62. Aptamer-siRNA Conjugate Directed Transcriptional Gene Silencing in HIV-1 Infected T Cells
2015
The primary standard of care against human immunodeficiency virus (HIV) -1 is highly active antiretroviral therapy which is plagued by toxicity and patient compliance. As such, alternative therapies are sought to provide sustained viral inhibition with reduced treatment intervention. One new potential approach, termed transcriptional gene silencing (TGS), involves the use of small non-coding RNAs (sncRNAs) to inhibit gene expression via the induction of concomitant silent-state epigenetic marks on histones and DNA. We previously demonstrated the ability to silence HIV-1 via TGS by targeting sites within the 5’ LTR promoter [1, 2]. However, the lack of efficient, systemic delivery of these RNAs to infected cells hinders successful clinical progression. To address this hurdle, we describe here the development of chimeric molecules based on the previously reported dual function of the gp120 (A-1) aptamer conjugated to 27-mer Dicer-substrate anti-HIV siRNA (dsiRNA) to deliver these sncRNAs to virally infected cells. The efficacy of such a system had been previously demonstrated both in vitro and in vivo to target selectively gp120-expressing cells and to deliver siRNAs for the inhibition of HIV-1 through post-transcriptional gene silencing (PTGS) [3]. To validate the ability for nuclear delivery of sncRNAs, we demonstrated, by fluorescent confocal microscopy, 15% nuclear delivery for our novel chimera-delivered dsiRNAs in a gp120 expressing CHO cells. Using nuclear-cytoplasmic fractionation of HIV-infected PBMCs, dsiRNAs targeting the susceptible regions within the 5’ LTR showed preferential nuclear localization when compared to a scrambled siRNAs. Additionally, two 800 nM doses at day 5 and 7 post-infection (p.i.) induced 10-fold suppression of viral p24 levels as measured at day 12 p.i. in CEM cells. The LTR-362 27-mer aptamer chimera proved more potent than A-1 tat/rev 27mer chimera, a positive control that inhibits HIV via PTGS. To validate the inhibition occurred via TGS, 5’-Azacytidine and trichostatin A were added at day 5 p.i, and resulted in transcriptional derepression by the A-1 LTR-263 27-mer, but not the A-1 Tat/Rev 27mer. Nuclear run-on analysis provided further confirmation of TGS inhibition. This is the first study to demonstrate RNA-induced TGS of an existing viral infection in both human PBMC and CEM cell lines following application of aptamer-siRNA chimeras.
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