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工程化血清抗性和热稳定的水疱性口炎病毒 G 糖蛋白用于假型逆转录病毒和慢病毒载体。

Engineering a serum-resistant and thermostable vesicular stomatitis virus G glycoprotein for pseudotyping retroviral and lentiviral vectors.

机构信息

Chemical and Biomolecular Engineering, Bioengineering and the Helen Wills Neuroscience Institute, University of California, Berkeley, CA 94720-3220, USA.

出版信息

Gene Ther. 2013 Aug;20(8):807-15. doi: 10.1038/gt.2013.1. Epub 2013 Jan 31.

DOI:10.1038/gt.2013.1
PMID:23364315
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC3735647/
Abstract

Vesicular stomatitis virus G glycoprotein (VSV-G) is the most widely used envelope protein for retroviral and lentiviral vector pseudotyping; however, serum inactivation of VSV-G pseudotyped vectors is a significant challenge for in vivo gene delivery. To address this problem, we conducted directed evolution of VSV-G to increase its resistance to human serum neutralization. After six selection cycles, numerous common mutations were present. On the basis of their location within VSV-G, we analyzed whether substitutions in several surface exposed residues could endow viral vectors with higher resistance to serum. S162T, T230N and T368A mutations enhanced serum resistance, and additionally K66T, T368A and E380K substitutions increased the thermostability of VSV-G pseudotyped retroviral vectors, an advantageous byproduct of the selection strategy. Analysis of a number of combined mutants revealed that VSV-G harboring T230N+T368A or K66T+S162T+T230N+T368A mutations exhibited both higher in vitro resistance to human serum and higher thermostability, as well as enhanced resistance to rabbit and mouse serum. Finally, lentiviral vectors pseudotyped with these variants were more resistant to human serum in a murine model. These serum-resistant and thermostable VSV-G variants may aid the application of retroviral and lentiviral vectors to gene therapy.

摘要

水疱性口炎病毒 G 糖蛋白（VSV-G）是最常用于逆转录病毒和慢病毒载体假型化的包膜蛋白；然而，VSV-G 假型化载体的血清失活是体内基因递送的一个重大挑战。为了解决这个问题，我们对 VSV-G 进行了定向进化，以提高其对人血清中和的抗性。经过六轮选择，出现了许多常见的突变。基于它们在 VSV-G 中的位置，我们分析了几个表面暴露的残基中的取代是否可以赋予病毒载体更高的血清抗性。S162T、T230N 和 T368A 突变增强了血清抗性，此外，K66T、T368A 和 E380K 取代提高了 VSV-G 假型逆转录病毒载体的热稳定性，这是选择策略的一个有利的附带产物。对一些组合突变体的分析表明，携带 T230N+T368A 或 K66T+S162T+T230N+T368A 突变的 VSV-G 既表现出体外对人血清更高的抗性和更高的热稳定性，又增强了对兔和鼠血清的抗性。最后，这些变体假型化的慢病毒载体在小鼠模型中对人血清具有更强的抗性。这些具有抗血清和热稳定的 VSV-G 变体可能有助于逆转录病毒和慢病毒载体在基因治疗中的应用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/a1f06b072c0e/nihms432227f8.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/c0aa1abb6ba4/nihms432227f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/e5c35f8c1982/nihms432227f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/a1f06b072c0e/nihms432227f8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/8e44eaf17db8/nihms432227f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/00510c3f0bcf/nihms432227f2a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/5356d82ab371/nihms432227f3a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/8ca26e3072b6/nihms432227f4a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/f7459351aa4f/nihms432227f5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/c0aa1abb6ba4/nihms432227f6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/e5c35f8c1982/nihms432227f7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/4fac/3735647/a1f06b072c0e/nihms432227f8.jpg

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