Genetic Modification of Insulin and Chromogranin A to Protect NOD Mice from Type-1 Diabetes

Background

Insulin and chromogranin A (ChgA) are autoantigens for CD4 T cells in the NOD mouse model of type-1 diabetes. Natural peptides from Insulin (B:9-23) and chromogranin A (WE14) are both weak antigens for diabetogenic CD4 T cells. Studies from our lab show that these peptides can be converted to super-agonists by modifying the amino acids at their C-terminus or N-terminus, respectively. These modified epitopes are 100-1,000,000 times more active than the natural peptides in stimulating insulin and ChgA specific CD4 T cells.

Methods

To see whether these modifications could be powerful tolerogens in vivo, we used CRISPR/CAS 9 technology to introduce them directly into the proinsulin-2 or chromogranin A gene in NOD mice embryos. The resultant mutant mice were bred to wildtype NOD mice and checked for the absence of mutations in the 5 most likely off target genes.

Results

NOD F1 mutant mice are now being used in long-term experiments to determine the fate of B:9-23 and WE14 specific T cells in these mice and the effect of the mutations on the development of diabetes. So far, the presence of the super-agonists do not accelerate diabetes in the mutant mice.