A self-processing ribozyme cassette: utility against human papillomavirus 11 E6/E7 mRNA and hepatitis B virus.
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 "SNIP(AA)" 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 SNIP(AA) cassette. SNIP(AA) 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. SNIP(AA) constructs targeted to HBV RNA were tested in vivo in a transgenic mouse model. SNIP(AA) 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 SNIP(AA) cassette against HBV.
Pan
Wei-Hua
WH
Department of Pathology, The Gittlen Cancer Research Institute, Hershey Medical Center, Pennsylvania State University, Hershey, PA 17033, USA.
Xin
Ping
P
Morrey
John D
JD
Clawson
Gary A
GA
D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D002460:Cell Line; D002478:Cells, Cultured; D004742:Enhancer Elements, Genetic; D015723:Gene Library; D018014:Gene Transfer Techniques; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D007150:Immunohistochemistry; D008081:Liposomes / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D008957:Models, Genetic; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000737:chemistry; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000378:metabolism*; D011061:Poly A; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012313:RNA / Q000737:chemistry; D016337:RNA, Catalytic / Q000737:chemistry*; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors; D014162:Transfection
C469553:E6 protein, Human papillomavirus type 11; C493190:E7 protein, human papillomavirus 11; D008081:Liposomes; D016376:Oligonucleotides, Antisense; D009856:Oncogene Proteins, Viral; D016337:RNA, Catalytic; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA