用于 rAAV 基因组滴度测定和成分分析的无扩增 CRISPR/Cas12a 分析法

IF 4.6 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL
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引用次数: 0

摘要

病毒基因组滴度是重组腺相关病毒(rAAV)基因治疗产品临床用药、生产和分析测试的重要指标。虽然定量 PCR 和数字 PCR 是量化 rAAV 基因组滴度的常用方法,但它们的准确性和稳健性不足。簇状规则间距短回文重复(CRISPR)-Cas12a 生物传感器正越来越多地应用于病毒检测,但目前还没有将其应用于基因治疗产品滴度测定的报道。本研究开发、优化了一种无扩增的 CRISPR-Cas12a 检测方法,并将其应用于 rAAV 基因组滴度测定。该检测方法在 4 × 109 和 1011 vg/mL 的检测范围内表现出较高的精确度和准确性。Cas12a 和 qPCR 检测结果之间无明显差异(p﹤0.05,t 检验)。此外,Cas12a 在单链和双链 DNA 底物上表现出相似的活性。根据这一特点,我们分别测定了正义链和负义链的滴度,结果显示它们的滴度与内部参考 AAV5-IN 的滴度存在显著差异。本研究首次报道了为测定滴度和分析 rAAV 基因组组成而开发的 Cas12a 检测方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

An amplification-free CRISPR/Cas12a assay for titer determination and composition analysis of the rAAV genome

An amplification-free CRISPR/Cas12a assay for titer determination and composition analysis of the rAAV genome

The viral genome titer is a crucial indicator for the clinical dosing, manufacturing, and analytical testing of recombinant adeno-associated virus (rAAV) gene therapy products. Although quantitative PCR and digital PCR are the common methods used for quantifying rAAV genome titer, they are limited by inadequate accuracy and robustness. The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a biosensor is being increasingly used in virus detection; however, there is currently no report on its application in the titer determination of gene therapy products. In the present study, an amplification-free CRISPR-Cas12a assay was developed, optimized, and applied for rAAV genome titer determination. The assay demonstrated high precision and accuracy within the detection range of 4 × 109 and 1011 vg/mL. No significant difference was observed between the Cas12a and qPCR assay results (p﹤0.05, t-test). Moreover, Cas12a exhibited similar activity on both single-stranded and double-stranded DNA substrates. Based on this characteristic, the titers of positive-sense and negative-sense strands were determined separately, which revealed a significant difference between their titers for an in-house reference AAV5-IN. This study presents the inaugural report of a Cas12a assay developed for the titer determination and composition analysis of the rAAV genome.

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来源期刊
Molecular Therapy-Methods & Clinical Development
Molecular Therapy-Methods & Clinical Development Biochemistry, Genetics and Molecular Biology-Molecular Biology
CiteScore
9.90
自引率
4.30%
发文量
163
审稿时长
12 weeks
期刊介绍: The aim of Molecular Therapy—Methods & Clinical Development is to build upon the success of Molecular Therapy in publishing important peer-reviewed methods and procedures, as well as translational advances in the broad array of fields under the molecular therapy umbrella. Topics of particular interest within the journal''s scope include: Gene vector engineering and production, Methods for targeted genome editing and engineering, Methods and technology development for cell reprogramming and directed differentiation of pluripotent cells, Methods for gene and cell vector delivery, Development of biomaterials and nanoparticles for applications in gene and cell therapy and regenerative medicine, Analysis of gene and cell vector biodistribution and tracking, Pharmacology/toxicology studies of new and next-generation vectors, Methods for cell isolation, engineering, culture, expansion, and transplantation, Cell processing, storage, and banking for therapeutic application, Preclinical and QC/QA assay development, Translational and clinical scale-up and Good Manufacturing procedures and process development, Clinical protocol development, Computational and bioinformatic methods for analysis, modeling, or visualization of biological data, Negotiating the regulatory approval process and obtaining such approval for clinical trials.
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