INTRODUCTION

Top Abstract Introduction Materials Results & Discussion References

Recombinant adenoviruses currently are used for a variety of purposes, including gene transfer in vitro, vaccination in vivo, and gene therapy (1-4). Several features of adenovirus biology have made such viruses the vectors of choice for certain of these applications. For example, adenoviruses transfer genes to a broad spectrum of cell types, and gene transfer is not dependent on active cell division. Additionally, high titers of viruses and high levels of transgene expression generally can be obtained.

Decades of study of adenovirus biology have resulted in a detailed picture of the viral life cycle and the functions of the majority of viral proteins (5, 6). The genome of the most commonly used human adenovirus (serotype 5) consists of a linear, 36-kb, double-stranded DNA molecule. Both strands are transcribed and nearly all transcripts are heavily spliced. Viral transcription units are conventionally referred to as early (E1, E2, E3, and E4) and late, depending on their temporal expression relative to the onset of viral DNA replication (6). The high density and complexity of the viral transcription units poses problems for recombinant manipulation, which therefore is usually restricted to specific regions, particularly E1, E2A, E3, and E4. In most recombinant vectors, transgenes are introduced in place of E1 or E3, the former supplied exogenously. The E1 deletion renders the viruses defective for replication and incapable of producing infectious viral particles in target cells; the E3 region encodes proteins involved in evading host immunity and is dispensable for viral production per se.

Two approaches traditionally have been used to generate recombinant adenoviruses. The first involves direct ligation of DNA fragments of the adenoviral genome to restriction endonuclease fragments containing a transgene (7, 8). The low efficiency of large fragment ligations and the scarcity of unique restriction sites have made this approach technically challenging. The second and more widely used method involves homologous recombination in mammalian cells capable of complementing defective adenoviruses ("packaging lines") (9, 10). Homologous recombination results in a defective adenovirus that can replicate in the packaging line (e.g., 293 or 911 cells) supplying the missing gene products (e.g., E1) (11). The desired recombinants are identified by screening individual plaques generated in a lawn of packaging cells (12). Though this approach has proven extremely useful, the low efficiency of homologous recombination, the need for repeated rounds of plaque purification, and the long times required for completion of the viral production process have hampered more widespread use of adenoviral vector technology.

The problems noted above have stimulated novel methods for generating adenoviral vectors. We report herein a strategy that builds on several technological and conceptual advances made in the last few years, including alternative systems for producing viral recombinants (13-16). In our system, the backbone vector, containing most of the adenoviral genome, is used in supercoiled form, obviating the need to enzymatically manipulate it. Second, the recombination is performed in Escherichia coli rather than in mammalian cells. Third, there are no ligation steps involved in generating the adenoviral recombinants, as the process takes advantage of the highly efficient homologous recombination machinery present in bacteria. Fourth, the vectors allow inclusion of up to 10 kb of transgene sequences and allow multiple transgenes to be produced from the same virus. Fifth, some of the new vectors contain a green fluorescent protein (GFP) gene incorporated into the adenoviral backbone, allowing direct observation of the efficiency of transfection and infection, processes that have been difficult to follow with adenoviruses in the past. These modifications resulted in highly efficient viral production systems that often can obviate the need for plaque purification and significantly decrease the time required to generate usable viruses.

	MATERIALS AND METHODS

Top Abstract Introduction Materials Results & Discussion References

Cell Culture, Medium, and Reagents. 293 cells (11) were purchased from Microbix Biosystems (Toronto, Canada) or from the American Type Culture Collection, and 911 cells (17) were kindly provided by Alex J. Van der Eb of the University of Leiden. These lines were maintained in growth medium [DMEM, Life Technologies, Gaithersburg, MD, supplemented with 10% fetal bovine serum (HyClone), 100 units/ml of penicillin, and 100 µg/ml of streptomycin] at 37°C in 5% CO₂.

Preparation of Competent Cells and Plasmid DNAs. To prepare electrocompetent BJ5183 bacteria (18), the cells were grown to an OD₅₅₀ of 0.8, then collected and washed twice with ice-cold 10% glycerol. Twenty-microliter aliquots of the electrocompetent BJ5183 cells were kept at 80°C. Electrocompetent DH10B cells were purchased from Life Technologies. To verify homologous recombination in bacteria, miniprep plasmid DNA was prepared by a standard alkaline lysis procedure. All other plasmids used in this study were prepared by CsCl-banding. Yields were 200-600 µg per 100 ml of Terrific Broth culture (Life Technologies) for plasmids larger than 30 kb (pAdEasy derivatives) and 400-1,000 µg for plasmids smaller than 15 kb (shuttle plasmid derivatives).

Establishment of an Adenoviral E4-Expressing Cell Line. A plasmid that constitutively expresses tet repressor in the same transcription unit as a geneticin-resistance marker was transfected into 911 cells. After growth in geneticin (0.4 mg/ml, Life Technologies), a clone stably expressing the tet repressor, 911tet, was chosen for further manipulation. A second vector that expressed adenoviral E4 under the control of tet responsive promoter was constructed by cloning a fragment containing adenoviral nucleotides 35,468-32,828 into the pBI vector (CLONTECH), resulting in pBI-E4. The pBI-E4 plasmid was cotransfected with linearized pCEP4 (Invitrogen) into 911tet cells. Stable clones were generated through selection in 0.4 mg/ml of geneticin, 0.1 mg/ml of hygromycin B (Calbiochem), and 100 ng/ml of doxycyclin (Sigma). A single clone, called 911-E4, was chosen for viral production based on its tight regulation of E4 protein expression. Expression of adenoviral E4 after removal of doxycyclin was confirmed by immunohistochemical analysis using a mAb against E4ORF6, kindly provided by P. Hearing (State University of New York, Stony Brook) (19).

Construction of Vectors for Homologous Recombination in Bacteria. The adenoviral plasmids (pAdEasy-1 and pAdEasy-2) and the shuttle vectors (pShuttle, pShuttle-CMV, pAdTrack, and pAdTrack-CMV) were constructed through multiple rounds of subcloning of PCR products or of restriction endonuclease fragments. All PCR-derived fragments were sequenced to confirm their predicted composition. Detailed information about the constructions is available from the authors upon request.

Vectors encoding both -galactosidase (-gal) and GFP.

Generation of Recombinant Adenoviral Plasmids by Homologous Recombination in E. coli. High competence of bacteria cells is desired to achieve efficient recombination. Typically, 0.5-1.0 µg of a shuttle vector plasmid (one-fifth of a miniprep) was linearized with PmeI, purified by phenol/chloroform extraction and ethanol precipitation, and mixed with 0.1 µg of supercoiled pAdEasy-1 or pAdEasy-2 in a total volume of 6.0 µl. Twenty microliters of electrocompetent E. coli BJ5183 cells were added, and electroporation was performed in 2.0 mm cuvettes at 2,500 V, 200 ohms, and 25 µF in a Bio-Rad Gene Pulser electroporator. The cells were immediately placed in 500 µl of L-Broth (Life Technologies) and grown at 37°C for 20 min. One hundred twenty-five microliters of the cell suspension then was inoculated onto each of four 10-cm Petri dishes containing L-agar plus 50 µg/ml of kanamycin. After 16-20 hr growth at 37°C, 10-25 colonies per dish generally were obtained. The smaller colonies (which usually represented the recombinants) were picked and grown in 2 ml of L-Broth containing 50 µg/ml of kanamycin. Clones were first screened by analyzing their supercoiled sizes on agarose gels, comparing them to pAdEasy-1 or pAdEasy-2 controls. Those clones that had inserts were further tested by restriction endonuclease digestions, generally PacI, SpeI, and BamHI. (Recombinations sometimes occurred between the plasmid Ori sequences shared between the shuttle and pAdEasy vectors; such recombinants were as useful as those generated by homologous recombination of the "left arm" sequences, but resulted in slightly different restriction patterns; see map in Fig. 1.) Once confirmed, supercoiled plasmid DNA was transformed into DH10B cells for large-scale amplification by electroporation. In such cases, 1.0 µl of plasmid DNA (100 ng) in 15 µl of water was mixed with 5.0 µl of electrocompetent DH10B cells in a total volume of 20.0 µl, and electroporation was performed as described above.

View larger version (27K): [in this window] [in a new window]   Fig. 1.   Schematic outline of the AdEasy system. The gene of interest is first cloned into a shuttle vector, e.g., pAdTrack-CMV. The resultant plasmid is linearized by digesting with restriction endonuclease PmeI, and subsequently cotransformed into E. coli BJ5183 cells with an adenoviral backbone plasmid, e.g., pAdEasy-1. Recombinants are selected for kanamycin resistance, and recombination was confirmed by multiple restriction endonuclease analyses. Finally, the linearized recombinant plasmid is transfected into adenovirus packaging cell lines, e.g., 911 or 293 cells. Recombinant adenoviruses typically are generated within 7-10 days. The "left arm" and "right arm" represent the regions mediating homologous recombination between the shuttle vector and the adenoviral backbone vector (see Materials and Methods for sequence composition). An, polyadenylation site; Bm, BamHI; RI, EcoRI; LITR, left-hand ITR and packaging signal; RITR, right-hand ITR; Sp, SpeI.

Production of Adenoviruses in Mammalian Cells. Approximately 1.5 × 10⁶ cells (911, 293, or 911E4) were plated in 25 cm² flasks 24 hr before transfection, by which time they reached 50-70% confluency. Cells were washed once with 3 ml of OptiMEM (Life Technologies), then 2.5 ml of OptiMEM was added to each flask and the flasks returned to the CO₂ incubator for 15-30 min before transfection. Four micrograms of recombinant adenoviral vector DNA, digested with PacI and ethanol-precipitated, were used for transfection of each 25 cm² flask. A transfection mix was prepared by adding 4 µg of linearized plasmid DNA and 20 µl of Lipofectamine (Life Technologies) to 500 µl of OptiMEM (Life Technologies) according to the manufacturer's instructions. After incubation at room temperature for 15-30 min, the transfection mix was added to the cells. After 4-6 hr at 37°C, the media containing the transfection mix was removed, and 6 ml of growth medium was added. For transfections of 911E4 cells, doxycyclin was removed from the growth media 24 hr after transfection. Transfected cells were monitored for GFP expression and collected 7-10 days after transfection by scraping cells off flasks and pelleting them along with any floating cells in the culture. All but 3 ml of the supernatant was removed. After three cycles of freezing in a methanol/dry ice bath and rapid thawing at 37°, 1 ml of viral lysate was used to infect 3-5 × 10⁶ cells in a 25 cm² flask. The efficiency of such infections could be conveniently followed with GFP. Three to four days later, viruses were harvested as described above. At this point, viral titers were often high enough to use for gene transfer experiments in cultured cells. To generate higher titer viral stocks, packaging cells were infected at a multiplicity of infection (MOI) of 0.1 to 1 and grown for 3-4 days, at which time viruses were harvested as described above. This process was repeated 1-3 times, with a final round using a total of 5 × 10⁸ packaging cells in fifteen 75 cm² flasks and an MOI of 1-5. After 3-5 days, 50% lysis was observed, and the resultant viruses were purified by CsCl banding; final yields were generally 10¹¹ to 10¹² plaque-forming units. Procedures for CsCl banding and viral plaquing are described in ref. 20.

	RESULTS AND DISCUSSION

Top Abstract Introduction Materials Results & Discussion References

Generation of Adenoviral Recombinants: General Considerations. The overall strategy developed here is diagrammed in Fig. 1 and involves three steps. First, the gene of interest is cloned into a shuttle vector (e.g., pAdTrack-CMV, Fig. 2). Second, the resultant construct is cleaved with a restriction endonuclease to linearize it and transformed together with a supercoiled adenoviral vector (e.g., pAdEasy-1) into E. coli strain BJ5183. Recombinants are selected with kanamycin and screened by restriction endonuclease digestion. Third, the recombinant adenoviral construct is cleaved with PacI to expose its inverted terminal repeats and transfected into a packaging cell line (e.g., 293 or 911 cells) (11, 17). In the past, validation of successful virus production at early stages of the process has been one of the most technically demanding aspects of adenoviral vector production. In some of the systems described here, the process of viral production can be directly and conveniently followed in the packaging cells by visualization of the GFP reporter that is incorporated into the viral backbone. After 7-10 days, viruses are harvested and either used directly for experimentation or amplified by infecting packaging cells.

View larger version (26K): [in this window] [in a new window]   Fig. 2.   Shuttle vectors and adenoviral plasmids. Abbreviations are defined in the legend to Fig. 1. See text for details.

Generation of Adenoviral Recombinants: Practical Considerations. The results obtained while generating a virus encoding -gal provides a representative example of the yields and practical considerations involved with the approach described herein. As described in Materials and Methods, a -gal cDNA was placed in the polylinker of pAdTrack-CMV to generate the shuttle vector pGFP+GAL. To make pAdEasy-GFP+GAL, 1 µg of linearized pGFP+GAL was cotransformed with 0.1 µg of supercoiled circular pAdEasy-1 into E. coli BJ5183 cells (see vector diagrams in Fig. 2). The transformation yielded 100 kanamycin-resistant clones, of which two-thirds contained recombinants based on the sizes of undigested miniprep plasmid DNA (Fig. 3A). Candidate clones were digested with several restriction endonucleases to verify proper recombination. As shown in Fig. 3B, the expected restriction fragments were generated in each case. For example, with BamHI, a 5.1-kb fragment containing the GFP gene was produced from pAdEasy-GFP+GAL (lane 3) in addition to the 11.7- and 21.7-kb fragments generated from pAdEasy-1 sequences (lane 2). When digested with PacI, a 3.0-kb fragment was produced (Fig. 3B, lane 6).

View larger version (81K): [in this window] [in a new window]   Fig. 3.   Generation of stable recombinants in bacterial cells. (A) DNA from recombinant pAdEasy-GFP+GAL constructs derived from homologous recombination of pAdTrack-CMV-gal and pAdEasy-1in BJ5183 cells was purified from minipreps. The DNA was analyzed in supercoiled form by electrophoresis through an 0.8% agarose gel and ethidium bromide staining. Lane 1, pAdEasy-1 control; lane 2, pAdTrack-GFP+GAL control; lanes 3-12, different pAdEasy-GFP+GAL clones. Based on the migration rates, the clones in lanes 3, 4, 6, 8, 9, 11, and 12 were potential valid recombinants. (B) Representative digestions with BamHI (lanes 1-3), PacI (lanes 4-6), and SpeI (lanes 7-9). Plasmids pAdTrack-CMV (lanes 1, 4, and 7), pAdEasy-1 (lanes 2, 5, and 8) and a pAdEasy-GFP+GAL recombinant (lanes 3, 6, and 9) are shown. * indicate the diagnostic fragments obtained with each enzyme.

View larger version (100K): [in this window] [in a new window]   Fig. 4.   Adenovirus-producing foci after transfection of 293 cells. PacI-digested pAdEasy-GFP-GAL was transfected into 293 cells and GFP expression was visualized by fluorescence microscopy at the indicated times thereafter. Comet-like adenovirus-producing foci became apparent at 4-5 days. No such foci were observed in the cells transfected with circular (i.e., not PacI-digested) pAdEasy-GFP-Gal.

View larger version (87K): [in this window] [in a new window]   Fig. 5.   Adenoviral titer monitored by GFP expression. Linearized pAdEasy-GFP+GAL was transfected into 293 cells as described in Fig. 4, and cells were harvested at the indicated times after transfection. Two percent of a freeze/thaw lysate of these cells was used to infect 293 cells, and fluorescence microscopy of the infected cells was performed 24 hr later. No viruses were generated in 12 days after the transfection of circular (i.e., not cleaved with PacI) pAdEasy-GFP+GAL (labeled "control").