Engineering AAV Vectors to Evade Antibody Neutralization

描述（由申请人提供）：基因治疗具有治疗和潜在治愈多种疾病的巨大潜力。然而，基因递送技术需要在安全性、效率和表达稳定性方面的显著改进，才能治疗大多数这些疾病。基于腺相关病毒（AAV）的载体已被证明在实验室和临床上都是非常有前途的，但它们仍然存在一些缺点。特别地，大多数人群已经暴露于AAV血清型2以及其他血清型，并且因此免疫系统被启动以中和AAV。AAV载体的抗体中和是一个既定的问题，并且细胞免疫应答也可能是一个挑战。我们将试图解决前一个问题，并将进一步研究涉及后者的基本机制。对于前者，我们已经开发了新的定向进化技术，以产生具有新特性的新的AAV突变体。具体地，产生在编码病毒外壳蛋白的衣壳基因中具有随机点突变的病毒的大文库，并且使用高通量筛选来选择具有新特性的变体。我们已经利用这种方法来产生具有改变的受体结合特性的变体，以及逃避抗体中和的变体，所述抗体极大地抑制野生型衣壳或外壳蛋白的AAV基因递送。我们将研究人类抗体逃避变体介导治疗基因红细胞生成素高效基因递送到携带抗腺相关病毒抗体的动物肌肉和肝脏的潜力。此外，虽然抗体中和AAV是一个既定的问题，但关于AAV与免疫系统其他组分的相互作用知之甚少。因此，将在体外和体内研究补体[和T细胞]免疫中和该病毒的基本机制。总而言之，病毒自然地为自己的目的而进化，这并不总是满足人类治疗的需要。因此，在这项工作中开发的将病毒再进化为增强的人类治疗剂的新方法将对增强的病毒基因递送载体（包括替代的AAV血清型以及其他载体）的分子工程产生广泛和普遍的影响。此外，它还将深入了解其他免疫系统成分对AAV的反应。

DESCRIPTION (provided by applicant): Gene therapy has vast potential for treating and potentially curing a wide variety of disorders. However, gene delivery technologies require significant improvements in safety, efficiency, and expression stability before the majority of these diseases can be treated. Vectors based on adeno-associated virus (AAV) have proven themselves to be highly promising, both in the laboratory and the clinic, but they still suffer from several shortcomings. In particular, the majority of the human population has been exposed to AAV serotype 2, as well as other serotypes, and as a result the immune system is primed to neutralize AAV. Antibody neutralization of AAV vectors is an established problem, and cellular immune responses may also be a challenge. We will attempt to solve the former problem and will further investigate basic mechanisms involved in the latter. For the former, we have developed novel directed evolution technology to generate new mutants of AAV with new properties. Specifically, large libraries of virus with random point mutations in the capsid gene encoding the viral coat protein are generated, and variants with novel properties are selected using high throughput screens. We have utilized this approach to generate variants with altered receptor binding properties, as well as variants that escape neutralization by antibodies that greatly inhibit AAV gene delivery by the wild type capsid or coat proteins. We will study the potential of human antibody evading variants to mediate high efficiency gene delivery of the therapeutic gene erythropoietin to the muscle and liver of animals carrying anti-AAV antibodies. In addition, while AAV neutralization by antibodies is an established problem, much less is known about AAV interactions with other components of the immune system. Therefore, the basic mechanisms of immune neutralization of this virus by complement [and T cells] will be investigated to both in vitro and in vivo. In summary, viruses have naturally evolved for their own ends, which do not always meet the needs of a human therapeutic. The novel approaches developed in this work to re-evolve viruses into enhanced human therapeutics will therefore have broad and general impact on the molecular engineering of enhanced viral gene delivery vehicles, including alternate AAV serotypes as well as other vectors. Furthermore, it will yield insights into the responses of other immune system components to AAV.