Identification of the role of SNARE proteins in rAAV vector production through interaction with the viral MAAP.

IF 4.6 2区医学 Q2 MEDICINE, RESEARCH & EXPERIMENTAL Molecular Therapy-Methods & Clinical Development Pub Date : 2024-12-05 eCollection Date: 2025-03-13 DOI:10.1016/j.omtm.2024.101392

Cagla Aksu Kuz, Kang Ning, Siyuan Hao, Shane McFarlin, Xiujuan Zhang, Fang Cheng, Jianming Qiu

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Abstract

Adeno-associated virus (AAV) expresses a membrane-associated accessory protein (MAAP), a small nonstructural protein, that facilitates AAV secretion out of the plasma membrane through an association with extracellular vesicles during AAV egress. Here, we investigated the host proteins that interact with AAV2 MAAP (MAAP2) using APEX2-mediated proximity labeling. We identified two SNARE proteins, Syntaxin 7 (STX7) and synaptosome-associated protein 23 (SNAP23), a vesicle (v-)SNARE and a target (t-)SNARE, respectively, that mediate intracellular trafficking of membrane vesicles aand exhibited associations with MAAP2 in HEK293 cells. We found that MAAP2 indirectly interacted with STX7 or SNAP23, and that the knockout of STX7 or SNAP23 not only enhanced rAAV secretion into the media but also increased total vector yield during rAAV vector production in HEK293 cells. Thus, our study revealed a practical approach for producing higher yields of rAAV vectors from the media, easing downstream processes in rAAV manufacturing.

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通过与病毒MAAP相互作用鉴定SNARE蛋白在rAAV载体生产中的作用。

腺相关病毒（AAV）表达一种膜相关辅助蛋白（MAAP），这是一种小的非结构蛋白，在AAV输出过程中通过与细胞外囊泡的结合促进AAV分泌出质膜。在这里，我们使用apex2介导的接近标记研究了与AAV2 MAAP （MAAP2）相互作用的宿主蛋白。我们鉴定了两个SNARE蛋白，Syntaxin 7 （STX7）和synaptosomes -associated protein 23 (SNAP23)，分别是囊泡（v-）SNARE和靶标（t-）SNARE，它们在HEK293细胞中介导细胞膜囊泡的胞内运输，并与MAAP2相关。我们发现MAAP2间接与STX7或SNAP23相互作用，敲除STX7或SNAP23不仅增强了rAAV向培养基的分泌，而且在HEK293细胞中产生rAAV载体时提高了总载体产量。因此，我们的研究揭示了一种从培养基中生产更高产量的rAAV载体的实用方法，简化了rAAV制造的下游过程。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

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.