AAV-Exosomes: Escaping Neutralizing Antibody and Enhancing Delivery

总结 基因治疗是治疗包括遗传性疾病在内的各种单基因疾病的一种有前途的方法。 心肌病和其他类型的心力衰竭。腺相关载体（AAV）是选择用于以下目的的载体： 将基因递送到心肌细胞用于长期表达并且由于它们在临床上的安全性。但 它们成功使用的重大挑战是由预先存在的抗体（NAb）以及 AAV施用后免疫力的随后发展。NAb防止AAV感染 靶细胞，大大降低了转导效率，从而降低了临床疗效。因此，为了提高基因 为更广泛的人群提供心血管治疗，必须制定策略， 绕过NAB。 外泌体是细胞外分泌的纳米囊泡，其在相邻的细胞间穿梭选择性生物分子。 遥远的细胞最近的研究表明，外泌体可以携带多种类型的病毒并保护它们 抗体中和的结果由载体外泌体保护的AAV的递送是一种有前途的方法， 在基于AAV的基因治疗中避免NAb中和。我们的体外和体内初步数据表明， AAV包封的外泌体（AAVExo）1）与游离的外泌体相比， AAV，2）更有效地将基因递送到心肌，3）保留病毒的心肌向性，以及4）保留 AAV介导的基因递送的治疗益处。 在这里，我们的目的是研究AAVExo逃避NAb的能力，并作为一个高效的基因， 用于心血管治疗的递送工具。我们已经开发了一种分离高纯度AAVExo的方法， 最小化来自游离AAV的污染。我们的中心假设是AAVExo屏蔽了AAV， 与游离AAV相比，避免NAb并增强基因递送至心肌。我们的主要目标是 为了全面了解AAVExo中和NAb的分子机制， 和2）确定AAVExo递送的治疗基因在临床前动物中的有益效果 心力衰竭的模型。目的1：表征AAVExo并确定其基因递送 体外NAb逃逸的有效性和分子机制。目的2：确定基因递送效率， AAVExo在体内的亲心性和NAb逃避机制。目的3：优化AAVExo纯化 使用NanoDLD微流控装置，并确定AAVExo-SERCA 2a在小的和 具有预先存在的NAb的心力衰竭的大型动物模型。

SUMMARY Gene therapy is a promising approach for the treatment of various monogenic diseases including inherited cardiomyopathies and other types of heart failure. Adeno-associated vectors (AAV) are vectors of choice for delivering genes to cardiomyocytes for long term expression and due to their safety in clinics. However, a significant challenge to their successful use is futility caused by pre-existing antibodies (NAbs) as well as subsequent development of immunity following AAV administration. NAbs prevent AAVs from infecting target cells, greatly reducing transduction efficiency, and thus, clinical efficacy. Therefore, to advance gene therapies for cardiovascular treatment for a wider population, it is essential to develop strategies to circumvent NAbs. Exosomes are extracellularly secreted nano-vesicles that shuttle selective biomolecules between neighboring and distant cells. Recent studies have shown that exosomes can carry several types viruses and shield them from antibody neutralization. Delivery of AAVs protected by carrier exosomes is a promising approach to circumvent NAb neutralization in AAV-based gene therapy. Our in vitro and in vivo preliminary data suggest that AAV-encapsulating exosomes (AAVExo) are 1) more resistant to NAb neutralization as compared to free AAVs, 2) more efficient in delivering genes to the myocardium, 3) preserves viral cardiotropism, and 4) retains the therapeutic benefits of AAV-mediated gene delivery. Here, we aim to investigate the ability of AAVExo to evade NAbs and serve as a highly efficient gene delivery tool for cardiovascular therapeutics. We have developed a method to isolate highly pure AAVExo with minimum contamination from free-AAVs. Our central hypothesis is that AAVExo shields AAVs to evade NAb and enhance gene delivery to the myocardium compared to free AAVs. Our major goal is to develop a comprehensive understanding of the molecular mechanisms of NAb neutralization by AAVExo, and 2) to determine the beneficial effects of therapeutic genes delivered by AAVExo in preclinical animal models of heart failure. The specific aims are: AIM 1: Characterize AAVExo and determine its gene delivery efficacy and molecular mechanisms of NAb evasion in vitro. AIM 2: Determine the gene delivery efficacy, cardiotropism and mechanisms of NAb evasion of AAVExo in vivo. AIM 3: Optimize AAVExo purification using a NanoDLD microfluidic device and determine the beneficial effect of AAVExo-SERCA2a in small and large animal models of heart failure with preexisting NAb.