Targeted dlivery of siRNA in vivo

Abstract

The exquisite sequence specificity of RNA interference (RNAi) poses challenges for its use as a therapeutic strategy against the highly mutable HIV-1. A single nucleotide substitution in the central target region (positions 8-12 of the 19 nucleotide target), can completely abolish silencing. Simultaneously targeting multiple highly conserved viral sequences is thought to be the efficient way of safeguarding against RNAi-induced viral escape. Thus the choice of conserved RNAi target sequences, where nucleotide changes would adversely affect viral fitness, is a critical factor in preventing RNAi-induced viral escape. Based on an exhaustive analysis of 625 HIV-1 isolates in the Los Alamos Database, we chose siRNAs targeting 2 highly conserved sequences in the Tat and Vif genes (>75% conservation) capable of wobble binding to most natural variants of HIV. We co-expressed these as artificial microRNAs from the PolII-driven miR-155-based vector and tested long-term antiviral activity in HeLa-CD4 cells infected with HIV-1. To test in vivo efficacy, we generated humanized mice by transplanting NOD/SCIDIL2rγc-/- mice with human hematopoietic stem cells and challenged them with HIV-1. Synthetic siRNAs specific to the Tat/Vif sequences were administered using a modified human T cell-targeting single chain antibody, scFvCD7 as a carrier. Disease progression was studied by monitoring CD4 T cell numbers by flow cytometry, ELISA for serum p24 and Amplicor test for plasma viral loads. Then co-expressed as artificial miRNA, the Vif/Tat sequences together mediated effective and sustained inhibition of HIV replication in HeLa-CD4 cells without virus breakout. Efficient silencing of reporter luciferase constructs bearing variant residues corresponding to those in natural HIV isolates revealed that the potent inhibition was attributable to the ability of the Tat/Vif sequences to tolerate wobble binding and mediate persistent silencing of the targets. Humanized mice treated with a combination of the synthetic Tat/Vif siRNAs delivered using a human T cell-specific antibody as carrier, were highly proficient in controlling virus replication and displayed viral loads below detection limits throughout the period of treatment in contrast to control mice. Co-targeting highly conserved viral sequences can provide cross-clade inhibition of HIV-1 and reduce/prevent viral escape demonstrating the feasibility of combinatorial RNAi for HIV therapy. Keywords: Humanized mice, single chain antibody, T cell, siRNA, miRNA

This work was supported by the Seoul R&BD grant, CR070027.