No abstract

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA) in the cell, and results in the silencing of homologous gene expression by the specific degradation of an mRNA containing the same sequence. dsRNA-mediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression. Synthetic 21-23 nucleotide (nt) small interfering RNAs (siRNAs) with 2-nt 3 0 overhangs were recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we show that synthetic siRNAs targeted against the viral structural Env proteins encoded by HIV-1 can specifically suppress the expression of HIV-1 genes. The siRNA-mediated RNAi also had advantages over antisense RNA-mediated inhibition, in terms of both the ease of designing effective antiviral agents and their potency. Especially, our best env-specific siRNAs, E7145 targeted to the central region of the V3 loop and E7490 targeted to the CD4 binding site of conserved regions on gp120, significantly inhibited the HIV-1 gene expression. Furthermore, E7145 and E7490 were effective against HIV-1 NL4-3 replication in PBMCs for a relatively long time (14 days). Therefore, the use of synthetic siRNAs provides a simple, rapid, and costeffective tool for new anti-HIV-1 gene therapeutics. Gene Therapy (2003) Keywords: RNA interference; small interfering RNAs; HIV-1 env genes; antiviral RNAi technology; PBMCs RNA interference (RNAi) or RNA silencing is an evolutionarily conserved phenomenon in which double-stranded RNA (dsRNA) induces the homologydependent degradation of the cognate mRNA. 1 RNAi is initiated by the RNase III-like nuclease Dicer, which promotes the processive cleavage of long dsRNAs into 21-to 23-nucleotide (nt) small interfering RNAs (siRNAs) with 2-nt 3 0 overhangs. [2][3][4][5][6] Subsequently, the siRNAs are incorporated into an RNA-induced silencing complex (RISC), identified in Drosophila, and the protein-RNA effector nuclease complex recognizes and destroys the target mRNAs. [7][8][9] It was recently found that the transfection of synthetic 21-23 nt siRNAs with 2-nt 3 0 overhangs into mammalian cells effectively inhibits the expression of the endogenous genes in a sequence-specific manner. 10-12 These small RNA duplexes, which are chemically synthesized mimics of Dicer products, are presumably incorporated into RISC and target their cognate substrates for degradation. Lee et al 13 have reported the inhibition of human immunodeficiency virus type 1 (HIV-1) replication by the expression of small interfering RNAs targeted against HIV-1 rev in human cells. In addition, Jacque et al 14 demonstrated the utility of siRNA-mediated RNAi for modulating HIV replication by synthetic siRNAs or plasmid-derived siRNAs targeted to various regions of the HIV-1 genome (TAR, vif, nef).We previously reported that long dsRNAs effectively inhibit HIV-1 replication in HIV-1 infected cells. 15 However, the utility of these long dsRNAs appeared to be limited, due to the nonspecific inhibi...

The RNA interference (RNAi) phenomenon is a recently discovered process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of mRNA containing the same sequence. We designed mammalian expression vectors that direct the synthesis of small interfering RNA (siRNA)-like transcripts and examined them for their siRNAmediated gene interference targeting the env gene (NL4-3:7490-7508, E7490). We constructed siRNA expression vectors for two different strands (sense and antisense; tandem promoter) and for siRNA expressed from the short hairpin RNA (shRNA). The inhibition efficacy on HIV-1 replication differed between these two vectors. Notably, the shRNA vector pU6-env-shRNA inhibited p24 production more effectively than the tandem promoter expression vector pU6-env-siRNA. Furthermore, we examined the ability of lentiviral vectors expressing shRNA to suppress HIV-1 expression in HIV-1-infected SupT1 cells. The envshRNA (E 7490) almost completely suppressed HIV-1 expression in infected cells for up to 15 days.

DNAzymes are easier to prepare and less sensitive to chemical and enzymatic degradation than ribozymes; however, a DNA enzyme expression system has not yet been developed. In this study, we exploited the mechanism of HIV-1 reverse transcription (RT) in a DNA enzyme expression system. We constructed HIV-1 RT-dependent lentiviral DNAzyme expression vectors including the HIV-1 primer binding site, the DNA enzyme, and either a native tRNA (Lys-3), tRMDtRL, or one of two truncated tRNAs (Lys-3), tRMDΔARMtRL or tRMD3′-endtRL. Lentiviral vector-mediated DNAzyme expression showed high levels of inhibition of HIV-1 replication in SupT1 cells. We also demonstrated the usefulness of this approach in a long-term assay, in which we found that the DNAzymes prevented escape from inhibition of HIV. These results suggest that HIV-1 RT-dependent lentiviral vector-derived DNAzymes prevent the emergence of escape mutations.

We designed a vector to produce single-stranded DNA (ssDNA). We used HIV-1 reverse transcription for the purpose of constructing a DNAzyme expression vector against the HIV-1 env V3 loop. Initiation of HIV-1 reverse transcription requires the formation of a complex containing the viral RNA, tRNALys and reverse transcriptase. The expression vector contains the HIV-1 primer binding site (PBS) and tRNALys at the 3' end of its RNA transcript, thus enabling to an ssDNA would be synthesized by HIV-1 reverse transcriptase. We have demonstrated that the DNAzyme expressed by the lentiviral vectors suppressed HIV-1 replication in SupT1 cells.

The RNA interference (RNAi) phenomenon is a recently observed process in which the introduction of a double-stranded RNA (dsRNA) into cells causes the specific degradation of an mRNA containing the same sequence. To study dsRNA-mediated gene interference targeted to the env gene (NL4-3: 7490-7508) in HIV-1 infected cells, we constructed tandem-type and hairpin-type siRNA expression vectors, which were under the control of two U6 promoters. We also constructed lentiviral-based siRNA expression vectors for further assessment of their antiviral activity in transduced cells. At both the transient plasmid and lentiviral-mediated RNA expression levels, the siRNA encoding the env fragment exhibited sequence-specific suppression of target gene expression and strongly inhibited (> or = 90%) HIV-1 infection in the cells, as compared to the antisense RNA expression vector. Targeting the HIV-1 env gene with siRNAs encoding the env gene fragment (7490-7508) might be an effective strategy for gene therapy applications in HIV-1/AIDS treatment and management.