长期核苷类似物治疗患者乙型肝炎共价闭合环状DNA残留库的

StephenW

Quantification and epigenetic evaluation of the residual pool of hepatitis B covalently closed circular DNA in long-term nucleoside analogue-treated patients
Fanny Lebossé  1   2   3 , Aurore Inchauspé  1   2 , Maëlle Locatelli  1   2 , Clothilde Miaglia  1   2   3 , Audrey Diederichs  1   2 , Judith Fresquet  1   2 , Fleur Chapus  1   2 , Kamal Hamed  4 , Barbara Testoni  5   6 , Fabien Zoulim  7   8   9
Affiliations
Affiliations

    1
    INSERM U1052-Cancer Research Center of Lyon (CRCL), Lyon, France.
    2
    University of Lyon, UMR_S1052, CRCL, Lyon, France.
    3
    Department of Hepatology, Croix Rousse Hospital, Hospices Civils de Lyon, Lyon, France.
    4
    Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA.
    5
    INSERM U1052-Cancer Research Center of Lyon (CRCL), Lyon, France. [email protected].
    6
    University of Lyon, UMR_S1052, CRCL, Lyon, France. [email protected].
    7
    INSERM U1052-Cancer Research Center of Lyon (CRCL), Lyon, France. [email protected].
    8
    University of Lyon, UMR_S1052, CRCL, Lyon, France. [email protected].
    9
    Department of Hepatology, Croix Rousse Hospital, Hospices Civils de Lyon, Lyon, France. [email protected].

    PMID: 33273565 DOI: 10.1038/s41598-020-78001-1

Abstract

Hepatitis B virus (HBV) covalently closed circular (ccc)DNA is the key genomic form responsible for viral persistence and virological relapse after treatment withdrawal. The assessment of residual intrahepatic cccDNA levels and activity after long-term nucleos(t)ide analogues therapy still represents a technical challenge. Quantitative (q)PCR, rolling circle amplification (RCA) and droplet digital (dd)PCR assays were used to quantify residual intrahepatic cccDNA in liver biopsies from 56 chronically HBV infected patients after 3 to 5 years of telbivudine treatment. Activity of residual cccDNA was evaluated by quantifying 3.5 kB HBV RNA (preC/pgRNA) and by assessing cccDNA-associated histone tails post-transcriptional modifications (PTMs) by micro-chromatin immunoprecipitation. Long-term telbivudine treatment resulted in serum HBV DNA suppression, with most of the patients reaching undetectable levels. Despite 38 out of 56 patients had undetectable cccDNA when assessed by qPCR, RCA and ddPCR assays detected cccDNA in all-but-one negative samples. Low preC/pgRNA level in telbivudine-treated samples was associated with enrichment for cccDNA histone PTMs related to repressed transcription. No difference in cccDNA levels was found according to serum viral markers evolution. This panel of cccDNA evaluation techniques should provide an added value for the new proof-of-concept clinical trials aiming at a functional cure of chronic hepatitis B.

StephenW

长期核苷类似物治疗患者乙型肝炎共价闭合环状DNA残留库的定量和表观遗传学评估
FannyLebossé1 2 3，AuroreInchauspé1 2，MaëlleLocatelli 1 2，Clothilde Miaglia 1 2 3，Audrey Diederichs 1 2，Judith Fresquet 1 2，Fleur Chapus 1 2，Kamal Hamed 4，Barbara Testoni 5 6，Fabien Zoulim 7 8 9
隶属关系
隶属关系

    1个
    INSERM U1052-法国里昂癌症研究中心（CRCL）。
    2
    里昂大学，UMR_S1052，CRCL，里昂，法国。
    3
    法国里昂平民医院，克鲁瓦鲁斯医院肝病科。
    4
    诺华制药公司，美国新泽西州东汉诺威。
    5
    INSERM U1052-法国里昂癌症研究中心（CRCL）。 [email protected]。
    6
    里昂大学，UMR_S1052，CRCL，里昂，法国。 [email protected]。
    7
    INSERM U1052-法国里昂癌症研究中心（CRCL）。 [email protected]。
    8
    里昂大学，UMR_S1052，CRCL，里昂，法国。 [email protected]。
    9
    法国里昂平民医院，克鲁瓦鲁斯医院肝病科。 [email protected]。

    PMID：33273565 DOI：10.1038 / s41598-020-78001-1

抽象

乙型肝炎病毒（HBV）共价闭合环状（ccc）DNA是负责停药后病毒持久性和病毒学复发的关键基因组形式。长期核苷酸（t）ide类似物治疗后，对残余肝内cccDNA水平和活性的评估仍然是一项技术挑战。定量（q）PCR，滚环扩增（RCA）和液滴数字（dd）PCR分析用于定量替比夫定治疗3至5年后56例慢性HBV感染患者肝活检中的残留肝内cccDNA。通过定量3.5 kB HBV RNA（preC / pgRNA）和通过微染色质免疫沉淀评估cccDNA相关的组蛋白尾部转录后修饰（PTM）来评估残留cccDNA的活性。长期替比夫定治疗导致血清HBV DNA抑制，大多数患者达到无法检测的水平。尽管通过qPCR评估的56位患者中有38位患者的cccDNA无法检测到，但RCA和ddPCR分析仅在一个阴性样品中检测到了cccDNA。替比夫定处理的样品中preC / pgRNA含量低与cccDNA组蛋白PTM富集有关，与转录抑制有关。根据血清病毒标志物的进化，没有发现cccDNA水平的差异。 cccDNA评价技术小组应为旨在有效治愈慢性乙型肝炎的新概念验证临床试验提供附加价值。

StephenW

https://www.nature.com/articles/s41598-020-78001-1.pdf