Nonviral Topical Transfection with Calprotectin in Periodontitis

钙卫蛋白非病毒局部转染治疗牙周炎

基本信息

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
项目类别：
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

项目摘要

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。牙周炎是一种非常普遍的疾病，其特征是牙周细菌感染组织。改进的、非侵入性牙周炎治疗方法的开发可能会对牙周炎产生影响美国和全世界的公共卫生。钙卫蛋白 (S100A8/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细胞质中。在小鼠模型中牙周炎，S100A8/A9 可以减缓牙周炎的进展并减少牙槽骨流失。我们假设体内钙卫蛋白特异性 mRNA 的有效递送和表达将导致减少炎症并减缓牙周炎的进展。在此，我们提出了一种新的治疗方法使用编码钙卫蛋白的 mRNA 构建体的局部递送来治疗牙周炎。我们将证明食物- 安全的聚合基因递送载体可以在体内局部递送治疗性 mRNA。为牢固树立这种方法，将使用动态光来表征具有 mRNA 的聚电解质复合物 (polyplexes) 散射、zeta 电位和溴化乙锭 (EtBr) 染料排除。优化多聚复合物的吸收和使用荧光显微镜通过 GFP 报告基因的表达来量化信息翻译和流式细胞术。增加复合物复合物的生物持久性并促进愈合感染组织，我们将开发一种纤维素水凝胶绷带。水凝胶将使用以下特征进行表征旋转盘流变学和拉伸测试。聚合物复合物加载到水凝胶中和从水凝胶中释放也将使用荧光测量或标记的聚合复合物进行定量。一旦达到理想的交货条件优化后，将使用小鼠模型评估修饰的钙卫蛋白表达的效果有或没有内源性钙卫蛋白表达的牙周炎。钙卫蛋白表达的影响将使用流式细胞术和 microCT 测量来量化炎症和牙槽骨丢失。使用负载复合物的水凝胶绷带有望提高转染效率，增加钙卫蛋白依赖性抗菌活性，并减少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治疗牙周炎的改进工具，并提高对局部基因传递和牙周炎生物学的理解。