Firstgeneration Adenoviral Vectors

Ads are excellent mammalian gene transfer vectors due to their ability to efficiently infect a variety of quiescent and proliferating cell types from various species to direct highlevel gene expression. Consequently, Ad vectors are extensively used as potential recombinant viral vaccines, for highlevel protein production in cultured cells and for gene therapy (for reviews, see 11-15). First-generation Ad vectors (FGAds) typically have foreign DNA inserted in place of early region 1 (E1). El-deleted vectors are replication deficient and are propagated in E1-complementing cells such as 293 (16). Typically, FGAds also have a deletion in the nonessential E3 region to maximize cloning capacity. The fundamental principle underlying all current methods of constructing FGAds is based on the discovery that up to 10% of Ad viral DNA molecules become circularized following infection of mammalian cells (17). This permitted cloning of the entire Ad genome as an infectious bacterial plasmid that could be manipulated with relative ease by standard molecular biology techniques. This Ad genomic plasmid could be stably propagated in Escherichia coli and was capable of generating infectious virus following transfection into permissive mammalian cells at efficiencies comparable to purified virion DNA. An excellent review by Danthinne and Imperiale (18) covers the plethora of methods for constructing FGAd. Although FGAd remain very useful for many applications, it has become clear that transgene expression in vivo is only transient. Several factors contribute to this, including strong innate and inflammatory responses to the vector (19,20), acute and chronic toxicity due to low level viral gene expression from the vector backbone (21), and generation of anti-Ad cytotoxic T-lymphocytes due to de novo viral gene expression (22-25) or processing of virion proteins (26). Although high-level transient transgene expression afforded by FGAd may be adequate, or even desirable, for many gene transfer and gene therapy applications, the toxicity and transient nature of expression kinetics renders these vectors unsuitable in cases where prolonged, stable expression is required

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