Horae Gene Therapy Center, UMass Chan Medical School, Worcester, Massachusetts, USA.
Department of Microbiology and Physiological Systems, UMass Chan Medical School, Worcester, Massachusetts, USA; and.
Hum Gene Ther. 2022 Nov;33(21-22):1187-1196. doi: 10.1089/hum.2022.143.
Recombinant adeno-associated viruses (rAAVs) are currently the most prominently investigated vector platform for human gene therapy. The rAAV capsid serves as a potent and efficient vehicle for delivering genetic payloads into the host cell, while the vector genome determines the function and effectiveness of these biotherapies. However, current production schemes yield vectors that may consist of heterogeneous populations, compromising their potencies. The development of next-generation sequencing methods within the past few years have helped investigators profile the diversity and relative abundances of heterogenous species in vector preparations. Specifically, long-read sequencing methods, like single molecule real-time (SMRT) sequencing, have been used to uncover truncations, chimeric genomes, and inverted terminal repeat (ITR) mutations in vectors. Unfortunately, these sequencing platforms may be inaccessible to investigators with limited resources, require large amounts of input material, or may require long wait times for sequencing and analyses. Recent advances with nanopore sequencing have helped to bridge the gap for quick and relatively inexpensive long-read sequencing needs. However, their limitations and sample biases are not well-defined for sequencing rAAV. In this study, we explored the capacity for nanopore sequencing to directly interrogate rAAV content to obtain full-length resolution of encapsidated genomes. We found that the nanopore platform can cover the entirety of rAAV genomes from ITR to ITR without the need for pre-fragmentation. However, the accuracy for base calling was low, resulting in a high degree of miscalled bases and false indels. These false indels led to read-length compression; thus, assessing heterogeneity based on read length is not advisable with current nanopore technologies. Nonetheless, nanopore sequencing was able to correctly identify truncation hotspots in single-strand and self-complementary vectors similar to SMRT sequencing. In summary, nanopore sequencing can serve as a rapid and low-cost alternative for proofing AAV vectors.
重组腺相关病毒 (rAAV) 是目前研究最为深入的人类基因治疗载体平台。rAAV 衣壳可作为将遗传有效载荷递送至宿主细胞的高效载体，而载体基因组决定了这些生物疗法的功能和效果。然而，目前的生产方案产生的载体可能包含异质群体，从而影响其效力。在过去几年中，新一代测序方法的发展帮助研究人员分析了载体制剂中异质物种的多样性和相对丰度。具体而言，长读测序方法，如单分子实时 (SMRT) 测序，已被用于揭示载体中的截短、嵌合基因组和反向末端重复 (ITR) 突变。不幸的是，这些测序平台可能对资源有限的研究人员来说无法获得，需要大量的输入材料，或者可能需要很长时间进行测序和分析。最近纳米孔测序的进展有助于填补快速且相对廉价的长读测序需求的空白。然而，它们在用于测序 rAAV 方面的局限性和样本偏倚尚不清楚。在这项研究中，我们探讨了纳米孔测序直接检测 rAAV 含量以获得完整基因组分辨率的能力。我们发现，纳米孔平台无需预先碎片化即可覆盖 ITR 到 ITR 之间的整个 rAAV 基因组。然而，碱基调用的准确性较低，导致大量错误碱基和假插入缺失。这些假插入缺失导致读取长度压缩；因此，基于读取长度评估异质性是不可取的，目前的纳米孔技术无法实现。尽管如此，纳米孔测序仍能够正确识别单链和自互补载体中的截短热点，与 SMRT 测序相似。总之，纳米孔测序可以作为一种快速且低成本的替代方法来验证 AAV 载体。
