概要

Construction of Adenoviral Vectors using DNA Assembly Technology

Published: June 16, 2022
doi:

概要

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 therapy1,2,3,4 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 types3,7,8.

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 vectors9,10,11,12. 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 plasmids14,15,16,17,18. 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 laboratory13,14,15,16,17,18,19.

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 …

開示

The authors have nothing to disclose.

Acknowledgements

This research was funded by Beijing Natural Science foundation (7204258), National Natural Science Foundation of China (82161138001, 82072266), CAMS Innovation Fund for Medical Sciences (2019-I2M-5-026), and the research and application on molecular tracing of essential respiratory pathogens in Beijing, by the Capital Health Development and Research of Special (2021-1G-3012).

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

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記事を引用
Zou, X., Zhu, Y., Li, C., Duan, Y., Zhang, L., Guo, X., Hou, W., Xie, Z., Lu, Z. Construction of Adenoviral Vectors using DNA Assembly Technology. J. Vis. Exp. (184), e64033, doi:10.3791/64033 (2022).

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