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

INSPIRE 技术增强 CRISPR 介导的体内基因组编辑的可行性

基本信息

批准号：
10324621
负责人：
Jordan Fong
金额：
$ 25.19万
依托单位：
GRADIENT MEDICAL, INC.
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10324621
关键词：
3-Dimensional 3D Print Address Animal Model Animals Antibodies CRISPR/Cas technology Cell Nucleus Cell membrane Cells Charge Clinical Clustered Regularly Interspaced Short Palindromic Repeats Collagen Computer software Custom DNA DNA Vaccines Data Defect Deoxyribonuclease I Dermal Development Devices Dose Electroporation Enzymes Epidermis Evaluation Event Family suidae Feasibility Studies Fibroblasts Gene Delivery Generations Genes Genetic Engineering Genome Guide RNA Hand Human In Vitro Injections Label Laboratories Malignant Neoplasms Mediating Messenger RNA Methods Modeling Muscle Muscle Contraction Pain Phase Physiologic pulse Plasmids Probability Process Proteins Protocols documentation Public Health Reagent Research Design Safety Series Serum Skin Subcutaneous Tissue System Techniques Technology Testing Time Tissue Viability Tissues Transfection Transistors Transmembrane Transport Transportation Vaccination Width base design efficacy study electric field endonuclease experimental study genome editing histological specimens improved in vivo in vivo Model millisecond minimally invasive nanoscale nanosecond novel portability prevent prototype silicon carbide simulation vaccine candidate vaccine delivery voltage

项目摘要

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.

项目摘要/摘要这一应用的长期目标是开发一种能够在体内进行基因工程的平台技术包括DNA疫苗接种和CRISPR基因精确编辑在内的技术。利用CRISPR面临的挑战体内技术是需要同时递送DNA内切酶、引导RNA和有效载荷DNA。集成纳秒脉冲皮内可逆电穿孔(INSPIRE)是一种新型的电穿孔技术用于增强这些成分的体内递送的技术。INSPIRE将大分子快速输送到通过使用超短电脉冲同时在细胞中诱导暂时性纳米级缺陷膜，并通过电动漂移驱动目标分子对抗浓度梯度。这项建议将在3D体外皮肤模型和猪的体内基因工程中展示INSPIRE的可行性真皮模型通过三个特定的目标。1)适合临床激励的手持式系统的研制治疗，2)在实验室模型中优化INSPIRE方案，以及3)INSPIRE方案的可行性在一个大型动物模型中。在目标1中，我们将构建一个适合临床使用的便携式脉搏发生系统高压组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蛋白导入细胞的方案模仿。在目标3中，我们将证明这些CRISPR组件通过猪体内传递的可行性真皮模型。在第一阶段的应用展示了在体内的可行性之后，第二阶段将专注于设计用于制作和启动旨在确定血清抗体水平的长期安全性/有效性研究用候选DNA疫苗转染后产生的。