Cardiac-targeted AAV5-S100A1 gene therapy protects against adverse remodeling and contractile dysfunction in post-ischemic herats

Background: Guided by long-term safety data for AAV5 in humans, our translational study investigated whether AAV5 effectively delivers genes to healthy and achieve therapeutic efficacy in dysfunctional human-sized hearts, using a clinically applicable mode of administration and vector dosages. Methods & Results: In a first biodistribution study, AAV5 (1?1013 vector genomes; vgs) - carrying the reporter gene luciferase (luc) - achieved broad and homogenous transduction of the left ventricle (LV) of healthy German farm pigs 30 days after percutaneous catheter-based retrograde intravenous delivery (CRID), as measured by vector genome and luc mRNA assessments. Both its myocardial and extra-cardiac distribution patterns were advantageous compared to AAV9-luc and AAV6-luc. Using the cardioprotective human gene S100A1 (hS100A1) in a subsequent therapy study, effective myocardial transduction with AAV5-hS100A1 (1?1013 vgcs) by CRID prevented LV myocardial infarction (MI) enlargement and improved systolic contractile performance after 3 months in a porcine model with chronic post-MI cardiac dysfunction, as assessed by serial cardiac magnetic resonance imaging. The use of a cardiac-biased promoter ensured cardiac-targeted hS100A1 expression without signs of clinical toxicity, as shown by RT-PCR analysis, clinical chemistry and blood count. A previously unknown anti-inflammatory effect of hS100A1 in damaged myocardium, detected and linked to the therapeutic actions by a cardiac transcriptome weighted gene coexpression network analysis, was confirmed using AAV5-hS100A1 therapy in a post-MI mouse model, as measured by RT-PCR and echocardiography. Conclusion: Providing the clinically relevant proof-of-concept for AAV5 to effectively transduce healthy and dysfunctional human-sized hearts, its clinical long-term safety, scalable producibility as well as low pre-existing immunity in humans may predestine AAV5 as an effective and safe gene carrier for a prevalent disease as chronic heart failure, using therapeutic genetic effectors, such as hS100A1 or others. Overall design: RNA-seq, WGCNA and enrichment analysis of myocardial tissue (anteromidventricular) of male farm pigs 14 weeks after LCX-myocardial infarction, AAV5-hS100A1 treated vs. AAV5-luc

背景：本研究以腺相关病毒5型（AAV5）在人类中的长期安全性数据为指导，开展转化研究，旨在评估采用临床可及的给药方式与载体剂量时，AAV5能否将基因有效递送至健康心脏，并在尺寸接近人类的功能异常心脏中实现治疗效果。

方法与结果：在首项生物分布研究中，携带报告基因荧光素酶（luciferase, luc）的AAV5（1×10¹³ 载体基因组，vgs）经皮导管逆行静脉给药（CRID）30天后，可在健康德国农场猪的左心室（LV）实现广泛且均一的转导，该结果通过载体基因组与luc mRNA检测得以验证。相较于AAV9-luc与AAV6-luc，其心肌与心外分布模式均更具优势。
在后续的治疗研究中，采用心脏保护性人源基因S100A1（hS100A1），通过CRID方式对AAV5-hS100A1（1×10¹³ vgcs）进行有效心肌转导，可在慢性心肌梗死（MI）后心功能异常的猪模型中，于3个月后阻止左心室梗死扩大并改善收缩功能，该效果通过系列心脏磁共振成像评估得到证实。通过使用心脏偏好型启动子，可确保hS100A1的心脏靶向表达，且未出现临床毒性迹象，该结果经逆转录聚合酶链反应（RT-PCR）分析、临床生化与血常规检测得以验证。
此前尚未被发现的hS100A1在受损心肌中的抗炎作用，通过心脏转录组加权基因共表达网络分析被检测到，并与治疗作用相关联；该作用在心肌梗死小鼠模型中经AAV5-hS100A1治疗得到证实，检测方式包括RT-PCR与超声心动图。

结论：本研究为AAV5能够有效转导健康及功能异常的尺寸接近人类的心脏提供了临床相关的概念验证；其临床长期安全性、可规模化生产性以及人类体内较低的预存免疫力，使得AAV5有望成为针对慢性心力衰竭这类常见疾病的有效且安全的基因载体，可搭载hS100A1或其他治疗性遗传效应分子。

整体实验设计：对左回旋支（LCX）心肌梗死14周后的雄性农场猪的前中部心室心肌组织进行RNA测序（RNA-seq）、加权基因共表达网络分析（WGCNA）及富集分析，实验组为AAV5-hS100A1处理组，对照组为AAV5-luc处理组。