AAV-Exosomes: Escaping Neutralizing Antibody and Enhancing Delivery

摘要 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很有前途的方法。 心肌病和其他类型的心力衰竭。腺相关载体(AAV)是用于 将基因输送到心肌细胞进行长期表达，并在临床上安全使用。然而，a 对它们成功使用的重大挑战是先前存在的抗体(Nabs)以及 甲型肝炎病毒接种后的后续免疫发展。NAB可预防AAVs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，要推进基因 要为更广泛的人群提供心血管治疗的治疗方法，必须制定战略 绕过Nabs。 外切体是细胞外分泌的纳米囊泡，在相邻的细胞之间穿梭选择性的生物分子。 和遥远的细胞。最近的研究表明，外切体可以携带几种类型的病毒并对它们进行屏蔽 来自抗体中和。载体外切体保护的AAVs的传递是一种很有前途的方法 避免AAV基因治疗中的NAB中和。我们的体外和体内初步数据表明 与游离相比，AAV包囊外体(AAVExo)对NAB中和的抵抗力更强 AAVs，2)更有效地将基因输送到心肌，3)保留病毒的向心性，以及4)保留 AAV介导的基因传递的治疗益处。 在这里，我们旨在研究AAVExo作为一个高效基因来逃避nabs的能力 心血管治疗的输送工具。我们开发了一种分离高纯度AAVExo的方法 最大限度地减少了不含AAVs的污染。我们的中心假设是AAVExo将AAVs屏蔽到 与游离的AAVs相比，它可以避开NAB并增强对心肌的基因输送。我们的主要目标是 为了全面了解AAVExo中和NAB的分子机制， 2)确定AAVExo携带的治疗性基因在临床前动物中的有益效果 心力衰竭的模型。具体目标是：目标1：鉴定AAVExo并确定其基因传递 体外NAB逃避的有效性和分子机制。目的2：确定基因传递效率， AAVExo的趋心性及其逃避NAB的体内机制目标3：优化AAVExo纯化 使用NanoDLD微流控装置测定AAVExo-SERCA2a在小鼠和小鼠中的益处 预先存在NAB的心力衰竭大型动物模型。

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.