PAR2 Deficiency By Targeted Delivery Of CRISPR-Cas9 System For The Treatment Of Inflammatory Diseases

Inflammation is a kind of defensive response accompanied by macromolecule exudation in blood vessels when the human body is stimulated,causing local edema and numerous inflammatory cytokine release.When the inflammatory response exceeds the normal range,it will evoke tissue damage such as paw edema and lung injury.In severe cases,it may even trigger cytokine storm leading to systemic inflammation and multi-functional organ dysfunction,such as COVID-19 emerged in recent years.However,it is still needed to explore potential molecular targets and underlying mechanisms due to the complicated and diverse causes of inflammatory diseases.Protease-activated receptor 2(PAR2)as a subfamily of G protein-coupled receptors(GPCRs)can be activated by proteases such as trypsin,which participates inflammatory responses via regulating intracellular signal transduction.Probing and utilizing the role of PAR2 in inflammation may realize the artificial intervention in the process of inflammation to avoid inflammatory diseases.Currently,PAR2 agonists are comparatively mature,whereas PAR2 inhibitors cannot achieve precise regulation.CRISPRCas9 system,as a novel gene editing tool,can replace inhibitors to achieve PAR2 gene knockout for exploring the role of PAR2 in inflammation.However,despite the simple preparation process,CRISPR-Cas9 plasmid also exhibited some problems such as large size,difficulty in cell uptake and easy degradation.Nanoparticles as common drug carriers can protect and accurately deliver nucleic acid to targeted sites,suggesting that it could be applied for CRISPR-Cas9 delivery system.Therefore,this project aims to link PAR2 to inflammation.We constructed PAR2 gene knockout via targeted delivery of CRISPR-Cas9 system,and then probed the novel role of PAR2 in inflammation and related mechanisms,indicating that the regulation of PAR2 may treat inflammatory diseases.The main contents are as follows:1.Successful construction of CRISPR-Cas9-PAR2 nanoparticles targeting inflammation(TAP/ PCAS9-PAR2).Firstly,serum albumin with high biocompatibility was selected as CRISPR-Cas9 delivery carrier,meanwhile collagen targeting peptide was introduced to achieve inflammation-targeting.By changing p H,stirring speed and time,temperature and composition,we selected the nanoparticle formula with the most excellent particle size,stability and dispersity.Additionally,the formula with the optimal N/P ratio with low toxicity and high efficiency was screened by cell transfection,uptake and toxicity experiments,which can consequently induce successful PAR2 gene knockout.2.Investigating the effects and molecular mechanisms of PAR2 gene knockout on inflammatory cells in vitro.We applied q PT-PCR,WB,NO detection and other experimental methods to detect the gene expression or protein secretion in inflammatory cells,revealing PAR2-mediated inflammatory responses through MAPK/NLRP3/Caspase-1/ l L-1? and MAPK/i NOS signaling pathway.3.Probing the anti-inflammatory effects and underlying mechanisms of edema and lung injury in mice after PAR2 gene knockout in vivo.We established the PAR2-specifc and non-specific paw edema models and found that PAR2 knockout could prevent both PAR2agonist-induced short-term paw edema and general paw edema.Moreover,a mouse model of lung injury was constructed to mimick lung injury caused by pneumonia,while PAR2 knockout possessed therapeutic effects for lung injury.Importantly,the related mechanism of PAR2 knockout in alleviating lung injury was uncovered with the involvement of NLRP3 and i NOS signalling,detected by ELISA,WB,BCA,cell count and other detection approaches.Thus,PAR2 may be a novel therapeutic target for pneumonia.