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ȣ - 490177 3 |
| Novel Replication-Defective Coxsackievirus Vector
Expressed Exofenous Genes without cytolysis in vitro |
| Department of Medicine, Sungkyunkwan University School of Medicine, Samsung Medical Center |
| Soo-Hyeon Yun, Byung-Kwan Lim, Chae-Ok Gil, Jae-Ok Shin, Sung-Uk Kwon, Seon Woon Kim, Dae-Kyung Cho, Seong-Hoon Lim, Hak Jin Kim, Sang-Chol Lee, Jin-Ho Choi, Seung-Woo Park, Duk-Kyung Kim, Eun-Seok Jeon |
Background: Replication-competent Coxsackievirus B3 (CVB3) has been used as a gene transfer vector to cultured cardiomyocytes and hearts. However, it lyses infected cells during its replication. In this study, we generated replication-defective CVB3 vector(s) and the transferred genes were expressed in the cells. Methods and Results: To make replication-defective CVB3 vector, we used two systems. 1) Transfection of whole (ΔP1) or partial (ΔVP0) CVB3 capsid protein deleted plasmids (pCVB3ΔVP0/P1) to the capsid protein (P1 or VP0) expressing HeLa cell lines. 2) Co-transfection of a helper plasmid encoded viral capsid protein VP0 or P1 (pCMV-VP0/P1) with pCVB3ΔVP0/P1 into 293T cell line. The deleted capsid protein region in pCVB3ΔVP0/P1 was replaced by the reporter (REP) gene (luciferase, renilla, GFP), resulting pCVB3-REP-ΔVP0/P1. The generated replication-defective viruses were concentrated by 30% sucrose cushion. The expressions of reporter gene(s) in infected HeLa cells and cultured rat neonatal myocytes were detected by western blot, and luciferase assay. Co-transfection was a more efficient to get encapsidated replication-defective viruses than transfection into capsid protein producing cell lines. Furthermore, P1 deletion was more effective than VP0 deletion to get higher titers of infectious replication-defective viruses (1.6 x 104 vs 10 PFU/ml by semi-quantitied luciferase assay). rCVB3-Luc-ΔP1 virus infected HeLa, 293T cells and the cultured myocytes, but not infect CHO cells. The infected cells expressed luciferase from 4 hours, reached their peaks during 8-16 hours, and decreased from 24 hours after infection. Cell survival assay showed no definite cytolysis of the infected cells. In summary, the generated replication-defective CVB3 vector can infect, and replicate in permissive cells and express the transferred genes without cytolysis. Conclusion: These data demonstrated, as a proof-of-thought, that replication-defective CVB3 virus vector could be generated and it could be used as a novel gene transfer vector for transient expression of exogenous genes in cultured cardiomyocyte and cell lines without cytolysis. Further in vivo experiments to transfer genes to the intact hearts are needed.
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