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.
