An efficient construction of conditionally replicating adenoviruses that target tumor cells with multiple factors.
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.
Nagano
S
S
Division of Gene Therapy and Regenerative Medicine, Cognitive and Molecular Research Institute of Brain Diseases, Kurume University, 67 Asahi-machi, Kurume, Fukuoka 830-0011, Japan.
Oshika
H
H
Fujiwara
H
H
Komiya
S
S
Kosai
K
K
D000256:Adenoviridae / Q000235:genetics*; D017500:Adenovirus E1A Proteins / Q000235:genetics; D019677:Artificial Gene Fusion / Q000379:methods*; D019149:Bioreactors; D002272:Carcinoembryonic Antigen / Q000378:metabolism; D045744:Cell Line, Tumor; D017931:DNA Primers; D004351:Drug Resistance / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D009369:Neoplasms / Q000628:therapy*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D014162:Transfection / Q000379:methods; D014779:Virus Replication / Q000235:genetics
D017500:Adenovirus E1A Proteins; D002272:Carcinoembryonic Antigen; D017931:DNA Primers