Foreign gene transfer to cardiomyocyte using a replication-defective recombinant coxsackievirus B3 without cytotoxicity.

BACKGROUND

Replication-competent coxsackievirus B3 (CVB3) has been used as a gene transfer vector for cultured cardiomyocytes and hearts in vivo. However, CVB3 induces cell lysis when it replicates in infected cells. In this study, we investigated whether a replication-defective rCVB3 vector could be generated and used as a noncytotoxic gene transfer vector for cardiomyocytes.

METHODS

We generated a replication-defective luciferase-expressing CVB3 plasmid. This recombinant cDNA and pCMV-P1 plasmids were amplified and cotransfected into Hek293 cells using transfection reagents. Replication-defective rLuCVB3 virus was recovered from the cells and cell culture supernatants for 3 days after transfection. The generated rLuCVB3 viruses were concentrated on a 30% sucrose cushion and semiquantified using a luciferase assay. In addition, foreign gene delivery by the rLuCVB3 was tested in cultured cardiomyocytes and intact mouse hearts after rLuCVB3 infection.

RESULTS

Luciferase was expressed in Hek293, HeLa cells and cardiomyocytes after rLuCVB3 infection. In addition, these cells did not show a significant cytopathic effect after 72 h. Luciferase protein expression or activity were detected for 3 days in the myocardium of rLuCVB3-infected mouse hearts without producing cytotoxicity or inflammation.

CONCLUSION

As a proof-of-concept, these data indicate that a replication-defective rCVB3 vector can be generated and used as a novel gene transfer system to transfect exogenous genes into cardiomyocytes without generating cytotoxicity.