RETROVIRAL VECTORS DESIGNED FOR TARGETED EXPRESSION OF RNA-POLYMERASEIII-DRIVEN TRANSCRIPTS - A COMPARATIVE-STUDY

Retroviral gene delivery systems for RNA polymerase II (RNA pol II)-ba sed promoters have been developed and are widely used in gene transfer studies. In contrast, gene delivery systems with RNA pol III-based ex pression cassettes have not been studied comprehensively, although the rapeutic applications (e.g., ribozymes, antisense, tripler RNA and RNA decoys) have been proposed. In this report, we describe retroviral ve ctors designed to optimize expression of short chimeric RNAs transcrib ed from a number of RNA pol III promoters. Our results show that all a nalysed RNA pol III expression cassettes (tRNA, U6, Ad VA1), regardles s of orientation, do not transcribe efficiently when located between t he retroviral long terminal repeats (LTRs). In contrast, high steady-s tate expression levels can be achieved by inserting the RNA pol III ex pression cassette into the U3 region of the LTR (double-copy design). Compared to human tRNA gene promoters (tRNA(Met), tRNA(Va1)), the huma n small nuclear RNA U6 gene (U6) and the adenovirus virus-associated R NA 1 (Ad VA1) gene promoters yielded higher expression levels. The maj ority of the chimeric U6-derived transcripts were detected in the nucl ear RNA fraction, and the VA1 and tRNA-driven transcripts were predomi nantly detected in the cytoplasmic compartments. This report is the fi rst comparative study of RNA pol III-driven promoters expressing short chimeric transcripts leading to an optimized retroviral-vector design .