Persistent and stable gene expression by a cytoplasmic RNA replicon based on a noncytopathic variant Sendai virus.
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.
Nishimura
Ken
K
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.
Segawa
Hiroaki
H
Biotherapeutic Research Laboratory and the National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan.
Goto
Takahiro
T
Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the.
Morishita
Mariko
M
Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the.
Masago
Akinori
A
Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan, the.
Takahashi
Hitoshi
H
Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan, the.
Ohmiya
Yoshihiro
Y
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.
Sakaguchi
Takemasa
T
Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Asada
Masahiro
M
Signaling Molecules Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, the.
Imamura
Toru
T
Signaling Molecules Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, the.
Shimotono
Kunitada
K
Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan, the.
Takayama
Kozo
K
Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the.
Yoshida
Tetsuya
T
Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan.
Nakanishi
Mahito
M
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.
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
D012367:RNA, Viral; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D016899:Interferon-beta