ISCHEMIC SKIN FLAP SURVIVAL USING AAV-FGF2 AND AAV-VEGF 165

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 这个项目的范围没有变化。 该项目将为基因治疗中的一项最新技术在重建外科领域开发一种新的应用。我们建议使用腺相关病毒载体，旨在使感染细胞精心设计强大的血液供应建立蛋白，即血管内皮生长因子、PDGF和FGF2。这种增强的血管网络似乎将缺血组织从死亡中拯救出来，允许构建更长、更大或更可靠的“皮瓣”(组织从一个解剖位置转移到另一个解剖位置，目的是闭合伤口或重建身体部分)。美国整形外科学会(www.PlatticSurgeery.org)编制的统计数据显示，仅去年一年，美国就有超过520万例整形手术。此外，该项目与改善伤口愈合的总体使命密切相关，可能适用于任何组织缺血的情况。 它建立在申请人(P刘)早先发表的工作的基础上，尽管他目前是罗杰威廉姆斯医院普罗维登斯的外科主席，但除了T32培训拨款外，从未接受过竞争性联邦资金。它没有得到指导，但将依赖于布朗大学的合作者和罗杰·威廉姆斯的关键意见，这些合作者精通那些对申请者来说是新的技术。验证的具体假设是：携带AAV基因的工程组织可以通过内皮祖细胞的募集来提高源自该组织的缺血皮瓣的存活率。除了测试每一种转基因的效果外，我们的方法还将利用病毒介导的基因转移的更高效率来评估VEGF+FGF2的组合，当通过脂质体以质粒形式传递时，它比以同样方式传递的单基因治疗更有效。我们提出了以下具体目标： 1)。通过使用AAV载体优化血管生成基因转移的时机和剂量，最大限度地提高皮瓣模型中的组织存活率，并评估联合使用VEGF和FGF2基因治疗的效果。 2)。开发一种作用机制来解释提高组织存活率的原因。 我们希望这种方法既有效又具有临床意义。解决目标2将有助于回答血管生物学中一个有争议的问题，即损伤修复中的新血液供应来自哪里？我们将利用siRNA的基因沉默方法，以及定位技术(IVIS)和将内皮祖细胞丰富的群体过继转移到缺血组织中来帮助找到答案。最后，一种新的便携式光谱设备ViOptix探头将有助于确定组织的实时生存能力。ViOptix探头测量含氧血红蛋白的近红外光谱随灌流的变化而发生的光谱位移。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. There has been no change in the scope of this project. This project will develop a novel application for a recent technique within gene therapy in the field of reconstructive surgery. We propose to use adeno-associated viral vectors designed to cause infected cells to elaborate potent blood supply-building proteins, namely VEGF, PDGF, and FGF2. This enhanced vascular network appears to rescue ischemic tissue from death, allowing "flaps" (tissue transferred from one anatomic location to another for the purpose of closing a wound or reconstructing parts of the body) to be constructed of longer length, greater size, or greater reliability. Statistics compiled by the American Society of Plastic Surgeons (www.plasticsurgery.org) tracked over 5.2 million reconstructive surgeries in the US last year alone. In addition, this project is germane to the overall mission of bettering wound healing, and may be applicable to any situation of tissue ischemia. It builds upon earlier, published work of the applicant (P Liu), who, though currently Chairman of Surgery at Roger Williams Hospital, Providence, RI, has never been the recipient of competitive Federal funding except a T32 training grant. It is not mentored, but will rely on the critical input from collaborators at Brown University and Roger Williams skilled in those techniques new to the applicant. The specific hypothesis tested is: Engineering tissue with AAV-delivered angiogenic genes can improve survival of ischemic flaps derived from that tissue via recruitment of endothelial progenitor cells. In addition to testing the effects of each of the transgenes, our approach will take advantage of the greater efficiency of viral-mediated gene transfer to assess the combination of VEGF + FGF2, which, when delivered via liposome in plasmid form, was more effective than single gene therapy delivered the same way. We propose the following specific aims: 1). Maximize tissue survival in a flap model by optimizing the timing and dosing of angiogenic gene transfers using AAV vectors, and assess the effects of combining VEGF and FGF2 gene therapy. 2). Develop a mechanism of action to account for enhanced tissue survival. We expect the approach to be both efficacious and clinically relevant. Addressing Aim 2 will help answer a controversial issue in vascular biology, namely, where does the new blood supply in injury repair come from? We will utilize siRNA methods of gene silencing to help get at that answer, as well as localization technology (IVIS) and adoptive transfer of endothelial progenitor cell-enriched populations into the ischemic tissue. Lastly, a new portable spectroscopic device, the ViOptix probe, measuring spectral shifts in the near infrared spectrum of oxygenated hemoglobin as a function of perfusion, will help determine real time tissue viability.