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.

抽象的。牙周炎是一种高度普遍的疾病，其特征是细菌感染的牙周感染 组织。改进的牙周炎的改进，非侵入性治疗可能对 美国和全球的公共卫生。钙骨蛋白酶（S100A8/A9）是一种抗菌蛋白 在牙龈上皮细胞和多形核白细胞的细胞质中表达。在鼠模型中 牙周炎，S100A8/A9减慢牙周炎的进展并减少肺泡骨质流失。我们 假设在体内有效递送和特异性mRNA的有效递送和表达将导致降低 炎症并减慢牙周炎的进展。在此，我们提出了一种新颖的方法来治疗 牙周炎使用编码钙染色素的mRNA构造的牙周炎。我们将证明食物 - 安全，聚合基因输送车辆可以局部递送体内治疗性mRNA。牢固建立 这种方法，将使用动态光来表征与mRNA的聚电解质复合物（多型） 散射，Zeta电位和溴化乙锭（ETBR）染料排除。息肉吸收的优化和 消息翻译将通过使用荧光显微镜表达GFP报告基因来量化。 和流式细胞仪。增加多链体复合物的生物学持久性并促进愈合 感染的组织，我们将发展一个纤维素水凝胶绷带。水凝胶将使用 旋转磁盘流变学和拉伸测试。从水凝胶中加载并释放的流传链体也将是 使用荧光测量或标记的多聚体定量。一旦理想的交货条件 优化，将使用鼠模型评估修饰的钙染色蛋白表达的效果 牙周炎，有或没有内源性钙染色素表达。钙曲蛋白表达对 使用流式细胞术和微分离测量值将量化炎症和肺泡骨损失。使用 预计载有多聚凝胶的水凝胶绷带将提高转染效率，提高 钙染色素依赖性抗菌活性，并减少小鼠中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改进的治疗牙周炎的工具， 并提高对局部基因递送和牙周炎生物学的了解。