Immunological impact of intracellular Streptococcus pyogenes Cas9 expression on human retinal pigment epithelium

CRISPR/Cas9 enables precise DNA sequence modifications and represents a potential cure for inherited retinal diseases (IRDs). It has been shown that Cas9 triggers immune responses which significantly impair gene editing efficacy. We aim to study the immunogenicity of plasmid-mediated gene transfer and expression of Cas9 on the human retinal pigment epithelium (RPE) in vitro. For this purpose, an immortalised RPE cell line was transfected with a self-designed Cas9-coding (spCas9) plasmid via cationic lipofection. The immunomodulatory surface marker expression and inflammatory cytokine concentrations were compared between spCas9 plasmid and non-coding (NC) plasmid transfected RPE cells to assure that potential immune responses could be ascribed to Cas9. To assess the immunogenicity of plasmid-mediated gene transfer, the same immunological parameters were compared between plasmid transfected and non-transfected RPE cells, which were exposed to either cationic lipids or plasmids only. Transfection of an spCas9 plasmid induced a significant IL 8 secretion (p = 0.004) in RPE cells, suggesting that the inflammatory response seen in the RPE cells was triggered by either the Cas9 DNA sequence, Cas9 mRNA, and/or the subsequently produced Cas9 protein. The supernatant of plasmid transfected RPE cells exhibited increased IL 6 (p < 0.001) and IL 8 levels (p < 0.001) when compared to non-transfected RPE cells. An upregulation of HLA-ABC (p < 0.001) and ICAM-1/CD54 expression (p < 0.001) as well as a reduced viability rate (85.2 %) of plasmid-transfected RPE was detected using FACS analysis. The data of this study indicates that Cas9 plasmid-mediated gene transfer has immunogenic potential. Further research is required to assess the role of pattern recognition receptors and signaling pathways in Cas9 pathogen detection, and the impact of the induced immune responses on gene editing efficacy. These results are highly relevant to the safety of CRISPR/Cas9-based gene therapy as a potential treatment for IRDs