GENE THERAPY APPROACH FOR ENGINEERING CRANIOFACIAL BONE

该项目的长期目标是开发基因治疗作为口腔和口腔外部位骨组织工程的一种可行方法。由于牙周病、肿瘤、创伤或重建手术导致的骨丢失是一个世界性的重大问题。由于目前或新兴的使用骨移植或重组蛋白的再生治疗都有严重的局限性，因此需要新的再生方法。基因疗法代表了一种刺激骨形成的令人兴奋的新选择。骨形态发生蛋白(BMPs)的基因被导入患者所需的部位进行骨再生。然后，受体细胞被转化为微型反应器，以产生持续水平的再生分子。对表达BMP7的工程化腺病毒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肉内诱导异位骨形成。本项目将检验两个假设：1)转导编码骨形态发生蛋白的腺病毒载体的宿主细胞将产生持续水平的再生分子，能够在异位和原位诱导大量新骨形成；2)BMP驱动的再生过程将概括发生在胚胎骨发育中的中心事件，并可以通过改变其他发育调节因子的表达来操纵。这些假说将通过实现下列特定目标来实现：1)构建表达BMPs的人腺病毒载体，并优化体内病毒载体和条件，2)比较单独表达BMP3、4和7的腺病毒以及联合表达BMP3、4和7的腺病毒的成骨活性，3)检测优化的病毒载体/基质组合的活性，目的2)在颅骨和长骨临界尺寸缺损模型中形成骨的能力，4)定义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因子来操纵BMP的反应。这些研究将极大地促进我们对BMP依赖的骨再生的理解，并为颅面和附件骨骼的骨再生开发一种可行的基因治疗方法。

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.