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

糖尿病是一种慢性疾病，在美国有3000多万人受到困扰。1型糖尿病(儿童期发病)和2型糖尿病(成年型)都与胰腺中的β细胞缺乏或受损有关。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一种必不可少的激素。工程化的胰岛素分泌细胞可以重建受损的胰岛β细胞功能，但所需的大量细胞对植入装置的尺寸、营养物质和氧气的供应构成了挑战，从而导致植入细胞的快速恶化。该项目旨在通过光遗传学手段，即结合遗传学和光学技术，提高工程贝塔细胞中胰岛素的产量。人类β细胞将经过基因工程改造，在近红外线窗口(NIRW)光激活时产生更多胰岛素。近红外光能穿透组织深处，比其他波长的光更能到达移植的贝塔细胞。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，这项技术将在糖尿病小鼠模型上进行测试。所产生的知识将为开发下一代有效治疗糖尿病的技术提供信息。与拟议研究交织在一起的教育活动将为STEM领域的新一代高中、本科生和研究生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普通公众。该项目的目标是设计具有增强葡萄糖刺激的胰岛素分泌(GSIS)的人β细胞，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。第一个进展表明，表达蓝光激活的腺苷环化酶(BPAC)以调节cAMP水平的啮齿动物β细胞显著(~3倍)增强GSIS，而不增加耗氧率。在BPAC-细胞移植后，接受蓝光照射的糖尿病小鼠表现出改善了葡萄糖耐量，降低了高血糖和更高的血浆胰岛素。BPAC原型的主要缺陷是由于蓝光穿透性差而导致的有限激活，由于BPAC在细胞内的错误定位而导致的非特异性cAMP效应，以及缺乏人β细胞。第二个进展是最近设计的一种由NIRW光激活的腺苷环化酶(NIRW-AC)，它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。研究计划是按照三个目标组织的。目的1设计一种在人β细胞中具有低暗活性、扩大光激活范围和优化表达的NIRW-AC。目标2是设计一种光诱导系统，用于局部的、靶向的cAMP水平的光激活。NIRW-AC的定位将被调整以反映天然ACS在β细胞中的定位，从而减少cAMP的非特异性影响，并改善β细胞的长期光遗传性能。目标3是用NIRW光控制增强GSIS来产生人β细胞。这将包括表达优化的NIRW-AC的工程化人类β细胞的生化和功能特征。目的4检测表达NIRW-AC的细胞在糖尿病小鼠模型中的作用。该项目的成果将是一种NIRW-AC，具有卓越的光激活、优化的空间定位和在人类β细胞中的表达，用于光基因增强型GSI。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.