Specific HIV-1 env gene silencing by small interfering RNAs in human peripheral blood mononuclear cells

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...

Lentiviral siRNAs targeting multiple highly conserved RNA sequences of human immunodeficiency virus type 1

“…7,8 These include siRNAs targeting host cell receptor CD4 and coreceptors CCR5 or CXCR4 to prevent viral entry, 9-14 as well as targeting viral genes such as tat, rev, gag and env to inhibit viral replication. [14][15][16][17][18][19] For gene therapy to be effective in HIV patients, efficient delivery and long-term stability are necessary. These are known limitations with RNA interference (RNAi) technology.…”

Gene therapy progress and prospects: Novel gene therapy approaches for AIDS

“…Other studies include those aimed at targeting the CXCR4 coreceptor, decreasing receptor expression on the cell surface and consequently lowering infectivity by HIV-1 45 or the V3 loop and CD4 binding domains of Env. 46 Some examples target both viral and host genes, for example, Tat and CCR5 (the main coreceptors in macrophage lines). 47 A combination of ribozyme and RNA decoy technologies also seems promising.…”

Stable RNA interference (RNAi) as an option for anti-bcr-abl therapy

“…Accordingly, both foreign genes encoded by infectious organisms and fusion genes resulting from chromosomal translocation have been analyzed by RNAi. [7][8][9][10][11][12][13][14][15][16][17][18] For example, short-term mRNA and protein reduction of the bcr-abl oncogene necessary for leukemic transformation in chronic myeloid and bcrabl+ acute lymphoblastic leukemia (CML, Ph+ALL), as well as inhibition of survival and proliferation of bcrabl+ cell lines have recently been described. [14][15][16] However, the application of RNAi for reverse genetics in mammalian cells is hampered by both the difficulty to deliver RNAi triggers to target cells and by the transient nature of RNAi in mammals.…”