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Updated: Aug 18, 2026
An Efficient Method for Adenovirus Production
Published on: June 10, 2021
An efficient construction of conditionally replicating adenoviruses that target tumor cells with multiple factors
S Nagano1, H Oshika, H Fujiwara
1Division of Gene Therapy and Regenerative Medicine, Cognitive and Molecular Research Institute of Brain Diseases, Kurume University, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan.
Abstract:
Despite the enormous potential of conditionally replicating adenoviruses (CRAs), the time-consuming and laborious methods required to construct CRAs have hampered both the development of CRAs that can specifically target tumors with multiple factors (m-CRA) and the efficient analysis of diverse candidate CRAs. Here, we present a novel method for efficiently constructing diverse m-CRAs. Elements involving viral replication, therapeutic genes, and adenoviral backbones were separately introduced into three plasmids of P1, P2, and P3, respectively, which comprised different antibiotic resistant genes, different ori, and a single loxP (H) sequence. Independently constructed plasmids were combined at 100% accuracy by transformation with originally prepared Cre and specific antibiotics in specific Escherichia coli; transfection of the resulting P1+2+3 plasmids into 293 cells efficiently generated m-CRAs. Moreover, the simultaneous generation of diverse m-CRAs was achieved at 100% accuracy by handling diverse types of P1+2 and P3. Alternatively, co-transfection of P1+3 and P2 plasmids into Cre-expressing 293 cells directly generated m-CRA with therapeutic genes. Thus, our three-plasmid system, which allows unrestricted construction and efficient fusion of individual elements, should expedite the process of generating, modifying, and testing diverse m-CRAs for the development of the ideal m-CRA for tumor therapy.
Insights
A new three-plasmid system enables rapid construction of multiple-factor conditionally replicating adenoviruses (m-CRAs). This innovation accelerates the development and testing of novel m-CRAs for effective tumor therapy.
Area of Science:
- Biotechnology
- Molecular Biology
- Oncology
Background:
- Conditionally replicating adenoviruses (CRAs) show promise for cancer therapy.
- Current CRA construction methods are slow and inefficient, hindering the development of advanced tumor-targeting CRAs (m-CRAs).
Purpose of the Study:
- To develop a novel, efficient method for constructing diverse multiple-factor CRAs (m-CRAs).
- To streamline the generation, modification, and testing of m-CRAs for improved tumor treatment strategies.
Main Methods:
- A three-plasmid system (P1, P2, P3) was designed, with each plasmid containing distinct viral replication, therapeutic gene, or adenoviral backbone elements.
- Plasmids were assembled with high accuracy in Escherichia coli using Cre recombinase and specific antibiotics, followed by transfection into 293 cells to generate m-CRAs.
- Simultaneous generation of diverse m-CRAs was achieved by combining different plasmid types.
Main Results:
- The three-plasmid system enabled 100% accurate construction of m-CRAs.
- Diverse m-CRAs were generated simultaneously and efficiently.
- The system allows for unrestricted construction and fusion of individual genetic elements.
Conclusions:
- The novel three-plasmid system significantly expedites the generation and analysis of m-CRAs.
- This method facilitates the development of optimized m-CRAs for targeted cancer therapy.
- The approach is adaptable for creating a wide range of therapeutic adenoviral vectors.
