Gene therapy for Glut1-deficient mouse using an adeno-associated virus vector with the human intrinsic GLUT1 promoter.
Gene therapy for Glut1-deficient mouse using an adeno-associated virus vector with the human intrinsic GLUT1 promoter.
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使用带有人类内在 GLUT1 启动子的腺相关病毒载体对 Glut1 缺陷小鼠进行基因治疗。
DOI：
10.1002/jgm.3013
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
Osaka H.
中科院分区：
文献类型：
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作者：
Nakamura S;Muramatsu SI;Takino N;Ito M;Jimbo EF;Shimazaki K;Onaka T;Ohtsuki S;Terasaki T;Yamagata T;Osaka H.
背景我们构建了腺相关病毒（AAV）载体，其中编码葡萄糖转运蛋白1（GLUT 1）的人SLC2A1基因在人内源性GLUT 1启动子（AAV-GLUT 1）下表达。我们研究了是否AAV-GLUT1管理可以导致功能改善inGLUT1缺陷mice. MethodsWe外推人类内源性GLUT1启动子序列从大鼠最小的Glut1启动子序列。我们产生了酪氨酸突变体AAV 9/3载体，其中人SLC2A1-myc-DDK在人GLUT 1启动子（AAV-GLUT 1）下表达。通过脑室内注射（1.85 × 1010 vg/小鼠或6.5 × 1010 vg/小鼠）向GLUT 1缺陷小鼠（GLUT 1 +/-小鼠）给予AAV ‐ GLUT 1。我们分析了外源性GLUT1 mRNA和蛋白质在脑和其他主要器官中的表达。我们还检查了脑微血管的改善，使用旋转杆和足迹测试的运动功能，以及血液和脑脊液（CSF）葡萄糖水平。结果外源性GLUT1蛋白在小鼠大脑皮层、海马和丘脑中有强表达，在海马和丘脑中表达量最高。主要表达于内皮细胞，部分表达于神经细胞和少突胶质细胞。侧脑室注射后，运动功能和CSF葡萄糖水平显著改善。外源性GLUT1的表达在脑室内注射AAV-GLUT1后在其他器官中未检测到，而在心脏内注射后在肝脏和肌肉组织中检测到。AAV-GLUT 1的局部中枢神经系统给药改善了GLUT 1缺陷小鼠的CSF葡萄糖水平和运动功能，并将脱靶效应降至最低。
BackgroundWe generated an adeno‐associated virus (AAV) vector in which the humanSLC2A1gene, 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 inGLUT1‐deficient mice.MethodsWe extrapolated human endogenous GLUT1 promoter sequences from rat minimal Glut1 promoter sequences. We generated a tyrosine‐mutant AAV9/3 vector in which humanSLC2A1‐myc‐DDKwas expressed under the human GLUT1 promoter (AAV‐GLUT1). AAV‐GLUT1 was administered toGLUT1‐deficient mice (GLUT1+/–mice) via intracerebroventricular injection (1.85 × 1010vg/mouse or 6.5 × 1010vg/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 × 1011vg/mouse).ResultsExogenous 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.ConclusionsExogenous 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 ofGLUT1‐deficient mice and minimized off‐target effects.