Sachie Nakamura1, Shin-Ichi Muramatsu2,3, Naomi Takino2, Mika Ito2, Eriko F Jimbo1, Kuniko Shimazaki4, Tatsushi Onaka5, Sumio Ohtsuki6, Tetsuya Terasaki7, Takanori Yamagata1, Hitoshi Osaka1. 1. Department of Pediatrics, Jichi Medical University, Tochigi, Japan. 2. Division of Neurology, Jichi Medical University, Tochigi, Japan. 3. Center for Gene and Cell Therapy, The Institute of Medical Science, The University of Tokyo, Japan. 4. Department of Neurosurgery, Jichi Medical University, Tochigi, Japan. 5. Division of Brain and Neurophysiology, Department of Physiology, Jichi Medical University, Tochigi, Japan. 6. Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan. 7. Division of Membrane Transport and Drug Targeting, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Abstract
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.
BACKGROUND: We generated an adeno-associated virus (AAV) vector in which the humanSLC2A1 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-deficientmice. METHODS: We extrapolated human endogenous GLUT1 promoter sequences from rat minimal Glut1 promoter sequences. We generated a tyrosine-mutant AAV9/3 vector in which humanSLC2A1-myc-DDK was expressed under the humanGLUT1 promoter (AAV-GLUT1). AAV-GLUT1 was administered to GLUT1-deficientmice (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 humanGLUT1 expression. Local central nervous system administration of AAV-GLUT1 improved CSF glucose levels and motor function of GLUT1-deficientmice and minimized off-target effects.
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