CRISPR用于沉默关键性乙型肝炎基因

StephenW

CRISPR Used to Silence Crucial Hepatitis B Gene         

Christina Bennett

   

November 12, 2019

   
                           The CRISPR gene-editing tool can be used to silence an important hepatitis B virus gene, a proof-of-concept in vitro study suggests.
"It's the first time we've seen CRISPR editing done in a hepatitis B model," said Douglas Dieterich, MD, director of the Institute of Liver Medicine and professor of medicine at the Icahn School of Medicine at Mount Sinai in New York City.
Hepatitis B can lead to liver disease and is the primary cause of hepatocellular carcinoma. In 2015, more than 250 million people around the world were infected with the virus, according to the World Health Organization.
For their study, investigator Hao Zhou, from The First Hospital of Jilin University in China and the Department of Medicine at the University of Minnesota in Minneapolis, and colleagues targeted the S gene. Zhou presented the findings at the Liver Meeting 2019 in Boston.

The S gene gives rise to the hepatitis B surface antigen, the presence of which indicates that a person is infected with the virus. "The question is whether it's the right target," Dieterich told Medscape Medical News.
Reducing the amount of the hepatitis B surface antigen is a "good idea" because that's what is believed to inhibit the immune system from clearing the virus. Doing so might help the immune system recover and clear the virus, "with a little help from some antivirals," explained Dieterich, who was not involved in the study.
However, "the surface is not the only DNA that's integrated into the host genome," he pointed out. "I think maybe a broader application might be necessary to actually get the hepatitis B genome out of the hepatocytes."
Zhou's team used a newer CRISPR approach, called CRISPR-STOP, for their gene-editing procedure.
CRISPR-STOP "The idea is that CRISPR-STOP can be as efficient as standard CRISPR editing, but it's safer," said Kiran Musunuru, MD, PhD, associate professor of cardiovascular medicine and genetics at Penn Medicine in Philadelphia, who was not involved in the study. Musunuru is cofounder of and senior scientific advisor at Verve Therapeutics, a company using gene editing to prevent cardiovascular disease.
The standard CRISPR-Cas9 approach requires a double-strand break in the genome, and the problem with that is it introduces the possibility for "mischief," he explained. "If you have more than one double-strand break occurring in the human genome at the same time, you have the potential for different parts of different chromosomes coming together in the wrong ways and then causing problems."
Instead of creating a double-strand break, CRISPR-STOP uses a base editor to chemically modify the DNA base from one base to another and introduce a stop codon into the target gene sequence, effectively hamstringing the ability of the target gene to produce a functional protein.
       This is a very nice, clean way to turn off a gene effectively.      

"This is a very nice, clean way to turn off a gene effectively," Musunuru told Medscape Medical News.
For their CRISPR-STOP procedure, Zhou's team first transduced liver cells infected with the hepatitis B virus using a base editor called AncBE4max. Next, to activate the base editor so that gene editing could begin, they transduced the cells with one of two lentivectors: one encoded for single-guide RNA that targets the S gene; and an empty one, which served as the control.
With the gene-editing approach, 71% of the liver cells that expressed the base editor gained the desired stop codon in the target gene.
"That's a very robust number," said Musunuru.
In addition, hepatitis B surface antigen secretion was reduced by 92% with the gene-editing approach.
The investigators report a high degree of conservativity for hepatitis B genotypes B, C, F, and H. Specifically, 94% of the S gene sequence was conserved for genotype B, 92% for genotype C, 91% for genotype F, and 71% for genotype H.
The Liver Meeting 2019: American Association for the Study of Liver Diseases (AASLD): Abstract 86. Presented November 10, 2019.
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StephenW

CRISPR用于沉默关键性乙型肝炎基因

克里斯蒂娜·贝内特（Christina Bennett）

2019年11月12日

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一项概念验证的体外研究表明，CRISPR基因编辑工具可用于沉默重要的乙型肝炎病毒基因。

“这是我们第一次看到在乙型肝炎模型中完成CRISPR编辑，”纽约市西奈山伊坎医学院的肝脏医学研究所所长，医学教授道格拉斯·迪特里里奇（Douglas Dieterich）医学博士说。

乙型肝炎可导致肝脏疾病，是肝细胞癌的主要原因。根据世界卫生组织的数据，2015年，全球有超过2.5亿人感染了该病毒。

对于他们的研究，来自吉林大学第一医院和明尼苏达州明尼苏达州大学医学系的研究人员郝浩及其同事靶向了S基因。周在波士顿举行的2019年肝脏会议上介绍了这些发现。

S基因产生乙型肝炎表面抗原，该表面抗原的存在表明一个人感染了该病毒。 Dieterich告诉Medscape医学新闻说：“问题是它是否是正确的目标。”

减少乙型肝炎表面抗原的量是一个“好主意”，因为据信这会抑制免疫系统清除病毒。不参与这项研究的迪特里里希解释说，这样做可能有助于免疫系统恢复并清除病毒，“在一些抗病毒药物的帮助下”。

但是，他指出：“表面并不是整合到宿主基因组中的唯一DNA。” “我认为也许可能需要应用才能从肝细胞中真正分离出乙肝基因组。”

Zhou的团队在其基因编辑过程中使用了一种新的CRISPR方法，称为CRISPR-STOP。
CRISPR停止

费城Penn Medicine心血管医学和遗传学副教授Kiran Musunuru医学博士说：“我们的想法是CRISPR-STOP可以与标准CRISPR编辑一样高效，但更安全。” Musunuru是Verve Therapeutics公司的联合创始人兼高级科学顾问，该公司使用基因编辑来预防心血管疾病。

他解释说，标准的CRISPR-Cas9方法需要在基因组中进行双链断裂，其问题在于它可能引发“恶作剧”。 “如果在人类基因组中同时发生多个双链断裂，则可能会导致不同染色体的不同部分以错误的方式聚集在一起，从而引起问题。”

CRISPR-STOP不会产生双链断裂，而是使用碱基编辑器将DNA碱基从一个碱基化学修饰为另一个碱基，并将终止密码子引入靶基因序列中，有效地抑制了靶基因产生功能性氨基酸的能力。蛋白。
这是关闭基因有效的非常好，干净的方法。

Musunuru告诉Medscape医学新闻说：“这是关闭基因有效的非常好，干净的方法。”

对于他们的CRISPR-STOP程序，Zhou的团队首先使用称为AncBE4max的基础编辑器转导了感染了乙型肝炎病毒的肝细胞。接下来，为了激活碱基编辑器以便开始基因编辑，他们用两种慢病毒载体之一转导了细胞：一种针对靶向S基因的单向导RNA编码；另一种针对S基因编码。还有一个空的作为控件。

通过基因编辑方法，表达基础编辑器的71％肝细胞在靶基因中获得了所需的终止密码子。

“这是一个非常强大的数字，”穆苏努鲁说。

此外，通过基因编辑方法，乙型肝炎表面抗原的分泌减少了92％。

研究人员报告说，乙型肝炎B，C，F和H基因型具有高度的保守性。特定的是，基因型B保守了94％的S基因序列，基因型C保守了92％，基因F保守了91％，71基因型H的％。

《 2019年肝脏会议：美国肝病研究协会》（AASLD）：摘要86。发表于2019年11月10日。

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yyjj777

还是割掉来的干净。这种折中的方案，是自己技术不过关。。。退而求其次的解决方案。

StephenW

回复 yyjj777 的帖子

这是一种发展中的治疗方法, 研究用途和实际治疗的前景非常光明.除非绝对安全和有效，否则将不使用治疗.

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