EXPRESSION OF HAMMERHEAD RIBOZYMES BY RETROVIRAL VECTORS TO INHIBIT HIV-1 REPLICATION - COMPARISON OF RNA LEVELS AND VIRAL INHIBITION

We have analyzed expression of anti-HIV-l hammerhead ribozymes in the context of retroviral vectors, To determine optimal vector designs for ribozyme expression, we compared three vectors, each of which contain ed the same pair of anti-HIV-l hammerhead ribozymes in tandem, Despite the presence of vastly different amounts of vector-derived flanking s equences, the ribozymes produced by each vector had similar cleavage a ctivity when assayed in vitro, The ribozyme vectors were packaged into amphotropic virion and used to transduce human CEM T lymphocytes, Ana lysis by Northern blot and RNAse protection assays demonstrated that t he highest steady-state levels of ribozyme-containing transcripts were produced by a vector in which the ribozymes were expressed under tran scriptional control of the vector MoMuLV LTR, Despite these difference s in the levels of ribozyme transcripts achieved by the vectors, their ability to confer resistance to HIV-1 replication was similar, Theref ore, other factors than the absolute levels of ribozymes play a role i n determining the effectiveness of ribozyme vectors to inhibit HIV-1. These may include structural features of the transcripts that affect t he 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 fa ctors may permit more effective application of ribozymes to inhibit ge ne expression.