Improvement Of Transkingdom RNAi(TkRNAi) Technology And Its Preliminary Application On Suppressing Replication Associated Key Genes Of Human Immunodeficiency Virus(HIV)

Transkingdom RNA interference(TkRNAi)is a novel approach for siRNA delivery,which employs the genetically engineered bacteria to produce siRNA in targeted mammalian cell.In this system,silencing shRNA are transcribed from a TransKingdom RNAi plasmid(TRIP)by T7 RNA polymerase inside non pathogenic bacteria that have been engineered to invade target cells.Following uptake into phagosomes,the bacteria are lyzed to release their silencing shRNA into the host cell cytoplasm.RNAi-mediated gene-silencing is then achieved through the canonical Dicer/RISC pathway.This novel approach has several advantages over delivery mediated by complexed siRNA and viral vectors.First,the TransKingdom system may provide a practical and clinically compatible way to achieve RNAi for medical indications.Second,the production of silencing shRNA by engineered non-pathogenic bacteria eliminates the siRNA manufacture issue,and significantly reduces the cost compared to other delivery technologies.Third,tkRNAi may have significant implications for high throughput functional genomics in mammalian systems.TkRNAi with long double-stranded RNA interference(dsRNA)can be used to interfere target genes in target cells.In the study long dsRNAi was designed to deliver interference to endogenous genes CTNNB1 and TUBA1B and the sequence was then cloned to dstrip(dsRNA trans-kingdom RNAi plasmid),thus forming dstrip-CTNNB1 and dstrip-TUBAlB which expresses the dsRNA.Treatment to target cell SW480 with those two plasmids was done via TransKingdom interference approach.qPCR and western blot tests showed that gene transcription levels and protein expression levels were constrained in the target gene,which proved that dsRNA was able to deliver interference to the RNA of target gene in target cells through Transkingdom interference approach.Based on the results,overlap extension was done on these two interference segments via PCR to form chimeric long dsRNA and was cloned into dstrip to form dstrip C-T(thereafter C represents CTNNB 1,T represents TUBA1B).Treatment to target cell SW480 with those two plasmids was done via TransKingdom interference approach.qPCR and western blot tests showed that gene transcription levels and protein expression levels were constrained in these two target genes,which proved that chimeric long dsRNA was also able to deliver interference to the RNA of multiple target genes in target cells through Transkingdom interference approach.Molecular biological approach was used to produce integrate TKRNAi,which was able to integrate the working components ofTKRNAi into BL21(DE3)chromosome,thus forming transkingdom interference bacteria.Infectious conditions of transkingdom interference bacteria were explored through experiments and results proved that transkingdom interference bacteria were able to deliver interference to target genes.Stable cell line of SW 480-nef with stable nef protein expression and stable cell line of SE480-gag with stable gag protein expression were constructed.dsRNAi was designed to deliver interference to nef and gag,the critical genes of HIV reproduction,thus forming dsTRIP-gag,dsTRIP-nef and dsTRIP-nef-gag.Treatment to stable cell lines with the above was done via TransKingdom interference approach.The results showed that protein expression levels were constrained in the target genes.Meanwhile,interference bacteria BL21(DE3)-dstrip-nef,BL21(DE3)-dstrip-gag and BL21(DE3)-dstrip-nef-gag were constructed through integration technology of transkingdom interference and experiments proved that these three transkingdom bacteria effectively constrained protein expression of target genes.Transkingdom interference technology was used on cell model and effectively delivered interference to the critical genes of HIV reproduction,suggesting that transkingdom interference technology is applicable to cure HIV disease.