GENE THERAPY APPROACH FOR ENGINEERING CRANIOFACIAL BONE

该项目的长期目标是发展基因治疗作为口腔和口腔外部位骨组织工程的可行方法。牙周病、肿瘤、创伤或重建手术引起的骨质流失是一个世界性的主要问题。由于目前或正在出现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。基因疗法代表了刺激骨形成的一个令人兴奋的新选择。骨形态发生蛋白（BMPs）基因被引入到患者所需的骨再生部位。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。该项目将测试两个假设：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，能够在异位和正位位点诱导大量新骨形成；2)bmp驱动的再生过程将重现胚胎骨发育过程中发生的中心事件，并可以通过改变其他发育调节因子的表达来控制。这些假设将通过实现以下具体目标来解决：1)构建表达BMPs的人腺病毒载体，优化基质载体和条件，在体内将病毒传递给细胞；2)在小鼠皮肤袋模型中比较单独表达BMPs 3、4和7的腺病毒和联合表达BMPs 3、4和7的腺病毒的成骨活性；3)在Aim 2中检测优化后的病毒载体/基质组合在颅骨和长骨临界尺寸缺陷模型中成骨能力的活性。4)定义BMP病毒转导后的早期事件，并利用BMP作用的已知上游和下游调节剂操纵BMP反应。这些研究将极大地促进我们对bmp依赖性骨再生的理解，并为颅面和阑尾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.