AASLD2018[403]针对α的新siRNA的抗病毒活性 乙型肝炎X基因超高

StephenW

403
Antiviral Activity of a New siRNA Targeting a
Hyper-Conserved Region in Hepatitis B X Gene
Transcripts: Preliminary Data.
Sheila Maestro1,2, Maria Francesca Cortese3,4, Carolina
González3, David Tabernero3,5, Mar Riveiro Barciela5,6,
Maria Buti5,7, Gloria González-Aseguinolaza1,2 and Francisco
Rodríguez-Frías3,5, (1)Gene Therapy and Regulation of Gene
Expression Program, Center for Applied Medical Research
(CIMA), (2)Instituto De Investigación Sanitaria De Navarra
(IdiSNA), (3)Biochemistry and Microbiology/Liver Pathology
Unit, Vall D’hebron University Hospital, (4)Liver Unit, Vall
D’hebron Research Institute, (5)Ciberehd, Instituto De Salud
Carlos III, (6)Department of Internal Medicine/Liver Unit, Vall
D’hebron University Hospital, (7)Hospital Universitari Vall
d’Hebron, Barcelona, Spain
Background: Functional cure of chronic hepatitis B (CHB)
by standard nucleos(t)ide analog-based treatment is rarely
achieved, with the risk of hepatocellular carcinoma (HCC)
remaining present. The hepatitis B X gene (HBX) plays a
key role in this process. Using next-generation sequencing
(NGS) a previous study by our group (González et al. World J
Gastroenterol 2018;24:2095) identified two hyper-conserved
regions in 5’ end of HBX [nucleotide (nt) 1255-1286; nt
1519-1603] regardless of the patients’ clinical stage or HBV
genotype. The aim of this study was to determine the antiviral
activity of a small interference RNA (siRNA) battery targeting
these regions. Methods: siRNAs were identified using the
iScore Designer. In order to evaluate their antiviral activity,
HepG2-NTCP cells were infected with purified HBV, and
48 hours post-infection they were transfected with siRNAs
(alone or in combination) using lipofectamine. A known siRNA
targeting X transcripts (siRNA4) and a non-specific scrambled
siRNA were used as controls. The expression of hepatitis
B X (HBx), core (HBc) and surface (HBs) antigen mRNAs
was determined by real time PCR at different time points.
Results: We identified 3 potential siRNAs (1-3) targeting
the described hyper-conserved regions. Mock-infected cells
were productively infected and expressed HBs, HBc and
HBx transcripts. siRNAs efficiently reduced gene expression
from 48h to 96h post-transfection (Figure). Of note, siRNA1
and siRNA4 were more potent inhibitors of viral expression
(percentage of HBx expression relative to scrambled siRNA:
18.6 and 29.5% for siRNA1 and siRNA4, respectively vs. 32.1
and 44% for siRNA2 and siRNA3 at 96h, Figure panel C). The
combination of siRNA1+4 further increased the inhibition of
gene expression with a percentage of HBV antigen expression
relative to scrambled siRNA of 27.5, 5.6 and 22% for HBc,
HBs and HBx, respectively at 96h (Figure). Conclusion: By
targeting a hyper-conserved region of HBX, siRNA1 efficiently
reduces HBV gene expression in vitro. The use of this siRNA
alone or in combination with other known siRNAs could help
to control HBV infection and prevent disease progression and
HCC development. Further in vitro and in vivo studies are
required to confirm these results. Funding: Instituto de Salud
Carlos III (grant PI15/00856) co-financed by the European
Regional Development Fund (ERDF).

StephenW

403
针对α的新siRNA的抗病毒活性
乙型肝炎X基因超高保守区
成绩单：初步数据。
Sheila Maestro1,2，Maria Francesca Cortese3,4，Carolina
González3，David Tabernero3,5，Mar Riveiro Barciela5,6，
Maria Buti5,7，GloriaGonzález-Aseguinolaza1,2和Francisco
Rodríguez-Frías3,5，（1）基因治疗和基因调控
表达程序，应用医学研究中心
（CIMA），（2）InstitutoDeInvestigaciónSanitariaDe Navarra
（IdiSNA），（3）生物化学和微生物学/肝脏病理学
单位，Vall D'hebron大学医院，（4）肝脏单位，Vall
D'hebron研究所，（5）Ciberehd，Instituto De Salud
卡洛斯三世，（6）瓦尔内科/肝脏科
D'hebron大学医院，（7）Universitari Vall医院
d'Hebron，巴塞罗那，西班牙
背景：慢性乙型肝炎（CHB）的功能性治愈
通过标准核苷（t）ide基于模拟的治疗很少
实现，患有肝细胞癌（HCC）的风险
还在场。乙型肝炎X基因（HBX）发挥作用
这个过程中的关键作用。使用新一代测序
（NGS）我们小组以前的一项研究（González等人，World J
Gastroenterol 2018; 24：2095）鉴定出两种超级保守的
HBX 5'端的区域[核苷酸（nt）1255-1286; NT
[1519-1603]无论患者的临床分期还是HBV
基因型。这项研究的目的是确定抗病毒药物
小干扰RNA（siRNA）电池靶向活性
这些地区。方法：用siRNA鉴定siRNAs
iScore Designer。为了评估他们的抗病毒活性，
HepG2-NTCP细胞用纯化的HBV感染，并且
感染后48小时，用siRNA转染它们
（单独或组合）使用lipofectamine。已知的siRNA
靶向X转录物（siRNA4）和非特异性杂乱的
siRNA用作对照。肝炎的表达
B X（HBx），核心（HBc）和表面（HBs）抗原mRNA
通过实时PCR在不同时间点确定。
结果：我们鉴定了3种潜在的siRNA（1-3）靶向
描述的超保守区域。模拟感染的细胞
有效地感染并表达HBs，HBc和HBs
HBx成绩单。 siRNA有效地降低了基因表达
转染后48h至96h（图）。值得注意的是，siRNA1
和siRNA4是更有效的病毒表达抑制剂
（HBx表达相对于乱序siRNA的百分比：
siRNA1和siRNA4分别为18.6和29.5％，而32.1
对于siRNA2和siRNA3在96h时为44％，图C）。该
siRNA1 + 4的组合进一步增强了对β1+的抑制作用
基因表达与HBV抗原表达的百分比
相对于HBc的乱序siRNA为27.5,5.6和22％，
HBs和HBx分别在96h（图）。结论：通过
有效地靶向HBX的超保守区域，siRNA1
在体外减少HBV基因表达。使用这种siRNA
单独或与其他已知siRNA组合可能有所帮助
控制HBV感染，预防疾病进展
HCC发展。进一步的体外和体内研究是
需要确认这些结果。资金来源：Instituto de Salud
Carlos III（拨款PI15 / 00856）由欧洲人共同出资
区域发展基金（ERDF）。