Gene Therapy (1999) 6, 1670–1678 1999 Stockton Press All rights reserved 0969-7128/99 $15.00 http://www.stockton-press.co.uk/gt Retrovirus vectors designed for efficient transduction of cytotoxic or cytostatic genes MUi1, M Takada1, T Arai1,2, K Matsumoto1, K Yamada3, T Nakahata3, T Nishiwaki4, Y Furukawa4, T Tokino4, Y Nakamura4 and H Iba1 1Department of Gene Regulation 3Department of Clinical Oncology and 4Laboratory of Molecular Medicine, Institute of Medical Science, University of Tokyo, Tokyo; and 2Tsukuba Research Laboratories, Eisai Co, Ltd, Tsukuba, Japan It is difficult to establish stable packaging cell lines produc- rying LacZ or p53 as the exogenous gene was introduced ing retrovirus vectors for the expression of anti-oncogenes into a previously constructed prepackaging cell lines PtG- with cytotoxic or cytostatic potential, because these genes S2, in which the expression of VSV-G is also designed to would also affect the growth of the packaging cell lines. To be initiated by the introduction of Cre recombinase, while overcome this problem, we designed a transcriptional unit the gag-pol gene is expressed continuously. After the intro- pBabeLPL for vector RNA production, in which the tran- duction of Cre recombinase by an adenovirus vector, LacZ- scription of the exogenous genes is completely suppressed or p53-expressing VSV-G-pseudotyped retrovirus vectors by the presence of a preceding insertion containing the with the designed structure were produced at high virus puromycin resistance gene (puro) and a poly(A) addition titers. The p53 virus was shown to be able to transduce signal. This insertion is flanked by a tandem pair of loxP, p53 into the entire population of several human cancer cell and is designed to be excised after the introduction of Cre lines and to induce their growth arrest at the G1 phase, recombinase, when transcription of the exogenous gene indicating that this vector-producing system will be advan- will be started from the 5Ј-LTR. The transcriptional unit car- tageous for human gene therapy. Keywords: retrovirus vector; Cre-loxP; p53; cell cycle arrest; VSV-G-pseudotypes Introduction Establishment of stable packaging cell lines for such retrovirus vectors is difficult, because the growth-sup- Retrovirus vectors based on murine leukemia virus pressing activity of these genes would cause problems (MLV) have been used as powerful gene delivery systems 1–3 in isolation of stable packaging cellular clones and their in basic research and human gene therapy. The intro- propagation in large-scale culture. Furthermore, the duced genes are stably integrated into the target cell gen- propagation step may potentially result in the accumu- ome and have the potential for long-term expression. lation of cellular populations carrying loss-of-function Recently, retrovirus vectors pseudotyped with the G pro- mutants of the tumor suppressor genes because of their tein of vesicular stomatitis virus (VSV-G) have been con- growth advantage. Such difficulties would lower the structed and shown to be advantageous in that they have quality and safety of the vectors, as discussed previously a much broader host range, often yielding higher trans- for an amphotropic retrovirus vector encoding p53.12 duction efficiency, than conventional amphotropic retro- virus vectors and that virus stocks can be concentrated To overcome these problems, we have designed a tran- by ultracentrifugation to give a very high titer scriptional unit for vector RNA production, pBabeLPL, in (approximately 1 × 109 IU/ml).4–7 Efficient production which the expression of the cytotoxic genes is completely systems for these vectors have recently been developed silenced during cloning and propagation of the packag- by us8 and by other groups,9–11 making use of the Cre- ing cell lines. When this transcriptional unit was intro- loxP-mediated recombination system and inducible duced into a prepackaging cell line PtG-S2, which we had 8 promoters, respectively. previously developed, the expression of both the Retrovirus vectors that can efficiently transduce anti- exogenous gene in the vector and the VSV-G gene was oncogenes or tumor suppressor genes with cytotoxic or induced in an all-or-nothing manner upon the introduc- cytostatic potential could be useful for gene therapy of tion of Cre recombinase in an adenovirus vector.13,14 We human cancer. Such vectors might be produced by transi- show here that pBabeLPL in combination with PtG-S2 ent transfection of the vector DNA into packaging cell functioned as designed and was able to produce high lines, but these systems are not suitable for the reproduc- titer virus stocks of VSV-G-pseudotyped retrovirus vec- ible preparation of certified vectors on a large scale. tor encoding p53. The virus stock transmitted the p53 gene into the entire population of several human tumor cell lines in a single transduction and induced cell cycle Correspondence: H Iba, Department of Gene Regulation, Institute of arrest of these cultures. Medical Science, University of Tokyo, 4–6–1 Shirokanedai, Minato-ku, Tokyo 108–8639, Japan Received 12 April 1999; accepted 8 June 1999 Retrovirus vectors for cytotoxic genes MUiet al 1671 Figure 1 (a) Schematic presentation of the system designed for the production of VSV-G-pseudotyped retrovirus vectors encoding cytotoxic or cytostatic genes. The transcriptional unit pBabeLPL-X contains both the puro gene and a poly(A) addition signal flanked with loxP sequences which are tandemly located. Before the introduction of Cre recombinase, the puro gene is transcribed from the vector 5Ј-LTR, while the X gene is completely silent because the RNA transcript terminates before its coding sequence. Arrows indicate the predicted transcript in each cell type. Cre recombinase excises the puro gene and poly(A) addition signal by site-specific recombination between the two loxP sequences, producing a proviral structure carrying the X gene with a loxP. This enzyme also excises the other insert carrying the neo gene, an mRNA-destabilizing signal and a poly(A) addition signal in the transcriptional unit, pCALNdLG and induces the transcription of the VSV-G gene, which initiates VSV-G-pseudotyped vector production. MoLTR, MoMLV long terminal repeat; ␺, packaging signal of retrovirus vector; puro, puromycin resistance gene; pA, polyadenylation signal; X gene, an arbitary gene; CAG, CAG promoter; neo, neomycin resistance gene; VSV-G, VSV-G-coding sequence; MoMLV gag-pol, MoMLV gag and pol genes; bsr, blasticidin resistance gene. (b, c) Predicted structural changes in pBabeLPL-lacZ (b) or pBabeLPL-p53 (c) before and after the introduction of Cre recombinase. The predicted lengths of the restriction fragments used for the chromosomal analysis are shown. K, KpnI sites; X, XbaI sites. Retrovirus vectors for cytotoxic genes MUiet al 1672 Results mixed populations of these transfectants of PtG-S2 pro- duced 2 × 104 IU/ml of LacZ-expressing retrovirus vec- Design of retrovirus vectors for the expression of tors around 5 to 7 days after the introduction of Cre cytotoxic or cytostatic genes recombinase (Figure 2a). The induction kinetics of the To overcome the difficulties in generating packaging cell vectors were slightly delayed compared with those of lines which produce retrovirus vectors expressing cyto- PtG-S2 harboring pMFGnlslacZ (PtG-S2 lacZ1) and the toxic or cytostatic genes, we first designed a transcrip- highest titer was about 1/50 of that of PtG-S2 lacZ1. The tional unit pBabeLPL-X (Figure 1a) for vector RNA pro- slight delay in the induction kinetics might be partially duction. While it has a usual MLV-based proviral DNA explained by the fact that two independent Cre-loxP structure, the exogenous gene X is preceded by an inser- recombination events are required for the virus tion carrying the puromycin resistance gene (puro) and a production in PtG-S2 harboring pBabeLPL-lacZ. poly(A) addition signal flanked by a tandem pair of loxP In order to improve the production of retrovirus sequences. The RNA transcript driven by the 5Ј-LTR would be expected to encode only the puro gene and to be terminated by the poly(A) addition signal present just before the X gene. Therefore, the X gene should not be transcribed from the 5Ј-LTR or from any other promoters, even when pBabeLPL-X is introduced into cells. In cells harboring pBabeLPL-X, the introduction of Cre recombi- nase will induce excision of the insertion (both the puro gene and the poly(A) addition signal) through loxP-spe- cific recombination and the transcription of the X gene from the 5Ј-LTR will be initiated. As the virus-producing cell system, we used the pre- packaging cell line PtG-S2, which we had previously developed.8 PtG-S2 constitutively expresses the gag and pol genes of MoMLV and also contains a Cre recombi- nase-inducible transcriptional unit, pCALNdLG, for the VSV-G gene (Figure 1a). Neither RCR nor adenovirus vector was detected in pseudotyped virus stocks of 1 × 107 IU produced from PtG-S2.8 After the introduction of an adenovirus vector encoding Cre recombinase, PtG- S2 harboring pBabeLPL-X would be expected to begin transcription of both full-length vector RNA driven by the 5Ј-LTR and VSV-G mRNA driven by a CAG pro- moter.15 Thereafter, virus vector carrying the X gene will be packaged and produced from the packaging cell line just generated from the prepackaging cell line. To develop this vector-producing system, we first used nlslacZ, the ␤-galactosidase gene with a nuclear localiz- ation signal, as a control gene (hereafter abbreviated as LacZ) and basic conditions for virus production, as well as the characteristics of the viral structure, were exam- ined in PtG-S2 harboring pBabeLPL-lacZ (Figure 1b). Using the conditions and procedures established for these cells, we further applied this system to a represen- tative tumor suppressor gene p53, with cytostatic and cytotoxic potential, by generating PtG-S2 harboring pBa- beLPL-p53 (Figure 1c).