Efficient Delivery of Plasmid DNA to Achieve Appropriate Transgene Expression

高效递送质粒 DNA 以实现适当的转基因表达

• 批准号: 9248339
• 负责人: RICHARD HELLER
• 金额: $ 35.39万
• 依托单位: OLD DOMINION UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9248339
• 关键词: Address; Adverse event; Antibodies; Biological Models; Blocking Antibodies; Blood specimen; CTLA4 gene; Cells; Cessation of life; Clinic; Clinical; Clinical Research; Clinical Trials; Combined Modality Therapy; Cytotoxic T-Lymphocyte-Associated Protein 4; DNA delivery; Death Rate; Disease-Free Survival; Distant; Dose; Effectiveness; Elements; Enrollment; Equilibrium; Event; Frequencies; Gene Delivery; Genes; Health; Immune; Immune response; Immunization; Immunosuppression; Immunotherapy; In complete remission; Incidence; Interleukin-12; Interleukin-15; Lead; Lesion; Life Expectancy; Location; Memory; Metastatic Melanoma; Methods; Modeling; Molecular Profiling; Mus; Muscle; Natural Immunity; Neoplasm Metastasis; Outcome; PDCD1LG1 gene; Patients; Pattern; Phase; Physiologic pulse; Plasmids; Protocols documentation; Regulatory T-Lymphocyte; Sampling; Signal Transduction; Site; Skin; System; T-Lymphocyte; Testing; Therapeutic; Therapeutic Effect; Tissues; Transgenes; Translating; Treatment Efficacy; Visceral; Work; adaptive immunity; advanced disease; cytokine; effective therapy; electric field; falls; gene therapy; immune checkpoint blockade; melanoma; mouse model; novel strategies; novel therapeutics; objective response rate; overexpression; phase 1 study; phase II trial; plasmid DNA; potential biomarker; preclinical study; public health relevance; response; success; synergism; therapeutic effectiveness; therapeutic evaluation; therapy outcome; transgene expression; treatment response; tumor; tumor microenvironment

DESCRIPTION: Delivery still remains as a barrier to achieving successful gene therapy. Administering gene delivery protocols in a manner that would allow better control over the expression pattern would enhance therapeutic outcomes. We have developed a delivery approach (gene electro transfer; GET) which utilizes pulsed electric fields that allows for controlled delivery. We have tested this approach as a means of delivering plasmids encoding immunostimulatory molecules. For immunotherapy, maintaining control over expression following plasmid delivery is critical to success as there is a fine balance between immunostimulation and immunosuppression. Manipulation of GET parameters can be used for controlled delivery of plasmid and will result in obtaining the appropriate transgene expression. The model system utilized to test this system is malignant melanoma which is a major health concern with no effective therapy for advanced disease. The incidence of melanoma continues to rise and it is estimated that there will be 76,690 new cases and 9,480 deaths in 2013. Melanoma is a good model for immunotherapy approaches as there is evidence demonstrating immune responsiveness including both innate and adaptive immunity. Recently, several new approaches have been tested as potential immunotherapies with some success. However, overall durable complete response rates (disease free survival) are low (<15%) and some of these therapies have significant adverse events documenting that there is still a need for more effective therapies. One potential new therapy is to deliver a plasmid encoding Interleukin-12 directly to the tumor to stimulate an immune response. The important criterion for success is administering IL-12 at the right dose and location. To address this, we have developed an effective means of delivering plasmid DNA utilizing GET. The hypothesis to be tested is: if appropriate delivery parameters are used to deliver plasmid IL-12 then a change in the tumor microenvironment will occur that will be associated with an appropriate therapeutic response. Therefore, it is critical to characterize the response and identify potential biomarkers that can signify proper delivery and expression. We also hypothesize that if an appropriate combination can be achieved then there will be an increased response at distant sites. The increased response rates together with boosting the immune response may lead to an effective therapy for metastatic melanoma due to a reduction of T-reg cells and enhanced activation of T-effector and memory cells. In this project, we will develop and test this approach in a mouse model and have the opportunity to determine how it correlates with samples obtained from an ongoing clinical trial. Thus, the work in this project is directly translatable. The following specific aim will be performed as part of this project. 1. Determine the influence expression profile has in inducing an effective anti-tumor response and determine if a specific pattern of response can be identified. 2. Evaluate expression patterns following delivery of plasmids encoding anti-PD1, anti-PD-L1 or anti-CTLA4. 3. Therapeutic efficacy of the approach in a mouse metastatic model.

描述：递送仍然是实现成功基因治疗的障碍。以允许更好地控制表达模式的方式施用基因递送方案将增强治疗结果。我们已经开发了一种递送方法（基因电转移; GET），其利用允许受控递送的脉冲电场。我们已经测试了这种方法作为递送编码免疫刺激分子的质粒的手段。对于免疫疗法，在质粒递送后保持对表达的控制对于成功至关重要，因为在免疫刺激和免疫抑制之间存在良好的平衡。GET参数的操作可用于质粒的受控递送，并将导致获得适当的转基因表达。用于测试该系统的模型系统是恶性黑色素瘤，这是一个主要的健康问题，对晚期疾病没有有效的治疗。黑色素瘤的发病率继续上升，估计2013年将有76，690例新发病例和9，480例死亡。黑色素瘤是免疫治疗方法的良好模型，因为有证据表明免疫应答性包括先天免疫和适应性免疫。最近，几种新的方法已经被测试为潜在的免疫疗法，并取得了一些成功。然而，总体持久完全缓解率（无病生存期）较低（<15%），并且这些疗法中的一些具有显著的不良事件，证明仍然需要更有效的疗法。一种潜在的新疗法是将编码白细胞介素-12的质粒直接递送到肿瘤以刺激免疫应答。成功的重要标准是以正确的剂量和位置给予IL-12。为了解决这个问题，我们开发了一种利用GET递送质粒DNA的有效方法。待检验的假设是：如果使用适当的递送参数递送质粒IL-12，则肿瘤微环境将发生变化，这将与适当的治疗反应相关。因此，关键是表征反应并鉴定可以表示适当递送和表达的潜在生物标志物。我们还假设，如果可以实现适当的组合，那么在远处的站点将有一个增加的响应。由于T-reg细胞的减少和T效应细胞和记忆细胞的增强激活，增加的应答率连同增强的免疫应答可能导致转移性黑素瘤的有效疗法。在这个项目中，我们将在小鼠模型中开发和测试这种方法，并有机会确定它如何与从正在进行的临床试验中获得的样本相关。因此，本项目中的工作是可直接翻译的。作为本项目的一部分，将实现以下具体目标。1.确定表达谱对诱导有效抗肿瘤应答的影响，并确定是否可以鉴定特定的应答模式。2.评价编码抗PD 1、抗PD-L1或抗CTLA 4的质粒递送后的表达模式。3.该方法在小鼠转移模型中的治疗功效。