Selection, characterization and application of new RNA HIV gp 120 aptamers for facile delivery of Dicer substrate siRNAs into HIV infected cells

Abstract

The envelope glycoprotein of human immunodeficiency virus (HIV) consists of an exterior glycoprotein (gp120) and a trans-membrane domain (gp41) and has an important role in viral entry into cells. HIV-1 entry has been validated as a clinically relevant anti-viral strategy for drug discovery. In the present work, several 2′-F substituted RNA aptamers that bind to the HIV-1BaL gp120 protein with nanomole affinity were isolated from a RNA library by the SELEX (Systematic Evolution of Ligands by EXponential enrichment) procedure. From two of these aptamers we created a series of new dual inhibitory function anti-gp120 aptamer–siRNA chimeras. The aptamers and aptamer–siRNA chimeras specifically bind to and are internalized into cells expressing HIV gp160. The Dicer-substrate siRNA delivered by the aptamers is functionally processed by Dicer, resulting in specific inhibition of HIV-1 replication and infectivity in cultured CEM T-cells and primary blood mononuclear cells (PBMCs). Moreover, we have introduced a ‘sticky’ sequence onto a chemically synthesized aptamer which facilitates attachment of the Dicer substrate siRNAs for potential multiplexing. Our results provide a set of novel inhibitory agents for blocking HIV replication and further validate the use of aptamers for delivery of Dicer substrate siRNAs.

In vitro and in vivo cleavage of HIV-1 RNA by new SOFA-HDV ribozymes and their potential to inhibit viral replication

“…In addition, the observed 90% decrease in RT suggests that the use of stronger promoters and additional combinations will further increase Rz concentration and overall efficacy against the virus. Together, the data presented here and the great potential of future delivery methods 13,41,49,50 suggest that SOFA-HDV Rzs constitute a very promising new avenue to control HIV replication.…”

Inhibition of HIV transmission in human cervicovaginal explants and humanized mice using CD4 aptamer-siRNA chimeras

“…This study builds on previous studies that used intravenous injection of PSMA-AsiCs to transfect human prostate cancer cells in an orthotopic mouse tumor model (28,29) or that used HIV gp120-AsiCs to transfect HIV-infected CD4 + cells in vitro and in vivo (30)(31)(32). Based on these studies, the CD4-AsiCs developed here likely could be used for systemic gene silencing in circulating immune cells.…”

“…Chimeric RNAs, composed of an siRNA fused to an aptamer (a structured RNA selected to bind a cell surface ligand with high affinity), provide an attractive alternative for in vivo gene knockdown (28)(29)(30)(31). Aptamer-siRNA chimeras (AsiCs) efficiently transfect and knock down gene expression in cells bearing the surface receptor recognized by the aptamer.…”

“…Intravenous injection of AsiCs incorporating aptamers targeting prostate surface membrane Ag (PSMA) silence target gene expression in orthotopic prostate cancer mouse xenografts (28,29). AsiCs containing an aptamer that recognizes HIV-gp120 inhibit HIV replication in already infected cells in vitro (30,31) and in vivo (32). However, to prevent HIV transmission, it might be better to inhibit de novo infection of uninfected cells.…”

Special delivery: targeted therapy with small RNAs

“…45,46 Similarly, a chimeric RNA containing an aptamer to HIV env protein specifically delivers siRNAs to HIV-infected CD4 + cells, which are otherwise refractory to transfection, and when joined with siRNAs that target viral genes can be used to inhibit HIV replication in vitro. 47,48 An siRNA-aptamer encoding a CD4 aptamer knocks down gene expression specifically in human CD4+ T cells, macrophages and dendritic cells. 49 Intravaginal administration of CD4 aptamer-siRNA chimeras designed to knock down CCR5 and/or viral genes can even prevent sexual transmission of HIV in humanized mice.…”