GENE THERAPY APPROACH FOR ENGINEERING CRANIOFACIAL BONE

本项目的长期目标是将基因治疗作为口腔和口外部位骨组织工程的可行方法。 由于牙周病、肿瘤、创伤或重建手术引起的骨丢失是一个主要的世界性问题。 由于目前或新兴的再生治疗使用骨移植或重组蛋白都有严重的局限性，有必要对新的再生方法。基因治疗是刺激骨形成的一种令人兴奋的新选择。 将骨形态发生蛋白（BMP）的基因导入患者的骨再生所需部位。 然后，细胞被转化为微型反应器，以产生持续水平的再生分子。 用表达BMP 7的工程腺病毒进行的初步研究支持这种方法的可行性，因为这种病毒可以在皮下和肌内部位诱导异位骨形成。 本项目将检验两个假设：1）用编码骨形态发生蛋白的腺病毒载体转导的宿主细胞将产生持续水平的再生分子，所述再生分子能够在异位和原位位点诱导大量新骨形成，2）BMP-驱动的再生过程将重演胚胎骨发育中发生的中心事件，并且可以通过改变其他发育相关基因的表达来操纵。调节因素。这些假设将通过实现以下具体目标来解决：1）构建表达BMP的人腺病毒载体并优化基质载体和体内病毒递送至细胞的条件，2）使用体内小鼠皮肤袋模型比较单独和组合表达BMP 3、4和7的腺病毒的成骨活性，3）检查在目的2中显示强成骨活性的优化的病毒载体/基质组合在颅骨和长骨临界尺寸缺损模型中形成骨的能力的活性，4）确定BMP病毒转导后的早期事件，并用已知的BMP作用的上游和下游调节剂操纵BMP应答。这些研究将极大地推进我们对BMP依赖性骨再生的理解，并开发出一种可行的基因治疗方法，用于颅面和无骨骨骼的骨再生。

The long term goal of this project is to develop gene therapy as a viable approach for the tissue engineering of bone at oral and extraoral sites. Bone loss due to periodontal disease, neoplasia, trauma or reconstructive surgery is a major worldwide problem. Because current or emerging regenerative treatments using either bone grafts or recombinant proteins all have severe limitations, there is a need for new regenerative approaches. Gene therapy represents an exciting new alternative for stimulating bone formation. Genes for bone morphogenetic proteins (BMPs) are introduced into the patient at the desired site for bone regeneration. Recipient cells are then converted into mini-reactors to produce sustained levels of regenerative molecules. Preliminary studies with an engineered adenovirus expressing BMP7 support the feasibility of this approach in that this virus can induce ectopic bone formation at both subcutaneous and intramuscular sites. Two hypotheses will be tested in this project: 1) Host cells transduced with adenovirus vectors encoding bone morphogenetic proteins will produce sustained levels of regenerative molecules capable of inducing substantial new bone formation at both ectopic and orthotopic sites, 2) The BMP-driven regenerative process will recapitulate central events occurring in embyonic bone development and can be manipulated by altering the expression of other developmentally regulated factors. These hypotheses will be addressed by achieving the following specific aims: 1) Construct human adenovirus vectors expressing BMPs and optimize matrix carriers and conditions for in vivo viral delivery to cells, 2) Compare the osteogenic activity of adenovirus expressing BMPS 3, 4, and 7 alone and in combination using an in vivo mouse skin pouch model, 3) Examine the activity of optimized viral vector/matrix combinations showing strong osteogenic activity in Aim 2 for ability to form bone in both cranial and long bone critical size defect models, 4) Define early events following BMP virus transduction and manipulate the BMP response with known upstream and downstream modulators of BMP action. These studies will greatly advance our understanding of BMP-dependent bone regeneration and develop a viable gene therapy approach for regenerating bone in the craniofacial and appendicular skeleton.