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全面的诱变图谱展示了 AAV2 基因中所有单密码子突变对 AAV 生产的影响。

Comprehensive mutagenesis maps the effect of all single-codon mutations in the AAV2 gene on AAV production.

机构信息

Wyss Institute for Biologically Inspired Engineering, Boston, United States.

Department of Genetics, Harvard Medical School, Boston, United States.

出版信息

Elife. 2024 Mar 27;12:RP87730. doi: 10.7554/eLife.87730.

DOI:10.7554/eLife.87730
PMID:38536879
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC10972557/
Abstract

Recombinant adeno-associated viruses (rAAVs) are the predominant gene therapy vector. Several rAAV vectored therapies have achieved regulatory approval, but production of sufficient rAAV quantities remains difficult. The AAV Rep proteins, which are essential for genome replication and packaging, represent a promising engineering target for improvement of rAAV production but remain underexplored. To gain a comprehensive understanding of the Rep proteins and their mutational landscape, we assayed the effects of all 39,297 possible single-codon mutations to the AAV2 gene on AAV2 production. Most beneficial variants are not observed in nature, indicating that improved production may require synthetic mutations. Additionally, the effects of AAV2 mutations were largely consistent across capsid serotypes, suggesting that production benefits are capsid independent. Our results provide a detailed sequence-to-function map that enhances our understanding of Rep protein function and lays the groundwork for Rep engineering and enhancement of large-scale gene therapy production.

摘要

重组腺相关病毒（rAAV）是主要的基因治疗载体。几种 rAAV 载体疗法已获得监管部门的批准，但大量生产 rAAV 仍然具有挑战性。AAV Rep 蛋白是基因组复制和包装所必需的，是提高 rAAV 生产的有前途的工程目标，但仍未得到充分探索。为了全面了解 Rep 蛋白及其突变景观，我们检测了对 AAV2 基因的所有 39,297 种可能的单密码子突变对 AAV2 生产的影响。大多数有益的变体在自然界中并未观察到，这表明提高产量可能需要合成突变。此外，AAV2 突变的影响在衣壳血清型之间基本一致，这表明产量的提高与衣壳无关。我们的研究结果提供了一个详细的序列到功能图谱，增强了我们对 Rep 蛋白功能的理解，并为 Rep 工程和大规模基因治疗生产的增强奠定了基础。

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