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


Viral-vector gene therapies use modified viruses as vehicles to introduce specific DNA sequences that encode genes, regulatory RNAs, or other therapeutic substrates into cells. This technology has long drawn interest for its potential advantages over traditional therapeutics. Nearly all gene therapies currently available use one of three vector types: recombinant adeno-associated-virus (rAAV) vectors, adenovirus vectors, or lentivirus vectors. However, problems can arise when a protein of interest has a large coding region or when delivery of a transgenic cassette is severely constrained by the modest packing capacity of AAV or LVV, 5 and 9 kb respectively. This challenge has been largely overcome by using helper-dependent adenoviral (HdAd) vectors; their use has led to considerable success in both fundamental science and clinical applications because they lack all viral genes and offer long-term transgenic expression, as well as a 36-kb packing capacity. Importantly, HdAd makes it feasible to answer enduring scientific problems that are impossible to address with the constrained packing capacity of rAAV or LVV.

The most effective HdAd production methods facilitate replication of the HdAd genome and the production of vector at high titers in producer cells (typically HEK293) by using, as a helper virus (HV), a first-generation Ad vector. Methods involving site-specific recombination (by either Cre/loxP or Flp/FRT) have thus been developed to prevent HV packaging, leaving more room for the HdAd genome in progeny vector particles . In these production systems, the HEK293 producer cells that express the Ad E1 proteins also express either a recombinase (Cre or Flp) in combination with a an HV packging sequence that is flanked by the appropriate sites (loxP or FRT). These vector systems and purification techniques produce HdAd with only minimal HV contamination (0.5% to 0.01%), making it possible to create HdAd vectors with titers comparable to those of first-generation Ad vectors.

Although HdAd can express numerous molecules that are larger than rAAV and LVV, current HdAd production systems prevent the use of HdAd vectors for genetic intersectional strategies that rely on Cre or Flp recombination for cell-type specific expressionA system that can manufacture HdAd without needing Cre or Flp is necessary for taking full advantage of the promise of this vector system. The Vika recombinase (Vika) from the bacterium Vibrio coralliilyticus demonstrates effective site-specific recombination in both bacterial and human cells, and it acts at a 34-bp palindromic target site (vox). Additional advantages of Vika is that it is substantially less genotoxic and cytotoxic than Cre.

In a new study published in Molecular Therapy – Methods and Clinical Development, researchers from the University of Iowa (Stacia Phillips, Paula Valino Ramos, Dr. Priyadharishini Veeraraghavan) led by Professor Samuel Young Jr. created the VikAD HdAd production system. This production platform was developed by creating a cell line that produces Vika and a form of Ad HV that includes the ψ flanked by vox sites. VikAD will make it possible to use HdAd both in vitro and in vivo, and in combination with genetic approaches that utilize Cre and Flp or other site-specific recombinases. Cell type-specific expression of genes and transgenic expression modules is not possible with rAAV or LVV, but the Young group found that this can be done by combining VikAd with intersectional genetic approaches. Additionally, their analysis of the HV contaminants in crude lysates produced in Vika 293d cells during VikAD synthesis revealed that levels of such contamination in HdAd preparations was comparable to that in the HdAd Cre production cell line, 116 cells. IN addition, they demonstrated that o excision of the ψ is similar in 116 and Vika293 cells, demonstrating that VikAD is an excellent producer systems with respect to excising the ψ. Furthermore, VikAD made it possible to produce the HdAd FLEX EGFP vector, which permits cell-type-specific expression in the context of Cre in vitro and in vivo. Thus, VikAD has the potential to significantly increase the utility of HdAd for research applications that rely on genetic intersectional techniques that employ site-specific recombinases (Cre, Flp, or others) in vitro and in vivo, which is not feasible with rAAV or LVV.

In a nutshell, the new VikAD system developed by Dr. Young and his colleagues is an innovative solution that addresses gene-therapy challenges from many angles. It is a promising advancement in the field of viral vectors, expanding the toolset available for studying the cellular and molecular processes that regulate cellular activity in vivo..


Phillips S. Ramos P. V. Veeraraghavan P. Young S. M. VikAD, a Vika site-specific recombinase-based system for efficient and scalable helper-dependent adenovirus production. Molecular Therapy: Methods & Clinical Development. 2022 March 10; 24: 117-126.

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