Design and Testing of Ribozymes for Cancer Gene Therapy
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DESIGN AND TESTING OF RIBOZYMES FOR CANCER GENE THERAPY
James S. Norris, Brian Hoel, Dale Voeks, Frideriki Maggouta, Michael Dahm1, Weihua Pan2, and Gary Clawson2 Department of Microbiology and Immunology Medical University of South Carolina 171 Ashley Avenue Charleston, SC 29425 1 HEXAL Gentech Forschungs GmbH Industriestr, 25 D-83607 Holzkirchen Germany 2Department of Pathology C7768 Penn State University 500 University Drive Hershey, Pennsylvania 17033
INTRODUCTION The following chapter will cover the subject of the development of a series of ribozymes that target the AC40 subunit of RNA polymerase I (RNA pol I), an essential cellular gene that, when inactivated, leads to cell death. The ribozymes are designed to be delivered in a virus or a liposome as a therapy for prostate cancer. In the course of development of these ribozymes it became clear that target selection in any given mRNA was subject to rules that were not easily defined. Therefore, the following chapter will describe the efforts of this laboratory to develop ribozymes against one of the essential subunits of RNA pol I first using an in vitro target selection system coupled with computer modeling followed by functional analyses of release and target cleavage activities.
RNA POLYMERASE I Yeast and higher eukaryotes contain three nuclear RNA polymerases. RNA pol I is used to transcribe rDNA genes, while the other two are involved in transcription of Cancer Gene Therapy: Past Achievements and Future Challenges, edited by Habib Kluwer Academic/Plenum Publishers, New York, 2000.
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messenger RNA, 5S, tRNAs, U6 and 7SK RNAs and certain viral RNAs. RNA pol I consists of at least 14 subunits of which ten appear to be essential for the function of the enzyme. Two subunits AC40 and AC 19 are thought to be essential and likely play a role in subunit assembly. The AC40 subunit is also shared by RNA pol III (Lanzendorfer et al., 1997). The mouse and human homologue of AC40 are cloned (Song et al., 1994; unpublished). Where studied, ribosomal RNA genes have been found to be tandemly arrayed with 100–1,000 copies spanning several million base pairs (Wallace and Birnstiel, 1966). RNA pol I transcribes these genes and the primary transcripts are processed to form 18S, 5.8S and 28S ribosomal RNA which form the core of the ribozyme. Ribosomal genes account for 40% of the total cellular transcription and 80% of the RNA content of the cell while constituting only 1% of the genome (Moss and Stefanovsky, 1995). Mature ribosomal RNA and ribosomal proteins form the ribosome complex. We hypothesized that any disruptions of ribosome complex formation would likely result in dysregulation of translation and/or death of the cell. The retinoblastoma gene product has been shown to disrupt RNA pol I mediated transcription by sequestering upstream binding factor UBF, thus down-regulating cellular proliferation via disruption of ribosome assembly (Cavanaugh et al., 1995; Voit, Schafer, and Grummit, 1997).
PROSTATE CANCER Prostate cancer is the now t
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