Gene Therapy (2000) 7, 70–74 2000 Macmillan Publishers Ltd All rights reserved 0969-7128/00 $15.00 www.nature.com/gt VIRAL TRANSFER TECHNOLOGY BRIEF COMMUNICATION Fas ligand gene-carrying adeno-5 AdEasy viruses can be efficiently propagated in apoptosis-sensitive human embryonic retinoblast 911 cells A Watzlik, Ch Dufter, M Jung, G Opelz and P Terness Department of Transplantation Immunology, Institute of Immunology, University of Heidelberg, INF-305, 69120-Heidelberg, Germany Recent reports have pointed out the difficulties in generating recombination (pShuttle-mFasL with pAdEasy-1) is perfor- recombinant adenoviral vectors expressing FasL using med first in bacteria and 911 cells are transfected thereafter eukaryotic cells. In the present study we cloned the murine with recombined AdEasy-mFasL, virus production starts FasL (mFasL) gene into the pCA14 or pShuttle vectors and immediately and the cells survive longer. The resulting recombined them with the adeno-5 virus backbone pBHG10 AdEasy-mFasL viruses are able to infect other eukaryotic or pAdEasy-1 in eukaryotic (911 cells) or prokaryotic (E. coli) cells and induce expression of functional mFasL. This study cells, respectively. Recombination of pCA14-mFasL with describes a method for efficient generation of recombinant pBHG10 in Fas-carrying 911 cells leads to rapid expression FasL adeno-5 viruses in eukaryotic cells. The method may of mFasL and cell death by apoptosis before virus replication be generally suitable for producing viruses carrying is initiated. The same effect is observed when 911 cells are deleterious genes. Gene Therapy (2000) 7, 70–74. transfected with the pCA14-mFasL shuttle vector only. If Keywords: FasL; adenovirus 5; vector; 911 cells; AdEasy system The Fas ligand (FasL) belongs to the tumor necrosis factor the graft (reviewed in Ref. 11). These results prompted (TNF) family, which includes TNF, lymphotoxin, TRAIL investigators to focus on the creation of immune-privi- (TNF-related apoptosis-inducing ligand), CD40 ligand, leged tissues for transplantation by inserting the FasL CD27 ligand, CD30 ligand and OX40 ligand.1 FasL gene into allogeneic donor cells (reviewed in Ref. 11). induces apoptosis resulting in the death of cells express- Other therapeutic applications of the FasL gene, such as ing the Fas receptor (APO-1, CD95). Whereas FasL is pre- the amelioration of rheumatoid arthritis12,13 or tumor dominantly found in activated T cells and natural killer regression,14 are also pursued. cells,2–4 its receptor is expressed in many other cell A vector system for expression of the FasL gene in types.5–7 eukaryotic cells is an important tool for experiments aim- The Fas/FasL system is involved in the regulation of ing to elucidate the physiological role of the FasL gene the immune response and contributes to the functional further. A functioning vector system would also have barrier which abrogates cytotoxic lymphocyte aggression practical implications with respect to therapeutic appli- in immune-privileged sites.8 Its role in immunoregulation cations. An attractive vector which has been successfully is mainly attributable to the induction of tolerance to used for the expression of genes in mammalian cells is autoantigens, down-regulation of lymphocyte expansion the adenovirus. Adenoviruses are relatively stable par- via cell–cell interaction, and the reduction of excess active ticles, their genome does not undergo high rate lymphocytes following an immune response.9 As shown rearrangement, and insertions of foreign genes are gener- by the experiments of Bellgrau et al10 the expression of ally maintained without change through successive repli- FasL in the Sertoli cells of testis is responsible for cation rounds. The adenovirus genome is easy to apoptosis of Fas-expressing T cells and thus may contrib- manipulate by recombinant DNA technology and the ute to the immune-privileged environment provided by virus replicates efficiently in permissive cells. Most this organ. It is also thought that FasL plays a pivotal role importantly, adenoviruses can infect a variety of different in creating an immune-privileged site in the anterior eye cell types, even resting cells.15 Moreover, Zhang et al16 chamber.8 Therefore, transplantation of foreign cells into showed that tolerance against the viruses can be induced the eye or testis usually results in prolonged survival of after their application in vivo. Larregina et al17 have recently shown that embryonic kidney 293 cells routinely used to generate E1-deleted adenoviral vectors do not survive the initial cotransfec- Correspondence: A Watzlik Because AW and ChD made an equal contribution to this work, tion with shuttle plasmid encoding the murine FasL they should both be regarded as the first author of this publication (mFasL) and adenoviral plasmid. Investigation of the rea- Received 14 May 1999; accepted 9 August 1999 son for massive cell death after cotransfection showed FasL Adeno-5 virus propagation in 911 cells A Watzlik et al 71 that 293 cells express the Fas receptor and respond with DNA fragmentation in Figure 2 illustrates that the apoptosis upon its occupation by mFasL. cause of cell death after transfection with pCA14-mFasL In the present work we describe a novel way to and pShuttle-mFasL is apoptosis. This is not surprising generate successfully mFasL gene-harbouring E1-deleted since the cells express the Fas receptor as demonstrated adenovirus type 5 in apoptosis-sensitive eukaryotic cells. by FACS analyses (Figure 3a) and efficiently transcribe In a first series of experiments we used the adenovirus- the FasL gene after transfection (Figure 3b). 5 system developed by Graham’s group,18,19 comprising Adeno-5 mFasL viruses produced in 911 cells are sup- a shuttle plasmid (eg pCA14) and an adenovirus-5 plas- posed to infect various eukaryotic cells followed by the mid (eg pBHG10) for recombination in permissive 293 or efficient expression of the mFasL gene. In order to dem- 911 cells. The mFasL gene was cloned into the pCA14 onstrate that, recombinant AdEasy-mFasL viruses were shuttle plasmid which contains the human cytomegalo- generated as described (titre: 109/ml). The correct mFasL virus immediate–early promoter (CMV).20 911 Cells, a insert of the recombinant construct was proven by human embryonic retinoblast cell line which permits pro- sequence analyses at the beginning and by PCR with liferation of E1-deleted adeno-5 viruses,21,22 were cotrans- mFasL- and adeno-5 virus-specific primers after each fected with 1 ␮g pCA14-mFasL and 1 ␮g pBHG10, an passage in 911 cells. Thereafter, COS cells were infected amount of DNA 10 times below that recommended by with AdEasy-mFasL viruses. They showed a positive the producer. Interestingly, 1–2 days later the cells mFasL band in RT-PCR with mFasL-specific primers showed typical morphological features of death (Figure 4). (Figure 1a). The same result was obtained upon transfec- mFasL molecules expressed on the membrane of tion with 1 ␮g pCA14-mFasL shuttle vector only AdEasy-mFasL-infected cells should be able to induce (Figure 1b). In a second series of experiments the aden- apoptosis of Fas-carrying cells. In the experiment ovirus-5 system developed by He et al,23 called AdEasy, depicted in Figure 5 COS cells were infected with was tested. The mFasL gene was first inserted into the AdEasy-mFasL viruses and thereafter coincubated with pShuttle plasmid containing the human CMV promoter. Jurkat cells, a human T cell line known to express Fas. Similarly to the previous experiment, transfection of 911 One day after coculture the Jurkat cells died as a cells with 1 ␮g pShuttle-mFasL resulted in cell death consequence of apoptosis. (Figure 1c). The AdEasy vector system has been designed In the article by Larregina et al17 several approaches for recombination of shuttle vector (pShuttle) with adeno- were proposed for the generation of recombinant adeno- virus-5 plasmid backbone (pAdEasy-1) in E. coli instead 5 viruses encoding for FasL. They include silencing of of eukaryotic cells. In the experiment depicted in FasL expression by regulation of promoter activity dur- Figure 1d, 911 cells were transfected with 1 ␮g linearized ing the development of the viral vectors in 293 cells, inhi- AdEasy-mFasL, the complete adenoviral sequence pre- bition of FasL-induced apoptosis, or growing the virus viously recombined in bacteria. In this case the cells on a novel cell line not expressing Fas which is stably showed only minimal signs of cytopathic effects after transfected with the E1 region gene. We show here that transfection. the AdEasy vector system, which allows recombination Figure 1 Death of 911 cells after transfection with mFasL-shuttle vector. 911 Cells (5 × 105) (IntroGene, Leiden, The Netherlands) were transfected via Effectene (Qiagen, Hilden, Germany) with plasmid DNA as follows: (a) 1 ␮g pCA14-mFasL and 1 ␮g pBHG10 adenoviral backbone vector (Microbix Biosystems, Toronto, Canada); (b) 1 ␮g pCA14-mFasL shuttle vector; (c) 1 ␮g pShuttle-mFasL shuttle vector (He et al23); or (d) 1 ␮g recombined AdEasy- mFasL. Transfection with pCA14-mFasL + pBHG10, pCA14-mFasL, and pShuttle-mFasL leads to cell death within 2 days as shown by loss of cell confluency and development of rounded cells. Transfection with recombined AdEasy-mFasL had no influence on cell growth and viability. Gene Therapy FasL Adeno-5 virus propagation in 911 cells A Watzlik et al 72 Figure 2 Induction of apoptosis after transfection of 911 cells with pCA14-mFasL or pShuttle-mFasL shuttle vectors. Cells (5 × 105) were incubated with 1 ␮g pCA14-mFasL, 1 ␮g pShuttle-mFasL or 1 ␮g recom- bined AdEasy-mFasL vector. Two days after transfection the cells were incubated over night at 4°Cin50␮g/ml propidium iodide staining buffer, and apoptosis (DNA fragmentation) was determined by measuring the percentage of subG2-peak in FACScan. Transfection with pCA14-mFasL resulted in 60%, with pShuttle-mFasL in 30% apoptotic cells versus only 10% in AdEasy-mFasL-transfected or untreated controls.