Construction Of Combind-RNAi Element Targeting To Conserved Regions Of HIV-1 And Cell Receptor Gene

RNA interference(RNAi) is a powerful approach to inhibit human immunodeficiency virus type 1(HIV-1) replication.However,HIV-1 can escape from RNAi-mediated antiviral therapy by selection of mutations in the targeted sequence. Simultaneous targeting of multiple highly conserved viral sequences has been suggested for a promise to avoid viral escape.In this study,we tested a multiple miRNA expression strategy which simultaneous targeting to HIV-1 and cell receptor.Therefore,we screened 165 different siRNAs targeting highly conserved regions.We identified multiple siRNAs that act as potent inhibitors of virus replication,including siRNA-vif37,siRNA-poll102 and siRNA-poll402 targeting to conserved sequences in the Vif and Pol genes.There used to expression siRNA in shRNA manner.However, it has been demonstrated that RNA polymeraseâ…¢shRNA expression cassettes usually results in high expression levels.This may not always be desired in a gene therapy setting because of increased toxicity due to saturation of the RNAi machinery with siRNAs.An attractive approach is to express siRNAs from miRNA transcript, such as miRNA can be expressed from an RNA polymeraseâ…¡promoter.This strategy is of particular interest for antiviral purposes because RNA polymeraseâ…¡promoter will allow lower and regulated expression,thereby reducing the risk of toxicity.In this study,we tested a microRNA(miRNA) expression strategy by inserting effective anti-HIV siRNA sequences in the mir-155 or mir-30a.Individual anti-HIV miRNAs that resemble the natural miRNA structures were optimized by varying the siRNA position in the hairpin stem to obtain maximal effectiveness.These artificial microRNA can mediated effective cross-clade inhibition.Furthermore,we also sceened 16 target against cell receptor gene CCR5,which is a key receptor for HIV infection and has been identified on CD4~+ lymphocytes in both blood and intestinal mucosa.The RNAi sequence was layed in mir-30a.Here,we got an efficient artificial microRNA named mir-CCR13A.Ideally,we linked these four efficient artificial microRNAs with three linkers, which came to a multi-miRNA element names miCVPP.The three linker sequences were derived from antisence sequence of env gene.Finally,we could expression of four different miRNAs from a singl vector.Fortunately,each of miRNAs can obtain maximal effectiveness against luciferase reporters and HIV-1.Thus,their combined expression resulted in a much stronger inhibition of virus production.We then transfered this multiple miRNA element into MT-4 cell by lentiviral vector.MT4-miCVPP was a positive cellline which transduced by lentivirus. miCVPP could be expression in MT4-miCVPP permanently.We showed that the four miRNAs was expressed respective.To understand the off-target effect of miCVPP,we analyzed the IFN response,cell cytotoxicity and proliferation of MT4-miCVPP.Furthermore,we also analyzed endogenous miRNA level in MT4-miCVPP.Reault shew that,our multi-miRNA have no off-target effect. Moreover,whereas HIV-1 could escape from a single miRNA,we now show that HIV-1 escape can be prevented when four miRNAs are simultaneously expressed in MT-4 cell.We demonstrated that HIV-1 replication can be efficiently inhibited by simultaneous expression of four antiviral miRNAs from the polycistronic miRNA transcript.These combined results indicated that a multiplex miRNA strategy may be a promising therapeutic approach to attack escape-prone viral pathogens.