Feasibility of INSPIRE Technology to Enhance CRISPR Mediated Genome Editors In Vivo

项目总结/摘要 该应用程序的长期目标是开发一种能够实现体内基因工程的平台技术 这些技术包括DNA疫苗接种和精确的CRISPR基因编辑。利用CRISPR的挑战 体内技术的另一个优点是需要同时递送DNA内切核酸酶、引导RNA和DNA片段。 有效载荷DNA集成纳秒脉冲皮内可逆电穿孔（INSPIRE）是一种新的 用于增强这些组分体内递送的技术。INSPIRE快速将大分子输送到 通过使用超短电脉冲同时在细胞中诱导暂时的纳米级缺陷 膜并通过电动漂移对抗浓度梯度驱动靶分子。这项建议 将在3D体外皮肤模型和猪模型中证明INSPIRE用于体内基因工程的可行性。 通过三个具体目标的真皮模型。1)适用于临床的手持式INSPIRE系统的研制 治疗，2）实验室模型中INSPIRE方案的优化，以及3）INSPIRE方案的可行性 在大型动物模型中。在目标1中，我们将构建一个适合临床使用的便携式脉冲发生系统 其中高压部件直接集成到手持施加器中。在目标2中，我们将优化 使用3D体外皮肤将CRISPR cas9质粒、cas9 mRNA和cas9蛋白递送到细胞中的方案 模仿在目标3中，我们将证明通过猪细胞在体内递送这些CRISPR组分的可行性。 真皮模型在第一阶段应用证明了体内可行性之后，第二阶段将专注于设计 用于生产和启动旨在确定抗体血清水平的长期安全性/有效性研究 在用DNA疫苗候选物转染后产生。

Project Summary / Abstract This applications long-term objective is to develop a platform technology enabling in vivo genetic engineering technologies including DNA vaccination and precision CRISPR gene editing. A challenge in utilizing CRISPR technologies in vivo is the need to simultaneously deliver the DNA endonuclease enzyme, guide RNA, and payload DNA. Integrated nanosecond pulse intradermal reversible electroporation (INSPIRE) is a novel technique for enhancing in vivo delivery of these components. INSPIRE rapidly transports large molecules into cells by using ultrashort electrical pulses to simultaneously induce temporary nanoscale defects in the cell membrane and drive the target molecules against concentration gradients via electrokinetic drift. This proposal will demonstrate the feasibility of INSPIRE for in vivo genetic engineering in a 3D in vitro skin model and a porcine dermal model via three specific aims. 1) Development of a Hand Held System Suitable for Clinical INSPIRE Treatments, 2) Optimization of INSPIRE Protocols in a Laboratory Model, and 3) Feasibility of INSPIRE Protocols in a Large Animal Model. In Aim 1 we will construct a portable pulse generation system suitable for clinical use with the high voltage components integrated directly into a hand held applicator. In Aim 2 we will optimize protocols for delivering CRISPR cas9 plasmid, cas9 mRNA, and cas9 protein into cells using a 3D in vitro skin mimic. In Aim 3 we will demonstrate the feasibility of delivering these CRISPR components in vivo via a porcine dermal model. Following this Phase I application demonstrating in vivo feasibility, Phase II will focus on design for manufacture and initiate long-term safety/efficacy studies designed to determine serum levels of antibodies produced following transfection with DNA vaccine candidates.