Thermal Assisted Gene Electro Transfer to the Skin

 描述：该研究计划的长期目标是开发高效的体内基因输送系统。这一具体项目的目标是开发一种改进的电转移系统，用于将质粒DNA输送到皮肤。皮肤很容易接触，这使得它成为基因治疗应用的极佳靶点，无论是直接治疗皮肤病还是利用皮肤作为仓库直接将蛋白质输送到血液循环进行全身治疗。皮肤电转移是一种简单、直接、体内传递基因用于治疗的方法，并且可以以微创的方式完成。我们以前已经开发了利用电转移来有效地将质粒DNA输送到皮肤的设备和方案。我们成功地展示了这种方法在DNA疫苗递送、伤口愈合和缺血方面的有效性。但仍有一些不足之处。目前方法的一个主要问题是，表达仅限于上皮层。此外，在某些情况下，实现递送所需的施加电压会导致细胞或组织损伤。解决这些问题是至关重要的，以便有效地翻译这种方法，并使这种方法适用于其他治疗应用，特别是那些需要提高表达的转基因血清水平的治疗应用。为了解决这些问题，我们建议将电转移与从外部来源施加的受控中热相结合。将皮肤温度提高到43摄氏度会增加细胞膜的流动性，同时降低皮肤内的电导率。这将允许以较低的电压传递质粒DNA，并使我们能够成功地将其传递到皮肤的更深层。我们假设，如果在中等温度下在组织内施加电转移脉冲，那么就有可能在较低的外加电场下实现传递，这将减少损伤和/或不适的可能性。我们进一步假设，如果使用受控的外生热和电转移的组合，那么将实现对场的更深渗透，从而在皮肤的更深层获得表达。作为该项目的一部分，将实现以下具体目标。1)评估外源受控加热和外加电场将质粒DNA输送到皮肤的组合，并确定最大表达水平的持续时间，并确定是否可以通过执行多个输送程序来延长这一时间；2)确定控制和/或靶向皮肤内输送的能力。将开展工作 在豚鼠模型中，将包括评估皮肤表达的深度；以及3)确定在前两个目标中建立的系统是否能够提供编码用于治疗目的的蛋白质的质粒。研究人员率先使用电穿孔进行体内给药，因此非常适合成功完成这项研究。

 DESCRIPTION: The long-range goal of this research program is the development of efficient in vivo gene delivery systems. The goal of this specific project is the development of an improved electro transfer system for the delivery of plasmid DNA to the skin. Skin is easily accessibility which makes it an excellent target for gene therapy applications whether it is for directly treatin cutaneous diseases or utilizing the skin as a depot for delivering proteins directly to the circulation for systemic therapy. Electro transfer of skin is a simple, direct, in vivo method to deliver genes for therapy and can be accomplished in a minimally invasive way. We have previously developed devices and protocols to utilize electro transfer to effectively deliver plasmid DNA to the skin. We successfully demonstrated the utility of this approach for delivery of DNA vaccines, wound healing and ischemia. There were however still some shortcomings. One major issue of the current approach is that expression is confined to the epithelial layer. In addition, the applied voltages needed to achieve delivery on some occasions caused cellular or tissue damage. It is critical to address these issues in order to effectively translate this approah as well as make this approach feasible for other therapeutic applications particularly those that require increased serum levels of the expressed transgene. To address these issues we propose to combine electro transfer with the application of controlled moderate heat that is applied from an exogenous source. Raising the temperature of the skin to 43oC would increase the fluidity of the cell membranes as well as decrease the conductance within the skin. This will allow for delivery of plasmid DNA at lower voltages and also enable us to successfully delivery to deeper layers of the skin. We hypothesize that if electro transfer pulses are applied within a tissue at a moderately elevated temperature then it will be possible to achieve delivery at lower applied electric fields which would decrease the potential for damage and/or discomfort. We further hypothesize that if a combination of controlled exogenous heat and electro transfer is used then deeper penetration of the fields will be achieved thereby obtaining expression in the deeper layers of the skin. The following specific aims will be performed as part of this project. 1 to evaluate the combination of exogenous controlled heat and applied electric fields for delivery of plasmid DNA to the skin and to determine the duration of maximal expression levels and to determine if this time can be increased by performing multiple delivery procedures; 2) to determine the ability to control and/or target the delivery within the skin. Work will be conducted in a guinea pig model and will include evaluating depth of expression within the skin; and 3) to determine if the system established in the first two aims can deliver plasmids coding for proteins applicable for therapeutic purposes. The investigators have pioneered the use of electroporation for in vivo delivery so are well suited to successfully complete the study.