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pubmed:abstractText |
Intracellular immunization is an anti-viral gene therapy strategy based on the introduction of DNA templates into cells to stably express genetic elements which inhibit viral gene expression and replication. We have recently developed an intracellular immunization strategy for human immunodeficiency virus (HIV) infection that uses RNA decoys. RNA decoys are short RNA oligonucleotides corresponding to the HIV trans activation response element (TAR) or Rev response element (RRE) sequences, which function by inhibiting the binding of the HIV regulatory proteins Tat and Rev to the authentic HIV RNA TAR and RRE regions, respectively. In this report we describe the characterization of potent RRE decoys containing the minimal 13-nucleotide primary Rev binding domain of the RRE. Using an improved tRNA cassette to express high levels of RRE transcripts in CEM cells, we found that this new generation of minimal RRE decoys were more potent inhibitors of HIV in isolated cell lines than previously described TAR or RRE decoys. CEM cells expressing RRE decoys exhibited diminished Rev function in cotransfection assays, confirming the specificity of inhibition of HIV by RRE decoys and indicating that the 13-nucleotide minimal Rev binding domain defined by using in vitro binding studies also binds Rev in vivo. Significant differences in the degree of HIV inhibition between individual CEM cell lines transduced with RRE decoy vectors which were not due to sequence alterations in the tRNA-RRE DNA template, differences in RRE decoy expression level, or endogenous variations in the resistance of CEM clonal cell lines to HIV were observed. In order to evaluate the efficacy of RRE decoys in a more realistic fashion than by comparison of individual clonal cell lines, polyclonal populations of transduced CEM cells were infected with HIV. By using a novel flow cytometric method for quantitating intracellular p24 expression, one version of the RRE decoys tested in this study was found to be capable of durably protecting polyclonal populations of CEM cells from HIV.
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