High-Throughput Quantification of In Vivo Adeno-Associated Virus Transduction with Barcoded Non-Coding RNAs.
High-Throughput Quantification of In Vivo Adeno-Associated Virus Transduction with Barcoded Non-Coding RNAs.
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DOI：
10.1089/hum.2018.253
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发表时间：
2019-08
影响因子：
4.2
通讯作者：
Meiyu Xu;Jia Li;Jun Xie;R. He;Q. Su;G. Gao;P. Tai
中科院分区：
文献类型：
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作者：
Meiyu Xu;Jia Li;Jun Xie;R. He;Q. Su;G. Gao;P. Tai
重组腺相关病毒（rAAV）已成为表达治疗性转基因的有利的基因递送载体。衣壳工程努力产生具有改善的转导效率、独特的组织特异性和降低的宿主免疫力的新型AAV是对现有rAAV目前无法满足的治疗需求的高需求的直接响应。通过定向进化方法、计算机模拟设计或从天然前病毒序列发现的新AAV衣壳最终需要在相关体内模型中进行广泛表征。因此，候选衣壳文库的定量筛选现在需要可靠的高通量测序方法。在这项研究中，我们已经开发了一个载体/转基因跟踪系统，采用索引的非编码RNA。具体地，表达高度稳定的RNA转录物的条形码化坚韧诱饵（bcTuD）可用作转导效率的读出。bcTuD的假发夹结构含有可变区，其适合于条形码插入，其可以通过靶扩增子测序检测。所描述的方法，命名为AAV-bcTuD筛选，提供了一种新的替代rAAV的体内评估，可以准确地量化载体基因组和组织中的转录丰度，如在肝脏和大脑感染中的演示。提供了概念验证以表明，对于混合载体注射中高达4 log的载体剂量范围，用该方法检测组织中的载体基因组和转录物是准确和一致的，表明该技术是稳健的、灵敏的，并且适用于体内衣壳性能的多重筛选。
Recombinant adeno-associated viruses (rAAVs) have become favorable gene delivery vehicles for expressing therapeutic transgenes. Capsid engineering efforts to produce novel AAVs with improved transduction efficiencies, unique tissue specificities, and reduced host immunities are a direct response to the high demand for treatment needs that preexisting rAAVs cannot currently fulfill. New AAV capsids discovered by directed evolution methods, in silico design, or from natural proviral sequences ultimately require extensive characterization in relevant in vivo models. Consequently, quantitative screening of candidate capsid libraries now requires reliable high-throughput sequencing approaches. In this study, we have developed a vector/transgene tracking system that employs the indexing of a non-coding RNA. Specifically, a barcoded Tough Decoy (bcTuD) that express highly stable RNA transcripts that can be used as readouts for transduction efficiency. The pseudo-hairpin structure of the bcTuD contains a variable region that is amenable to barcode insertion, which can be detected by target amplicon sequencing. The described approach, named AAV-bcTuD screening, offers a new alternative for in vivo assessment of rAAV that can accurately quantify vector genomes and transcript abundances in tissues, as exampled by the demonstration in liver and brain infections. Proof-of-concept is provided to show that vector genome and transcript detection in tissues with this method is accurate and consistent for a vector dose range of upwards to four logs in a mixed vector injection, showing that this technique is robust, sensitive, and applicable for multiplexed screening of capsid performance in vivo.