附属病院 とちぎ子ども医療センター

中村 幸恵

ナカムラ サチエ  (Sachie Nakamura)

基本情報

所属
自治医科大学 医学部小児科

J-GLOBAL ID
201401088746698154
researchmap会員ID
B000238506

外部リンク

主要な経歴

 4

論文

 11
  • Sachie Nakamura, Yasushi Ito, Hiroko Hayakawa, Shiho Aoki, Takanori Yamagata, Hitoshi Osaka
    Molecular genetics and metabolism reports 34 100954-100954 2023年3月  
    OBJECTIVE: We assessed the usefulness of flow cytometry as a functional assay to measure glucose transporter 1 (GLUT1) levels on the surface of red blood cells (RBCs) from Japanese patients with glucose transporter 1 deficiency syndrome (Glut1DS). METHODS: We recruited 13 genetically confirmed Glut1DS patients with a solute carrier family 2 member 1 (SLC2A1) mutation (eight missense, one frameshift, two nonsense, and two deletion) and one clinically suspected Glut1DS-like patient without an SLC2A1 mutation, and collected whole blood with informed consent. We stained pelleted RBCs (1 μL) from the patients with a Glut1.RBD ligand and anti-glycophorin A antibody, which recognizes a human RBC membrane protein, and analyzed the cells using flow cytometry. RESULTS: Relative GLUT1 levels quantified by flow cytometry in 11 of 13 patients with definite Glut1DS were 90% below those of healthy controls. Relative GLUT1 levels were not reduced in two of 13 Glut1DS patients who had a missense mutation and no intellectual disability and one Glut1DS-like patient without an SLC2A1 mutation. Relative GLUT1 levels were significantly reduced in Glut1DS patients with an SLC2A1 mutation, more severe intellectual disability, and spasticity. CONCLUSIONS: This method to detect GLUT1 levels on RBCs is simple and appears to be an appropriate screening assay to identify severe Glut1DS patients in the early stage before the development of irreversible neurologic damage caused by chronic hypoglycorrhachia.
  • Shinichi Kumagai, Takeshi Nakajima, Kuniko Shimazaki, Takeharu Kakiuchi, Norihiro Harada, Hiroyuki Ohba, Yoshiyuki Onuki, Naomi Takino, Mika Ito, Makoto Sato, Sachie Nakamura, Hitoshi Osaka, Takanori Yamagata, Kensuke Kawai, Shin-Ichi Muramatsu
    The journal of gene medicine 25(1) e3457 2023年1月  
    BACKGROUND: The delivery of adeno-associated virus (AAV) vectors via the cerebrospinal fluid (CSF) has emerged as a valuable method for widespread transduction in the central nervous system. Although infusion into the cerebral ventricles is a common protocol in preclinical studies of small animals, the cisterna magna has been recognized as an alternative target for clinical studies because it can be reached in a less invasive manner using an intrathecal catheter via the subarachnoid space from a lumbar puncture. METHODS: We evaluated the early distribution of fluorine-18-labeled AAV9 vectors infused into the lateral ventricle or cisterna magna of four non-human primates using positron emission tomography. The expression of the green fluorescent protein was immunohistochemically determined. RESULTS: In both approaches, the labeled vectors diffused into the broad arachnoid space around the brain stem and cervical spinal cord within 30 min. Both infusion routes efficiently transduced neurons in the cervical spinal cord. CONCLUSIONS: For gene therapy that primarily targets the cervical spinal cord and brainstem, such as amyotrophic lateral sclerosis, cisterna magna infusion would be a feasible and effective administration method.
  • Sachie Nakamura, Hitoshi Osaka, Shin-Ichi Muramatsu, Naomi Takino, Mika Ito, Eriko F Jimbo, Chika Watanabe, Shuji Hishikawa, Takeshi Nakajima, Takanori Yamagata
    Gene therapy 28(6) 329-338 2021年6月  
    Glucose transporter 1 deficiency syndrome (GLUT1DS) is caused by haplo-insufficiency of SLC2A1, which encodes GLUT1, resulting in impaired hexose transport into the brain. Previously, we generated a tyrosine-mutant AAV9/3 vector in which SLC2A1 was expressed under the control of the endogenous GLUT1 promoter (AAV-GLUT1), and confirmed the improved motor function and cerebrospinal fluid glucose levels of Glut1-deficient mice after cerebroventricular injection of AAV-GLUT1. In preparation for clinical application, we examined the expression of transgenes after intra-cisterna magna injection of AAV-GFP (tyrosine-mutant AAV9/3-GFP with the CMV promoter) and AAV-GLUT1. We injected AAV-GFP or AAV-GLUT1 (1.63 × 1012 vector genomes/kg) into the cisterna magna of pigs to compare differential promoter activity. After AAV-GFP injection, exogenous GFP was expressed in broad areas of the brain and peripheral organs. After AAV-GLUT1 injection, exogenous GLUT1 was expressed predominantly in the brain. At the cellular level, exogenous GLUT1 was mainly expressed in the endothelium, followed by glia and neurons, which was contrasted with the neuronal-predominant expression of GFP by the CMV promotor. We consider intra-cisterna magna injection of AAV-GLUT1 to be a feasible approach for gene therapy of GLUT1DS.
  • Yoshie Kurokawa, Hitoshi Osaka, Takeshi Kouga, Eriko Jimbo, Kazuhiro Muramatsu, Sachie Nakamura, Yuki Takayanagi, Tatsushi Onaka, Shin-Ichi Muramatsu, Takanori Yamagata
    Human gene therapy 32(11-12) 589-598 2021年6月  
    Niemann-Pick disease type C1 (NPC1) is a fatal congenital neurodegenerative disorder caused by mutations in the NPC1 gene, which is involved in cholesterol transport in lysosomes. Broad clinical manifestations of NPC1 include liver failure, pulmonary disorder, neurological deficits, and psychiatric symptoms. The main cause of death in NPC1 patients involves central nervous system (CNS) dysfunction; there is no essential treatment. We generated a tyrosine-mutant adeno-associated virus (AAV) 9/3 vector that expresses human NPC1 under a cytomegalovirus (CMV) promoter (AAV-CMV-hNPC1) and injected it into the left lateral ventricle (5 μL) and cisterna magna (10 μL) of Npc1 homo-knockout (Npc1-/-) mice. Each mouse received total 1.35 × 1011 vector genome on days 4 or 5 of life. AAV-treated Npc1-/- mice (n = 11) had an average survival of >28 weeks, while all saline-treated Npc1-/- mice (n = 11) and untreated Npc1-/- mice (n = 6) died within 16 weeks. Saline-treated and untreated Npc1-/- mice lost body weight from 7 weeks until death. However, the average body weight of AAV-treated Npc1-/- mice increased until 15 weeks. AAV-treated Npc1-/- mice also showed a significant improvement in the rotarod test performance. A pathological analysis at 11 weeks showed that cerebellar Purkinje cells were preserved in AAV-treated Npc1-/- mice. In contrast, untreated Npc1-/- mice showed an almost total loss of cerebellar Purkinje cells. Combined injection into both the lateral ventricle and cisterna magna achieved broader delivery of the vector to the CNS, leading to better outcomes than noted in previous reports, with injection into the lateral ventricles or veins alone. In AAV-treated Npc1-/- mice, vector genome DNA was detected widely in the CNS and liver. Human NPC1 RNA was detected in the brain, liver, lung, and heart. Accumulated unesterified cholesterol in the liver was reduced in the AAV-treated Npc1-/- mice. Our results suggest the feasibility of gene therapy for patients with NPC1.
  • Sachie Nakamura, Shin-Ichi Muramatsu, Naomi Takino, Mika Ito, Eriko F Jimbo, Kuniko Shimazaki, Tatsushi Onaka, Sumio Ohtsuki, Tetsuya Terasaki, Takanori Yamagata, Hitoshi Osaka
    The journal of gene medicine 20(4) e3013 2018年4月  査読有り
    BACKGROUND: We generated an adeno-associated virus (AAV) vector in which the human SLC2A1 gene, encoding glucose transporter type 1 (GLUT1), was expressed under the human endogenous GLUT1 promoter (AAV-GLUT1). We examined whether AAV-GLUT1 administration could lead to functional improvement in GLUT1-deficient mice. METHODS: We extrapolated human endogenous GLUT1 promoter sequences from rat minimal Glut1 promoter sequences. We generated a tyrosine-mutant AAV9/3 vector in which human SLC2A1-myc-DDK was expressed under the human GLUT1 promoter (AAV-GLUT1). AAV-GLUT1 was administered to GLUT1-deficient mice (GLUT1+/- mice) via intracerebroventricular injection (1.85 × 1010 vg/mouse or 6.5 × 1010 vg/mouse). We analyzed exogenous GLUT1 mRNA and protein expression in the brain and other major organs. We also examined improvements of cerebral microvasculature, motor function using rota-rod and footprint tests, as well as blood and cerebrospinal fluid (CSF) glucose levels. Additionally, we confirmed exogenous GLUT1 protein distribution in the brain and other organs after intracardiac injection (7.8 × 1011 vg/mouse). RESULTS: Exogenous GLUT1 protein was strongly expressed in the cerebral cortex, hippocampus and thalamus. It was mainly expressed in endothelial cells, and partially expressed in neural cells and oligodendrocytes. Motor function and CSF glucose levels were significantly improved following intracerebroventricular injection. Exogenous GLUT1 expression was not detected in other organs after intracerebroventricular injection of AAV-GLUT1, whereas it was detected in the liver and muscle tissue after intracardiac injection. CONCLUSIONS: Exogenous GLUT1 expression after AAV-GLUT1 injection approximated that of physiological human GLUT1 expression. Local central nervous system administration of AAV-GLUT1 improved CSF glucose levels and motor function of GLUT1-deficient mice and minimized off-target effects.

MISC

 3
  • Akira Morimoto, Yukiko Oh, Sachie Nakamura, Yoko Shioda, Tomomi Hayase, Toshihiko Imamura, Kazuko Kudo, Shinsaku Imashuku
    CYTOKINE 97 73-79 2017年9月  
    Objective: Langerhans cell histiocytosis (LCH) is characterized by immature dendritic cell proliferation, infiltration of LCH lesions by various inflammatory cells, and a lesional cytokine storm. It is classified into three groups on the basis of disease extent, namely, multisystem with risk-organ involvement (MS +), multisystem without risk-organ involvement (MS -), and single-system (SS) disease. We comprehensively analyzed whether serum levels of cytokines/chemokines reflect the disease extent. Methods: Serum samples from 52 children with LCH (eight, 25, and 19 with MS +, MS -, and SS, respectively) and 34 control children were analyzed quantitatively for 48 humoral factors. DNA samples extracted from biopsied LCH lesions from 12 patients were tested for BRAF V600E status. Results: The LCH patients had significantly higher serum levels of IL-1Ra, IL-3, IL-6, IL-8, IL-9, IL-10, IL12, IL-13, IL-15, IL-17, IL-18, TNF-alpha, G-CSF, M-CSF, MIF, HGF, VEGF, CCL2, CCL3, CCL7, CXCL1, and CXCL9 than the controls by univariate analysis. Of these IL-9, IL-15 and MIF were significant by multivariate analysis; but not differed between MS and SS diseases. MS disease associated with significantly higher IL-2R, IL-3, IL-8, IL-18, M-CSF, HGF, CCL2, CXCL1, and CXCL9 levels than SS disease by univariate analysis. Of these, CCL2 and M-CSF were significant by multivariate analysis. IL-18 levels were significantly higher in MS + disease than MS disease. The LCH patients with BRAF V600E mutation had higher serum levels of CCL7. Conclusion: Numerous inflammatory cytokines and chemokines play a role in LCH. Of those, more specific ones reflect the disease extent (MS vs. SS and MS + vs. MS -) or the BRAF V600E mutation status. It is thought that the most responsible cytokines and chemokines involved in the poor outcome may become future candidate therapeutic targets in LCH.
  • Sachie Nakamura, Hitoshi Osaka, Shin-ichi Muramatsu, Naomi Takino, Mika Ito, Shiho Aoki, Eriko F. Jimbo, Kuniko Shimazaki, Tatsushi Onaka, Sumio Ohtsuki, Tetsuya Terasaki, Takanori Yamagata
    MOLECULAR THERAPY 25(5) 108-108 2017年5月  
  • 門田 行史, 中瀬 葉子, 中村 幸恵, 早瀬 朋美, 翁 由紀子, 柏井 良文, 郡司 勇治, 今村 俊彦, 森本 哲, 桃井 真里子
    小児科臨床 64(9) 2047-2051 2011年9月  
    急性リンパ性白血病/非ホジキンリンパ腫の初発症状の5%に眼球突出があり、視力の保持の観点からは重要な所見である。今回我々は視力について異なる転帰をとった2例を経験した。症例1は2歳の男児。主訴は右眼球突出。眼症状以外の所見なく、CTで眼窩内腫瘤があり、発症1ヵ月時に摘出術を実施し、病理像はsmall round cell tumorであった。骨髄穿刺で芽球を67%認め、B前駆型急性リンパ性白血病(ALL)と診断し、発症後8週目から化学療法を開始した。眼窩の残存腫瘤は速やかに消失し寛解したが、右側の視力は光覚弁であった。症例2は6ヵ月の女児。主訴は左眼球突出。MRIで眼窩内腫瘤を認めた。胸水貯留があり、胸腔穿刺でB前駆型非ホジキンリンパ腫と診断し、発症3週間目にALLに準じた化学療法を開始した。速やかに眼窩腫瘤は消失、寛解し、視力は保たれた。眼窩腫瘤の原因鑑別診断には急性白血病/非ホジキンリンパ腫を鑑別する必要がある。(著者抄録)

共同研究・競争的資金等の研究課題

 4