Molecular Therapy: Oncolytics (Dec 2021)
A one-step construction of adenovirus (OSCA) system using the Gibson DNA Assembly technology
- Na Ni,
- Fang Deng,
- Fang He,
- Hao Wang,
- Deyao Shi,
- Junyi Liao,
- Yulong Zou,
- Hongwei Wang,
- Piao Zhao,
- Xue Hu,
- Connie Chen,
- Daniel A. Hu,
- Maya Sabharwal,
- Kevin H. Qin,
- William Wagstaff,
- David Qin,
- Bryce Hendren-Santiago,
- Rex C. Haydon,
- Hue H. Luu,
- Russell R. Reid,
- Le Shen,
- Tong-Chuan He,
- Jiaming Fan
Affiliations
- Na Ni
- Ministry of Education Key Laboratory of Diagnostic Medicine, and Department of Clinical Biochemistry, The School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Fang Deng
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Pathophysiology, and Key Laboratory of High Altitude Medicine, College of High Altitude Military Medicine, Army Medical University, Chongqing, China
- Fang He
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Gastroenterology, Blood Transfusion, and Orthopaedic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Hao Wang
- Ministry of Education Key Laboratory of Diagnostic Medicine, and Department of Clinical Biochemistry, The School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Deyao Shi
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Orthopaedic Surgery, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China
- Junyi Liao
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Gastroenterology, Blood Transfusion, and Orthopaedic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Yulong Zou
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Orthopaedic Surgery, the Second Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Hongwei Wang
- Division of Research and Development, Decoding Therapeutics, Inc., Mt Prospect, IL 60056, USA
- Piao Zhao
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Gastroenterology, Blood Transfusion, and Orthopaedic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Xue Hu
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Departments of Gastroenterology, Blood Transfusion, and Orthopaedic Surgery, the First Affiliated Hospital of Chongqing Medical University, Chongqing 400016, China
- Connie Chen
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Daniel A. Hu
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Maya Sabharwal
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Kevin H. Qin
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- William Wagstaff
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- David Qin
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Bryce Hendren-Santiago
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Rex C. Haydon
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Hue H. Luu
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Russell R. Reid
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Surgery Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Le Shen
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Surgery Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, IL 60637, USA
- Tong-Chuan He
- Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Department of Surgery Section of Plastic Surgery, The University of Chicago Medical Center, Chicago, IL 60637, USA; Corresponding author: Tong-Chuan He, MD, PhD, Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, 5841 South Maryland Avenue, MC3079 Chicago, IL 60637, USA.
- Jiaming Fan
- Ministry of Education Key Laboratory of Diagnostic Medicine, and Department of Clinical Biochemistry, The School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China; Molecular Oncology Laboratory, Department of Orthopaedic Surgery and Rehabilitation Medicine, The University of Chicago Medical Center, Chicago, IL 60637, USA; Corresponding author: Jiaming Fan, MD, PhD Ministry of Education Key Laboratory of Diagnostic Medicine, Department of Clinical Biochemistry, School of Laboratory Medicine, Chongqing Medical University, Chongqing 400016, China.
- Journal volume & issue
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Vol. 23
pp. 602 – 611
Abstract
Adenovirus (Ad) is a non-enveloped linear double-stranded DNA virus with >50 serotypes in humans. Ad vectors have been used as gene delivery vehicles to express transgenes, small interfering RNAs (siRNAs) for gene silencing, or CRISPR/Cas and designer nucleases for genome editing. Although several methods are used to generate Ad vectors, the Ad-making process remains technically challenging and time consuming. Moreover, the Ad-making techniques have not been improved for the past two decades. Gibson DNA Assembly (GDA) technology allows one-step isothermal DNA assembly of multiple overlapping fragments. Here, we developed a one-step construction of Ad (OSCA) system using GDA technology. Specifically, we first engineered several adenoviral recipient vectors that contain the ccdB suicide gene flanked with two 20-bp unique sequences, which serve as universal sites for GDA reactions in the Ad genome ΔE1 region. In two proof-of-principle experiments, we demonstrated that the GDA reactions were highly efficient and that the resulting Ad plasmids could be effectively packaged into Ads. Ad-mediated expression of mouse BMP9 in mesenchymal stem cells was shown to effectively induce osteogenic differentiation both in vitro and in vivo. Collectively, our results demonstrate that the OSCA system drastically streamlines the Ad-making process and should facilitate Ad-based applications in basic, translational, and clinical research.