EX VIVO DEVELOPMENT OF TRANSFECTED ORAL MUCOSAL GRAFTS

我们的长期目标是生产一种“智能”口腔粘膜移植物，离体，将用于重建主要的口腔缺损，继发于肿瘤切除，创伤事件或发育障碍。目前，口腔粘膜在主要重建过程中的供应有限。其他替代品如皮肤会带来缺点，如存在附件结构和不同的角化模式，这可能导致口腔功能受损。该提案计划从角化粘膜收获人口腔角化细胞，在体外扩增它们，并将它们接种到细胞的非免疫原性真皮等同物AllodDerm/TM（已批准用于人）上，以产生具有与天然口腔粘膜相似的解剖学和处理特性的粘膜等同物。离体产生的口腔粘膜复合物（EVPOMC）将在无血清和转化的辐照饲养细胞的环境中开发，使得其可用于移植回自体人受体。EVPOMC的口腔角质形成细胞将用质粒转染，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”口腔粘膜移植装置。我们将通过组织学和免疫组织化学评价血管长入EVPOMC，以评估器械中插入质粒的功能。该设备的安全性和有效性，这是FDA批准所必需的，将在SCID小鼠和小型猪中进行测试。我们的具体目标是：1.表征离体产生的人口腔粘膜复合物。2.定义用VEGF的cDNA质粒转染口腔角质形成细胞的最佳参数。3.开发动物模型，以a）证明转染的口腔粘膜装置的安全性（SCID小鼠），和B）证明转染的口腔粘膜装置可以成功移植到口腔内部位（小型猪）。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.