Development of cell-based therapeutics for diabetes using genome-editing technologies

Abstract

The current stem cell-derived beta cells for the ultimate treatment of diabetes face several limitations. 1) They exhibit insufficient glucose-stimulated insulin secretion (GSIS) compared to human islets. 2) Although cells are encapsulated in a macroencapsulation device to eliminate immune rejection, foreign body reaction (FBR) occurs in response to the device, leading to the formation of a fibrotic capsule around the device. This capsule obstructs substance transport, resulting in cell death. 3) Cells within the device experience insufficient oxygen supply, especially problematic for beta cells with high oxygen consumption, making them prone to apoptosis under hypoxic conditions. Research aims to overcome these limitations. Using CRISPR/Cas9 technology, functional genes are introduced into stem cells, which are then differentiated to produce enhanced stem cell-derived beta cells. Editing was attempted not only at the existing safe harbor loci but also in the exploration of new candidate safe harbor genes to introduce various functional genes. A system was established by fusing a secretion peptide to nanoluciferase, enabling the external secretion of proteins. This laid the groundwork for the external secretion of anti-fibrosis proteins and GSIS-enhancing proteins. Additionally, a hypoxia-responsive protein expression system was developed using the hypoxia response element (HRE). Based on this system, anti-apoptotic functions of XIAP and BCL-xL were confirmed under hypoxia. Furthermore, an efficient edited cell sorting system using GFP fragment complementation was established, minimizing genetic footprint while effectively isolating edited cells with approximately 20% efficiency. Presently, these method is employed for the generation of therapeutic cells. These findings are expected to provide new perspectives for the development of next-generation diabetes cell therapies.