Exploiting cis-acting replication elements to direct hepatitis C virus-dependent transgene expression.
We describe here a novel targeting gene therapy strategy to direct gene expression responsive to hepatitis C virus (HCV). The goal was approached by engineering a construct containing the antisense sequence of the transgene and internal ribosome entry site of encephalomyocarditis virus flanked by 5'- and 3'-end sequences of HCV cDNA that contain cis-acting replication elements. Thus, expression of the transgene is only promoted when the minus-strand RNA has been synthesized by the functional replication machinery present in infected cells. Reporter assay and strand-specific reverse transcription-PCR showed selective transgene expression in Huh-7 cells harboring an autonomously replicating HCV subgenome but remaining silent in uninfected cells. Furthermore, using the cytosine deaminase suicide gene as a transgene coupled with recombinant adenovirus delivery, we demonstrated that cytosine deaminase was specifically expressed in replicon cells, resulting in marked chemosensitization of replicon cells to the cytotoxic effects of flucytosine. This new targeting strategy could be extended to other single-stranded RNA viruses encoding the unique RNA-dependent RNA polymerase that has no parallel in mammalian cells.
Zhang
Jing
J
Research & Development Center, FUSO Pharmaceutical Industries, LTD., 2-3-30 Morinomiya, Joto-ku, Osaka 536-8523, Japan. j-zhang@fuso-pharm.co.jp
Yamada
Osamu
O
Sakamoto
Takashi
T
Yoshida
Hiroshi
H
Araki
Hiromasa
H
Shimotohno
Kunitada
K
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D002460:Cell Line; D002461:Cell Line, Transformed; D002470:Cell Survival / Q000187:drug effects; D003588:Cytopathogenic Effect, Viral / Q000187:drug effects; D043525:Cytosine Deaminase / Q000096:biosynthesis / Q000235:genetics*; D004257:DNA Polymerase II / Q000096:biosynthesis / Q000235:genetics; D004686:Encephalomyocarditis virus / Q000235:genetics; D005437:Flucytosine / Q000494:pharmacology; D015967:Gene Expression Regulation, Viral; D041722:Genes, Transgenic, Suicide; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors; D016174:Hepacivirus / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000235:genetics; D034741:RNA, Small Interfering / Q000235:genetics; D012367:RNA, Viral / Q000096:biosynthesis; D012093:Replicon / Q000235:genetics*; D014162:Transfection; D017361:Viral Nonstructural Proteins / Q000037:antagonists & inhibitors / Q000235:genetics; D012324:RNA-Dependent RNA Polymerase
D043525:Cytosine Deaminase; D004257:DNA Polymerase II; D005437:Flucytosine; D008156:Luciferases; D034741:RNA, Small Interfering; D012367:RNA, Viral; D017361:Viral Nonstructural Proteins; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase