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J. Biol. Chem., Vol. 282, Issue 37, 27383-27391, September 14, 2007

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Persistent and Stable Gene Expression by a Cytoplasmic RNA Replicon Based on a Noncytopathic Variant Sendai Virus^*

Ken Nishimura, Hiroaki Segawa, Takahiro Goto^¶, Mariko Morishita^¶, Akinori Masago^||, Hitoshi Takahashi^**, Yoshihiro Ohmiya, Takemasa Sakaguchi, Masahiro Asada^¶¶, Toru Imamura^¶¶, Kunitada Shimotono^**, Kozo Takayama^¶, Tetsuya Yoshida, and Mahito Nakanishi¹

From the Biotherapeutic Research Laboratory and the ^¶¶Signaling Molecules Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan, the Japan Society for Promotion of Science, 6 Ichibancho, Chiyoda-ku, Tokyo 102-8471, Japan, the ^¶Department of Pharmaceutics, Hoshi University, 2-4-41 Ebara, Shinagawa, Tokyo 142-8501, Japan, the ^||Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan, the ^**Department of Viral Oncology, Institute for Virus Research, Kyoto University, 53 Kawahara-cho, Shogo-in, Sakyo-ku, Kyoto 606-8507, Japan, the 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 Department of Virology, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8551, Japan

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 10⁴ 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.

Received for publication, March 8, 2007 , and in revised form, June 18, 2007.

^* This work was supported in part by grants from the National Institute of Advanced Industrial Science and Technology (AIST) (to M. N.), from the Ministry of Health and Welfare of Japan (to M. N.), and from the Japan Society for Promotion of Science (to K. N.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1 and S2 and supplemental Figs. S1-S3.

¹ To whom correspondence should be addressed: Biotherapeutic Research Laboratory, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Higashi, Tsukuba, Ibaraki 305-8562, Japan. Tel.: 81-29-861-3040; Fax: 81-29-861-2798; E-mail: mahito-nakanishi{at}aist.go.jp.

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