2430-P: Inducing Beta-Cell Maturity through Selective Activation of Endogenous Gene Regulatory Networks

Introduction and Objective: Stem cell derived β-cells (sBC) exhibit an immature phenotype, potentially obstructing clinical efficacy. Bioinformatic analyses reveal differential or absent expression of key β-cell transcription factors (TFs) between sBC and primary human β-cells. We hypothesize upregulating specific TFs simultaneously will facilitate sBC maturation. Methods: We engineered human pluripotent stem cells (hPSCs) with a GFP endogenous insulin reporter to constitutively express dCas9-VPR, a dCas9 fused with transcriptional activators (Fig. 1A, B). Verifying function, we delivered 3 guide RNAs (sgRNAs) targeting distinct sites upstream of the insulin promoter, confirmed normal karyotype, and pluripotency marker expression. (Fig. 1C-E). Using established protocols, we differentiated Cas9VPR sBC, established a cryobank, and a protocol for sgRNA delivery in thawed sBC. (Fig. 1F-H). Results: Upon Insulin sgRNA transfection in Cas9VPR hPSC Clone #5, we observed increasing insulin expression within 72 hours. (Fig. 1C) Endogenous PDL1 expression in Cas9VPR sBC post PDL1 sgRNA delivery was comparable to 72-hour IFN-γ treated sBC controls (Fig. 1H). Conclusion: We have selected 13 TFs with low or absent expression in sBC, procured gRNAs, and aim to conduct detailed analysis upon upregulation. We anticipate these experiments will provide detailed insights into human β-cell maturation and help to establish mature sBC for clinical efforts. Disclosure A. T. Kratz: None. H. Russ: Other - Advisory panel and consultant, stock holder; Current; Sernova, Corp. Other - Scientific founder, Consultant, Advisory panel, stock holder; Current; Tolerance Bio. Consultant; Current; Guidepoint consulting, Axxon Advisors. Funding The work in the Russ lab is or was supported by R01DK120444, R01DK132387 and R01NS122911, a new investigator award from NIDDK/HIRN RRID: SCR₀14393; UC24 DK104162, and SRA grants 2-SRA-2023-1313-S-B and 3-SRA-2023-1367-S-B by Breakthrough T1D (BT1D). ATK is supported by the NIH NIDDK T32 Training Program in Type 1 Diabetes and Biomedical Engineering T32 DK10876.