A cell-based glucose sensing and insulin delivery system

描述(申请人提供)：治疗1型糖尿病的理想细胞疗法是一种细胞来源，这种细胞可以根据生理信号分泌大量胰岛素，但缺乏免疫原性，从而避免移植排斥反应的风险。我们发现，可以用转基因小鼠技术改造垂体中间叶(IL)的细胞，将其移植到糖尿病NOD小鼠体内，产生足够数量的胰岛素(INS)来治疗糖尿病，但与Betacells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，它们被设计为表达高水平的关键葡萄糖敏感成分GLUT2和葡萄糖激酶(GK)。尽管这些动作赋予了令人印象深刻的血糖感知能力，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存活的必要性。我们最近开发了一种转基因小鼠，在ILins组织中表达生理水平的GLUT2和GK。同时，我们创建了可诱导的病毒载体系统，使我们能够滴定GK(和GLUT2)在ILINS细胞中的表达水平。总之，这些系统将使我们能够确定葡萄糖感应和葡萄糖刺激的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，而KATP通道或GLP-1受体是连接葡萄糖代谢和胰岛素分泌的关键。这项建议的目标是： 1.将B细胞KATP通道复合体工程表达到表达GK和GLUT2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，实时测量单细胞内胰岛素的分泌动力学和代谢。 3.评估GLP-1受体的引入是否将提高LLINs细胞对葡萄糖的感知和反应能力，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影响，并评估生物工程LLINs细胞在自身免疫性糖尿病环境中的表现。 这个项目是利普斯博士、电生理学家科林·利奇博士和分析化学家罗伯特·肯尼迪博士之间的多学科合作。利普斯博士是一名成熟的1型糖尿病研究人员，也是一名分子生物学家，而罗伯特·肯尼迪博士则是一名分析化学家，他开创了在单细胞中实时评估胰岛功能的创新技术。这些研究的结果可能对开发用于治疗糖尿病的胰岛素递送系统中基于闭环细胞的传感器具有重要意义。

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.