Introduction: Protection against the human immune system is required to realize the therapeutic potential of stem cell (SC)-islet cells for the treatment of type 1 diabetes. Various groups have attempted to address this challenge by engineering immune-evasive human embryonic stem cells (hESCs) that can be differentiated into various cell types. In many cases, over-expression of the tolerogenic molecules at safe harbor loci is lost due to transgene silencing. We recently addressed this issue by targeting Firefly luciferase (Luc2) to the constitutively expressed GAPDH locus of hESCs (GAPluc), followed by successful differentiation into SC-islet cells that maintain Luc2 expression. Thus, in this study we used a similar engineering strategy to generate hypoimmunogenic SC-islet cells.

Methods: Using CRISPR/Cas9 we introduced Luc2 and the B2M::HLA-E fusion to the GAPDH locus of hESCs, followed by CRISPR knockout of endogenous B2M to eliminate class I HLA expression. Using sequential antibiotic selection of GAPDH-targeted and FACS sorting of HLA-ABC^-/- hESCs, we generated a hypoimmunogenic hESC line (BEC) that was differentiated into SC-islet cells. SC-islet cell immunogenicity was assessed in in vitro PBMC and NK cell co-culture assays using cells isolated from allogeneic human peripheral blood (n=5 donors), with Luc2 expression serving as a reporter of cell viability. Wild-type (WT) and B2M^-/- SC-islet cells served as controls. To assess SC-islet cell survival in vivo, 5x10⁶ WT and BEC SC-islet cells were transplanted under the kidney capsule of HLA-A2 mismatched humanized NSG-(K^b D^b)^null (IA^null) mice (n=4/group). Graft survival was assessed via bioluminescence imaging using the IVIS Spectrum.

Results: Differentiation of WT, B2M^-/- and BEC hESCs into SC-islet cells resulted in a similar yield (~20%) of Nkx6.1⁺/C-peptide⁺ SC-beta cells. HLA-E and Luc2 expression correlated throughout the course of differentiation, with a ~10-fold decrease in expression in hESCs vs CD49a-enriched SC-beta cells. No significant difference in Luc2 expression was observed in CD49a-enriched SC-beta cells derived from all gene-modified hESC lines. In in vitro PBMC co-culture assays, B2M^-/- and BEC SC-islet cells were significantly protected from PBMC cytotoxicity by comparison to WT. In in vitro NK cell co-culture assays, BEC SC-islet cells were significantly protected against NK cell cytotoxicity by comparison to B2M-/-. Additionally, following transplant into the kidney capsule of humanized NSG-(K^b D^b)^null (IA^null) mice, BEC SC-islet cells showed a significant survival advantage by comparison to WT at 35 days post transplantation.

Conclusion: In this study we show that targeting transgenes to constitutively expressed loci results in persistent transgene expression throughout the course of islet cell differentiation. Furthermore, our engineering strategy resulted in the generation of hypoimmunogenic SC-islet cells that evade immune rejection both in vitro and in vivo.