Division of Genetic Therapeutics, Jichi Medical School1 and CREST, Japan Science and Technology Corporation (JST)2, 3311-1 Yakushiji, Minamikawachi-machi, Tochigi 329-0498, Japan
Avigen Inc., 1201 Harbor Bay Parkway, #1000, Alameda, CA 94502, USA3
Laboratory of Molecular Genetics, Institute for Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan4
Author for correspondence: Keiya Ozawa (at Jichi Medical School). Fax +81 285 44 8675. e-mail kozawa{at}ms.jichi.ac.jp
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Abstract |
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In this report, we investigated the adenovirus-mediated Cre/loxP system (Kanegae et al., 1995 ) as an inducible genetic switch to overcome the toxicity of the Rep proteins. In theory, cells are transfected with a plasmid in which a `stuffer' sequence, flanked by a pair of loxP recombination target sites, is placed between the CAG promoter and the sequence encoding the AAV proteins. When the cells are infected by a recombinant adenovirus expressing Cre recombinase, the sequence between the two loxP sites should be deleted and the target gene should be activated by the CAG promoter. At the same time, adenoviruses supply helper functions for virus replication. As a result, toxic gene expression can be controlled by Cre-expressing adenovirus vectors.
The structures of the plasmids used in this study are shown in Fig. 1. The SalIEcoRI fragment of the CAG promoter sequence from pUC-CAGGS, which consists of a variant chicken actin promoter with a CMV-IE enhancer, was blunt-ended with Klenow fragment, ligated with HindIII linkers and then inserted into the HindIII site of plox2 (pCAGLL) (Niwa et al., 1991
; Orban et al., 1992
). Plasmid plox2 was provided by J. D. Marth (University of California, San Diego, USA) and pUC-CAGGS by Jun-ichi Miyazaki (Tohoku University, Japan). The HindIII fragment of pSV2bsr, containing the blasticidin S-resistance gene (bsr), was subcloned into the HindIII site of pCMV to give pCMVbsr (Urabe et al., 1997
). The XhoINotI fragment of pCMVbsr, which carries the bsr gene and SV40 polyadenylation signal, was blunt-ended, ligated with BamHI linkers and inserted into the BamHI site of pCAGLL (pCAGLBL). The p5 promoter-deleted BsaI fragment from pIM45 (nt 2854458 of the AAV genome), which contains the rep and cap genes (McCarty et al., 1991
) and the polyadenylation signal sequence, was isolated, blunt-ended and then inserted into the SmaI site of pCAGLBL to complete pCAGLBLAAV.
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Expression profiles for the AAV proteins are shown in Fig. 2. Cell extracts from untreated CAGLBLAAV did not show any recognizable Rep and Cap proteins. However, upon infection with AdCre (m.o.i. of 1), Rep78/68 were strongly induced and Rep52/40 were weakly expressed (Fig. 2a
). Induction of Cap proteins was negligible (Fig. 2b
). Wild-type adenovirus did not show this effect by itself. Of 15 clones tested, nine showed significant induction of Rep78/68 expression.
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To determine the function of the large Rep proteins induced by the Cre/loxP system, we measured the packaging capacity of these cell lines. For this purpose, the AAV vector plasmid (pAAV-LacZ) was used, which contains a lacZ gene under the control of the CMV-IE promoter between ITRs. The titres of AAV-LacZ were determined by quantitative DNA dot-blot hybridization. The AAV-LacZ stock (50 µl) was first treated with 70 U DNaseI for 1 h and then with 500 µg/ml proteinase K for 1 h, before being extracted with phenolchloroform. The isolated DNA and standard DNA (a 1·8 kb ClaIPvuII fragment located within the LacZ coding sequence) were denatured, applied to a nylon membrane (Hybond-N+; Amersham) with a manifold dot-blot apparatus (Schleicher & Schuell), cross-linked by UV light and hybridized with a lacZ gene probe radiolabelled with a random primer labelling kit (Amersham). The filter was washed and exposed to an imaging plate (Fuji Photo Film). The blots were counted directly and analysed on a BAS-2000 imaging system (Fuji). The number of AAV vector genomes present was determined by comparison with the standard DNA. Transfectants of CAGLBLAAV showed a titre of 5·0x106 particles per 10 cm dish. Following the addition of the pR52/40Cap plasmid, the packaging efficiency increased 100-fold to 5·5x108 particles per 10 cm dish (Table 1). After culturing this cell line for 1 month, the titre had decreased to 4·6x107 particles per 10 cm dish, accompanied by a decrease in Rep52/40 and Cap expression (data not shown). Two further experiments showed similar results (data not shown). The packaging efficiency of these cell lines was much lower than that of a standard transfection protocol using pIM45 (about 1011 vector particles per 10 cm dish). Functional titres assayed by X-Gal staining were approximately 104-fold less than particle titres in our experiments.
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One of the problems hampering the clinical application of AAV vectors is the difficulty of large-scale production. To avoid complicated processes in vector production, suitable packaging cell lines are desirable. Several cell lines have been reported utilizing different inducible gene expression systems (Clark et al., 1996 ; Hölscher et al., 1994
; Inoue & Russell, 1998
; Tamayose et al., 1996
; Yang et al., 1994
). Although recent reports showed great improvements in titres, further improvement is still required. Our system will offer an alternative strategy for the development of the novel AAV-packaging cell lines.
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Acknowledgments |
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Footnotes |
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References |
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Received 18 March 1999;
accepted 19 May 1999.