Use of lentiviral vectors for local gene transfer to the rat hemimandible

Local gene transfer has become a viable method for differentially expressing proteins to study their function. Among the various viral vectors available, lentiviral-based vectors are distinctive in that they integrate at high efficiency in the chromosomes of cells, they do not induce an inflammatory reaction, they have a wide tropism and can be concentrated to high titers. Objectives: The aim of the present study was to evaluate such vectors for the delivery of genetic material through a “surgical window” in the mandibular bone. Methods: This approach consists of drilling a hole in the bone on the buccal aspect of rat hemimandibles and connecting to it an osmotic minipump for local infusion of biological agents. A 24-hour minipump (220 µl) was used to deliver lentiviral vectors driven by PGK or EF-1 promoters and encoding the Green Fluorescent Protein (GFP). The infectivity of the vectors was tested on calvarial osteogenic cell cultures prior to use in vivo. Tissues injected with 0.1 ml of the preparation served as a positive control for extended delivery and contralateral tissues as negative controls. At 72 hours following administration of the vector, animals were sacrificed, and tissues were examined for the presence of GFP by fluorescence microscopy. Results: Both vectors resulted in transgene expression in vitro and following in vivo injection, the EF-1 vector generally giving a more intense signal. Fluorescence was detected in cells surrounding the surgical site and along the apical portion of the incisor. Contralateral tissues exhibited background signal only. Conclusions: These results demonstrate that lentiviral vectors are effective for the direct transfer of recombinant DNA to cells of the tooth organ and surrounding periodontal tissues, and validate the use of the experimental system for studies of molecular events underlying calcified tissue formation. Supported by the Canadian Institutes of Health Research.