Antisense RNA‐Mediated Inhibition of the Replication of the Human Immunodeficiency Virus Type 1.

The acquired immunodeficiency syndrome (AIDS) is associated with infection by human immunodeficiency viruses (HIV)' that show tropism predominantly for hematopoietic cell lineages (e.g., lymphocytes and macrophages) which differentiate from bone marrow stem cells.One way of making hematopoietic cells resistant to replication of HIV-1 could be the intracellular expression of an antivirally active RNA or protein, a therapeutic principle that has been termed "intracellular immunization."2Tools for this therapeutic principle include antisense nucleic acids (for review see ref.3), particularly the intracellular expression of antisense RNA.2,4In HIV-1, antiviral effects of intracellular antisense RNA expression were demonstrated?-'even though inhibition under most of the experimental conditions was not complete.After microinjection into human cells of infectious proviral HIV-1 DNA together with antisense RNA expression plasmids or in vitro synthesized antisense RNA, the extent of virus production (measured by HIV-1 antigen ELISA) indicates inhibitory effects.Different target regions for antisense RNA ranging in size from about 80 nts to more than 3,000 nts were tested.Sequences from the 5'-leader/gag region and from the first coding tat and rev exons exhibited strongest inhibition in this transient assay as well, when stably expressed in antisense orientation in human T-lymphoid Jurkat cell clones (FIG.1).In the latter model system, inhibition of HIV-1 replication can be expressed as a shift of virus spread in the culture along the time axis in antisense RNA producing Jurkat clones versus control cells; that is, infection finally broke through under the applied high infective HIV-1 doses4 However, when the dose of infectious particles was smaller than 0.1 per cell, we did not observe virus production for up to 5 weeks in one of the pAR6-derived clones.It is reasonable to assume that two characteristics of the subgenomic or subgenic target sequences contribute to the extent of inhibition in general and specifically in the case of HIV-I: functional elements (e.g., translational start sites, splice sites) and secondary structure motifs that were shown in prokaryotic systems to influence the hybridization kinetics between the antisense and the target strand in vitro.This was found to correlate with antisense RNA-mediated biological effects?In analogy, we measured hybridization kinetics in vitro between pAR6-derived antisense RNA and the complementary strand (pSR6) as a model target RNA.Comparison of the