A novel replication-defective HSV-1 vector for regulatable gene delivery to wounds

Abstract

Abstract Introduction: Gene delivery of growth factors to wounds can accelerate healing but has so far not been achieved with any control over the level of transgene expression. Herpes viral vectors are efficient vehicles for in vivo gene delivery and have the advantage of being able to establish long-term gene expression in sensory neurons that are known to interact with the healing wound. To increase safety and control of in vivo gene-transfer we constructed a novel replication-deficient viral vector that for the first time allows efficient regulation of transgene expression in a dose-dependent manner by a HSV-1 recombinant system. Methods: QR9TOhEGF is a novel replication-defective virus that encodes hEGF under control of the tetO-containing CMV promoter (T-REx, Invitrogen) and the tetracycline repressor protein tetR. Without tetracycline, tetR binds to tetO, leading to effective repression of hEGF expression, while in the presence of tetracycline this repression is released. Vero cells were infected with QR9TOhEGF in the presence and absence of tetracycline. Porcine full-thickness wounds sealed with a polyvinyl chamber were infected with QR9TOhEGF in the presence and absence of tetracycline. Wound fluid was assayed at 24 and 48h using hEGF-ELISA. Results: hEGF concentration (pg/ml) at 24 and 48hrs following infection shows up to at least 400-fold regulation. Sensitivity limit: 3pg/ml Vero cellsT−T+Fold regulationp Value (unpaired t-test)24 h613602260.009648 h3.514004000.0003In vivo2.06 × 10 7 PFU/wound24 h841120.028148 h6365.80.0241EGF-ELISA of conditioned medium (Vero cells) and wound fluid (In vivo) at 24 and 48 hours, in the presence and absence of tetracycline. Lower limit of sensitivity: 3pg/ml. Conclusions: We have constructed an HSV-1 vector that allows the highest degree of regulation of gene expression in a single vector system. The feasibility of regulatable gene transfer to wounds has been demonstrated. Our method of in vivo delivery is currently being optimized to achieve higher expression.