CRISPR/Cas9-Mediated Epigenome Editing of the IL-10 Gene for Targeted Whole Organ Gene Therapy for Lung Transplant

• Published in: The Journal of heart and lung transplantation Vol. 40; no. 4; p. S52
• Main Authors: Mesaki, K., Juvet, S., Guan, Z., Hu, J., Davidson, A.R., Cypel, M., Liu, M., Keshavjee, S.
• Format: Journal Article
• Language: English
• Published: Elsevier Inc 01.04.2021
• ISSN: 1053-2498; 1557-3117
• DOI: 10.1016/j.healun.2021.01.1867

Anti-inflammatory and immunomodulatory gene therapy has shown promise in lung transplant. CRISPR/Cas9 technology unleashed the possibility of making gene therapy persistent by radically altering gene expression through precise DNA targeting. We envisioned activation of the endogenous IL-10 gene, which codes for this anti-inflammatory cytokine, using epigenome editing via CRISPR/Cas9 in the lung. This study investigated the efficacy and specificity in vitro and in rat lungs. Adenoviral vectors were developed expressing a Cas9 activator and either one or two gRNAs targeting the promoter region of the IL-10 gene in the genome (Fig. A). Lewis rats were divided into three groups (Fig. B, n=5-10), trans-bronchially administered adenovirus (1 × 108 PFU/rat) under standard transplant triple immunosuppression, and assessed after 72 hours. For specificity, primary lung fibroblasts treated with adenovirus were analyzed at 48 hours using chromatin immunoprecipitation (ChIP) of Cas9 followed by qPCR designed against the on- and off-target regions. The two-gRNA group showed significantly increased IL-10 gene expression compared to the diluent group (relative expression: 19.0±10.8 vs 1.3±1.2, p=0.001) and the single gRNA group (2.9±0.78, p=0.0009) (Fig. C). There was no significant increase in TNFα or IL-6 gene expression or Wet/Dry ratio in the two-gRNA group compared to the diluent group (Fig. D, E) suggesting minimal vector-related inflammation. ChIP-qPCR demonstrated enriched binding of Cas9 to on-target sites (Fig. F, fold enrichment; 17.5±1.7, p=0.02, 18.5±5.8, p=0.02, respectively) compared to the control region whereas the binding to off-target sites were not significantly enriched. We have achieved targeted in vivo whole organ activation of the IL-10 gene via CRISPR/Cas9-mediated epigenome editing in the lung. This study demonstrates the feasibility of this strategy for optimizing and immunologically enhancing donor organs for transplant.