Ribozyme-mediated in vitro cleavage of transcripts arising from the major transforming genes of human papillomavirus type 16.
Human papillomaviruses (HPV) have been strongly implicated as important cofactors in the development of several human malignancies, particularly anogenital carcinomas. Products arising from the E6 and E7 open reading frames (ORFs) from HPV-16, a type commonly associated with human cervical carcinoma, are essential for viral transformation. Unfortunately, a highly effective treatment for this infection is not available. To develop a novel treatment for this disease, ribozymes were designed to cleave all transcripts encoding HPV-16 E6 and E7 ORFs in proximity to their translational start sites ("AUG"). Cleavage sites for Rz110 and Rz558 occur immediately 3' to nucleotides 110 and 558 of the viral genomic DNA, respectively. Oligonucleotides corresponding to these ribozymes were synthesized and inserted into a eucaryotic viral vector derived from the nonpathogenic parvovirus, adeno-associated virus. Ribozyme transcription from this vector, termed CWRT7:SVN, is under control of both the highly active Rous sarcoma virus long terminal repeat and bacteriophage T7 promoters. T7 transcripts of the E6 and E7 ribozymes efficiently cleaved their cognate targets in vitro under a variety of conditions, including physiological temperature. These results may provide the basis for the development of a ribozyme-based, gene therapeutic treatment for HPV-associated diseases.
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Division of Pediatrics, City of Hope National Medical Center, Duarte, California 91010, USA.
Chatterjee
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Brar
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Wong
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D001358:Avian Sarcoma Viruses / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D001483:Base Sequence; D004274:DNA, Recombinant / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D000229:Dependovirus / Q000235:genetics; D004353:Drug Evaluation, Preclinical; D005091:Exons / Q000235:genetics; D015316:Genetic Therapy; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000235:genetics; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D016366:Open Reading Frames; D027383:Papillomaviridae; D050725:Papillomavirus E7 Proteins; D030361:Papillomavirus Infections / Q000628:therapy; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000494:pharmacology* / Q000627:therapeutic use; D012333:RNA, Messenger / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D012097:Repressor Proteins; D013379:Substrate Specificity; D013696:Temperature; D014158:Transcription, Genetic; D014412:Tumor Virus Infections / Q000628:therapy
D004274:DNA, Recombinant; D004279:DNA, Viral; C068651:E6 protein, Human papillomavirus type 16; D016376:Oligonucleotides, Antisense; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; D012097:Repressor Proteins; C059731:oncogene protein E7, Human papillomavirus type 16