A simplified system for generating recombinant adenoviruses

Abstract

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.

Acknowledgments

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