CONSTRUCTION AND CHARACTERIZATION OF LCK-SPECIFIC AND FYN-SPECIFIC TRANSFER-RNA-RIBOZYME CHIMERAS

Two src-family protein tyrosine kinases (PTKs), p56(lck) and p59(fyn), are thought to play an important role in the antigen-specific T cell receptor (TCR)/CD3-initiated signaling pathway, but their relative con tribution to these events is not clearly defined. Here, we have explor ed the potential of catalytic RNA molecules, or ribozymes, as tools fo r selectively inhibiting expression of the corresponding target genes in T cells. Several lck- or fyn-specific hammerhead ribozymes were syn thesized, cloned into a bacterial transcription vector, and found to d isplay specific catalytic activity in vitro. In order to achieve stabl e high-level ribozyme expression in intact cells, selected ribozymes w ere subsequently cloned into a retroviral vector (DC-T5T) immediately downstream of a tRNA(met) transcription unit. Upon retroviral transduc tion of a human leukemic T cell line (Jurkat), two out of four chimeri c tRNA:ribozymes, fyn-1 and lck-1, were stably expressed at levels of similar to 10,000 or similar to 25,000 copies/cell, respectively. Ribo zyme expression was associated with a reduction of up to 80% (lck) or 61% (fyn) in endogenous target mRNA by comparison to the corresponding transcript levels in control clones transfected with vector alone. By contrast, expression of the corresponding target proteins was not red uced, suggesting a post-transcriptional compensatory mechanism that in creases translation or stability of the p56(lck) and/or p59(fyn) prote ins.