A self-processing ribozyme cassette: Utility against human papillomavirus 11 E6/E7 mRNA and hepatitis B virus

Wei Hua Pan, Ping Xin, John D. Morrey, Gary A. Clawson

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

We have been developing a self-processing triple-ribozyme cassette, which consists of two cis-acting hammerhead ribozymes flanking an internal, trans-acting hammerhead ribozyme (ITRz). Here, the single ITRz was replaced by two contiguous ITRz (dITRz), and a short poly(A) tail was designed onto the 3′ end of the liberated dITRz, to produce the "SNIPAA" cassette. Self-processing of the cassette appeared to proceed efficiently in cells: The only region of the cassette identified in cells was the liberated dITRz, with approximately 10-20% of the dITRz found within the nucleus. We tested this reagent against two therapeutically important targets, human papillomavirus 11 E6/E7 mRNA and hepatitis B virus (HBV). Library selection protocols were utilized to define accessible target sites, and ribozymes targeted to these sites were very active in vitro. Pairs of the selected ribozymes were then inserted into the SNIPAA cassette. SNIPAA constructs targeted to the E6/E7 mRNA were tested in cell culture using a cotransfection approach. Significant reductions were produced in E6/E7 target, with 80-90% reductions observed at 5 days following cotransfection. SNIPAA constructs targeted to HBV RNA were tested in vivo in a transgenic mouse model. SNIPAA constructs were packaged in liposomes, which were targeted to hepatocytes using asialofetuin, and administered ip. After 2 weeks, a >80% reduction in viral liver DNA was observed. Immunohistochemical staining for core antigen showed a similar decrease in the number of hepatocytes staining positively, compounded by a concomitant loss of residual staining intensity. These results demonstrate the in vivo utility of the self-processing SNIPAA cassette against HBV.

Original languageEnglish (US)
Pages (from-to)596-606
Number of pages11
JournalMolecular Therapy
Volume9
Issue number4
DOIs
StatePublished - Apr 2004

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Human papillomavirus 11
Catalytic RNA
Hepatitis B virus
Messenger RNA
Staining and Labeling
Hepatocytes
Viral DNA
Liposomes
Transgenic Mice
Libraries
Cell Culture Techniques
RNA
Antigens
hammerhead ribozyme
Liver

All Science Journal Classification (ASJC) codes

  • Molecular Medicine
  • Molecular Biology
  • Genetics
  • Pharmacology
  • Drug Discovery

Cite this

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title = "A self-processing ribozyme cassette: Utility against human papillomavirus 11 E6/E7 mRNA and hepatitis B virus",
abstract = "We have been developing a self-processing triple-ribozyme cassette, which consists of two cis-acting hammerhead ribozymes flanking an internal, trans-acting hammerhead ribozyme (ITRz). Here, the single ITRz was replaced by two contiguous ITRz (dITRz), and a short poly(A) tail was designed onto the 3′ end of the liberated dITRz, to produce the {"}SNIPAA{"} cassette. Self-processing of the cassette appeared to proceed efficiently in cells: The only region of the cassette identified in cells was the liberated dITRz, with approximately 10-20{\%} of the dITRz found within the nucleus. We tested this reagent against two therapeutically important targets, human papillomavirus 11 E6/E7 mRNA and hepatitis B virus (HBV). Library selection protocols were utilized to define accessible target sites, and ribozymes targeted to these sites were very active in vitro. Pairs of the selected ribozymes were then inserted into the SNIPAA cassette. SNIPAA constructs targeted to the E6/E7 mRNA were tested in cell culture using a cotransfection approach. Significant reductions were produced in E6/E7 target, with 80-90{\%} reductions observed at 5 days following cotransfection. SNIPAA constructs targeted to HBV RNA were tested in vivo in a transgenic mouse model. SNIPAA constructs were packaged in liposomes, which were targeted to hepatocytes using asialofetuin, and administered ip. After 2 weeks, a >80{\%} reduction in viral liver DNA was observed. Immunohistochemical staining for core antigen showed a similar decrease in the number of hepatocytes staining positively, compounded by a concomitant loss of residual staining intensity. These results demonstrate the in vivo utility of the self-processing SNIPAA cassette against HBV.",
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A self-processing ribozyme cassette : Utility against human papillomavirus 11 E6/E7 mRNA and hepatitis B virus. / Pan, Wei Hua; Xin, Ping; Morrey, John D.; Clawson, Gary A.

In: Molecular Therapy, Vol. 9, No. 4, 04.2004, p. 596-606.

Research output: Contribution to journalArticle

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