慢性乙型肝炎患者循环 HBV RNA 检测和定量测定的分子标准

IF 9.5 1区 医学 Q1 GASTROENTEROLOGY & HEPATOLOGY JHEP Reports Pub Date : 2024-05-25 DOI:10.1016/j.jhepr.2024.101124
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引用次数: 0

摘要

背景& 目的循环中的 HBV RNA 被认为是反映共价闭合环状 DNA(cccDNA)转录活性的生物标志物,可能有助于评估 HBV 治疗活性。目前已提出了不同的研究测定方法,尽管已开发出两种基于 PCR 的研究用测定方法,但缺乏标准化方案是一个重要的限制因素。在这里,我们设计并生成了一种稳定的克隆细胞系,它能产生一种基于 RNA 的标准,用于校准基于 PCR 的循环 HBV RNA 检测。结果携带 YMAA 和 Y63F 双突变的克隆细胞系(Huh7-3D29)在培养过程中表现出高 RNA 分泌表型,其分泌的 HBV DNA 残留量可忽略不计。密度梯度离心显示,Huh7-3D29 细胞分泌的大部分 HBV RNA 在裸盖体和病毒样颗粒中被检测到,只有少数在小的胞外囊泡中被检测到。5'RACE 产物的纳米孔测序显示,Huh7-3D29 细胞分泌的 HBV RNA 大部分以 pgRNA 和 pgRNA 衍生的剪接 RNA 的 5' 端为起始。最后,Huh7-3D29 细胞显示出很高且可升级的分泌 RNA 产量,一个烧瓶在 9 天内可生成 1,300 条标准曲线。结论:我们生成了一种克隆细胞系,它能产生大量 HBV RNA,同时污染的 HBV DNA 数量极低,是校准 HBV RNA 检测的稳定 RNA 标准来源。影响和意义:目前已开发出几种研究性检测方法和两种仅供研究使用的检测方法,用于检测和量化循环中的 HBV RNA,它是共价闭合环状 DNA 转录活性和 HBV 新疗法靶标参与的新兴生物标志物。循环 HBV RNA 定量缺乏独特的分子标准是一个重要的局限。在这里,我们描述了一种稳定克隆细胞系的产生和分泌,这种基于 RNA 的标准包含 HBV 患者血清中发现的所有 HBV RNA 类型(如 pgRNA、HBx 转录本)。这种新的 RNA 标准可用于校准所有基于 PCR 的循环 HBV RNA 定量检测方法,从而以非侵入性的方式评估具有转录活性的 cccDNA 池的大小以及旨在治愈 HBV 感染的新策略的活性。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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A molecular standard for circulating HBV RNA detection and quantification assays in patients with chronic hepatitis B

Background & Aims

Circulating HBV RNAs have been proposed as a biomarker that reflects the transcriptional activity of covalently closed circular DNA (cccDNA) and may help to evaluate HBV treatment activity. Different research assays have been proposed and, although two PCR-based research use only investigational assays have been developed, the lack of standardized protocols represents an important limitation. Here we have designed and generated a stable clonal cell line producing an RNA-based standard for the calibration of PCR-based circulating HBV RNA assays.

Methods

HBV RNA-producing Huh7-derived stable cell lines were generated by transfecting pTriEX plasmids containing 1.1 unit length HBV DNA genomes carrying mutations in the catalytic site (YMAA mutation) and the TP domain (Y63F) of the polymerase, and the ε-loop of the pregenomic (pg)RNA (mutation A1G).

Results

The clonal cell line (Huh7-3D29), carrying a double YMAA and Y63F mutation, displayed, and maintained over several passages in culture, a high RNA secretion phenotype with negligible residual secreted HBV DNA. Density gradient centrifugation showed that most of the secreted HBV RNA from Huh7-3D29 cells was detected in naked capsid and virion-like particles and only a minority in small extracellular vescicles. Nanopore sequencing of 5’RACE products shows that the majority of the Huh7-3D29-secreted HBV RNAs start at the 5' end of pgRNA and pgRNA-derived spliced RNAs. Finally, Huh7-3D29 cells showed a high and up-scalable secreted RNA yield allowing 1,300 standard curves in 9 days from one flask.

Conclusion

We generated a clonal cell line that produces high quantities of HBV RNAs with very low quantities of contaminating HBV DNAs, representing a stable source of RNA standard for HBV RNA assay calibration.

Impact and implications:

Several investigational assays and two research use only assays have been developed to detect and quantify circulating HBV RNAs, an emerging biomarker of covalently closed circular DNA transcriptional activity and target engagement by new HBV treatments. The lack of a unique molecular standard for circulating HBV RNA quantification represents an important limitation. Here we describe the generation of a stable clonal cell line producing and secreting an RNA-based standard containing all the HBV RNA species found in HBV patients’ sera (e.g. pgRNA, HBx transcripts). This new RNA standard can be used to calibrate all PCR-based assays for circulating HBV RNA quantification to evaluate, in a non-invasive manner, the size of the transcriptionally active cccDNA pool and the activity of novel strategies aimed at curing HBV infection.

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来源期刊
JHEP Reports
JHEP Reports GASTROENTEROLOGY & HEPATOLOGY-
CiteScore
12.40
自引率
2.40%
发文量
161
审稿时长
36 days
期刊介绍: JHEP Reports is an open access journal that is affiliated with the European Association for the Study of the Liver (EASL). It serves as a companion journal to the highly respected Journal of Hepatology. The primary objective of JHEP Reports is to publish original papers and reviews that contribute to the advancement of knowledge in the field of liver diseases. The journal covers a wide range of topics, including basic, translational, and clinical research. It also focuses on global issues in hepatology, with particular emphasis on areas such as clinical trials, novel diagnostics, precision medicine and therapeutics, cancer research, cellular and molecular studies, artificial intelligence, microbiome research, epidemiology, and cutting-edge technologies. In summary, JHEP Reports is dedicated to promoting scientific discoveries and innovations in liver diseases through the publication of high-quality research papers and reviews covering various aspects of hepatology.
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Contents Editorial Board page Copyright and information Contents ALT levels, alcohol use, and metabolic risk factors have prognostic relevance for liver-related outcomes in the general population
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