A dual function TAR Decoy serves as an anti-HIV siRNA delivery vehicle

Abstract

The TAR RNA of HIV was engineered as an siRNA delivery vehicle to develop a combinatorial therapeutic approach. The TAR backbone was found to be a versatile backbone for expressing siRNAs. Upon expression in human cells, pronounced and specific inhibition of reporter gene expression was observed with TARmiR. The resulting TARmiR construct retained its ability to bind Tat and mediate RNAi. TARmiR was able to inhibit HIV gene expression as a TAR decoy and by RNA interference when challenged with infectious proviral DNA. The implications of this dual function therapeutic would be discussed.

Figure 1

Inhibition of target gene expression by siRNAs expressed from the TAR microRNA backbone. (A) siRNA sequence targeting the earlier reported site II of HIV rev was folded in silico and the configurations that retained the correct Tat binding region were selected for further studies. Configurations I & II have the lower single nucleotide bulge removed or both the bulges retained respectively. Configuration III is the anti-site II Rev shRNA with the 9 nt loop reported earlier by us[28]. Arrows indicate single nucleotide bulge (B) Target site corresponding to the site II of HIV rev is cloned in the 3' untranslated region of the renilla luciferase ORF in the siCHECK vector. The three configurations of siRNA were invitro transcribed and treated with Calf intestinal alkaline phosphatase. The CIP treated RNA were then cotransfected with the psiCHECK plasmid having the Rev Target site. Dramatic inhibition of reporter gene expression is observed with all three configurations. The configuration where the lower bulge is removed is three-fold more potent than even the anti-Rev shRNA. (C) An siRNA targeting TGF-β is expressed in a similar fashion from the TAR miRNA backbone and co-transfected with siCHECK plasmid having the TGF-β target site. ~70% inhibition is observed with this construct suggesting that the TAR miRNA is a versatile backbone for expressing siRNA. All siCHECK assays are a mean of three experiments. CFI and CFII = Configuration I and Configuration II.

Figure 2

anti- rev TARmiR configuration I can function as siRNA in RNA interference: HEK 293 cells were co-transfected with CMV RevEGFP and the anti-HIV TARmiR configuration I. Potent inhibition of EGFP expression is observed in these cells suggesting that the siRNA sized fragments released from the TARmiR backbone can demonstrate RNAi even when the target site is present within the ORF.

Figure 3

Gel mobility shift assay: anti-HIV TARmiR configuration I or II was transcribed in vitro, end labeled and was allowed to bind to a peptide corresponding to the arginine rich region of Tat that is responsible for binding TAR [23]. A mobility shift (arrow) clearly demonstrates Tat peptide binding to the TARmiR. As a control the fusion peptide HA-2 of influenza was used to bind the TARmiR configuration I. No shift in mobility is observed with HA2.

Figure 4

Inhibition of HIV gene expression by TARmiR constructs. (A) Generation of U6-TARmiR PCR constructs. An PCR expression cassette with the U6 promoter driving the expression of anti-rev TARmiR, anti-TGF-β TARmiR or anti-Rev shRNA is generated as described earlier by us [24]. (B) HEK 293 cells are co-transfected with infectious proviral DNA pNL4-3 and U6anti-Rev TARmiR configuration I, U6 anti-TGF-β TARmiR or U6anti-Rev shRNA. Pronounced inhibition is observed with both, the anti-Rev shRNA and anti-Rev TARmiR. Inhibition is also observed with anti-TGF-β TARmiR. This could be due to the presence of an intact TAT binding bulge which serves in the capacity of a TAR decoy.