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首页> 外文期刊>Human gene therapy methods. >Fast-Seq: A Simple Method for Rapid and Inexpensive Validation of Packaged Single-Stranded Adeno-Associated Viral Genomes in Academic Settings
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Fast-Seq: A Simple Method for Rapid and Inexpensive Validation of Packaged Single-Stranded Adeno-Associated Viral Genomes in Academic Settings

机译：FAST-SEQ：一种简单的方法，用于在学术环境中快速且廉价地验证包装的单链腺相关病毒基因组

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摘要

Adeno-associated viral (AAV) vectors have shown great promise in gene delivery as evidenced by recent FDA approvals. Despite efforts to optimize manufacturing for good manufacturing practice (GMP) productions, few academic laboratories have the resources to assess vector composition. One critical component of vector quality is packaged genome fidelity. Errors in viral genome replication and packaging can result in the incorporation of faulty genomes with mutations, truncations, or rearrangements, compromising vector potency. Thus, sequence validation of packaged genome composition is an important quality control (QC), even in academic settings. We developed Fast-Seq, an end-to-end method for extraction, purification, sequencing, and data analysis of packaged single-stranded AAV (ssAAV) genomes intended for non-GMP preclinical environments. We validated Fast-Seq on ssAAV vectors with three different genome compositions (CAG-GFP, CAG-tdTomato, EF1 alpha-FLuc), three different genome sizes (2.9, 3.6, 4.4kb), packaged in four different capsid serotypes (AAV1, AAV2, AAV5, and AAV8), and produced using the two most common production methods (Baculovirus-Sf9 and human HEK293), from both common commercial vendors and academic core facilities supplying academic laboratories. We achieved an average genome coverage of >1,400x and an average inverted terminal repeat coverage of >280x, despite the many differences in composition of each ssAAV sample. When compared with other ssAAV next-generation sequencing (NGS) methods for GMP settings, Fast-Seq has several unique advantages: Tn5 transposase-based fragmentation rather than sonication, 125xless input DNA, simpler adapter ligation, compatibility with commonly available inexpensive sequencing instruments, and free open-source data analysis code in a preassembled customizable Docker container designed for novices. Fast-Seq can be completed in 18h, is more cost-effective than other NGS methods, and is more accurate than Sanger sequencing, which is generally only applied at 1-2xsequencing depth. Fast-Seq is a rapid, simple, and inexpensive methodology to validate packaged ssAAV genomes in academic settings.
机译：adeno相关的病毒（AAV）向量在基因交付中显示出很大的希望，如最近的FDA批准所证明。尽管努力优化良好的制造实践（GMP）制造业的制造业，但很少有学术实验室具有评估载体组成的资源。矢量质量的一个关键组分是包装的基因组保真度。病毒基因组复制和包装中的误差可导致突变，截断或重排，损害载体效力的故障基因组。因此，即使在学术环境中，包装基因组组合物的序列验证也是重要的质量控制（QC）。我们开发了Fast-SEQ，一种用于非GMP临床前环境的包装的单链AAV（SSAAV）基因组的提取，纯化，排序和数据分析的端到端的方法。我们在SSAAV载体上验证了具有三种不同基因组组成的SSAAV载体（CAG-GFP，CAG-TDTOMATO，EF1α-FLUC），三种不同的基因组大小（2.9,3.6,4.4KB），包装在四种不同的衣壳血清型（AAV1， AAV2，AAV5和AAV8），并在普通商业供应商和学术核心设施供应学术实验室的两个最常见的生产方法（Baculovirus-SF9和人HEK293）生产。尽管每个SSAAV样本的组成差异，但我们实现了> 1,400x> 1,400x的平均基因组覆盖率> 1,400x和平均倒置终端重复覆盖范围。与GMP设置的其他SSAAV下一代测序（NGS）方法进行比较时，FAST-SEQ具有几种独特的优势：TN5基于转座酶的碎片而不是超声处理，125 X输入DNA，更简单的适配器连接，与常用廉价测序仪器的兼容性，并为新人设计的预装可自定义的Docker容器中的免费开源数据分析代码。 Fast-SEQ可以在18小时内完成，比其他NGS方法更具成本效益，并且比Sanger测序更准确，这通常仅在1-2×序列深度施加。 FAST-SEQ是一种快速，简单，廉价的方法，可在学术环境中验证包装的SSAAV基因组。

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