Rescue, Amplification, and Large-Scale Production of Helper-Dependent Adenoviral Vectors

Abstract

INTRODUCTIONThis protocol describes in detail the rescue, amplification, and large-scale production of helper-dependent adenoviral vectors (HDAds) for gene transfer and gene therapy.Production of HDAd can be divided into three parts: (1) rescue, which involves converting pHDAd (plasmid form) to HDAd (viral form); (2) amplification, in which the amount of HDAd is increased by serial coinfections (passages) of the producer cells with the HDAd and the helper virus; and (3) large-scale production to generate large quantities of HDAd. HDAds (also referred to as gutless, gutted, mini, fully deleted, high-capacity, Δ, pseudo) are deleted of all virus-coding sequences. HDAds retain the advantages of early-generation Ad vectors including high-efficiency in vivo transduction and high-level transgene expression. However, the absence of viral gene expression in transduced cells permits long-term transgene expression in the absence of chronic toxicity. Moreover, the deletion of the viral sequences permits a cloning capacity of ∼37 kb. This allows for the delivery of whole-genomic loci, multiple transgenes, and large cis-acting elements to enhance, prolong, and regulate transgene expression. In addition, because the vector genome exists episomally in transduced cells, the risks of germ-line transmission and insertional mutagenesis leading to oncogenic transformation are negligible.