Genetic Engineering of Human Skin Substitutes

描述（由申请方提供）：烧伤伤口感染是导致患者发病和死亡的主要并发症。局部抗菌剂对角质形成细胞具有细胞毒性，并损害培养的皮肤替代物的植入。本提案的目的是产生具有抗微生物活性的治疗性人类皮肤替代物，用于治疗严重烧伤患者。在过去的十年中，已经出现了关于人源性内源性抗微生物肽的令人兴奋的研究。作为肽的一个家族，人β防御素对革兰氏阳性和革兰氏阴性细菌具有广泛的抗微生物性质。NIKS人角质形成细胞系是遗传均一、非致瘤性、无病原体的人角质形成细胞的一致来源，其将被遗传工程化以表达增强水平的广谱防御素家族成员hBD-3。我们的具体目标是1）开发稳定的遗传修饰的NIKS细胞克隆，并在单层培养中评估hBD-3的表达水平和抗微生物活性。2)使用稳定的hBD-3克隆生产器官型培养物，并根据表达水平、生长特性、形成分层表皮的能力和抗菌活性进行表征。3)为安全和有效性进行动物移植。

DESCRIPTION (provided by applicant): Burn wound infection is a major complication leading to patient morbidity and mortality. Topical antimicrobials are cytotoxic to keratinocytes and compromise engraftment of cultured skin substitutes. The goal of this proposal is to generate a therapeutic human skin substitute with antimicrobial activity for use in the treatment of severely burned patients. In the past decade there has been emerging excitement regarding human derived endogenous antimicrobial peptides. One family of peptides, human beta defensins has broad antimicrobial properties against gram positive and gram negative bacteria. The NIKS human keratinocyte cell line is a consistent source of genetically uniform, non-tumorigenic, pathogen free human keratinocytes that will be genetically engineered to express enhanced levels of the broad spectrum defensin family member, hBD-3. Our specific aims are to 1) Develop stable genetically modified NIKS cell clones and evaluate expression levels of hBD-3 and antimicrobial activity in monolayer culture. 2) Produce organotypic cultures using stable hBD-3 clones and characterize based on expression levels, growth properties, ability to form stratified epidermis, and antimicrobial activity. 3) Perform animal grafting for safety and efficacy.