Nonviral Topical Transfection with Calprotectin in Periodontitis

钙卫蛋白非病毒局部转染治疗牙周炎

• 批准号: 10064626
• 负责人: William S Boyle
• 金额: $ 3.39万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-12-03 至 2021-05-02
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10064626
• 关键词: Address; Adult; Affect; Alveolar Bone Loss; Bacterial Infections; Bandage; Binding; Biocompatible Materials; Biological; Biology; Biomedical Engineering; Cellulose; Charge; Clinical Sciences; Complex; Custom; Cytoplasm; Data; Development; Diffusion; Disease; Dyes; Engineering; Environment; Epithelial Cells; Exclusion; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Food; Formulation; Gel; Gene Delivery; Genetic Variation; Gingiva; Goals; Homidium Bromide; Hydrogel Bandage; Hydrogels; Immune; Infection; Inflammation; Ionic Strengths; Knowledge; Label; Lead; Leukocyte L1 Antigen Complex; Measurement; Mentorship; Messenger RNA; Methods; Minnesota; Modeling; Mouth Diseases; Mus; Natural Immunity; Nucleic Acids; Oral cavity; Periodontal Diseases; Periodontal Infection; Periodontal Pocket; Periodontitis; Phenotype; Polymers; Property; Proteins; Public Health; Reporter; Reporter Genes; Research; Rheology; Role; S100A8 gene; Severities; Small Interfering RNA; Surface; Testing; Therapeutic; Tissues; Tooth Tissue; Topical agent; Topical application; Training; Transfection; Translations; Treatment Efficacy; Trehalose; United States; Universities; alveolar bone; antimicrobial; base; biomaterial compatibility; career; career development; dysbiosis; experience; experimental study; healing; improved; in vivo; intraepithelial; light scattering; mRNA Expression; mRNA delivery; microCT; microbiome; mouse model; neutrophil; non-viral gene delivery; novel; novel therapeutic intervention; oral tissue; overexpression; phosphoethanolamine; research and development; senescence; tool; translational scientist; uptake; zeta potential

Abstract. Periodontitis is a highly prevalent condition characterized by a bacterial infection of periodontal tissues. The development of improved, non-invasive treatments for periodontitis could have implications for public health in the United States and worldwide. Calprotectin (S100A8/A9) is an antimicrobial protein that is expressed in the cytoplasm of gingival epithelial cells and polymorphonuclear leukocytes. In a murine model of periodontitis, S100A8/A9 slows the progression of periodontitis and reduces alveolar bone loss. We hypothesize that efficient delivery and expression of calprotectin-specific mRNA in vivo will lead to reduced inflammation and slow the progression of periodontitis. Herein, we propose a novel method to treat periodontitis using topical delivery of an mRNA construct encoding calprotectin. We will demonstrate that food- safe, polymeric gene delivery vehicles can topically deliver the therapeutic mRNA in vivo. To firmly establish this approach, polyelectrolyte complexes (polyplexes) with mRNA will be characterized using dynamic light scattering, zeta potential, and ethidium bromide (EtBr) dye exclusion. Optimization of polyplex uptake and message translation will be quantified by expression of a GFP reporter gene using fluorescence microscopy and flow cytometry. To increase biological persistence of the polyplexes complex and promote healing of infected tissue, we will develop a cellulosic hydrogel bandage. The hydrogel will be characterized using spinning disk rheology, and tensile testing. Polyplex loading into and release from the hydrogel will also be quantified using fluorescence measurements or labeled polyplexes. Once ideal delivery conditions are optimized, the effects of modified calprotectin expression will be assessed using a murine model for periodontitis with and without endogenous calprotectin expression. The impact of calprotectin expression on inflammation and alveolar bone loss will be quantified using flow cytometry and microCT measurements. Use of the polyplex-loaded hydrogel bandages are expected to improve transfection efficiency, increase calprotectin-dependent antimicrobial activity, and reduce the signs of experimental periodontitis in our mouse model. If successful, this project has the capacity to provide clinicians with improved tools to treat periodontitis, and improve understanding of topical gene delivery and the biology of periodontitis.

抽象。牙周炎是一种高度流行的疾病，其特征在于牙周组织的细菌感染。 组织中牙周炎改良的非侵入性治疗方法的发展可能对 在美国和全世界的公共卫生。钙卫蛋白（S100 A8/A9）是一种抗菌蛋白， 表达于牙龈上皮细胞和多形核白细胞的胞浆中。鼠模型中 S100 A8/A9可减缓牙周炎的进展并减少牙槽骨丢失。我们 假设钙卫蛋白特异性mRNA在体内有效递送和表达将导致降低的 牙周炎的症状有哪些？在这里，我们提出了一种新的方法来治疗 使用编码钙卫蛋白的mRNA构建体的局部递送治疗牙周炎。我们会证明食物- 安全的聚合基因递送载体可以在体内局部递送治疗性mRNA。牢固树立 在这种方法中，将使用动态光来表征与mRNA的复合物（聚合复合物 散射、ζ电位和溴化乙锭（EtBr）染料排除。复合物摄取的优化和 使用荧光显微镜通过GFP报告基因的表达来定量信息翻译 和流式细胞仪为了增加复合物复合物的生物持久性并促进 感染的组织，我们将开发一种纤维素水凝胶绷带水凝胶将使用以下来表征： 旋转盘流变学和拉伸测试。聚合复合物加载到水凝胶中和从水凝胶中释放也将是有利的。 使用荧光测量或标记的复合物定量。一旦理想的交付条件 优化后，修饰的钙卫蛋白表达的作用将使用鼠模型进行评估， 有和没有内源性钙卫蛋白表达的牙周炎。钙卫蛋白表达对 使用流式细胞术和microCT测量来定量炎症和牙槽骨损失。使用 的聚合复合物负载的水凝胶绷带预期改善转染效率，增加 钙卫蛋白依赖的抗菌活性，并减少我们的小鼠实验性牙周炎的迹象 模型如果成功，该项目有能力为临床医生提供更好的工具来治疗牙周炎， 提高对局部基因传递和牙周炎生物学的理解。