Nanopore Methods for Determining Capsid Viability

测定衣壳活力的纳米孔方法

• 批准号: 10414621
• 负责人: Kimberly Ritola
• 金额: $ 57.26万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 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/10414621
• 关键词: Nanopore; Methods; Determining; Capsid; Viability

Nanopore Methods for Determining Capsid Viability ABSTRACT AAV (adeno-associated virus) vectors are quickly becoming an important therapeutic tool for treatment of a variety of diseases, resulting in a significant increase in the number of applications to the FDA for approvals for treatment of various diseases. Precise characterization of these AAVs is extremely important for process control and determination of the effectiveness of the final product for both production efficiency and regulatory/quality reasons. We have developed nano-fluidic, synthetic pore-based sensors capable of detecting and quantifying various nanoparticles. These existing sensors can be modified for application to capsid characterization. Demonstrated applications of this technology include DNA and RNA sequencing, SARS-CoV-2 virus detection from saliva (including viral load and infectivity classification) using resistive pulse sensing (RPS), and analysis of ligase detection reaction (LDR) products utilizing Time-of-Flight (TOF) capabilities. Specifically for this project, we will use the nanopore technology to detect capsids and characterize them as either full or empty (Aim 1) and we will extend the sensor capabilities to address high-throughput strategies for inline analysis (Aim 2). Finally, we will implement the sensors into AAV production, validate their responses against established QCAs and use sensor data to optimize production processes (Aim 3). We expect these developments to result in sensor systems that will help to improve AAV production efficiencies, improve final product infectivity, and provide important quality assurance data.

用于测定衣壳活力的纳米孔方法 摘要 AAV（腺相关病毒）载体正迅速成为治疗肿瘤的重要工具。 治疗各种疾病，导致患者数量大幅增加。 向FDA申请批准治疗各种疾病。精确 这些AAV的表征对于过程控制和确定 最终产品在生产效率和监管/质量方面的有效性 原因我们已经开发出纳米流体，合成孔为基础的传感器，能够检测 以及对各种纳米颗粒进行定量。这些现有的传感器可以被修改以应用于 衣壳特征这项技术的应用包括DNA和RNA 测序、唾液SARS-CoV-2病毒检测（包括病毒载量和感染性 分类），并分析连接酶检测反应 (LDR)利用飞行时间（TOF）功能的产品。对于这个项目，我们将 使用纳米孔技术检测衣壳，并将其表征为满或空 (Aim 1）我们将扩展传感器功能，以解决高通量策略， 在线分析（目标2）。最后，我们将把传感器应用到AAV生产中，验证 他们对已建立的QCA的反应，并使用传感器数据来优化生产 过程（目标3）。我们希望这些发展将导致传感器系统，这将有助于 提高AAV生产效率，提高最终产品感染性，并提供重要的 质量保证数据。