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

• 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
• 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

 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（通过应用提供）：电子产品是一种用于体内细菌，真核细胞和组织的标准实验室技术。我们的实验室是最早通过将临床前肿瘤，肌肉和皮肤传递到治疗应用中的体内质粒电穿孔的人之一，也是第一个进行了兽医和临床基因治疗试验的。体内电穿孔已成为一种临床分子递送的一项良好接受的技术。目前，在NIH的临床试验中注册了将近100次临床试验。GOV数据库用于药物和核酸的电穿孔。我们观察到，当使用各种电穿孔方案将对照骨干pDNA电穿孔到不同的肿瘤类型中时，可能会发生完全的肿瘤回归。回归之前是促炎性mRNA和蛋白质的产生。我们的初步结果暗示了DNA特异性识别受体（PRR）的激活，该受体通常在诱导这些促炎蛋白的诱导中为防御病原体侵袭而起作用。这些传感器都在免疫和非免疫细胞类型中发现。这些传感器在非免疫细胞（例如肿瘤细胞）中的激活可能有助于我们在先前的研究中观察到的炎症和回归，并可能影响基因治疗。例如，感染可能会提高抗肿瘤疗法或感染性疾病疫苗的效率，但被靶向健康组织的蛋白质替代疗法中断。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。因此，在此提案中，我们将阐明在几种正常细胞类型和肿瘤细胞中的不同电穿孔方案后，在非免疫细胞中的PRR激活，在3维微环境中检查潜在的激活，并将这种模式与随后的体内效应相关。我们的长期目标是能够根据推定的PRR激活和体内效应来预测哪些电穿孔参数应用于特定的组织和治疗应用。 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在质粒DNA电穿孔及其对肿瘤，肌肉和皮肤组织的基因疗法的潜在影响之后。