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增强 CNS 基因传递的结构引导 AAV 衣壳进化策略
Nature Protocols
(
IF
14.8
)
Pub Date : 2023-09-21
, DOI:
10.1038/s41596-023-00875-y
Trevor J Gonzalez
1
,
Aaron Mitchell-Dick
2
,
Leo O Blondel
2
,
Marco M Fanous
2
,
Joshua A Hull
2
,
Daniel K Oh
2
,
Sven Moller-Tank
3
,
Ruth M Castellanos Rivera
3
,
Jorge A Piedrahita
4
,
Aravind Asokan
1,
2,
5
Affiliation
- Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA.
- Department of Surgery, Duke University School of Medicine, Durham, NC, USA.
- Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
- North Carolina State University College of Veterinary Medicine, Raleigh, NC, USA.
- Department of Biomedical Engineering, Duke University, Durham, NC, USA.
在过去的5年里,我们的实验室系统地开发了一种结构引导的文库方法来进化新的腺相关病毒(AAV)衣壳,其具有改变的组织向性、更高的转导效率和逃避预先存在的体液免疫的能力。在这里,我们提供了一个详细的协议,描述了两种不同的进化策略,使用结构不同的 AAV 血清型作为模板,以提高体内 CNS 基因转移效率为例。我们概述了我们策略的四个主要组成部分:(i) AAV 衣壳文库的结构引导设计,(ii) AAV 文库生产,(iii) 单个动物模型与多个动物模型中的文库循环,然后是 (iv) 对先导 AAV 载体的评估体内候选者。该协议持续约 95 天,不包括体内基因表达分析,并且可能会根据用户体验、资源和实验设计而有所不同。当前协议的一个显着属性是专注于为生物医学研究人员提供 3D 结构信息,以指导 AAV 衣壳上精确“热点”的进化。此外,该协议概述了 AAV 库进化的两种不同方法,包括单个物种中腺病毒启用的感染性循环和跨物种方式的非感染性循环。值得注意的是,我们的工作流程可以与其他基于 RNA 转录本的文库策略无缝合并,并针对组织特异性衣壳选择进行定制。总体而言,本文概述的程序可用于扩展 AAV 载体工具包,用于动物模型的遗传操作和人类基因疗法的开发。
"点击查看英文标题和摘要"
Structure-guided AAV capsid evolution strategies for enhanced CNS gene delivery
Over the past 5 years, our laboratory has systematically developed a structure-guided library approach to evolve new adeno-associated virus (AAV) capsids with altered tissue tropism, higher transduction efficiency and the ability to evade pre-existing humoral immunity. Here, we provide a detailed protocol describing two distinct evolution strategies using structurally divergent AAV serotypes as templates, exemplified by improving CNS gene transfer efficiency in vivo. We outline four major components of our strategy: (i) structure-guided design of AAV capsid libraries, (ii) AAV library production, (iii) library cycling in single versus multiple animal models, followed by (iv) evaluation of lead AAV vector candidates in vivo. The protocol spans ~95 d, excluding gene expression analysis in vivo, and can vary depending on user experience, resources and experimental design. A distinguishing attribute of the current protocol is the focus on providing biomedical researchers with 3D structural information to guide evolution of precise ‘hotspots’ on AAV capsids. Furthermore, the protocol outlines two distinct methods for AAV library evolution consisting of adenovirus-enabled infectious cycling in a single species and noninfectious cycling in a cross-species manner. Notably, our workflow can be seamlessly merged with other RNA transcript-based library strategies and tailored for tissue-specific capsid selection. Overall, the procedures outlined herein can be adapted to expand the AAV vector toolkit for genetic manipulation of animal models and development of human gene therapies.
更新日期:2023-09-22