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的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该项目的范围没有变化。 该项目将为重建手术领域的基因治疗中的最新技术开发一种新的应用。我们建议使用腺相关病毒载体，旨在使感染的细胞精心制作有效的血液供应建设蛋白，即VEGF，PDGF和FGF 2。这种增强的血管网络似乎可以将缺血组织从死亡中拯救出来，允许“皮瓣”（从一个解剖位置转移到另一个解剖位置以闭合伤口或重建身体部分的组织）被构造成更长的长度，更大的尺寸或更高的可靠性。美国整形外科医生协会（www.plasticsurgery.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，并可适用于任何组织缺血的情况。 它建立在申请人（P Liu）早期发表的工作的基础上，虽然目前是RI普罗维登斯罗杰威廉姆斯医院的外科主席，但除了T32培训补助金外，从未获得过竞争性联邦资金。它不是指导，但将依赖于关键的投入，从合作者在布朗大学和罗杰威廉姆斯熟练的那些技术新的申请人。测试的具体假设是：用AAV递送的血管生成基因工程化组织可以通过募集内皮祖细胞来改善源自该组织的缺血性皮瓣的存活。除了测试每种转基因的效果外，我们的方法将利用病毒介导的基因转移的更高效率来评估VEGF + FGF 2的组合，当以质粒形式通过脂质体递送时，其比以相同方式递送的单一基因疗法更有效。我们提出以下具体目标： 1）。通过使用AAV载体优化血管生成基因转移的时间和剂量，最大限度地提高皮瓣模型中的组织存活率，并评估VEGF和FGF 2基因治疗组合的效果。 2）。开发一种作用机制，以提高组织存活率。 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？我们将利用基因沉默的siRNA方法来帮助找到答案，以及定位技术（IVIS）和内皮祖细胞富集群体过继转移到缺血组织中。最后，一种新的便携式光谱设备，ViOptix探头，测量氧合血红蛋白的近红外光谱中的光谱变化作为灌注的函数，将有助于确定真实的时间组织活力。

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.