Periodontal disease is a leading cause of tooth loss in adults. In preclinical studies, the applicant and colleagues have shown that platelet-derived growth factor-BB (PDGF) used alone, or in combination with other growth factors, promotes regeneration of the periodontal attachment apparatus. A rate limiting factor has been the targeted delivery of growth factors (Gfs) to the wound site. Recent advances in DNA delivery systems for transient gene expression offer the potential for the efficient expression of recombinant proteins for transient periods of time in selected tissues. The long term goal of this research is to utilize PDGF-mediated gene transfer to promote periodontal wound healing in vivo. The study plan has three main objectives. The first objective is to construct replication deficient SV40 and adenoviral transient expression vectors containing the PDGF genes. Several vector constructs will be utilized in these studies: PDGF-B (c-sis); a negative control (the loss of function PDGF mutant (PDGF-1308); and lacZ. The applicant has constructed the loss of function mutant and has verified the expression of the mutant transgene at both the RNA and protein levels. The second objective is to use the engineered viral vectors for gene transfer experiments in primary cultures of rat osteoblasts, periodontal ligament fibroblasts, and gingival fibroblasts. The in vitro transduction efficiency of the viral vectors will be assessed initially by measuring lacZ expression. Southern and western blotting will be used for transgene and corresponding protein assessment. The effects of the transgenes on cell growth, differentiation and matrix synthesis will be evaluated. Activation of PDGF-beta receptors due to PDGF-B gene transfer will be monitored in situ using activation-state specific antibodies raised against the PDGF-beta receptor. The applicant has shown the specificity of the antibodies in cells following PDGF beta-receptor activation by PDGF-BB, as well as in constitutively activated PDGF-beta receptors (v-sis transformed cells). The third objective is to utilize the appropriate viral vectors tested in vitro to deliver the transgenes to periodontal wounds in rats. The delivery of the transgenes will be tested by using in vivo microseeding at the time of periodontal surgery. The applicant has demonstrated the targeted transient expression of a lacZ/adenoviral recombinant vector to periodontal wounds in rats. The transgene expression will be evaluated by in situ hybridization, beta-receptor activation and reporter gene assessment. The effects of the PDGF transgenes on periodontal wound healing will be measured by quantitative histomorphometry and fluorochrome bone labeling. The applicant hopes that these studies will provide important information on the use of growth factor-mediated gene transfer to affect periodontal repair, as well as to give insight into designing gene therapy strategies for oral wound healing.
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