VikAD, a Vika site-specific recombinase-based system for efficient and scalable helper-dependent adenovirus production

Abstract

Recombinant viral vectors have become integral tools for basic in vivo research applications. Helper-dependent adenoviral (HdAd) vectors have a large packaging capacity of ∼36 kb of DNA that mediate long-term transgene expression in vitro and in vivo . The large carrying capacity of HdAd enables basic research or clinical applications requiring the delivery of large genes or multiple transgenes, which cannot be packaged into other widely used viral vectors. Currently, common HdAd production systems use an Ad helper virus (HV) with a packaging signal (Ψ) that is flanked by either loxP or FRT sites, which is excised in producer cells expressing Cre or Flp recombinases to prevent HV packaging. However, these production systems prevent the use of HdAd vectors for genetic strategies that rely on Cre or Flp recombination for cell-type-specific expression. To overcome these limitations, we developed the VikAD production system, which is based on producer cells expressing the Vika recombinase and an HV that contains a Ψ flanked by vox sites. The availability of this production system will greatly expand the utility and flexibility of HdAd vectors for use in research applications to monitor and manipulate cellular activity with increased specificity.

The use of adenoviral vectors in gene therapy and vaccine approaches

“…This system provided lower levels of helper contaminations, around 0,02 - 0,1%, and improved vector production ( Palmer and Ng, 2003 ). Other recombinase systems were also explored, such as the FLP/frt system ( Ng et al, 2001 ) and the Vika recombinase system ( Phillips et al, 2022 ). However, none of these approaches completely eliminates the presence of the helper virus.…”

Application of a split‐Cre system for high‐capacity adenoviral vector amplification

“…The packaging signal is thus cleaved only in the HV genome, whereas the HC‐AdV genome can be encapsidated giving rise to infective particles. The most widely used method for cleavage is based on the Cre/LoxP system, [ 7–9 ] although alternatives employing other recombinases such as the Flp/FRT [ 10,11 ] or the Vika/vox [ 12 ] systems have been described. One current bottleneck is the need of packaging cells stably expressing not only E1, but also high levels of the recombinase.…”