A cell-based glucose sensing and insulin delivery system

描述（由申请人提供）：1型糖尿病的理想的基于细胞的疗法将是细胞的来源，所述细胞可以响应于生理线索而分泌大量胰岛素，但缺乏免疫原性，从而避免移植物排斥的风险。我们已经发现，垂体中叶（IL）细胞可以用转基因小鼠技术进行工程改造，以产生足够量的胰岛素（ins），当植入糖尿病NOD小鼠时，治疗糖尿病，但与Betacell相反，ILins细胞逃避免疫攻击。在将葡萄糖敏感特性引入这些细胞的最初尝试中，它们被工程化以表达高水平的关键葡萄糖敏感组分GLUT 2和葡萄糖激酶（GK）。虽然这些操作赋予了令人印象深刻的葡萄糖传感能力，但这些基因的高水平表达也与细胞毒性有关。最近对转基因小鼠的研究强调了密切优化GK基因表达以确保细胞活力的必要性。我们最近开发了一种在ILins组织中表达生理水平的GLUT 2和GK的转基因小鼠系。同时，我们已经创建了可诱导的病毒载体系统，其将允许我们滴定IL ins细胞中GK（和GLUT 2）的表达水平。总之，这些系统将使我们能够确定葡萄糖传感和葡萄糖刺激胰岛素分泌的最佳基因剂量水平。然而，ILins细胞不表达KATP通道或GLP-1受体，其对于将葡萄糖代谢偶联至胰岛素分泌至关重要。本提案的目标是： 1.将B细胞KATP通道复合物工程化表达到表达GK和GLUT 2的ILins细胞中; 2.评估这些细胞感知和分泌胰岛素的能力。确定是否可以通过改变GK表达来进一步优化这些细胞的性能。使用新型分析仪器在单细胞中真实的实时测量胰岛素分泌动力学和代谢。 3.评估GLP-1受体的引入是否会改善ILins细胞感知和响应葡萄糖的能力，以及是否独立于KATP通道途径 4.评估这些操作对体内葡萄糖稳态的生理作用，并评估生物工程化的ILins细胞在自身免疫性糖尿病背景下的性能。 该项目是Lipes博士，一位成熟的1型糖尿病研究人员和分子生物学家，Colin Leech博士，一位专家电生理学家和Robert Kennedy博士之间的多学科合作，Robert Kennedy博士是一位分析化学家，他开创了单细胞和真实的时间评估胰岛功能的创新技术。这些研究的结果可能有助于开发一个闭环的细胞为基础的传感器在胰岛素输送系统治疗糖尿病。

DESCRIPTION (provided by applicant): An ideal cell-based therapy for type 1 diabetes would be a source of cells that could secrete abundant amounts of insulin in response to physiological cues but that lacks immunogenicity, thereby avoiding the risk of graft rejection. We have discovered that cells in the intermediate lobe (IL) of the pituitary could be engineered with transgenic mouse techniques to produce sufficient amounts of insulin (ins) to cure diabetes when implanted into diabetic NOD mice but, in contrast to Betacells, the ILins cells evaded immune attack. In initial attempts to introduce glucose-sensing properties into these cells, they were engineered to express high levels of the key glucose sensing components, GLUT2 and glucokinase (GK). Although these maneuvers conferred impressive glucose sensing capabilities, the high-level expression of these genes was also associated with cellular toxicity. More recent work with transgenic mice has underscored the need to closely optimize GK gene expression to ensure cell viability. We have recently developed a line of transgenic mice that express physiological levels of GLUT2 and GK in ILins tissues. In parallel, we have created inducible viral vector systems that will allow us to titrate the expression levels of GK (and GLUT2) in ILins cells. Together, these systems will enable us to determine the optimal gene dosage levels for glucose sensing and glucose-stimulated insulin secretion. However, ILins cells do not express KATP channels or GLP-1 receptors that are critical for coupling glucose metabolism to insulin secretion. The goal of this proposal is to: 1. Engineer expression of the B cell KATP channel complex into ILins cells that express GK and GLUT2; 2. Evaluate the ability these cells to sense and secrete insulin. Determine whether the performance of these cells can be further optimized by altering GK expression. Use novel analytical instrumentation to measure insulin secretory dynamics and metabolism in single cells and in real time. 3. Assess whether the introduction of GLP-1 receptors will improve the ability of lLins cells to sense and respond to glucose, in addition to, and independent of the KATP channel pathway 4. Assess the physiological effect of these manipulations on glucose homeostasis in vivo and evaluate the performance of the bioengineered lLins cells in the setting of autoimmune diabetes. This project is a multidisciplinary collaboration between Dr. Lipes, an established type 1 diabetes researcher and a molecular biologist, Dr. Colin Leech, an expert electrophysiologist, and Dr. Robert Kennedy, an analytical chemist who has pioneered innovative technology for evaluating islet function in single cells and in real time. The result of these studies could contribute importantly to the development of a closed-loop cell based sensor in insulin delivery system for the treatment of diabetes.