Associate Professor Institute of Science Tokyo, United States
Abstract: Human induced pluripotent stem cells (iPSCs) offer powerful potential for applications in disease modeling and regenerative medicine. However, existing genome engineering tools fall short in enabling the large-scale, precise modifications required for these advanced applications. Our proprietary genome engineering platform, Geno-Writing™, overcomes these limitations by enabling (1) bi-allelic modification of endogenous genomic loci up to 100 kb and (2) integration and stable expression of up to 12 transgenes in human iPSCs within two months (Nature Communications., 13, 4219, 2022; unpublished). This unprecedented capability to perform swift and large-scale genomic alterations and complex functional enhancements represents a paradigm shift in iPSC engineering. As a proof of concept, we are applying the Geno-Writing™ platform to develop iPSCs for cell therapy in Type 1 Diabetes, focusing on two key aspects: (1) enhancing immune tolerance and (2) improving differentiation efficiency into pancreatic β cells. For immune tolerance, we precisely engineer the human leukocyte antigen (HLA) locus using Geno-Writing™, directly deleting only HLA genes while preserving the expression of adjacent protein genes. Conventional strategies, such as disrupting B2M or CIITA, can abolish HLA protein expression but may also impair other essential cellular functions due to the pleiotropic roles of these genes. Moreover, such approaches may not eliminate the potential re-expression of HLA molecules in specific organs, posing a risk of immune rejection after transplantation. Our HLA-specific editing avoids these pitfalls, generating truly HLA-null hypoimmune iPSCs. To enhance β cell differentiation, we identified candidate genes using our proprietary gene algorithm and generated their biallelic knockouts in iPSCs. These engineered clones having only single gene deletion demonstrated up to 10% improvement in β cell yield or a 6-fold increase in insulin secretion upon differentiation. These results highlight the power of the Geno-Writing™ platform as a highly effective technology for developing next-generation allogeneic iPSC-derived cell therapies.
Funding Source: This work was supported by internal funding.
