Genome editing of PAR2 through targeted delivery of CRISPR-Cas9 system for alleviating acute lung inflammation via ERK/NLRP3/IL-1β and NO/iNOS signalling

Abstract

Excessive and uncontrollable inflammatory responses in alveoli can dramatically exacerbate pulmonary disease progressions through vigorous cytokine releases, immune cell infiltration and protease-driven tissue damages. It is an urgent need to explore potential drug strategies for mitigating lung inflammation. Protease-activated receptor 2 (PAR2) as a vital molecular target principally participates in various inflammatory diseases via intracellular signal transduction. However, it has been rarely reported about the role of PAR2 in lung inflammation. This study applied CRISPR-Cas9 system encoding Cas9 and sgRNA (pCas9-PAR2) for PAR2 knockout and fabricated an anionic human serum albumin-based nanoparticles to deliver pCas9-PAR2 with superior inflammation-targeting efficiency and stability (TAP/pCas9-PAR2). TAP/pCas9-PAR2 robustly facilitated pCas9-PAR2 to enter and transfect inflammatory cells, eliciting precise gene editing of PAR2 in vitro and in vivo. Importantly, PAR2 deficiency by TAP/pCas9-PAR2 effectively and safely promoted macrophage polarization, suppressed pro-inflammatory cytokine releases and alleviated acute lung inflammation, uncovering a novel value of PAR2. It also revealed that PAR2-mediated pulmonary inflammation prevented by TAP/pCas9-PAR2 was mainly dependent on ERK-mediated NLRP3/IL-1β and NO/iNOS signalling. Therefore, this work indicated PAR2 as a novel target for lung inflammation and provided a potential nanodrug strategy for PAR2 deficiency in treating inflammatory diseases.

Keywords

• Protease-activated receptor 2 (PAR2)
• CRISPR-Cas9
• Gene editing
• Inflammation
• Acute lung inflammation
• NLRP3