公募シンポジウム

公募シンポジウムアデノ随伴ウイルスベクターの最先端　ー基礎研究から臨床応用まで

7月7日（金）　10:40-12:40　Room C 2SY③-1 細胞種特異的AAVベクターの開発 -マウスからマーモセットまで Development of AAV vectors targeting a specific cell population - from mouse to marmoset 平井　宏和^1,2 1. 群馬大学　脳神経再生医学, 2. 群馬大学　ウイルスベクター開発研究センター Hirokazu Hirai^1,2 1. Dept. of Neurophysiol., Gunma Univ., Maebashi, Japan, 2. Gunma Univ., Viral Vector Core Adeno-associated virus (AAV) vectors can infect both neurons and glial cells, and allow efficient and long-term expression of foreign genes, making them ideal gene therapy vectors for neuropsychiatric diseases. Many CNS diseases affect specific cell populations. For example, Parkinson’s disease impairs Dopaminergic neurons in the Substantia nigra, many types of Spinocerebellar ataxia damage cerebellar Purkinje cells, and Alzheimer’s disease compromises microglia in the cerebral cortex. Thus, cell type-specific AAVs are desirable as gene therapy vectors to achieve efficient therapeutic outcome with minimal toxicity. We have developed AAV vectors specifically targeting pan-neuron, excitatory or inhibitory neuron, Purkinje cell, astrocyte and microglia. Some of the promoters have been proved available also in common marmosets, non-human primates. In this presentation, we introduce availability of those cell type-specific AAVs for basic/translational research as well as therapeutic interventions.		7月7日（金）　10:40-12:40　Room C 2SY③-2 マウスを用いたウイルス遺伝学的アプローチによる周産期オキシトシン神経活動解析 Insights into maternal oxytocin neural activities using viral genetic approaches in mice 幸長　弘子^1,2, 矢口　花紗音², 宮道　和成² 1. 京都大学大学院　生命　生体制御学, 2. 理研　比較コネクトミクス研究チーム Hiroko Yukinaga^1,2, Yaguchi Kasane², Miyamichi Kazunari² 1. Lab. of Bioimaging and Cell Signal., Grad. Sch. of Biostudies, Kyoto Univ., Kyoto, Japan, 2. Lab. for Comparative Connectomics, Riken BDR, Kobe, Japan Oxytocin (OT) is responsible for uterine contraction during parturition and milk ejection during lactation. The pulsatile release of OT is generated by unique activity patterns of OT neurons in the hypothalamus. Previous studies characterized putative OT neurons in rats based on electrophysiological characteristics but not on OT gene expression. To better understand maternal OT neuron activity and make use of cell-type-specific neuroscience toolkits, we developed a mouse model using viral genetic approaches and fiber photometry. A sharp photometric peak of OT neurons was observed at around 520 seconds after suckling stimuli from pups. The amplitude of the peak was dynamically modulated by the experience and history of lactation patterns, suggesting the intrinsic plasticity of maternal OT neurons. By activating inhibitory neurons in the bed nucleus of the stria terminalis, the pre-synaptic partners of OT neurons, we identified a modulatory circuit of pulsatile activities of OT neurons. Additionally, we established a simple Cre-free method for monitoring OT neurons by a virus vector that drives GCaMP6s under a mouse oxytocin mini-promoter. Collectively, our study illuminates the temporal dynamics of maternal neural activities of OT neurons, which serve as a basis for uncovering molecular/circuit mechanisms of maternal life-stage transitions.

7月7日（金）　10:40-12:40　Room C 2SY③-3 AAVベクターを活用した脊髄小脳失調症共通の発症機序解明と治療薬探索 Elucidation of pathogenic mechanism and therapeutic strategies commonly for spinocerebellar ataxias using AAV vectors 関　貴弘姫路獨協大学　薬学部　薬理学研究室 Takahiro Seki Dept. Pharmacol., Fac. Pharm. Sci. Himeji Dokkyo Univ. Spinocerebellar ataxia (SCA) is a group of autosomal dominant neurodegenerative diseases, which is characterized by the progressive motor dysfunction and cerebellar atrophy. SCA is classified into SCA1-49 by the differences in the loci of causal genes. Although common functions of SCA causal proteins have not been identified, we assume that there are molecular mechanisms commonly for various SCAs. Therefore, we attempted to identify the phenotypes and molecular alterations for SCA using AAV-mediated gene transfer of several SCA-causing genes. The expression of SCA-causing proteins commonly induced the dendritic shrinkage of Purkinje cells (PCs) in primary cerebellar cultures. Additionally, SCA-causing proteins commonly triggered the impairment of chaperone-mediated autophagy (CMA), one of the proteolytic pathways in primary cultured PCs. Importance of CMA in SCA pathogenesis is validated by the finding that miRNA-mediated knockdown of LAMP2A, a CMA-related protein, in cerebellar neurons mimics the phenotypes of SCA model mice, including the progressive motor impairment and cerebellar neurodegeneration. We have recently demonstrated that D-cysteine activates CMA in cerebellar neurons, alleviates the dendritic shrinkage in SCA model PCs, and prevents motor impairment in SCA model mice. Therefore, CMA would be a novel therapeutic target commonly for various types of SCA.		7月7日（金）　10:40-12:40　Room C 2SY③-4 非ヒト霊長類を用いたAAVベクター研究の新展開 Development in novel adeno-associated virus vectors suitable for gene transduction into the nonhuman primate brain 高田　昌彦京都大学　ヒト行動進化研究センター、犬山、日本 Masahiko Takada Center for the Evolutionary Origins of Human Behavior, Kyoto University, Inuyama, Japan Recent emphasis has been placed on gene transduction mediated through recombinant adeno-associated virus (AAV) vector to manipulate activity of neurons and their circuitry in nonhuman primates. Here, we introduce two mosaic AAV vectors that have been developed for highly-efficient transfer of foreign genes into the nonhuman primate brain. First, we created the AAV2.1 vector of which capsid was composed of capsid proteins derived from both of AAV1 and AAV2. Following its intracortical injection in macaques, this vector was found to exhibit simultaneously the superiority of transgene expression and neuron specificity to other serotypes of vectors, including AAV1 and AAV2 vectors. Second, we created the AAV9.2 vector of which capsid was composed of capsid proteins derived from both of AAV2 and AAV9. Through its intravenous administration in marmoset neonates, this vector was seen to display neuron-specific, brain-wide expression of foreign genes. In this presentation, some applicability of such novel vectors is also introduced.

7月7日（金）　10:40-12:40　Room C 2SY③-5 遺伝性神経疾患に対するAAVベクターによる治療 Gene therapy using AAV vector for genetic neurological disorders 小坂　仁自治医科大学　小児科 Hitoshi Osaka Dept. of Pediatrics, Jichi Medical University, Shimotsuke, Japan Majorities of genetic neurological disorders are caused by loss of function and are good targets for gene therapy. Recently, we examined an adeno-associated virus (AAV) vector to treat 8 patients with Aromatic L-amino acid decarboxylase deficiency(Kojima et al., Brain 2019). Glucose transporter 1 deficiency syndrome (GLUT1DS) is an autosomal dominant disorder caused by the haploinsufficiency of SLC2A1, a gene encoding GLUT1 that is expressed in all brain areas. We have established a functional assay for glucose transporter activity (Nakamura et al., Mol Genet Metab. 2015). We generated an AAV vector, AAV-glut1-SLC2A1, involving SLC2A1 under its intrinsic promoter, and injected this vector into the cerebral ventricles of the GLUT1+/- model mouse. One injection improved cerebrospinal fluid glucose levels and motor functions to comparable to wild-type levels, as assessed by rota-rod tests (Nakamura et al., J Gene Med. 2018). We injected AAV-GLUT1 into the cisterna magna of pigs. After injection, exogenous GLUT1 was expressed in the wide areas of the central nervous system (CNS). Intra-cisterna magna injection appears to be a feasible approach for gene therapy of GLUT1DS (Nakamura et al., Gene Ther. 2021). Patients with rare genetic neurological diseases comprise a large proportion of unmet medical needs. AAV gene therapy can provide great promises for these patients and families.