Derepression of the IL-10 Gene by CRISPR-Cas Genome Editing as a Therapeutic Strategy for Persistent Immunomodulation of Donor Lungs for Transplantation

Abstract

<h3>Purpose</h3> Anti-rejection immunosuppression is a persistent challenge in lung transplantation. We envisioned creating less immunogenic donor organs to obviate or reduce the need for immunosuppression. To induce persistent expression of the immunomodulatory cytokine IL-10, we aimed to harness CRISPR-Cas-mediated genome editing. We hypothesized that mutating the regulatory region of the IL-10 could induce persistent IL-10 upregulation with practicality in organ modification. To prove our concept, we investigate the efficacy and persistence of this approach in vitro. <h3>Methods</h3> Plasmids encoding Streptococcus pyogenes Cas9 (SpCas9), a puromycin cassette, and a single guide RNA (gRNA), designed to bind the promoter of the rat IL-10 gene, were delivered into a rat lung epithelial cell line. Transfected cells were enriched using puromycin and assessed for mutations and gene expressions after 14 and 28 days. The treatment group (targeting gRNA group) was compared to a no gRNA group and a non-targeting gRNA group. <h3>Results</h3> Insertions/deletions at the target site were detected in 77±20 % of alleles at day 14 and 87±11% at day 28 in the treatment group (Fig. 1A). The treatment group showed enhanced rat IL-10 expression while other groups showed mostly undetectable IL-10 expression (457±338- and 865+573- fold increase compared to negative control at day 14 and 28 respectively, Fig.1B). Importantly, IL-10 upregulation lasted for 28 days despite a decline in SpCas9 expression (Fig. 1C) suggesting an effective and persistent expression after one round of genome editing. <h3>Conclusion</h3> We have demonstrated that CRISPR-Cas9-mediated genome editing at the promoter region induces stable IL-10 expression. Targeted editing outside of the coding region potentially leads to a safer application of whole organ gene editing. Our findings expand the potential of genome editing towards engineering optimized donor organs for transplantation.