Cure of Congenital Purpura Fulminans via Expression of Engineered Protein C Through Neonatal Genome Editing in Mice.

IF 7.4 1区 医学 Q1 HEMATOLOGY Arteriosclerosis, Thrombosis, and Vascular Biology Pub Date : 2024-11-07 DOI:10.1161/ATVBAHA.123.319460
Tomoki Togashi, Nemekhbayar Baatartsogt, Yasumitsu Nagao, Yuji Kashiwakura, Morisada Hayakawa, Takafumi Hiramoto, Takayuki Fujiwara, Eriko Morishita, Osamu Nureki, Tsukasa Ohmori
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Abstract

Background: PC (protein C) is a plasma anticoagulant encoded by PROC; mutation in both PROC alleles results in neonatal purpura fulminans-a fatal systemic thrombotic disorder. In the present study, we aimed to develop a genome editing treatment to cure congenital PC deficiency.

Methods: We generated an engineered APC (activated PC) to insert a furin-cleaving peptide sequence between light and heavy chains. The engineered PC was expressed in the liver of mice using an adeno-associated virus vector or CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat-associated 9)-mediated genome editing using an adeno-associated virus vector in vivo.

Results: The engineered PC could be released in its activated form and significantly prolonged the plasma coagulation time independent of the cofactor activity of PS (protein S) in vitro. The adeno-associated virus vector-mediated expression of the engineered PC, but not wild-type PC, prolonged coagulation time owing to the inhibition of activated coagulation FV (factor V) in a dose-dependent manner and abolished pathological thrombus formation in vivo in C57BL/6J mice. The insertion of EGFP sequence conjugated with self-cleaving peptide sequence at Alb locus via neonatal in vivo genome editing using adeno-associated virus vector resulted in the expression of EGFP in 7% of liver cells, mainly via homology-directed repair, in mice. Finally, we succeeded in improving the survival of PC-deficient mice by expressing the engineered PC via neonatal genome editing in vivo.

Conclusions: These results suggest that the expression of engineered PC via neonatal genome editing is a potential cure for severe congenital PC deficiency.

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通过新生小鼠基因组编辑表达工程蛋白 C 治疗先天性富贵性紫癜
背景:PC(蛋白C)是一种由PROC编码的血浆抗凝剂;PROC两个等位基因的突变会导致新生儿紫癜--一种致命的全身性血栓性疾病。在本研究中,我们旨在开发一种基因组编辑治疗方法,以治愈先天性 PC 缺乏症:方法:我们生成了一种工程化 APC(活化 PC),在轻链和重链之间插入了呋喃分解肽序列。利用腺相关病毒载体或CRISPR/Cas9(簇状规则间距短回文重复序列/簇状规则间距短回文重复序列相关9)介导的基因组编辑技术,在小鼠肝脏中表达工程化的PC:结果:工程 PC 能以活化形式释放,在体外显著延长血浆凝固时间,不受 PS(蛋白 S)辅助因子活性的影响。在 C57BL/6J 小鼠体内,腺相关病毒载体介导表达的工程 PC(而非野生型 PC)以剂量依赖的方式抑制了活化凝血因子 FV(因子 V),从而延长了凝血时间,并消除了病理性血栓的形成。通过使用腺相关病毒载体进行新生儿体内基因组编辑,在 Alb 基因座上插入与自裂解肽序列连接的 EGFP 序列,主要通过同源定向修复,在小鼠 7% 的肝细胞中实现了 EGFP 的表达。最后,我们通过新生儿基因组编辑在体内表达工程PC,成功改善了PC缺陷小鼠的存活率:这些结果表明,通过新生儿基因组编辑表达工程化 PC 有可能治愈严重的先天性 PC 缺乏症。
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来源期刊
CiteScore
15.60
自引率
2.30%
发文量
337
审稿时长
2-4 weeks
期刊介绍: The journal "Arteriosclerosis, Thrombosis, and Vascular Biology" (ATVB) is a scientific publication that focuses on the fields of vascular biology, atherosclerosis, and thrombosis. It is a peer-reviewed journal that publishes original research articles, reviews, and other scholarly content related to these areas. The journal is published by the American Heart Association (AHA) and the American Stroke Association (ASA). The journal was published bi-monthly until January 1992, after which it transitioned to a monthly publication schedule. The journal is aimed at a professional audience, including academic cardiologists, vascular biologists, physiologists, pharmacologists and hematologists.
期刊最新文献
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