New Methods and Technologies
J RNAi Gene Silenc (February 2006), 2(1), 146-153
Published online: 13 January 2006
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Long-term transgene expression and inhibition of HIV-1 replication by a Cre/loxP-EBNA-1/oriP HIV-1-dependent ribozyme vector: Applications for HIV-1 gene therapy
Takashi Nagawa †§, Yuichiro Habu ‡¶§, Norihiko Matsumoto †, Naoko Miyano-Kurosaki †‡, and Hiroshi Takaku †‡*
† Department of Life and Environmental Sciences, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
‡ High Technology Research Center, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino, Chiba 275-0016, Japan.
¶ Japanese Foundation for AIDS Prevention, Toranomon, Minato-ku, Tokyo, 105-0001, Japan
§ Joint first authors
*Correspondence to: Hiroshi Takaku, Email: hiroshi. firstname.lastname@example.org, Tel: +81 47 478 0407, Fax: +81 47 471 8764
Received: 01 November 2005, Revised: 10 December 2005, Accepted: 15 December 2005
© Copyright The Authors
The cleavage of target mRNA by ribozymes is being exploited as a means of gene silencing in nucleic-acid-based therapies. We previously established an HIV-1-dependent ribozyme-expression vector system, based on Cre-loxP technology with an LTR-gag-p17 promoter as a molecular switch for use in acute HIV-1 infection. The simultaneous expression of the Cre protein and loxP homologous recombination induced a high level of HIV-1-replication inhibition, but ribozyme expression was transient. In the current study, we overcame this limitation by inserting EBNA-1 and oriP genes from the Epstein-Barr virus (EBV) into the vector. When this plasmid was introduced into HeLa CD4 + cells, we observed long-term expression of both the EGFP reporter gene and the ribozyme. Moreover, HIV-1 replication was inhibited in the long-term in transfected cells. These data suggest that the HIV-1-dependent ribozyme-expression vector containing EBNA-1/oriP sequences would be a useful tool in HIV-1 gene therapy applications.
KEYWORDS: Cre/loxP recombination, EBNA-1/oriP, gene therapy, HIV-1, ribozyme