通过新型双荧光联免疫吸附测定法量化腺相关病毒载体的全颗粒和空颗粒

IF 4.6 2区 医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL
Sereirath Soth, Mikako Takakura, Masahiro Suekawa, Takayuki Onishi, Kiichi Hirohata, Tamami Hashimoto, Takahiro Maruno, Mitsuko Fukuhara, Yasuo Tsunaka, Tetsuo Torisu, Susumu Uchiyama
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引用次数: 0

摘要

腺相关病毒(AAV)载体因其低免疫原性和非致病性而成为最先进的基因治疗平台之一。AAV载体空颗粒和AAV载体全颗粒(FPs)的浓度都是重要的质量属性,前者不含DNA,也不显示任何疗效。本研究建立了一种双重荧光联结免疫吸附测定(dFLISA),它使用两种荧光染料在一次分析中定量检测囊壳和基因组滴度。在 dFLISA 中,AAV 粒子的捕获、病毒盖蛋白的检测以及病毒基因组的释放和检测都在同一孔中进行。我们证明,用 dFLISA 测定的囊膜和基因组滴度与分析超速离心法测定的滴度相当。dFLISA 测定的 FP 比值与预期值非常一致。此外,我们还发现 dFLISA 可以定量检测粗样品的基因组和囊膜滴度。dFLISA 可以很容易地进行修改,以检测其他 AAV 载体血清型和不同基因组长度的 AAV 载体。这些特点使 dFLISA 成为未来开发基于 AAV 的基因疗法的重要工具。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Quantification of full and empty particles of adeno-associated virus vectors via a novel dual fluorescence-linked immunosorbent assay
The adeno-associated virus (AAV) vector is one of the most advanced platforms for gene therapy because of its low immunogenicity and non-pathogenicity. The concentrations of both AAV vector empty particles, which do not contain DNA and do not show any efficacy, and AAV vector full particles (FPs), which contain DNA, are important quality attributes. In this study, a dual fluorescence-linked immunosorbent assay (dFLISA), which uses two fluorescent dyes to quantify capsid and genome titers in a single analysis, was established. In dFLISA, capture of AAV particles, detection of capsid proteins, and release and detection of the viral genome are performed in the same well. We demonstrated that the capsid and genomic titers determined by dFLISA were comparable with those of analytical ultracentrifugation. The FP ratios determined by dFLISA were in good agreement with the expected values. In addition, we showed that dFLISA can quantify the genomic and capsid titers of crude samples. dFLISA can be easily modified for measuring other AAV vector serotypes and AAV vectors with different genome lengths. These features make dFLISA a valuable tool for the future development of AAV-based gene therapies.
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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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