生物
遗传增强
腺相关病毒
病毒载体
病毒学
基因传递
基因
计算生物学
医学
载体(分子生物学)
心脏病学
重组DNA
遗传学
作者
Li Ni,Larry Scott,Hannah M. Campbell,Xiaolu Pan,Katherina M. Alsina,Julia O. Reynolds,Leonne E. Philippen,Mohit Hulsurkar,William R. Lagor,Na Li,Xander H.T. Wehrens
出处
期刊:Circulation Research
[Ovid Technologies (Wolters Kluwer)]
日期:2019-01-18
卷期号:124 (2): 256-262
被引量:50
标识
DOI:10.1161/circresaha.118.313811
摘要
Somatic overexpression in mice using an adeno-associated virus (AAV) as gene transfer vectors has become a valuable tool to analyze the roles of specific genes in cardiac diseases. The lack of atrial-specific AAV vector has been a major obstacle for studies into the pathogenesis of atrial diseases. Moreover, gene therapy studies for atrial fibrillation would benefit from atrial-specific vectors. Atrial natriuretic factor (ANF) promoter drives gene expression specifically in atrial cardiomyocytes.To establish the platform of atrial specific in vivo gene delivery by AAV-ANF.We constructed AAV vectors based on serotype 9 (AAV9) that are driven by the atrial-specific ANF promoter. Hearts from mice injected with AAV9-ANF-GFP (green fluorescent protein) exhibited strong and atrial-specific GFP expression without notable GFP in ventricular tissue. In contrast, similar vectors containing a cardiac troponin T promoter (AAV9-TNT4-GFP) showed GFP expression in all 4 chambers of the heart, while AAV9 with an enhanced chicken β-actin promoter (AAV-enCB-GFP) caused ubiquitous GFP expression. Next, we used Rosa26mT/mG (membrane-targeted tandem dimer Tomato/membrane-targeted GFP), a double-fluorescent Cre reporter mouse that expresses membrane-targeted tandem dimer Tomato before Cre-mediated excision, and membrane-targeted GFP after excision. AAV9-ANF-Cre led to highly efficient LoxP recombination in membrane-targeted tandem dimer Tomato/membrane-targeted green fluorescent protein mice with high specificity for the atria. We measured the frequency of transduced cardiomyocytes in atria by detecting Cre-dependent GFP expression from the Rosa26mT/mG allele. AAV9 dose was positively correlated with the number of GFP-positive atrial cardiomyocytes. Finally, we assessed whether the AAV9-ANF-Cre vector could be used to induce atrial-specific gene knockdown in proof-of-principle experiments using conditional JPH2 (junctophilin-2) knockdown mice. Four weeks after AAV9-ANF-Cre injection, a strong reduction in atrial expression of JPH2 protein was observed. Furthermore, there was evidence for abnormal Ca2+ handling in atrial myocytes isolated from mice with atrial-restricted JPH2 deficiency.AAV9-ANF vectors produce efficient, dose-dependent, and atrial-specific gene expression following a single-dose systemic delivery in mice. This vector is a novel reagent for both mechanistic and gene therapy studies on atrial diseases.
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