降低慢病毒载体用于β-地中海贫血基因治疗的转录通读率。

IF 3.2 4区 医学 Q2 BIOTECHNOLOGY & APPLIED MICROBIOLOGY
Jiahui Wu, Yuan Chen, Wenchen Shen, Jingzhi Zhang, Fanyi Zeng
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引用次数: 0

摘要

背景:LentiGlobin BB305是一种自身失活的慢病毒载体,携带人β-珠蛋白表达盒,用于治疗β-地中海贫血。首先,将鸡cHS4基因座控制区(Locus Control Region)的2 × 250 bp片段作为绝缘体放置在其ΔU3位置,在法国团队领导的第一次临床试验后将其移除,以避免剪接异常等。这一行为可能导致β-珠蛋白启动子驱动的转录通读率风险显著增加,在临床试验中构成生物安全风险。方法:在本研究中,设计了一种读透还原剂(C-U+或WPRE),放置在β-珠蛋白基因的3' UTR上。在mRNA水平和慢病毒制备滴度的蛋白表达水平上检测增强子活性和/或转录读透率(EATRT)。结果:我们发现元素(C-U+或WPRE)的插入分别有效降低了53%和41%的EATRT。C-U+对病毒包效率的影响较小。此外,插入C-U+或WPRE后,蛋白表达水平无显著差异。结论:本研究结果表明,在BB305的polyA序列前插入C-U+或WPRE可以在不影响其表达效果和病毒制备滴度的情况下降低EATRT率。因此,我们提出了一个替代改进的更安全的慢病毒载体β-地中海贫血临床试验。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

Reducing the transcriptional read-through rate of a lentiviral vector for β-thalassemia gene therapy

Reducing the transcriptional read-through rate of a lentiviral vector for β-thalassemia gene therapy

Reducing the transcriptional read-through rate of a lentiviral vector for β-thalassemia gene therapy

Background

LentiGlobin BB305 is a self-inactivating lentiviral vector carrying a human β-globin expressing cassette for treating β-thalassemia. Initially, a 2 × 250 bp chicken Locus Control Region fragment of cHS4, functioning as an insulator, was placed at its ΔU3, which was removed after the first clinical trial led by a French team to avoid abnormal splicing, etc. This action could potentially lead to an increasing risk of the transcriptional read-through rate driven by the β-globin promoter to a significant level, posing a biosafety risk in clinical trials.

Methods

In the present study, a read-through reducing agent (C-U+ or WPRE) was designed to be placed at the 3′ UTR of the β-globin gene. The Enhancer Activities and/or Transcriptional Read-Through (EATRT) rate at the mRNA level and the protein expression level regarding lentiviral preparation titer were examined.

Results

We found that the insertion of the element (C-U+ or WPRE) reduced the EATRT effectively by 53% or 41%, respectively. C-U+ has less impact on virus package efficiency. Furthermore, there was no significant difference in the protein expression level after the C-U+ or WPRE insertion.

Conclusions

The results of the present study show that inserting C-U+ or WPRE before the polyA sequence of the BB305 would reduce the EATRT rate at no cost of its expressing efficacy and viral preparation titers. Thus, we present an alternative improvement for a safer lentiviral vector for β-thalassemia clinical trials.

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来源期刊
Journal of Gene Medicine
Journal of Gene Medicine 医学-生物工程与应用微生物
CiteScore
6.40
自引率
0.00%
发文量
80
审稿时长
6-12 weeks
期刊介绍: The aims and scope of The Journal of Gene Medicine include cutting-edge science of gene transfer and its applications in gene and cell therapy, genome editing with precision nucleases, epigenetic modifications of host genome by small molecules, siRNA, microRNA and other noncoding RNAs as therapeutic gene-modulating agents or targets, biomarkers for precision medicine, and gene-based prognostic/diagnostic studies. Key areas of interest are the design of novel synthetic and viral vectors, novel therapeutic nucleic acids such as mRNA, modified microRNAs and siRNAs, antagomirs, aptamers, antisense and exon-skipping agents, refined genome editing tools using nucleic acid /protein combinations, physically or biologically targeted delivery and gene modulation, ex vivo or in vivo pharmacological studies including animal models, and human clinical trials. Papers presenting research into the mechanisms underlying transfer and action of gene medicines, the application of the new technologies for stem cell modification or nucleic acid based vaccines, the identification of new genetic or epigenetic variations as biomarkers to direct precision medicine, and the preclinical/clinical development of gene/expression signatures indicative of diagnosis or predictive of prognosis are also encouraged.
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