉による内側前頭前皮質でのCASKの発現抑制で成体マウスの社会行動が阻害される。）. 信州大学, 2019, 博士論文. 博士（医学）, 甲第1222号, 令和01年09月30日授与.doctoral thesi

Huntingtin-lowering strategies for Huntington's disease.

INTRODUCTION: Huntington's disease (HD) is an incurable, autosomal dominant neurodegenerative disease caused by an abnormally long polyglutamine tract in the huntingtin protein. Because this mutation causes disease via gain-of-function, lowering huntingtin levels represents a rational therapeutic strategy. AREAS COVERED: We searched MEDLINE, CENTRAL, and other trial databases, and relevant company and HD funding websites for press releases until April 2020 to review strategies for huntingtin lowering, including autophagy and PROTACs, which have been studied in preclinical models. We focussed our analyses on oligonucleotide (ASOs) and miRNA approaches, which have entered or are about to enter clinical trials. EXPERT OPINION: ASO and mRNA approaches for lowering mutant huntingtin protein production and strategies for increasing mutant huntingtin clearance are attractive because they target the cause of disease. However, questions concerning the optimal mode of delivery and associated safety issues remain. It is unclear if the human CNS coverage with intrathecal or intraparenchymal delivery will be sufficient for efficacy. The extent that one must lower mutant huntingtin levels for it to be therapeutic is uncertain and the extent to which CNS lowering of wild-type huntingtin is safe is unclear. Polypharmacy may be an effective approach for ameliorating signs and symptoms and for preventing/delaying onset and progression

The Role of Estrogen Receptor β in the Dorsal Raphe Nucleus on the Expression of Female Sexual Behavior in C57BL/6J Mice

17β-Estradiol (E2) regulates the expression of female sexual behavior by acting through estrogen receptor (ER) α and β. Previously, we have shown that ERβ knockout female mice maintain high level of lordosis expression on the day after behavioral estrus when wild-type mice show a clear decline of the behavior, suggesting ERβ may be involved in inhibitory regulation of lordosis. However, it is not identified yet in which brain region(s) ERβ may mediate an inhibitory action of E2. In this study, we have focused on the dorsal raphe nucleus (DRN) that expresses ERβ in higher density than ERα. We site specifically knocked down ERβ in the DRN in ovariectomized mice with virally mediated RNA interference method. All mice were tested weekly for a total of 3 weeks for their lordosis expression against a stud male in two consecutive days: day 1 with the hormonal condition mimicking the day of behavioral estrus, and day 2 under the hormonal condition mimicking the day after behavioral estrus. We found that the level of lordosis expression in ERβ knockdown (βERKD) mice was not different from that of control mice on day 1. However, βERKD mice continuously showed elevated levels of lordosis behavior on day 2 tests, whereas control mice showed a clear decline of the behavior on day 2. These results suggest that the expression of ERβ in the DRN may be involved in the inhibitory regulation of sexual behavior on the day after behavioral estrus in cycling female mice

The pathogenic exon 1 HTT protein is produced by incomplete splicing in Huntington’s disease patients

We have previously shown that exon 1 of the huntingtin gene does not always splice to exon 2 resulting in the production of a small polyadenylated mRNA (HTTexon1) that encodes the highly pathogenic exon 1 HTT protein. The level of this read-through product is proportional to CAG repeat length and is present in all knock-in mouse models of Huntington’s disease (HD) with CAG lengths of 50 and above and in the YAC128 and BACHD mouse models, both of which express a copy of the human HTT gene. We have now developed specific protocols for the quantitative analysis of the transcript levels of HTTexon1 in human tissue and applied these to a series of fibroblast lines and post-mortem brain samples from individuals with either adult-onset or juvenile-onset HD. We found that the HTTexon1 mRNA is present in fibroblasts from juvenile HD patients and can also be readily detected in the sensory motor cortex, hippocampus and cerebellum of post-mortem brains from HD individuals, particularly in those with early onset disease. This finding will have important implications for strategies to lower mutant HTT levels in patients and the design of future therapeutics

Gene Therapy 2017: Progress and Future Directions

Introduction: Gene therapy has changed dramatically in the 28 years since the first human gene transfer experiment in 1989. Alipogene tiparvovec, GlyberaR®, a recombinant adeno-associated virus (rAAV) product for lipoprotein lipase deficiency, and Strimvelis®, a lentivirus vector for severe combined immune deficiency are approved in Europe. An rAAV2 product for a congenital form of blindness is currently under review in the United States, likely to be followed by numerous other gene therapies

Therapeutic strategies for Huntington’s disease

PURPOSE AND REVIEW: Huntington's disease is a fatal autosomal dominant neurodegenerative disorder caused by a trinucleotide expansion in the HTT gene, and current therapies focus on symptomatic treatment. This review explores therapeutic approaches that directly target the pathogenic mutation, disrupt HTT mRNA or its translation. RECENT FINDINGS: Zinc-finger transcription repressors and CRISPR-Cas9 therapies target HTT DNA, thereby preventing all downstream pathogenic mechanisms. These therapies, together with RNA interference (RNAi), require intraparenchymal delivery to the brain in viral vectors, with only a single delivery potentially required, though they may carry the risk of irreversible side-effects. Along with RNAi, antisense oligonucleotides (ASOs) target mRNA, but are delivered periodically and intrathecally. ASOs have safely decreased mutant huntingtin protein (mHTT) levels in the central nervous system of patients, and a phase 3 clinical trial is currently underway. Finally, orally available small molecules, acting on splicing or posttranslational modification, have recently been shown to decrease mHTT in animal models. SUMMARY: Huntingtin-lowering approaches act upstream of pathogenic mechanisms and therefore have a high a priori likelihood of modifying disease course. ASOs are already in late-stage clinical development, whereas other strategies are progressing rapidly toward human studies

Safe and Efficient Silencing with a Pol II, but not a Pol lII, Promoter Expressing an Artificial miRNA Targeting Human Huntingtin

Huntington\u27s disease is a devastating, incurable neurodegenerative disease affecting up to 12 per 100,000 patients worldwide. The disease is caused by a mutation in the Huntingtin (Htt) gene. There is interest in reducing mutant Huntingtin by targeting it at the mRNA level, but the maximum tolerable dose and long-term effects of such a treatment are unknown. Using a self-complementary AAV9 vector, we delivered a mir-155-based artificial miRNA under the control of the chicken β-actin or human U6 promoter. In mouse brain, the artificial miRNA reduced the human huntingtin mRNA by 50%. The U6, but not the CβA promoter, produced the artificial miRNA at supraphysiologic levels. Embedding the antisense strand in a U6-mir-30 scaffold reduced expression of the antisense strand but increased the sense strand. In mice treated with scAAV9-U6-mir-155-HTT or scAAV9-CβA-mir-155-HTT, activated microglia were present around the injection site 1 month post-injection. Six months post-injection, mice treated with scAAV9-CβA-mir-155-HTT were indistinguishable from controls. Those that received scAAV9-U6-mir-155-HTT showed behavioral abnormalities and striatal damage. In conclusion, miRNA backbone and promoter can be used together to modulate expression levels and strand selection of artificial miRNAs, and in brain, the CβA promoter can provide an effective and safe dose of a human huntingtin miRNA

Functional Insights Into Novel Regulators of Plasma Lipids:STAP1 and GPR146

Elucidating the mechanisms how novel genes regulate plasma lipids promises to expand the current understanding of the origins of dyslipidemias and atherosclerotic cardiovascular diseases (ASCVD). New insights can also offer possibilities to develop additional treatment strategies to alleviate ASCVD, currently the largest cause of death worldwide. This thesis offers insights into how STAP1 (Signal Transducing Adaptor Family Member 1) and GPR146 (G-protein coupled receptor 146) regulate plasma lipids. Our functional studies into STAP1, a previously proposed Familial Hypercholesterolemia (FH) gene, show that STAP1 does not regulate LDL-cholesterol in mice or humans. Consequently, we have proposed to delist STAP1 as an FH gene. For GPR146, we studied common and rare variants and their associated plasma lipid profiles in large population cohorts. Our findings support a novel role for GPR146 in human hypolipidemia, with carriers of GPR146 loss-of-function variants exhibiting an overall beneficial cardiometabolic risk profile. It remains to be demonstrated whether genetic or pharmaceutical inhibition of GPR146 confers atheroprotection in humans. We also found that GPR146 expression is negatively associated with SR-B1 protein levels. However, our experimental findings indicated that SR-B1 probably is not the causal driver of this phenotype. In conclusion, our studies suggest that hepatic GPR146 inactivation might constitute a potential therapeutic strategy to reduce plasma cholesterol and atherosclerosis independently of the LDLR and SR-B1. However, the current biological understanding of GPR146 functions and interactions, hepatic and extrahepatic, remains incomplete; therefore, additional efforts to clarify its true potential as a drug target are required