Preparation of Helper-Dependent Adenoviral Vectors
Adenoviruses (Ads) are excellent mammalian gene transfer vectors because of their ability to infect efficiently a wide variety of quiescent and proliferating cell types from various species to direct high-level gene expression. Consequently, Ad vectors are extensively used as potential recombinant viral vaccines, for high-level protein production in cultured cells and for gene therapy (1, 2, 3, 4). First-generation Ad vectors typically have foreign DNA inserted in place of early region 1 (E1). E1-deleted vectors are replication deficient and are propagated in E1-complementing cells such as 293 (5). Although these vectors 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 (6,7), acute and chronic toxicity caused by low-level viral gene expression from the vector backbone (8), and generation of anti-Ad cytotoxic T-lymphocytes caused by de novo viral gene expression (9, 10, 11, 12) or processing of virion proteins (13). Although high-level transient transgene expression afforded by first-generation Ad vectors may be adequate, or even desirable, for many gene transfer and gene therapy applications, the transient nature of expression kinetics renders these vectors unsuitable when prolonged, stable expression is required.
KeywordsVortex Sucrose Glycerol Recombination Titration
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