Abstract

Gene therapy and vaccine development need more novel adenovirus vectors. Here, we attempt to provide strategies to construct adenovirus vectors based on restriction-assembly for researchers with little experience in this field. Restriction-assembly is a combined method of restriction digestion and Gibson assembly, by which the major part of the obtained plasmid comes from digested DNA fragments instead of PCR products. We demonstrated the capability of restriction-assembly in manipulating the genome of simian adenovirus 1 (SAdV-1) in this study. A PCR product of the plasmid backbone was combined with SAdV-1 genomic DNA to construct an infectious clone, plasmid pKSAV1, by Gibson assembly. Restriction-assembly was performed repeatedly in the steps of intermediate plasmid isolation, modification, and restoration. The generated adenoviral plasmid was linearized by restriction enzyme digestion and transfected into packaging 293 cells to rescue E3-deleted replication-competent SAdV1XE3-CGA virus. Interestingly, SAdV1XE3-CGA could propagate in human chronic myelogenous leukemia K562 cells. The E1 region was similarly modified to generate E1/E3-deleted replication-defective virus SAdV1-EG. SAdV1-EG had a moderate gene transfer ability to adherent mammalian cells, and it could efficiently transduce suspension cells when compared with the human adenovirus 5 control vector. Restriction-assembly is easy to use and can be performed without special experimental materials and instruments. It is highly effective with verifiable outcomes at each step. More importantly, restriction-assembly makes the established vector system modifiable, upgradable and under sustainable development, and it can serve as the instructive method or strategy for the synthetic biology of adenoviruses.

Highlights

  • Adenoviruses are non-enveloped viruses with an icosahedral nucleocapsid, which packs a genome of linear double-stranded DNA of 26−48 kb

  • The methods of polymerase chain reaction (PCR), restriction enzyme digestion, and Gibson assembly were used for intermediate plasmid modification and adenoviral plasmid cloning (Figures 1 and 2)

  • Adenoviral vectors have been extensively used in vaccine development and gene therapy

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Summary

Introduction

Adenoviruses are non-enveloped viruses with an icosahedral nucleocapsid, which packs a genome of linear double-stranded DNA of 26−48 kb. Adenoviridae is divided into five genera, of which Masteradenovirus infects mammals [1]. Adenoviruses have been isolated from many mammals, such as humans, monkeys, bovines, and mice. Human adenoviruses contain more than 100 types (http://hadvwg.gmu.edu/, accessed on 5 February 2022), which belong to seven species (HAdV-A to -G) [1,2]. HAdV-5, as a prototype of HAdV-C, has been intensively studied and reconstructed as gene transfer vectors. Adenoviral vectors have special properties when compared with other viral vectors [3,4]

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