Anti-HBV Effects And Mechanism Of A Novel Gene Editing System Combined With RNAi And CRISPR/Cas Technologies

ObjectivesChronic hepatitis B(CHB),a disease caused by hepatitis B virus(HBV),is a global public health problem and the main cause of liver failure,liver cirrhosis,and hepatocellular carcinoma.Benefit from the rapid progress in the treatment of hepatitis B,“functional cure” can be achieved,though only in very small part of individuals withHBV infection.High levels ofHBV replication and gene expression induce immune tolerance,and enhancing cellular and humoral immunity will improve the control ofHBV infection.While The steady-state level ofHBV covalently closed circular DNA(ccc DNA)in the nucleus of hepatocytes is the source of persistent infection,which cannot be directly degraded by current treatments.While gene editing technology can directly cleave genomic DNA.The purpose of the study was to explore the anti-HBV and immunostimulatory effects of msiRNAs with unpaired uridine bulges in the passenger strand and CRISPR/Cas gene-editing systems targetingHBV DNA genomes,and a novel complex gene editing system combined withRNAi and CRISPR/Cas technologies.To explore new strategies for the clearance ofHBV.MethodsmsiRNAs targeting theHBV S and X genes were designed and named msiHBs and msiHBx,respectively.HepG2 cells were cotransfected with siRNA or msiRNA and theHBV replication-competent plasmid p HY106-wta or p HY106-X15.HepG2.215 cells were transfected with siRNA or msiRNA.The levels ofHBs Ag,HBe Ag,and the cytokines TNF-α,IFN-α,IFN-β,IL-1α,and IL-6in the culture supernatant were detected by ELISA.The levels of intracellularHBVRNA,nuclearHBV replication intermediates,andHBV DNA in the supernatants were measured by quantitative PCR.The levels ofHBV replication intermediates were detected by Southern blotting.Peripheral blood mononuclear cells were transfected with siRNA or msiRNA,and the levels of secreted cytokines IFN-α and IFN-β were detected by ELISA.The bioactivity of type I IFNs in the supernatants was detected by the virus protection assay.sgRNA targeting theHBV C、S and X genes were designed and named sg C1,sg C2,sg C3,sg S2,sg S3,sg S4,sg X1,sg X2 and sg X3,respectively.Each sgRNA was transcribed in vitro and combined with recombinant Cas9 protein.HBV DNA fragment amplified by PCR was cut extracellular by combination of sgRNA and Cas9,and the cut efficiency was detected by gel electrophoresis.The plasmids contain Cas-C3,Cas-S4,or Cas-X1 targetingHBV DNA were constructed using px459-Cas9-puro plasmid as the vector,and then co-transfected into HepG2 cells withHBV replication plasmid p HY106-wta.On the other hand,HepG2.2.15 cells were infected by CRISPR/Cas lentivirus(Lenti-Cas-C3,Lenti-Cas-S4 and Lenti-Cas-X1).The levels ofHBs Ag andHBe Ag in the culture supernatants were detected by ELISA.The levels of intracellularHBc Ag were detected by Western blotting.The levels ofHBV replication intermediates were detected by quantitative PCR and Southern blotting.HBV DNA mutation rate of HepG2.2.15 cells infected with Lenti-Cas-C3 was detected by T7 EI and sequencing.The plasmid contains shHBx targetingHBV X gene and double sgRNA/Cas targetingHBV C and X genes were constructed using px458-Cas9-EGFP plasmid as the vector,and then co-transfected into HepG2 cells withHBV replication plasmid p HY106-wta.The levels ofHBs Ag andHBe Ag in the culture supernatants were detected by ELISA.The levels of intracellularHBs Ag andHBc Ag,were detected by Western blotting.ResultsmsiHBx treatment led to a significant decrease inHBs Ag(to a negative level)andHBV DNA(95.5%)in the supernatants and intrahepatocellularHBV replication intermediates(89.8%)in HepG2 cells with transientHBV replication and in HepG2.2.15 cells.There was no significant difference between msiHBx and siHBx in terms of the reduction inHBV proteins andHBV replication(P>0.05).Compared with siHBx,msiHBx treatment of HepG2 cells transfected with theHBV replication-competent plasmid led to a significant increase in the levels of the antiviral cytokine TNF-α(3.3-fold),IFN-α(1.4-fold),and IFN-β(2.5-fold)(P<0.01),without upregulation of the proinflammatory cytokines IL-1αand IL-6.The virus protection assay results showed msiHBx-mediated type I IFNs effectively protected L929 cells against ECMV infection.Each sgRNA/Cas9 transcribed in vitro was effective in cuttingHBV DNA(the efficiency was higher than 50%).Cas-C3,Cas-S4 and Cas-X1 displayed higher cut efficiency,reaching 68.7%,64.6%,and 54.4%,respectively,and the combination of these three sgRNA/Cas9 completely cutHBV DNA(100%).In theHBV transient replication system,sgRNA/Cas9 significantly reduced the levels ofHBs Ag andHBe Ag(80%),HBc Ag(60.9%),and nuclearHBV replication intermediates(67%),with statistically significant differences compared with the negative control group(P<0.001).In HepG2.2.15 cells,there was no statistical difference in the level ofHBs Ag between the experimental groups and the negative control group(P>0.05),but the level ofHBe Ag in Lenti-Cas-C3 and Lenti-Cas-X1 groups was significantly decreased(P<0.01).In all the experimental groups,intracellularHBc Ag decreased significantly(up to about 50.4%).HBV DNA mutation rate of HepG2.2.15 cells infected with Lenti-Cas-C3 was about 53%.Compared with the vector group,the level ofHBs Ag andHBe Ag of the shHBx-Cas-C/X grop were reduced by 2.3 times and 2.5 times respectively.Compared with the single target(Cas-C)group,the levels ofHBs Ag of the double target(Cas-C/X and shHBx-Cas-C/X)group were reduced by 2 times and 4.1 times respectively.Compared with dual target(Cas-C/X)group,the level ofHBs Ag andHBe Ag of shHBx-Cas-C/X group were reduced by 2.1 times and 2 times respectively.The results of Western blotting showed that compared with the vector group,the levels ofHBs Ag andHBc Ag in all experimental groups were significantly reduced,and that in the shHBx-Cas-C/X group were the lowest.ConclusionsmsiHBx could effectively inhibitHBV expression and replication,and induce an antiviral innate immune response without proinflammatory activation.The dualRNAi and immunostimulatory activity of msiRNAs may play an important role in the control ofHBV infection.CRISPR/Cas could effectively inhibitHBV expression and replication,and remove the source ofHBV replication and persistent infection from the genetic level.Combination of gene-editing and immunoregulatory therapy could up-regulate/restore the anti-HBV immune response to promote the clearance of the remainingHBV DNA after gene editing.The in vivo effects of thisRNAi-CRISPR/Cas complex gene editing system onHBV infection is worthy of further study,so as to explore a new strategy for achieving a complete cure ofHBV infection.