Collaborative Research: Near-infrared light-controlled beta-cells

合作研究：近红外光控制的β细胞

• 批准号: 2015855
• 负责人: Mark Gomelsky
• 金额: $ 29.94万
• 依托单位: University of Wyoming
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2024-03-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2015855&HistoricalAwards=false
• 关键词: Collaborative; Research; Near; infrared; light

Diabetes is a chronic disease afflicting over 30 million people in the United States. Both type 1 (childhood onset) and type 2 (adult onset) diabetes are associated with the deficiency or impairment in beta-cells in the pancreas. These are the only cells in the body that secrete insulin, an essential hormone for blood glucose regulation. Engineered insulin-producing cells can reconstitute the impaired pancreatic beta-cell function, however, the large number of required cells poses challenges on the implantation device size, and supply of nutrients and oxygen, which results in rapid deterioration of implanted cells. This project aims to enhance insulin production in engineered beta-cells via optogenetic means, i.e., using a combination of genetics and optics. Human beta cells will be genetically engineered to produce more insulin when activated by near-infrared window (NIRW) light. NIRW light penetrates deep through tissues and can reach transplanted beta-cells better than light of other wavelengths. The enhanced function of beta-cells means that fewer cells need to be transplanted to correct high blood glucose. Once developed, the technique will be tested in a mouse model of diabetes. The knowledge generated will inform the development of the next generation of technologies for efficient diabetes treatments. Educational activities, which are intertwined with the proposed research, will provide ample training opportunities for a new generation of high school, undergraduate and graduate students in STEM fields. The knowledge and associated technologies generated through this work will be disseminated to the scientific community and the general public through online media, presentations, and publications.The goal of this project is to engineer human beta-cells with enhanced glucose-stimulated insulin secretion (GSIS) in response to near-infrared window (NIRW) light. The project capitalizes on two recent advances from the collaborating laboratories. The first advance is demonstrating that rodent beta-cells expressing a blue light-activated adenylate cyclase (bPAC) for modulation of cAMP levels significantly (~3-fold) enhanced GSIS with no increase in oxygen consumption rate. After bPAC--cell transplantation, diabetic mice subjected to blue light display improved glucose tolerance, lower hyperglycemia and higher plasma insulin. The key deficiencies of the bPAC-prototype were limited activation due to poorly penetrating blue light, nonspecific cAMP effects due to intracellular mislocalization of bPAC, and the lack of human Beta-cells. The second advance is a recently engineered adenylate cyclase activated by NIRW light (NIRW-AC) that will help overcome these deficiencies as it penetrates more deeply through mammalian tissue than blue light. The Research Plan is organized under three objectives. OBJECTIVE 1 is to design a NIRW-AC with low dark activity, improved photoactivation range and optimized expression in human beta-cells. OBJECTIVE 2 is to engineer a light inducible system for localized, target-specific photoactivation of cAMP levels. The localization of the NIRW-AC will be tuned to mirror the localization of native ACs in beta-cells thereby reducing the nonspecific effects of cAMP and improving the long-term optogenetic performance of beta-cells. OBJECTIVE 3 is to generate human beta-cells with NIRW light controlled enhancement of their GSIS. This will encompass the biochemical and functional characterization of the engineered human beta-cells expressing the optimized NIRW-AC. OBJECTIVE 4 is to test the performance of the NIRW-AC-expressing cells in a murine model of diabetes. The project’s deliverable will be a NIRW-AC with superior photoactivation, optimized spatial positioning and expression in human beta-cells for optogenetically enhanced GSIS.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

糖尿病是一种慢性疾病，在美国困扰着超过3000万人。1型（儿童期发病）和2型（成人期发病）糖尿病都与胰腺中β细胞的缺陷或损伤有关。这些细胞是体内唯一分泌胰岛素的细胞，胰岛素是调节血糖的必需激素。工程化的胰岛素产生细胞可以重建受损的胰腺β细胞功能，然而，所需的大量细胞对植入装置的尺寸以及营养物质和氧气的供应提出了挑战，这导致植入细胞的快速退化。该项目旨在通过光遗传学手段增强工程β细胞中的胰岛素生产，即，利用遗传学和光学的结合。 人类β细胞将被基因工程改造，当被近红外窗口（NIRW）光激活时，会产生更多的胰岛素。 NIRW光可以穿透组织，比其他波长的光更好地到达移植的β细胞。 β细胞功能的增强意味着需要移植更少的细胞来纠正高血糖。 一旦开发出来，该技术将在糖尿病小鼠模型中进行测试。 所产生的知识将为下一代有效糖尿病治疗技术的开发提供信息。教育活动与拟议的研究交织在一起，将为新一代的高中，本科和研究生在STEM领域提供充足的培训机会。通过这项工作产生的知识和相关技术将通过在线媒体，演示文稿和出版物传播给科学界和公众。该项目的目标是工程化人类β细胞，增强葡萄糖刺激的胰岛素分泌（GSIS），以响应近红外窗口（NIRW）光。 该项目利用了合作实验室的两项最新进展。第一个进展是证明表达蓝光激活的腺苷酸环化酶（bPAC）以调节cAMP水平的啮齿动物β细胞显著（~3倍）增强GSIS而不增加耗氧速率。在bPAC-1细胞移植后，接受蓝光照射的糖尿病小鼠显示出改善的葡萄糖耐量、较低的高血糖和较高的血浆胰岛素。bPAC原型的关键缺陷是由于蓝光穿透性差导致的有限激活、由于bPAC的细胞内错误定位导致的非特异性cAMP效应以及缺乏人β细胞。 第二个进展是最近工程化的腺苷酸环化酶，由近红外线光（NIRW-AC）激活，这将有助于克服这些缺陷，因为它比蓝光更深入地穿透哺乳动物组织。 研究计划有三个目标。 目的1是设计一种具有低暗活性、改善光活化范围和优化在人β细胞中表达的NIRW AC。目的2是设计一种光诱导系统，用于局部的、靶向特异性的cAMP水平的光活化。NIRW-AC的定位将被调整以反映β细胞中天然AC的定位，从而减少cAMP的非特异性作用并改善β细胞的长期光遗传学性能。 目的3是产生具有近红外光控制的GSIS增强的人β细胞。 这将包括表达优化的NIRW-AC的工程化人β细胞的生物化学和功能表征。 目的4是测试NIRW-AC表达细胞在糖尿病小鼠模型中的性能。该项目的交付成果将是一个具有上级光活化、优化空间定位和在人类β细胞中表达的NIRW-AC，用于光遗传学增强GSIS。该奖项反映了NSF的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。