Construction of Adenoviral Vectors using DNA Assembly Technology

Summary

In this study, an infectious clone of human adenovirus type 7 (HAdV-7) was constructed, and an E3-deleted HAdV-7 vector system was established by modifying the infectious clone. This strategy used here can be generalized to make gene transfer vectors from other wild-type adenoviruses.

Abstract

Adenoviral vectors have been used as a gene transfer tool in gene therapy for more than three decades. Here, we introduce a protocol to construct an adenoviral vector by manipulating the genomic DNA of wild-type HAdV-7 by using a DNA assembly method. First, an infectious clone of HAdV-7, pKan-Ad7, was generated by fusing the viral genomic DNA with a PCR product from plasmid backbone, comprising of the kanamycin-resistant gene and the origin of replication (Kan-Ori), through DNA assembly. This was done by designing a pair of PCR primers, that contained ~25 nucleotides of the terminal sequence of HAdV-7 inverted terminal repeat (ITR) at the 5′ end, a non-cutter restriction enzyme site for HAdV-7 genome in the middle, and a template-specific sequence for PCR priming at the 3′ end. Second, an intermediate plasmid-based strategy was employed to replace the E3 region with transgene-expressing elements in the infectious clone to generate an adenoviral vector. Briefly, pKan-Ad7 was digested with dual-cutter restriction enzyme Hpa I, and the fragment containing the E3 region was ligated to another PCR product of plasmid backbone by Gibson assembly to construct an intermediate plasmid pKan-Ad7HpaI. For convenience, restriction-assembly was used to designate the plasmid cloning method of combined restriction digestion and assembly. Using restriction-assembly, the E3 genes in pKan-Ad7HpaI was replaced with a green fluorescent protein (GFP) expression cassette, and the modified E3 region was released from the intermediate plasmid and restored to the infectious clone to generate an adenoviral plasmid pKAd7-E3GFP. Finally, pKAd7-E3GFP was linearized by Pme I digestion and used to transfect HEK293 packaging cells to rescue recombinant HAdV-7 virus. To conclude, a DNA assembly-based strategy was introduced here for constructing adenoviral vectors in general laboratories of molecular biology without the need of specialized materials and instruments.

Introduction

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.

Protocol

NOTE: Adenoviruses are classified as Biosafety Level 2 (BSL-2); all the steps to use the viruses were carried out in a biosafety level 2 laboratory. The wild-type HAdV-7 was isolated in 2017 from the nasopharyngeal aspirate specimen of a 10-month-old infant who was hospitalized with acute respiratory tract infection in Beijing Children's Hospital20. The virus stocks were stored at -80 °C. 1. Extraction of HAdV-7 genomic DNA Seed 2.0…

Representative Results

The strategy for the construction of an infectious clone of HAdV-7 is shown in Figure 1 and Figure 2. Two infectious clone plasmids were randomly selected and identified by BstZ17 I, BamH I, and EcoR V, respectively. The results showed that the fragments were consistent with the expected size (Figure 2A), indicating that the plasmids were constructed correctly. Comet-foci could be seen in the cells 12 days after the Pme I-linearized…

Discussion

Different adenoviruses have various tissue tropisms, and the prevalence of host pre-existing immunity against different adenoviruses can fluctuate intensively in human beings²⁴, which attracts the interest in constructing novel adenoviral vectors for gene therapy or vaccine development. However, the establishment of a new adenoviral vector system remains cumbersome for generic laboratories of molecular biology.

Here, we introduced a protocol for generating vectors from …

Materials

1.5 mL polypropylene microcentrifuge Tube	Axygen	MCT-150-C	Storage of virus
15 mL polypropylene centrifuge tubes	Corning	430790	Storage of virus
150 mm TC-treated culture dishes	Corning	430599	Growth of HEK29E cells
20 K MWCO dialysis cassette	ThermoFisher Scientific	66005	Dialysis of virus
Acetic acid	Amresco	714	Extraction of DNA
Afl II	NEB	R0520	Digestion
Agarose	Takara	5260	Electrophoresis
Age I	NEB	R0552	Digestion
Asc I	NEB	R0558	Digestion
BamH I	NEB	R0136	Digestion
Benzonase Nuclease	Sigma	E8263-25KU	Purification of virus
BsrG I	NEB	R0575	Digestion
Cell lifter	Corning	3008	Scrape off the cells
CsCl	Sigma	C3032	Purification of virus
DNA gel recovery kit	Zymo	D4045	Recovery of DNA
Dulbecco’s modified Eagle’s medium (DMEM)	Cytiva	SH30022.01	HEK293 cells medium
E.coli TOP10 competent cells	TIANGEN BIOTECH (BEIJING) CO.,LTD.	CB-104	Transformation of assembly product
EcoR V	NEB	R3195	Digestion
EDTA	Thermo Fisher Scientific	R3104	Extraction of DNA
Fetal bovine serum (FBS)	Cytiva	SV30208.02	HEK293 cells culture
Genomic DNA Clean and concentrator kit	Zymo	D4065	Purification of DNA
Glycerol	Shanghai Macklin Biochemical Co., Ltd	G810575	Dialysis of virus
HEK293 cells	ATCC	CRL-1573	Amplification of virus
High-Fidelity DNA Polymerase	NEB	M0491	PCR
Hind III	NEB	R3104	Digestion
Kanamycin sulfate	Amresco	408	Selection of plasmid
Kpn I	NEB	R3142	Digestion
Lambda/HindIII DNA marker	Takara	3403	Electrophoresis
LB broth	BD	240230	LB plate for bacteria
LB medium	Solarbio Life Science	L1010	Medium for bacteria
MgCl2	Sigma	63068	Dialysis of virus
Microcentrifuge	Thermo Fisher Scientific	Sorvall Legend Micro 21R	Extraction of DNA
NaCl	Sigma	S5886	Dialysis of virus
Nde I	NEB	R0111	Digestion
NEBuilder HiFi DNA Assembly Master Mix	NEB	E2621	DNA assembly
Nhe I	NEB	R0131	Digestion
Phosphate Buffered Saline	Cytiva	SH30256.01	Washing of cells
Pipette	Thermo Fisher Scientific	Matrix	Aspirate the medium
Plasmid Maxprep Kit	Vigorous Biotechnology Beijing Co., Ltd.	N001	Extraction of DNA
Plasmid Miniprep Kit	TIANGEN BIOTECH (BEIJING) CO.,LTD.	DP103	Extraction of DNA
Pme I	NEB	R0560	Digestion
Potassium acetate	Amresco	698	Extraction of DNA
Protease K	Thermo Fisher Scientific	AM2542	Extraction of DNA
pShuttle-CMV	Stratagene	240007	PCR template
RNase	Beyotime	D7089	Extraction of DNA
Sal I	NEB	R0138	Digestion
Sbf I	NEB	R3642	Digestion
SDS	Amresco	227	Extraction of DNA
Swinging-bucket rotor	HITACHI	S52ST	Purification of virus
T-25 cell flask	Corning	430639	Growth of HEK29E cells
T-75 cell flask	Corning	430641	Growth of HEK29E cells
Transfection reagent	Polyplus-transfection	114-15	Transfection
Transmission electron microscope	FEI	TECNAI 12	Obsevation of virus
Tris-HCl	Amresco	234	Dialysis of virus
Ultracentrifuge	HITACHI	Himac CS120GXII	Purification of virus