IMPROVED ACCUMULATION AND ACTIVITY OF RIBOZYMES EXPRESSED FROM A TRANSFER-RNA-BASED RNA-POLYMERASE-III PROMOTER

RNA polymerase III (pol III) transcripts am abundant in all cells. The refore, pol III promoters may be ideal for expressing high levels of e xogenous RNAs, such as antisense RNAs, decoy RNAs and ribozymes, in ma ny different cell types. We have improved accumulation of recombinant RNAs expressed from a human met(i) tRNA-derived pol III promoter >100- fold by modifying the 3' terminus of the transcripts to hybridize to t he 5' terminus. This terminal duplex includes the 8 nt leader sequence present in the primary wild-type met(i) tRNA transcript that is norma lly removed during processing to the mature tRNA. Expression of an ant i-HIV ribozyme was analyzed in cells stably transduced with retroviral vectors encoding pol III transcription units containing this modifica tion. High accumulation of recombinant pol III ribozyme transcripts wa s observed in all cell lines tested. Due to the enhanced transcript ac cumulation, 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 c leavage activity. The improved pol III transcription units reported he re may be useful for expressing a variety of functional and therapeuti c RNAs.