EX VIVO DEVELOPMENT OF TRANSFECTED ORAL MUCOSAL GRAFTS

我们的长期目标是生产一种“智能”的口腔粘膜移植物，用于重建继发于肿瘤切除、创伤事件或发育障碍的主要口腔缺陷。目前，用于重大重建手术的口腔黏膜供应有限。其他替代方案，如皮肤，会带来缺点，如附件结构的存在和不同的角化模式，可能导致口腔功能的妥协。该方案计划从角质化的粘膜中获取人口腔角质形成细胞，在体外扩增，并将其植入细胞性、非免疫原性的皮肤等效物AllodDerm/TM（已批准用于人类），以生产具有与天然口腔粘膜相似解剖和处理特性的粘膜等效物。体外生产的口腔黏膜复合材料（EVPOMC）将在无血清的环境中开发，并转化辐照饲养细胞，以便用于移植回自体人类受体。EVPOMC的口腔角化细胞将被质粒转染，使用一种新的方法来改变细胞膜粘度，使它们过度分泌血管内皮生长因子（VEGF），从而使其成为一种“智能”口腔粘膜移植设备。我们将通过组织学和免疫组织化学来评估血管向EVPOMC的生长情况，以评估插入的质粒在设备中的功能。该装置的安全性和有效性将在SCID小鼠和小型猪身上进行测试，这是FDA批准的必要条件。我们的具体目标是：1。表征体外生产的人口腔粘膜复合物。2. 确定用VEGF cDNA质粒转染口腔角化细胞的最佳参数。3. 建立动物模型，a)证明转染的口腔粘膜装置（SCID小鼠）的安全性，b)证明转染的口腔粘膜装置可以成功地移植到口腔内部位（迷你猪）。在这项研究中，口腔粘膜装置的转染和移植将使我们能够收集必要的临床前数据，用于提交给监管机构，将这项技术转化为早期I期人体临床试验。

Our long-term goal is to produce a "smart" oral mucosal graft, ex vivo, that will be used for reconstruction of major oral defects that are seen secondary to oncologic resection, traumatic events or developmental disturbances. At present, oral mucosa is in limited supply for use in major reconstructive procedures. Other alternatives such as skin introduce disadvantages, such as the presence of adnexal structures and a different pattern of keratinization that can lead to a compromise in oral function. This proposal plans to harvest human oral keratinocytes from keratinized mucosa, expand them in vitro, and seed them onto a cellular, non- immunogenic dermal equivalent, AllodDerm/TM (already approved for human use), to produce a mucosal equivalent with similar anatomic and handling properties as native oral mucosa. The ex vivo produced oral mucosal composite (EVPOMC) will be developed in an environment free of serum and transformed irradiated feeder cells so that it can be used for grafting back into autogenous human recipients. The oral keratinocytes of the EVPOMC will be transfected with a plasmid, using a novel approach to alter cell membrane viscosity, to cause them to over secrete vascular endothelial growth factor (VEGF), thus making this a "smart" oral mucosal grafting device. We will evaluate vascular ingrowth into the EVPOMC, by histology and immunohistochemistry, to assess the functionality of the inserted plasmid in the device. The safety and efficacy of the device, which is necessary for FDA approval, will be tested in SCID mice and minipigs. Our specific aims are: 1. Characterize the ex vivo produced human oral mucosal composite. 2. Define optimal parameters for transfection of oral keratinocytes with a cDNA plasmid for VEGF. 3. Develop animal models to a) demonstrate the safety of the transfected oral mucosal device (SCID mouse), and b) show that the transfected oral mucosal device can be grafted successfully to an intra-oral site (minipig). The transfection and grafting of oral mucosal device, in this investigation, will allow us to gather the necessary preclinical data, to use for submission to regulatory agencies, for the translation of this technology into early Phase I human clinical trials.