AASLD2020[825]GAPMERS的基因沉默：一种新型治疗工具 在HBV感染中

StephenW

825
GENE SILENCING BY GAPMERS: A NEW THERAPEUTIC TOOL
IN HBV INFECTION.
Maria Francesca Cortese1,2, Selene Garcia-Garcia1,2, Rosario
Casillas1,2, Rosa Lopez-Martinez1, Beatriz Pacin Ruiz2, Sara
Sopena1,2, David Tabernero1,3, Roser Maria Ferrer-Costa4, Mar
Riveiro Barciela5,6, Maria Asuncion Buti Ferret7,8 and Francisco
Rodriguez-Frías1,2,7, (1)Biochemistry and Microbiology/Liver
Pathology Unit, Vall D’hebron University Hospital, (2)Liver
Unit, Vall D’hebron Research Institute, (3)Liver Pathology
Unit Departments of Biochemistry and Microbiology, Vall
d’ Hebron University Hospital and Universitat Autonoma
De Barcelona, Barcelona, Spain, University Hospital Vall
D’hebron, (4)Department of Biochemistry, Vall D’hebron
University Hospital, Vall D’hebron University Hospital, (5)Liver
Unit, Internal Medicine Department. Hospital Universitari Vall
d’Hebron, Barcelona, Spain, University Hospital Vall D’hebron,
(6)Ciberehd, Instituto Carlos III, Madrid, (7)Centro De
Investigación Biomédica En Red, Enfermedades Hepáticas y
Digestvas (CIBERehd), Instituto De Salud Carlos III, Madrid,
Spain, (8)Liver Unit, Internal Medicine Department. Hospital
Universitari Vall d’Hebron, Barcelona, Spain
Background: Hepatitis B Virus (HBV) infection cannot
be eradicated due to the persistence of cccDNA, which
continuously supports the intracellular expression of viral
proteins Of note, a little but consistent percentage of
successfully treated patients with chronic hepatitis B develops
hepatocarcinoma, reinforcing the need of new therapeutic
strategy HBV X gene (HBX), due to its co-terminal localization,
could be an optimal target for a gene therapy strategy Here
we present a new system of viral gene silencing based on
antisense locked nucleic acid (LNA) Gapmer (GP) that
targets HBX hyperconserved regions Methods: HepG2-
NTCP cells were treated with DMSO 2 5% at least 14 days
and later infected with HBV at 500 multiplicities of genome
equivalents by spinoculation at 37ºC for 1h and incubating
overnight Infected cells were treated with GP (GP1 and/or
GP4 at 50nM, Qiagen) and/or siRNA (50nM) using TransIT-X2
(Mirus) at 48h or 5d post infection (pi) A scrambled control
gapmer was used in each experiment Cells and supernatants
were collected after 72h of treatment Pregenomic RNA
(pgRNA) was extracted from cells through RNAeasy Plus
mini kit (Qiagen) and quantified by RT-qPCR using TaqMan
probe (Roche, Lightcycler® 480) HBsAg and HBeAg were
quantified in supernatants by chemilumiscent enzyme assay
(Roche, COBAS® 8000) Results: Mock-untreated infected
cells productively expressed HBV reaching a peak of 4log
of pgRNA (copies/ng), 16IU/mL of HBeAg and 2U/mL of
HBsAg. Early treatment (at 48hpi) with GP efficiently inhibited
HBV pgRNA related to the scrambled, reaching an average
of 75 2±4 5, 54 1±19 2 and 54 3±15 5% for GP1, GP4 and
GP1+GP4, respectively Similar percentages of inhibition
related to the scrambled were obtained for protein expression
(HBeAg = 79 3±8 3, 60 2±13 8, 70 4±11 6%; HBsAg =
71 1±10 2, 60 8±15 3; 64 8±8 7% for GP1, GP4, GP1+4
respectively) The inhibition was also maintained when
treatment was carried out at 5dpi, reaching a percentage
of 81% for GP1, 78% for GP4 and 69% for siRNA Of note,
co-treatment with GP4 and siRNA partially increased the
inhibition up to 93 3% Conclusion: Gapmers targeting
hyper-conserved regions in HBX gene efficiently inhibit HBV
expression in vitro at either early or late treatment The cotreatment
of gapmers with siRNA targeting the same regions
could be a valuable strategy to inhibit HBV expression at both
nuclear and cytoplasmic level Funding: Instituto de Salud
Carlos III (grant PI18/01436), co-financed by the European
Regional Development Fund (ERDF)
Disclosures:
Mar Riveiro Barciela – AbbVie and Gilead: Speaking and Teaching
Maria Asuncion Buti Ferret – Abbvie, Gilead,Janssen: Advisory Committee
or Review Panel; Abbvie, Gilead,Janssen: Grant/Research Support; Abbvie,
Gilead,Janssen: Speaking and Teaching
The following people have nothing to disclose: Maria Francesca Cortese,
Selene Garcia-Garcia, Rosario Casillas, Rosa Lopez-Martinez, Beatriz Pacin
Ruiz, Sara Sopena, David Tabernero, Roser Maria Ferrer-Costa, Francisco
Rodriguez-Frías

StephenW

825
GAPMERS的基因沉默：一种新型治疗工具
在HBV感染中。
玛丽亚·弗朗西斯·科蒂斯1,2，塞琳娜·加西亚·加西亚1,2，罗萨里奥
卡西利亚斯1,2，罗莎·洛佩兹-马丁内斯1，比阿特丽斯·帕辛·鲁伊斯2，萨拉
Sopena1,2，David Tabernero1、3，Roser Maria Ferrer-Costa4、3月
Riveiro Barciela5,6，Maria Asuncion Buti Ferret7,8和Francisco
Rodriguez-Frías1,2,7，（1）生化与微生物学/肝脏
Vall D’hebron大学医院病理科，（2）肝
Vall D’hebron研究所单位，（3）肝病理学
瓦尔生物化学与微生物学系
d'希伯伦大学医院和Autonoma大学
De Barcelona，西班牙巴塞罗那，瓦尔大学医院
D'hebron，（4个）Vall D’hebron生物化学系
瓦莱德黑布伦大学医院大学医院，（5）肝
内科医学科。瓦尔大学医院
d'Hebron，西班牙巴塞罗那，大学医院Vall D'hebron，
（6）马德里卡洛斯三世学院的塞伯雷德（7）
生物医学研究会（EnfermedadesHepáticasy）
Digestvas（CIBERehd），马德里萨洛德·卡洛斯三世研究所，
西班牙，（8）内科肝病科。医院
Universitari Vall d’Hebron，西班牙巴塞罗那
背景：乙肝病毒（HBV）感染不能
由于cccDNA的持久性而被根除，
持续支持病毒的细胞内表达
值得注意的蛋白质
成功治疗的慢性乙型肝炎患者发展
肝癌，增加了对新疗法的需求
策略HBV X基因（HBX），由于其共末端定位，
可能是基因治疗策略的最佳靶标
我们提出了一种基于病毒基因沉默的新系统
反义锁核酸（LNA）Gapmer（GP）
靶向HBX超保守区方法：HepG2-
用5％的DMSO 2处理NTCP细胞至少14天
后来又感染了500个基因组的HBV
在37℃旋转接种1小时并孵育的当量
过夜将感染的细胞用GP（GP1和/或
使用TransIT-X2在50nM的GP4（Qiagen）和/或siRNA（50nM）
（Mirus）感染后48h或5d（pi）加扰对照
在每个实验中使用gapmer细胞和上清液
处理72小时后收集前基因组RNA
（pgRNA）通过RNAeasy Plus从细胞中提取
迷你试剂盒（Qiagen），并使用TaqMan通过RT-qPCR进行定量
探针（Roche，Lightcycler®480）的HBsAg和HBeAg
通过化学发光酶法定量上清液
（罗氏，COBAS®8000）结果：未经治疗的模拟感染
细胞有效表达的HBV达到4log的峰值
pgRNA（份数/ ng），16IU / mL的HBeAg和2U / mL的
乙肝表面抗原GP的早期治疗（48hpi）被有效抑制
与加扰的HBV pgRNA有关，达到平均值
GP1，GP4和75的75 2±4 5，54 1±19 2和54 3±15 5％
GP1 + GP4分别具有相似的抑制百分比
获得与加扰相关的蛋白质表达
（HBeAg = 79 3±8 3，60 2±13 8，70 4±11 6％; HBsAg =
71 1±10 2，60 8±15 3；对于GP1，GP4，GP1 + 4，为64 8±8 7％
分别）当
处理以5dpi进行，达到一定百分比
GP1为81％，GP4为78％，siRNA为69％
与GP4和siRNA共同治疗可部分增加
抑制高达93 3％结论：靶向Gapmers
HBX基因的超保守区有效抑制HBV
在早期或晚期治疗中的体外表达
siRNA靶向相同区域的gapmers的合成
可能是在两个方面抑制HBV表达的有价值的策略
核和细胞质水平资助：Salud研究所
卡洛斯三世（赠款PI18 / 01436），由欧洲共同资助
区域发展基金（ERDF）
披露事项：
Mar Riveiro Barciela – AbbVie和Gilead：演讲和教学
玛丽亚·亚松森·布蒂·费雷特（Maria Asuncion Buti Ferret）–阿卜维，吉利德，詹森：咨询委员会
或审查小组； Abbvie，Gilead，Janssen：资助/研究支持；阿伯维
吉利德·詹森（Gilead，Jansen）：口语和教学
以下人无话可说：Maria Francesca Cortese，
塞琳娜·加西亚·加西亚（Selene Garcia-Garcia），罗萨里奥·卡西利亚斯（Rosario Casillas），罗莎·洛佩兹·马丁内斯（Rosa Lopez-Martinez）
Ruiz，Sara Sopena，David Tabernero，Roser Maria Ferrer-Costa，Francisco
罗德里格斯·弗里亚斯