Engineering AAV Vectors to Evade Antibody Neutralization

基因疗法在治疗和潜在治愈各种疾病方面具有巨大的潜力。然而， 基因传递技术需要显著提高安全性、效率和表达稳定性 在这些疾病的大部分能够被治疗之前。基于腺相关病毒(AAV)的载体 在实验室和临床上都证明了自己是非常有前途的，但他们仍然遭受着 有几个缺点。特别是，大多数人类人口都接触到了AAV。 血清型2以及其他血清型，因此免疫系统准备好中和AAV。 AAV载体的抗体中和是一个既定的问题，细胞免疫反应也可能 成为一个挑战。我们将尝试解决前面的问题，并将进一步研究基本机制 牵涉到后者。对于前者，我们开发了新颖的定向进化技术来生成 具有新特性的AAV新突变体。具体地说，具有随机点突变的大型病毒库 产生编码病毒外壳蛋白的衣壳基因，并且具有新特性的变体是 使用高吞吐量筛网进行选择。我们已经利用这种方法生成了带有更改的变体 受体结合特性，以及逃脱抗体中和的变异体 AAV基因由野生型衣壳蛋白或外壳蛋白传递。我们将研究人类抗体的潜力 避开突变体以介导治疗性基因促红细胞生成素的高效基因传递到肌肉 以及携带抗AAV抗体的动物的肝脏。此外，虽然抗体中和AAV是一种 已确定的问题，关于AAV与免疫的其他成分的相互作用知之甚少 系统。因此，补体[和T细胞]免疫中和该病毒的基本机制 将从体外和体内两方面进行研究。总而言之，病毒是为它们自己而自然进化的。 目的，这并不总是满足人类治疗的需要。在这方面开发的新方法 因此，将病毒重新进化为增强的人类疗法的工作将对 增强型病毒基因传递载体的分子工程，包括替代AAV血清型，如 以及其他媒介。此外，它还将深入了解其他免疫系统的反应。 组件连接到AAV。

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.