Single–gene knockout of RNLS or HIVEP2 are insufficient to protect β–cell spheroids from allo– and xeno–rejection

Abstract Type 1 diabetes can be cured by β–cell replacement in principle, yet recurrent autoimmunity and transplantation barriers rapidly destroy implanted cells. Genome–wide CRISPR screening by Cai et al. highlighted RNLS and HIVEP2 as candidate genes, but their value outside an autoimmune setting is unknown. Here, it was evaluated whether single-gene knockout of RNLS or HIVEP2 could similarly protect β-cell grafts against allo- and xenogeneic rejection. Murine β–TC–6 and human EndoC–βH1 cell lines were genetically edited using CRISPR-Cas9 to knockout RNLS or HIVEP2, and editing efficiencies were confirmed via T7 endonuclease I assay and TIDE analysis. Functional characterization indicated that RNLS deletion modestly impaired glucose-stimulated insulin secretion in murine cells, whereas HIVEP2 deletion showed no functional alterations in either cell line. For in vivo assessment, genetically edited β-cell spheroids were subcutaneously transplanted into CD-1 mice to model allo- (murine β-cells) and xenogeneic (human β-cells) rejection scenarios. Bioluminescence imaging revealed no protective effects of RNLS or HIVEP2 deletion, with grafts from both knockout groups displaying identical rejection kinetics compared to controls. These findings indicate that single-gene deletions of RNLS or HIVEP2 are insufficient for conferring meaningful protection against allo- or xenorejection, highlighting the necessity for combinatorial genome editing strategies or complementary biomaterial-based immunomodulation to achieve effective and sustained β-cell graft survival. Competing Interest Statement The authors have declared no competing interest.