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Nucleic Acids Research, 1995, Vol. 23, No. 12 2259-2268
© 1995

RNA

Improved accumulation and activity of ribozymes expressed from a tRNA-based RNA polymerase III promoter

James D. Thompson*, David F. Ayers, Terra A. Malmstrom, Timothy L. McKenzie, Louis Ganousis, Bharat M. Chowrira, Larry Couture and Dan T. Stinchcomb

Ribozyme Pharmaceuticals Inc. 2950 Wilderness Place, Boulder, CO 80301, USA

* To whom correspondence should be addressed

Received February 1, 1995. Revised April 26, 1995. Accepted April 26, 1995.

RNA polymerase III (pol III) transcripts are abundant in all cells. Therefore, pol III promoters may be ideal for expressing high levels of exogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in many different cell types. We have improved accumulation of recombinant RNAs expressed from a human met1 tRNA-derived pol III promoter >100-fold by modifying the 3' terminus of the transcripts to hybridize to the 5' terminus. This terminal duplex Includes the 8 nt leader sequence present in the primary wild-type met) tRNA transcript that is normally removed during processing to the mature tRNA. Expression of an anti-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modification. High accumulation of recombinant pol III ribozyme transcripts was observed in all cell lines tested. Due to the enhanced transcript accumulation, ribozyme cleavage activity was readily detectable in total RNA extracted from stably transduced human T cell lines. One pol III transcription unit, termed ‘TRZ’, was optimized further for ribozyme cleavage activity. The improved pol III transcription units reported here may be useful for expressing a variety of functional and therapeutic RNAs.


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