Expression of Hammerhead Ribozymes by Retroviral Vectors to Inhibit HIV-1 Replication: Comparison of RNA Levels and Viral Inhibition

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Abstract
We have analyzed expression of anti-HIV-1 hammerhead ribozymes in the context of retroviral vectors. To determine optimal vector designs for ribozyme expression, we compared three vectors, each of which contained the same pair of anti-HIV-1 hammerhead ribozymes in tandem. Despite the presence of vastly different amounts of vector-derived flanking sequences, the ribozymes produced by each vector had similar cleavage activity when assayed in vitro. The ribozyme vectors were packaged into amphotropic virion and used to transduce human CEM T lymphocytes. Analysis by Northern blot and RNAse protection assays demonstrated that the highest steady-state levels of ribozyme-containing transcripts were produced by a vector in which the ribozymes were expressed under transcriptional control of the vector MoMuLV LTR. Despite these differences in the levels of ribozyme transcripts achieved by the vectors, their ability to confer resistance to HIV-1 replication was similar. Therefore, other factors than the absolute levels of ribozymes play a role in determining the effectiveness of ribozyme vectors to inhibit HIV-1. These may include structural features of the transcripts that affect the antisense effects of the ribozyme constructs, the actual catalytic activity of the ribozymes, their RNA folding, the binding of proteins, and the intracellular localization. Greater understanding of these factors may permit more effective application of ribozymes to inhibit gene expression.