HSCs Reprogramming Strategy Based On CRISPR/dCas9 Targeted Delivery System And Its Application In The Treatment Of Hepatic Fibrosis

BackgroundLiver is the only substantive organ that can be regenerated and plays a vital role in the process of substance metabolism in the body.Hepatic fibrosis is a pathological process in which a variety of chronic pathogenic factors act on the liver to cause tissue damage and repair,leading to a large accumulation of liver extracellular matrix（ECM）.Its pathogenesis is complex,and there is no effective anti-fibrotic therapy at present.A variety of cells in the liver are involved in the hepatic fibrogenesis.To study the molecular mechanism of hepatic fibrosis,more reports have focused on hepatic stellate cells（HSCs）.When the liver is injured by various acute and chronic injuries,HSCs are activated and transformed into proliferating myofibroblasts（MFs）,which generate excessive ECM and lead to hepatic fibrosis.As a new gene therapy method,gene editing technology may cure diseases that previously lacked effective intervention measures.With the rapid development of the technology in recent years,gene therapy has entered a new era.As the new generation of gene editing tools,the CRISPR/Cas9 system has become a revolutionary technology and has been widely used.CRISPR/Cas9 technology can knock in,knock out,inhibit,and activate genes,and the CRISPR/d Cas9-SAM system has a highly efficient transcription factor activation function.Gene delivery is crucial for gene therapy,which offers hope for previously incurable diseases.Exosome is a kind of extracellular vesicle,which can carry a series of complex bioactive substances such as proteins,DNA,m RNA,and mi RNAs.Exosome is widely involved in the information transmission in vivo,and it is the natural carrier of communication between cells.The biological characteristics of exosomes make them safe and effective potential gene delivery carriers.Compared with other gene delivery systems,exosomes have natural advantages such as being able to cross biological barriers,being capable of genetic engineering modification,and being multifunctional.Exosome surface proteins,such as CD63,CD9 and Lamp2b,are usually used as exosome engineered target proteins.Engineered exosomes give these transport nanoparticles the ability to target and deliver materials to specific cells or tissues.In recent years,exosomes have become a hot topic in the field of medical research.ObjectiveThis study aims to induce the transformation of HSCs into hepatocyte-like cells and prevent hepatic fibrosis via exosome-mediated CRISPR/d Cas9 targeted delivery.Methods1.Construction of the CRISPR/d Cas9 system for inducing the transformation of HSCs into hepatocyte-like cells and exosome delivery system for targeting HSCs:The CRISPR/d Cas9-SAM system was constructed to activate the hepatocellular specific transcription factors HNF4α/HGF1/FOXA2 in HSCs.AML12 cells-derived exosomes were extracted and verified to construct the Lamp2b-RBP4 engineered exosome delivery system targeting HSCs.Finally,q PCR and Western blot were used to detect the loading efficiency of the CRISPR/d Cas9-SAM system in exosomes.2.In vitro transformation of HSCs into hepatocyte-like cells was induced by CRISPR/d Cas9 system:Immunofluorescence was used to compare the ability of HSCs to take up exosomes,and the efficiency of exosomes in vitro delivery of CRISPR/d Cas9-SAM system to HSCs was examined.Cell morphology and markers were used to identify primary mouse hepatocytes and HSCs.Finally,under different treatments,the CRISPR/d Cas9-SAM system was verified to induce HSCs to transform into hepatocyte-like cells in vitro.3.In vivo exosome-mediated targeted delivery of CRISPR/d Cas9 system to prevent hepatic fibrosis:In vivo animal imaging,immunofluorescence,q PCR,and Western blot experiments confirmed the effective delivery of CRISPR/d Cas9-SAM to HSCs via Lamp2b-RBP4 exosomes.Mouse hepatic fibrosis model induced by CCL₄ was established,and the engineered exosomes were injected into mice through the tail vein.The changes of hepatic fibrosis were detected by H&E,Sirius red and Masson staining.The expression of HSCs and hepatocytes specific proteins in vivo was analyzed by immunofluorescence,Western blot,and q PCR.Finally,hepatic fibrosis factors and liver function were detected to verify the reversal of hepatic fibrosis.Results1.The CRISPR/d Cas9 system for inducing the transformation of HSCs into hepatocyte-like cells and the exosome delivery system targeting HSCs were successfully constructed.Firstly,the CRISPR/d Cas9-SAM system was successfully constructed to activate the hepatocellular specific transcription factors HNF4α/HGF1/FOXA2 in HSCs,and its activation function was verified in HSCs.Secondly,the Lamp2b-RBP4 engineered exosome delivery system targeting HSCs was successfully constructed,and it was confirmed that the morphology,particle size distribution and specific proteins of engineered exosomes did not change significantly.Finally,it was verified that exosomes could effectively load the CRISPR/d Cas9-SAM system.2.In vitro CRISPR/d Cas9 system successfully induced the transformation of HSCs into hepatocyte-like cells.In vitro experiments,firstly,it was proved that HSCs could efficiently absorb AML12 cells-derived exosomes,and exosomes could effectively deliver the CRISPR/d Cas9-SAM system to HSCs in vitro.Secondly,the primary mouse hepatocytes were round-shaped with HNF4αprotein in the nucleus,and the primary mouse HSCs were star-shaped with CRBP1 protein in the cytoplasm.Finally,the CRISPR/d Cas9-SAM system successfully induced the transformation of HSCs into hepatocyte-like cells in vitro.3.In vivo exosome-mediated targeted delivery of CRISPR/d Cas9 system reverses hepatic fibrosis.In vivo experiments,firstly,it was verified that Lamp2b-RBP4 exosomes can effectively deliver the CRISPR/d Cas9-SAM system to HSCs in vivo.Secondly,in the CCL₄-induced mouse hepatic fibrosis model,the reduction of hepatic fibrosis was confirmed by liver tissue staining after the engineered exosomes were injected into the mice through the tail vein.Finally,it was verified that the CRISPR/d Cas9-SAM system induced the transformation of HSCs into hepatocyte-like cells in vivo,and hepatic fibrosis factors and liver function further confirmed the reversal of hepatic fibrosis.ConclusionsAfter the engineering of Lamp2b-RBP4,the AML12 cells-derived exosomes can targeted deliver the CRISPR/d Cas9 system to HSCs,inducing the transformation of HSCs into hepatocyte-like cells and prevent hepatic fibrosis.This study is expected to provide a new strategy for gene therapy of hepatic fibrosis.