DNA-specific pattern recognition receptor activation following DNA electroporation

DNA 电穿孔后 DNA 特异性模式识别受体激活

基本信息

批准号：
9051482
负责人：
Loree C Heller
金额：
$ 32.99万
依托单位：
OLD DOMINION UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-02-01 至 2021-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9051482
关键词：
3-Dimensional Bacteria Binding Cells Clinical Clinical Trials Coculture Techniques Communicable Diseases Country Cultured Tumor Cells Cytosol DNA Databases Development Disease Electroporation Endosomes Eukaryotic Cell European Union Gene therapy trial Goals Histocompatibility Testing Immune In Vitro Inflammation Laboratories Location Malignant Neoplasms Mammalian Cell Messenger RNA Methods Molecular Muscle Myomatous neoplasm Normal Cell Nucleic Acids Oncogenes Pattern Pattern recognition receptor Pharmaceutical Preparations Plasmids Production Proteins Protocols documentation Receptor Activation Research Skin Skin Tissue Solid Neoplasm Techniques Testing Therapeutic Tissues Transfection Treatment Efficacy Tumor Tissue Vaccination Vaccines Vertebral column base cell type chemotherapeutic agent clinical practice enzyme replacement therapy gene therapy gene therapy clinical trial in vivo neoplastic cell pathogen plasmid DNA pre-clinical pre-clinical research public health relevance receptor binding research study response sensor tumor

项目摘要

DESCRIPTION (provided by applicant): Electroporation is a standard laboratory technique for transfection of bacteria, eukaryotic cells, and tissues in vivo. Our laboratories were among the first to develop in vivo plasmid electroporation for therapeutic applications by delivery to preclinical tumors, muscle, and skin and the first that conducted veterinary and clinical gene therapy trials. In vivo electroporation is becoming a well-accepted technique for clinical molecular delivery. Currently, nearly 100 clinical trials are registered in the NIH's clinicaltrials.gov database for electroporation of drugs and nucleic acids. We observed that complete tumor regression can occur when control backbone pDNA is electroporated into different tumors types using various electroporation protocols. Regression is preceded by the production of proinflammatory mRNAs and proteins. Our preliminary results implicate the activation of DNA-specific pattern recognition receptors (PRRs), which normally function for defense against pathogen invasion, in the induction of these proinflammatory proteins. These sensors are found in both immune and non-immune cell types. Activation of these sensors in non-immune cells, such as tumor cells, may contribute to the inflammation and regression that we observed in our previous research and may influence gene therapy efficacy. For example, inflammation may enhance the efficacy of an anti-tumor therapy or an infectious disease vaccine, but interfere with a protein replacement therapy targeting a healthy tissue. Our hypothesis is that DNA-specific PRR activation in non-immune cell types may influence the therapeutic potential of DNA electroporation. Therefore, in this proposal, we will elucidate the activation of PRRs in non-immune cells following different electroporation protocols in several normal cell types and in tumor cells, examine potential activation in a 3-dimensional microenvironment, and relate this pattern to subsequent in vivo effects. Our long-term goal is to be able to predict, based on putative PRR activation and in vivo effects, what electroporation parameters should be used for a specific tissue and therapeutic application. To test our hypothesis, we will conduct our experiments within the following specific aims: Specific Aim 1. Determine the DNA sensor activation pattern in response to plasmid DNA electroporation using various protocols in non-immune cell types Specific Aim 2. Determine effect of 3-dimensional culture and co-cultures of tumor and normal cells on the DNA sensor activation pattern in response to plasmid DNA electroporation Specific Aim 3. Determine the in vivo DNA sensor activation pattern after plasmid DNA electroporation and its potential influence on gene therapies delivered to tumor, muscle, and skin tissues.

描述（由申请人提供）：电穿孔是一种用于体内转染细菌、真核细胞和组织的标准实验室技术，我们的实验室是最早开发用于通过递送至临床前肿瘤、肌肉和组织的治疗应用的体内质粒电穿孔的实验室之一。皮肤和第一个进行兽医和临床基因治疗试验的体内电穿孔技术正在成为一种广为接受的临床分子传递技术，目前已注册了近 100 项临床试验。在 NIH 的药物和核酸电穿孔数据库中，我们观察到，当使用各种电穿孔方案将对照主链 pDNA 电穿孔到不同的肿瘤类型中时，可以发生肿瘤的完全消退。消退之前会产生促炎性 mRNA 和蛋白质。我们的初步结果表明，在诱导这些促炎蛋白的过程中，DNA 特异性模式识别受体 (PRR) 的激活通常起到防御病原体入侵的作用。这些传感器存在于免疫和免疫系统中。非免疫细胞类型中的这些传感器的激活可能会导致我们在之前的研究中观察到的炎症和消退，并可能影响基因治疗的功效，例如，炎症可能会增强疗效。抗肿瘤疗法或传染病疫苗，但会干扰针对健康组织的蛋白质替代疗法。我们的假设是，非免疫细胞类型中的 DNA 特异性 PRR 激活可能会影响 DNA 电穿孔的治疗潜力。在本提案中，我们将阐明在几种正常细胞类型和肿瘤细胞中采用不同电穿孔方案后非免疫细胞中 PRR 的激活，检查 3 维微环境中的潜在激活，并将这种模式与随后的体内效应联系起来。为了能够根据假定的 PRR 激活和体内效应来预测特定组织和治疗应用应使用哪些电穿孔参数。为了检验我们的假设，我们将在以下特定目标内进行实验：特定目标 1。在非免疫细胞类型中使用各种方案确定 DNA 传感器激活模式对质粒 DNA 电穿孔的响应具体目标 2. 确定肿瘤和正常细胞的 3 维培养和共培养对 DNA 传感器激活模式的影响质粒 DNA 电穿孔具体目标 3. 确定质粒 DNA 电穿孔后的体内 DNA 传感器激活模式及其对肿瘤、肌肉和皮肤组织基因治疗的潜在影响。