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: 8817124
• 负责人: RICHARD HELLER
• 金额: $ 37.15万
• 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/8817124
• 关键词: Address; Adverse event; Antibodies; Biological Markers; Biological Models; Blocking Antibodies; Blood specimen; CTLA4 gene; Cells; Cessation of life; Clinic; Clinical; Clinical Research; Clinical Trials; Combined Modality Therapy; DNA delivery; Death Rate; Disease-Free Survival; Distant; Dose; Effectiveness; Elements; Enrollment; Equilibrium; Event; Frequencies; Gene Delivery; Gene Transfer; 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; 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 correction; gene therapy; melanoma; mouse model; novel strategies; phase 1 study; phase II trial; plasmid DNA; preclinical study; public health relevance; response; success; therapeutic effectiveness; transgene expression; 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：分娩仍然是实现成功基因疗法的障碍。以一种可以更好地控制表达模式的方式管理基因递送方案将增强治疗结果。我们已经开发了一种输送方法（基因电转移； GET），该方法利用了允许控制交付的脉冲电场。我们已经测试了这种方法，作为传递编码免疫刺激分子的质粒的一种手段。对于免疫疗法，在免疫刺激和免疫抑制之间存在良好的平衡，维持质粒递送后表达的控制至关重要。操纵获取参数可用于控制质粒的递送，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，没有有效的晚期疾病治疗。黑色素瘤的发病率继续升高，据估计，2013年将有76,690例新病例和9,480例死亡。黑色素瘤是免疫疗法方法的良好模型，因为有证据表明免疫反应，包括先天性和适应性免疫。最近，已将几种新方法作为潜在的免疫疗法进行了测试，并有所成功。但是，总体耐用的完整应答率（无疾病生存率）较低（<15％），其中一些疗法的不良事件记录了仍然需要更有效的疗法。一种潜在的新疗法是将编码白介素12的质粒直接传递到肿瘤中，以刺激免疫反应。成功的重要标准是在正确的剂量和位置管理IL-12。为了解决这个问题，我们开发了一种有效的方法来传递利用GET的质粒DNA。要测试的假设是：如果使用适当的递送参数来传递质粒IL-12，则会发生改变与适当的治疗反应有关的肿瘤微环境。因此，至关重要的是表征响应并确定可以表示适当传递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，那么远处的响应将会增加。由于T-REG细胞的减少以及T-效应器和记忆细胞的激活增强，增加的反应率和增强免疫反应可能会导致有效的转移性黑色素瘤治疗。在这个项目中，我们将在小鼠模型中开发和测试这种方法，并有机会确定其与正在进行的临床试验中获得的样品的相关性。因此，该项目的工作是可以直接翻译的。将作为该项目的一部分执行以下特定目标。 1。确定影响表达曲线在诱导有效的抗肿瘤反应方面具有，并确定是否可以鉴定出特定的响应模式。 2。评估编码抗PD1，抗PD-L1或抗CTLA4的质粒传递后的表达模式。 3。在小鼠转移模型中该方法的治疗功效。