Eurola, S. | bioRxiv Science

bioRxiv2023

RFX6 haploinsufficiency predisposes to diabetes through impaired beta cell functionality

Regulatory factor X 6 (RFX6) is indispensable for pancreatic endocrine development and differentiation. The RFX6 protein-truncating variant p.His293LeufsTer7 is significantly enriched in the Finnish population with almost 1:250 individuals as a carrier. Importantly, the FinnGen study indicates a high predisposition for heterozygous carriers to develop type 2 diabetes (T2D) and gestational diabetes. To understand the role of this variant in {beta}-cell development and function, we generated allelic series of isogenic pluripotent stem cell models and directed them into pancreatic islet lineages (SC-islets). Expectedly, in-vitro models of the homozygous RFX6-/- variant failed to generate pancreatic endocrine cells, recapitulating the phenotype in Mitchell-Riley syndrome. Notably, heterozygous RFX6+/- derived SC-islets showed reduced {beta}-cell maturation markers and calcium oscillations, resulting in defective insulin secretion, without affecting {beta}-cell number or insulin content. The reduced insulin secretion is sustained during in-vivo implantation studies, consistent with the susceptibility of the carriers to develop diabetes. TeaserModeling RFX6-assocciated neonatal and type-2 diabetes using allelic series stem cell-derived islets in-vitro and in-vivo.

cell biology↗

bioRxiv2023

CRISPR/Cas9 DNA synthesis disruption as a tool to control cell proliferation in vitro and in vivo

Engineered cells hold great promise for regenerative medicine and gene therapy. However, living cell products entail a fundamental biological risk of unwanted growth. Here, we describe a novel metabolic safety system to control cell proliferation without added genetic elements. We inactivated a key enzyme for nucleotide metabolism, TYMS, in several cell lines, thus obtaining cells that proliferate only when supplemented with exogenous thymidine but fail to replicate in its absence. Under supplementation, TYMS-/- pluripotent stem cells proliferate normally, produce teratomas and differentiate into potentially therapeutic cell types such as pancreatic beta cells. After differentiation, the postmitotic cells do not require thymidine to function, as seen by prolonged in vivo production of human insulin in implanted mice. Hence, this method allows robust cell culture and manufacture while mitigating the risk of uncontrolled growth of transplanted cells. One Sentence Summary: Genetic disruption of DNA synthesis prevents unwanted proliferation in cell therapies without affecting cell function.

bioengineering↗