Construction of Adenoviral Vectors using DNA Assembly Technology

Over the past three decades, recombinant adenoviral vectors have been widely used in vaccine development and gene therapy^1,2,3,4 as well as in basic research due to their outstanding biological properties, such as high gene transduction efficiency, non-integration to the host genome, the manipulative viral genome, and the ease of large-scale production.

Currently, the most commonly used adenoviral vectors are constructed based on human adenovirus 5 (HAdV-5)^5,6. Although HAdV-5 vector-mediated transduction provides encouraging results, preclinical and clinical applications have revealed several disadvantages, (e.g., high pre-existing anti-vector immunity within the human population and low transduction efficiency in cells lacking the coxsackievirus and adenovirus receptor (CAR)). To circumvent these problems, there has been a great interest to construct vectors based on other human or mammalian adenovirus types^3,7,8.

Until now, the most popular method to construct an adenoviral vector is homologous recombination in bacteria⁵. Such bacterial strains must express recombinases, which can affect the stability or amplification of the plasmids they bear. Some strains are even commercially unavailable. Recently, methods based on other principles, including bacterial artificial chromosomes, direct cloning, or direct DNA assembly, have been employed to generate infectious clones of adenovirus or recombinant adenoviral vectors^9,10,11,12. However, these methods are somewhat unfriendly to researchers with little experience in this field.

In 2018, the process of constructing an adenovirus infectious clone is simplified in the laboratory by directly ligating the virus genome with a PCR product carrying plasmid backbone through Gibson assembly¹³. After that, the methods of restriction digestion and Gibson assembly are combined together to load transgenes to existing adenoviral plasmids^{14,15,16,17,18}. For the sake of convenience, restriction-assembly is used hereafter to refer to the method of combined restriction enzyme digestion and Gibson assembly. Strategies were further developed to construct adenoviral vectors from infectious clones by using restriction-assembly¹⁹. The essence of restriction-assembly is to include fragments excised from plasmids as much as possible in a DNA assembly reaction, while short PCR products serve as linkers or patches for plasmid modification. At the same time, the number of fragments included is kept as low as possible. Such efforts reflect the payoff; the possibility of unwanted mutations caused by PCR or DNA assembly can be minimized, and the success rate can be improved. Conclusively, a pipeline from a wild-type adenovirus to an adenoviral vector has been set up in the laboratory^{13,14,15,16,17,18,19}.

Here, we attempt to introduce these methods by providing examples of constructing an HAdV-7 infectious clone and an E3-deleted replication-competent HAdV-7 vector.