Restriction-Assembly: A Solution to Construct Novel Adenovirus Vector

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.

CELO Fiber1 Knob Is a Promising Candidate to Modify the Tropism of Adenoviral Vectors

“…An intermediate plasmid-based strategy was employed to modify the fiber gene in adenoviral genome [ 28 , 29 ]. In brief, a fragment, which contained the region to be modified, was excised from adenoviral genome to form a smaller intermediate plasmid.…”

“…An intermediate plasmid-based strategy was employed for FAdV-4 fiber substitution, and we called the cloning method of combined restriction digestion and Gibson assembly restriction-assembly [ 28 , 29 ]. Briefly, two unique cutter (AvrII/HindIII) sites were chosen after restriction analysis of the intermediate plasmid pMD-FAV4Fs in silico, which flanked fiber2 knob region.…”

HEHR: Homing Endonuclease-Mediated Homologous Recombination for Efficient Adenovirus Genome Engineering

“…For example, fowl adenovirus 4 genome was cloned into a pBR322 plasmid backbone using Gibson Assembly [ 34 ]. Moreover, site-directed modification of an adenoviral vector was also achieved by combination of Gibson Assembly and restriction-ligation [ 43 , 44 ]. However, compared to LLHR that is suitable for any targeting region for seamless modification, adenoviral vector engineering utilizing assembly methods is much more limited to locations that have manageable restriction enzyme sites in the near region.…”

The Adenovirus Vector Platform: Novel Insights into Rational Vector Design and Lessons Learned from the COVID-19 Vaccine

“…Recent methods utilize modular assembly of complete viral genomes and advanced recombineering techniques for cloning of complete adenovirus genomes from different sources and further genetic modification ( Figure 2 ). For modular assembly, the inserts, for instance complex transgenes [ 24 ] or even the complete adenovirus genomes [ 25 ], are ligated using the Gibson assembly technique with isothermal DNA assembly technique to seamlessly fuse DNA fragments with short overlaps [ 26 , 27 , 28 , 29 ]. The Gibson Assembly reaction is carried out under isothermal conditions using three enzymatic activities: a 5′ exonuclease generates long overhangs, a polymerase fills in the gaps of the annealed single strand regions, and a DNA ligase seals the nicks of the annealed and filled-in gaps [ 26 , 30 ].…”

“…Gibson assembly has already been used to clone the fowl adenovirus 4 genome into a pBR322 plasmid backbone [ 31 ]. Moreover, site-directed modification of an adenoviral vector genomes has been achieved by combination of Gibson Assembly and restriction mediated-ligation [ 28 ]. However, insertions or gene replacement directly in the adenoviral genome can be performed by Gibson Assembly only if unique restriction sites delimiting the region to be modified are already present, which substantially limits the applicability of this technique.…”