title: Persistent and stable gene expression by a cytoplasmic RNA replicon based on a noncytopathic variant Sendai virus.
abstract: Persistent and stable expression of foreign genes has been achieved in mammalian cells by integrating the genes into the host chromosomes. However, this approach has several shortcomings in practical applications. For example, large scale production of protein pharmaceutics frequently requires laborious amplification of the inserted genes to optimize the gene expression. The random chromosomal insertion of exogenous DNA also results occasionally in malignant transformation of normal tissue cells, raising safety concerns in medical applications. Here we report a novel cytoplasmic RNA replicon capable of expressing installed genes stably without chromosome insertion. This system is based on the RNA genome of a noncytopathic variant Sendai virus strain, Cl.151. We found that this variant virus establishes stable symbiosis with host cells by escaping from retinoic acid-inducible gene I-interferon regulatory factor 3-mediated antiviral machinery. Using a cloned genome cDNA of Sendai virus Cl.151, we developed a recombinant RNA installed with exogenous marker genes that was maintained stably in the cytoplasm as a high copy replicon (about 4 x 10(4) copies/cell) without interfering with normal cellular function. Strong expression of the marker genes persisted for more than 6 months in various types of cultured cells and for at least two months in rat colonic mucosa without any apparent side effects. This stable RNA replicon is a potentially valuable genetic platform for various biological applications.
authors.affiliation: Biotherapeutic Research Laboratory and the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan; Japan Society for Promotion of Science, 6 Ichibancho, Chiyoda-ku, Tokyo 102-8471, Japan, the.
authors.forename: Ken
authors.initials: K
authors.lastname: Nishimura
authors.affiliation: Biotherapeutic Research Laboratory and the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
authors.forename: Hiroaki
authors.initials: H
authors.lastname: Segawa
authors.affiliation: Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Goto
authors.affiliation: Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the.
authors.forename: Mariko
authors.initials: M
authors.lastname: Morishita
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan, the.
authors.forename: Akinori
authors.initials: A
authors.lastname: Masago
authors.affiliation: Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan, the.
authors.forename: Hitoshi
authors.initials: H
authors.lastname: Takahashi
authors.affiliation: Resarch Institute for Cell Engineering, National Institute of Advanced Industrial Science and Technology (AIST), 1-8-31 Midorigaoka, Ikeda, Osaka 563-8577, Japan, and the.
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Ohmiya
authors.affiliation: Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
authors.forename: Takemasa
authors.initials: T
authors.lastname: Sakaguchi
authors.affiliation: Signaling Molecules Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, the.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Asada
authors.affiliation: Signaling Molecules Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, the.
authors.forename: Toru
authors.initials: T
authors.lastname: Imamura
authors.affiliation: Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan, the.
authors.forename: Kunitada
authors.initials: K
authors.lastname: Shimotono
authors.affiliation: Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the.
authors.forename: Kozo
authors.initials: K
authors.lastname: Takayama
authors.affiliation: Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Yoshida
authors.affiliation: Biotherapeutic Research Laboratory and the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan. Electronic address: mahito-nakanishi@aist.go.jp.
authors.forename: Mahito
authors.initials: M
authors.lastname: Nakanishi
mesh_terms: D000818:Animals; D016466:CHO Cells; D002645:Chickens; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism*; D005822:Genetic Vectors; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics; D016899:Interferon-beta / Q000096:biosynthesis; D008297:Male; D010641:Phenotype; D012367:RNA, Viral / Q000096:biosynthesis; D051381:Rats; D017208:Rats, Wistar; D012093:Replicon / Q000235:genetics*; D029082:Sendai virus / Q000235:genetics*; D015398:Signal Transduction
chemical_list: D012367:RNA, Viral; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016899:Interferon-beta