Efficient Delivery of Plasmid DNA to Achieve Appropriate Transgene Expression

描述：递送仍然是实现成功基因治疗的障碍。以一种能够更好地控制表达模式的方式管理基因传递方案将提高治疗效果。我们已经开发了一种递送方法（基因电转移；GET），它利用脉冲电场，允许控制递送。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手段。对于免疫治疗来说，在质粒递送后保持对表达的控制是成功的关键，因为在免疫刺激和免疫抑制之间有一个很好的平衡。操纵GET参数可以用于控制质粒的传递，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方法。黑色素瘤的发病率继续上升，估计2013年将有76 690例新病例和9 480例死亡。黑色素瘤是免疫治疗方法的一个很好的模型，因为有证据表明免疫反应性包括先天免疫和适应性免疫。最近，已经测试了几种新的方法作为潜在的免疫疗法，并取得了一些成功。然而，总体持久完全缓解率（无病生存率）很低（<15%），其中一些疗法有明显的不良事件，表明仍需要更有效的疗法。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施用IL-12。为了解决这个问题，我们开发了一种利用GET传递质粒DNA的有效方法。要测试的假设是：如果使用适当的传递参数来传递质粒IL-12，那么将发生肿瘤微环境的变化，这将与适当的治疗反应相关。因此，表征反应和识别潜在的生物标志物是至关重要的，这些生物标志物可以表示适当的传递和表达。我们还假设，如果能够实现适当的组合，那么在遥远的地点将会有更大的反应。由于T-reg细胞的减少和t效应细胞和记忆细胞的激活增强，反应率的提高和免疫反应的增强可能导致转移性黑色素瘤的有效治疗。在这个项目中，我们将在小鼠模型中开发和测试这种方法，并有机会确定它与正在进行的临床试验中获得的样本之间的关系。因此，这个项目的作品是可以直接翻译的。以下具体目标将作为该项目的一部分执行。1. 确定表达谱在诱导有效的抗肿瘤反应方面的影响，并确定是否可以确定特定的反应模式。2. 评估传递编码抗pd1、抗pd - l1或抗ctla4的质粒后的表达模式。3. 该方法在小鼠转移模型中的治疗效果。

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.