Thermal Assisted Gene Electro Transfer to the Skin

热辅助基因电转移至皮肤

• 批准号: 9230219
• 负责人: RICHARD HELLER
• 金额: $ 34.43万
• 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: 2015-05-01 至 2019-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9230219
• 关键词: Address; Affect; Animals; Blood Circulation; Cavia; Cell Membrane Permeability; Cell Survival; Cell membrane; Cells; Clinical Trials; Code; DNA Vaccines; DNA delivery; Development; Devices; Electroporation; Epithelial; Erythropoietin; Factor IX; Gene Targeting; Gene Transfer; Genes; Goals; In Vitro; Ischemia; Kinetics; Lasers; Methods; Modeling; Nature; Normal tissue morphology; Oncogenes; Pain; Penetration; Physiologic pulse; Plasmids; Procedures; Proteins; Protocols documentation; Research; Research Personnel; Serum; Skin; Skin Temperature; Source; System; Systemic Therapy; Techniques; Temperature; Testing; Therapeutic; Time; Tissues; Transgenes; Translating; Ultrasonography; Work; Wound Healing; cytotoxicity; electric field; electric impedance; enzyme replacement therapy; fluidity; gene delivery system; gene therapy; improved; in vivo; minimally invasive; plasmid DNA; programs; public health relevance; skin disorder; success; targeted delivery; therapeutic protein; vaccine delivery; voltage

 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.

 描述：本研究计划的长期目标是开发有效的体内基因递送系统。这个特定项目的目标是开发一种改进的电转移系统，用于将质粒DNA递送到皮肤。皮肤是容易接近的，这使其成为基因治疗应用的极好靶点，无论是用于直接治疗皮肤疾病还是利用皮肤作为将蛋白质直接递送到循环中用于全身治疗的储库。皮肤电转移是一种简单、直接的体内基因转移方法，可以以微创的方式完成。我们之前已经开发了利用电转移将质粒DNA有效递送到皮肤的设备和方案。我们成功地证明了这种方法用于递送DNA疫苗、伤口愈合和缺血的实用性。然而，仍有一些不足之处。当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，以便有效地转化这种方法，以及使这种方法对于其他治疗应用是可行的，特别是那些需要增加表达的转基因的血清水平的治疗应用。为了解决这些问题，我们建议将联合收割机电转移与从外源施加的受控适度热的应用相结合。将皮肤的温度升高到43摄氏度会增加细胞膜的流动性，并降低皮肤内的电导率。这将允许在较低电压下递送质粒DNA，并且还使我们能够成功地递送到皮肤的更深层。我们假设，如果在适度升高的温度下在组织内施加电转移脉冲，则可以在较低的施加电场下实现递送，这将降低损伤和/或不适的可能性。我们进一步假设，如果使用受控的外源热和电转移的组合，则将实现场的更深穿透，从而在皮肤的更深层中获得表达。作为本项目的一部分，将实现以下具体目标。1）评价外源受控热和施加电场的组合以将质粒DNA递送至皮肤，并确定最大表达水平的持续时间，并确定该时间是否可以通过进行多次递送程序来增加; 2）确定控制和/或靶向皮肤内递送的能力。将开展工作 在豚鼠模型中，并将包括评估皮肤内的表达深度;和3）确定在前两个目的中建立的系统是否可以递送编码适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递送，因此非常适合成功完成这项研究。