Comparative Analyses of Intracellularly Expressed Antisense RNAs as Inhibitors of Human Immunodeficiency Virus Type 1 Replication

Abstract

The antiviral activities of intracellularly expressed antisense RNAs complementary to the human immunodeficiency virus type 1 (HIV-1) pol, vif, and env genes and the 3′ long terminal repeat (LTR) sequence were evaluated in this comparative study. Retroviral vectors expressing the antisense RNAs as part of the Moloney murine leukemia virus LTR promoter-directed retroviral transcript were constructed. The CD4+ T-cell line CEM-SS was transduced with retroviral constructs, and Northern blot analyses showed high steady-state antisense RNA expression levels. The most efficient inhibition of HIV-1 replication was observed with theenv antisense RNA, followed by the polcomplementary sequence, leading to 2- to 3-log10 reductions in p24 antigen production even at high inoculation doses (4 × 104 50% tissue culture infective doses) of the HIV-1 strain HXB3. The strong antiviral effect correlated with a reduction of HIV-1 steady-state RNA levels, and with intracellular Tat protein production, suggesting that antisense transcripts act at an early step of HIV-1 replication. A lower steady-state antisense RNA level was detected in transduced primary CD4+ lymphocytes than in CEM-SS cells. Nevertheless, replication of the HIV-1 JR-CSF isolate was reduced with both the pol and envantisense RNA. Intracellularly expressed antisense sequences demonstrated more pronounced antiviral efficacy than thetrans-dominant RevM10 protein, making these antisense RNAs a promising gene therapy strategy for HIV-1.

Assessment of an anti-HIV-1 combination gene therapy strategy using the antisense RNA and multimeric hammerhead ribozymes

“…The binding of this RNA to the unspliced HIV-1 RNA occurs over 1553 nts and to the singly and completely spliced RNAs over 196 nts. Antisense RNAs over 800 nts in length were previously reported to inhibit HIV-1 replication better than their smaller counterparts (9,11,27). Therefore, the AS Psi-gag RNA is expected to act primarily at the level of the unspliced RNA.…”

RNA‐based gene therapy for HIV infection

“…One study has found that an RRE decoy, a double ribozyme, and the Rev M10 trans-dominant negative protein all display similar inhibition when introduced into stem cells challenged with various virus strains [68]. Another study has shown that env and pol antisense sequences delay viral replication in transduced peripheral blood lymphocytes at a dose of virus at which Rev M10 exhibits no effect [69]. A comparison of a single chain antibody against Rev with a tRNA-RRE ribozyme fusion has shown much stronger ef®cacy of the protein-targeted antiviral [70].…”

A combination anti-HIV-1 gene therapy approach using a single transcription unit that expresses antisense decoy and sense RNAS and trans-dominant negative mutant gag and env proteins

“…Antisense RNAs spanning 1000 nucleotides or more within this region conferred similar protection (6), although the best result was obtained when the 1430-nucleotide-long region was targeted. Several other antisense RNAs spanning a region of similar or greater length within the HIV-1 RNA were since developed (7,8).…”

“…These antisense RNAs were shown to act at a pre-splicing step (7), and were also shown to be packaged, yielding noninfectious progeny virus (8).…”