P162 Progress towards gene editing of HLA-DRB1∗04:01 by CRISPR/Cas9

Abstract

The HLA-DRB1 locus represents the greatest genetic risk factor for susceptibility for rheumatoid arthritis (RA). We have refined this genetic association to a single amino acid polymorphism at position 71 that is associated with both susceptibility or resistance, depending on the presence of a basic or acidic residue. Mutating DRβ1*04:01 at position 71 from lysine to glutamic acid (K71E) completely abrogates the preferential binding of citrullinated vimentin66–78 and citrullinated α-enolase11–25 peptides compared to the native forms of these peptides, and eliminates binding of collagen258–272. This single amino acid substitution renders the arthritogenic peptide-binding profile of DRβ1*04:01 nearly identical to that of the resistant allele, DRβ1*04:02. The strong correlation between peptide binding and genetic susceptibility to RA suggests that CRISPR/Cas9 could be used to specifically edit position 71 of DRB1*04:01 and eliminate the binding preference for citrullinated peptides, thus attenuating the disease. The CRISPR/Cas9 system was utilized to specifically edit the DRB1*04:01 gene. We created two guide sequences (208/fwd and 185/rev) and prepared Lentivirus constructs to test their specificity in T2 cell lines expressing different human HLA molecules. Since indels are often formed by Cas9 editing, the loss of HLA-DR expression was used as an indication that the guides were specific. Since HLA-DRB1*04:01K71E has not been seen in nature, we produced a DRB1*04:01K71E transgenic mouse to demonstrate that this modified allele is not conditionally lethal. This mouse will also be used to demonstrate that stem cells from DRB1*04:01K71E mice transplanted into HLA-DRB1*04:01 mice do not cause graft-vs-host disease and can render mice resistant to RA. Next, DRβ1*04:01 T2 cells were transduced with lentiCRISPR 208/fwd or 185/rev. HLA-DR expression decreased from 95% to 20% or 12%, respectively. No loss of HLA-DR expression was seen on DRβ1*04:02 or DRβ1*08:01 control cells, demonstrating specificity of the guide RNAs. Experiments using homologous directed repair to introduce the K71E gene edit are in process. The CRISPR/Cas9 system can be used to specifically edit DRB1*04:01 suggesting that gene editing could be a potential treatment for RA.