Controlled Delivery of Plasmid DNA via Low-Temperature Ion Deposition

这项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微创系统，用于交付 将DNA质粒化到皮肤。皮肤很容易接近，这使它成为基因治疗的极佳靶点 无论是用于直接治疗皮肤病还是利用皮肤作为仓库输送 蛋白质直接进入血液循环进行系统治疗。利用皮肤的易访问性 开发一种简单直接的体内传递DNA和DNA的非接触性方法至关重要 可以以最小的侵入性方式完成。我们一直在努力开发这种方法，并 先前开发的利用电转移进行此传递的设备和协议。而这些设备 已经有效地工作并且能够以相对非侵入性的方式实现这一点，仍然有必要 在电极和组织靶标之间有接触。此外，需要实现的施加电压 在某些情况下分娩可能会导致细胞或组织损伤或潜在的不适。这是至关重要的 开发一种替代方法，可以像电转移一样工作，但不需要接触。一个 另一个考虑是开发一种方法和/或设备，允许更好地控制交付和 转向一种更可预测、更可重现的表达模式。为达致这个目标，我们建议 利用非热常压等离子体设备，可以渗透细胞并促进质粒化 DNA摄取。我们假设投放是通过离子沉积在靶材表面实现的。 如果离子沉积水平得到控制，那么表达水平就可以控制。这个 有待进一步开发和评估的新型等离子体装置是基于纳秒脉冲空气等离子体的。vbl.使用 这种方法将使我们能够开发一种可以由电池供电的小型便携式设备。这将是一个 非接触式输送装置，可最大限度地减少或消除潜在的不适和/或细胞损伤。这个 以下具体目标将作为该项目的一部分执行。1)评价非热大气 用于受控产生可沉积在组织表面的离子的等离子体设备；2)评估NTAP 产生不同水平的离子的装置，用于将质粒DNA输送到皮肤并确定持续时间 最大表达水平，并确定是否可以通过执行多次传递来增加这一时间 程序；以及3)确定在前两个目标中建立的系统是否能够传递编码的质粒 治疗性蛋白质。研究人员在开发非热等离子体设备方面拥有丰富的经验。 和基因转移非常适合成功完成这项研究。

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 minimally invasive 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 treating cutaneous diseases or utilizing the skin as a depot for delivering proteins directly to the circulation for systemic therapy. To take advantage of the easy accessibility of the skin it is critical to develop non-contact approaches that are a simple and direct in vivo method to deliver DNA and can be accomplished in a minimally invasive way. We have been working on developing such approaches and previously developed devices and protocols that utilized electrotransfer for this delivery. While these devices have worked effectively and can accomplish this in a relatively non-invasive manner, it is still necessary to have contact between the electrodes and the tissue target. In addition, the applied voltages needed to achieve delivery on some occasions may cause cellular or tissue damage or potential discomfort. It is critical to develop an alternative approach that can work as well as electrotransfer but do it without the contact. An additional consideration is to develop an approach and/or device that will allow for better control of delivery and move towards a more predictable and reproducible pattern of expression. To accomplish this, we propose to utilize a non-thermal atmospheric pressure plasma device that can permeabilize cells and facilitate plasmid DNA uptake. We hypothesize that delivery is achieved by ion deposition on the surface of the target tissue and that if the level of ion deposition is regulated then expression levels can be controlled. The novel plasma device to be further developed and evaluated is based on nanosecond pulsed air plasmas. Using this approach will allow us to develop a small portable device that could be battery operated. This will be a non-contact delivery device that will minimize or eliminate potential discomfort and/or cellular damage. The following specific aims will be performed as part of this project. 1) To evaluate non-thermal atmospheric plasma devices for controlled production of ions that can be deposited on tissue surface; 2) To evaluate NTAP devices producing various level of ions 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; and 3) to determine if the system established in the first two aims can deliver plasmids encoding therapeutic proteins. The investigators have extensive expereince in developing non-thermal plasma devices and gene transfer so are well suited to successfully complete the study.