Engineered BacNav and BacCav for Improved Excitability and Contraction

专为改善兴奋性和收缩性而设计的 BacNav 和 BacCav

基本信息

批准号：
10611385
负责人：
Nenad Bursac
金额：
$ 47.75万
依托单位：
DUKE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-05-01 至 2026-01-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10611385
关键词：
3-Dimensional Action Potentials Address Adult Adverse effects Animal Model Anti-Arrhythmia Agents Arrhythmia Brugada syndrome Calcium Calcium Channel Cardiac Cardiac Electrophysiologic Techniques Cardiac Myocytes Cause of Death Cell Culture Techniques Cells Chromosome Mapping Codon Nucleotides Computer Simulation Congenital Abnormality Defect Developed Countries Disease Disease model Echocardiography Electrocardiogram Electrophysiology (science)Engineering Fibrosis Foundations Functional disorder Future Genes Genetic Genetic Engineering Goals Heart Heart Diseases Heart failure Human Human Engineering Human body Impairment In Situ In Vitro Ions Kinetics Left Loss of Heterozygosity Mammalian Cell Maps Measurement Mechanics Mediating Membrane Methods Modeling Mus Muscle Contraction Mutate Mutation Myocardial Infarction Myocardial Ischemia Myocardium Neonatal Optics Orthologous Gene Pathology Permeability Play Potassium Channel Predisposition Preparation Property Rattus Regulation Role Short QT syndrome Sick Sinus Syndrome Site-Directed Mutagenesis Slice Sodium Sodium Channel Speed Syndrome System Testing Therapeutic Therapeutic Effect Tissue Model Tissues Variant Ventricular Viral Viral Vector Virus Work adeno-associated viral vector biophysical properties candidate identification cardiac tissue engineering extracellular gene therapy heart function hemodynamics improved in silico in vitro Model in vivo in vivo evaluation loss of function mutation mouse model novel overexpression patch clamp pharmacologic prevent recombinant viral vector sudden cardiac death trafficking transgene expression voltage

项目摘要

Impaired cardiomyocyte excitability and contractile function represent important targets for preventing the occurrence of sudden cardiac death and progression of heart failure. Growing mechanistic understanding of cardiac pathologies and increasingly safe and effective methods to deliver viruses to human body make gene therapies an attractive strategy for combatting various heart diseases. Specifically, the ability to genetically, in a stable fashion, directly augment sodium or L-type calcium current in cardiomyocytes could directly enhance cell excitability and contractility and counteract occurrence of electrical abnormalities in a variety of heart diseases. However, cardiac Na+ or L-type Ca2+ channel genes are too large to be effectively delivered by therapeutic viruses including adeno-associated viral (AAV) vectors. To address this challenge, we propose to develop a novel AAV-based therapy that leverages engineering of much smaller prokaryotic voltage-gated sodium (BacNav) and calcium (BacCav) channel genes. Our preliminary results show that genetically engineered BacNav channels can improve cardiomyocyte excitability and action potential conduction in in vitro and in silico models of rat and human fibrotic heart tissues. Furthermore, we demonstrate successful cardiomyocyte-specific AAV9 delivery of BacNav channels in healthy murine hearts without any adverse effects on cardiac electrophysiology or contractile function. Building on these promising results, we propose to: 1) identify engineered BacNav variants with specific mutations and trafficking motifs that maximize cardiomyocyte excitability and action potential speed by utilizing in vitro cell culture, ex vivo heart slice preparations, and computer simulations and 2) engineer new variants of BacCav, which alone or in combination with BacNav can augment not only cardiomyocyte excitability but also contractile strength, which will be studied using engineered 3D heart tissue models in vitro. Finally, we will exploit murine models of impaired cardiac tissue excitability (genetic loss of cardiac Na+ current (SCN5A+/- )) or contractile dysfunction (myocardial infarction) to explore which of the identified BacNav and BacCav genes delivered by AAV vector will induce optimal long-term therapeutic effects in vivo. If successful, these studies will create a foundation for the future mechanistic studies of prokaryotic channel regulation in mammalian cardiomyocytes and will guide testing of the engineered BacNav and BacCav channel therapies in large animal models of heart disease.

心肌细胞兴奋性和收缩功能受损是预防心肌细胞损伤的重要目标。心源性猝死的发生和心力衰竭的进展。不断加深对机制的理解心脏病和日益安全有效的将病毒输送到人体的方法使基因疗法是对抗各种心脏病的有吸引力的策略。具体来说，从基因角度来说，能力稳定的方式，直接增强心肌细胞内钠或L型钙电流，可直接增强细胞兴奋性和收缩性并抵消各种心脏病中电异常的发生。然而，心脏 Na+ 或 L 型 Ca2+ 通道基因太大，无法通过治疗有效传递病毒，包括腺相关病毒（AAV）载体。为了应对这一挑战，我们建议开发一个基于 AAV 的新型疗法，利用更小的原核电压门控钠工程 (BacNav) 和钙 (BacCav) 通道基因。我们的初步结果表明，基因工程 BacNav 通道可以在体外和计算机模型中改善心肌细胞的兴奋性和动作电位传导大鼠和人类纤维化心脏组织。此外，我们成功证明了心肌细胞特异性 AAV9 在健康小鼠心脏中传递 BacNav 通道，不会对心脏电生理产生任何不利影响或收缩功能。基于这些有希望的结果，我们建议：1）识别工程 BacNav 变体具有特定的突变和运输基序，可最大限度地提高心肌细胞的兴奋性和动作电位速度通过利用体外细胞培养、离体心脏切片制备和计算机模拟，2) 设计新的 BacCav 的变体，单独或与 BacNav 组合不仅可以增强心肌细胞的兴奋性还有收缩强度，将使用工程 3D 心脏组织模型进行体外研究。最后，我们将利用心脏组织兴奋性受损的小鼠模型（心脏Na+电流的遗传损失（SCN5A+/- )) 或收缩功能障碍（心肌梗死），以探索已识别的 BacNav 和 BacCav 基因中的哪一个由AAV载体递送的药物将在体内诱导最佳的长期治疗效果。如果成功的话，这些研究将为未来哺乳动物原核通道调节机制研究奠定基础心肌细胞，并将指导大型动物中工程化 BacNav 和 BacCav 通道疗法的测试心脏病模型。