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Engineering AAV Vectors to Evade Antibody Neutralization

设计 AAV 载体以逃避抗体中和

基本信息

批准号：
7849654
负责人：
DAVID V SCHAFFER
金额：
$ 43.63万
依托单位：
UNIVERSITY OF CALIFORNIA BERKELEY
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-06-08 至 2011-05-31
项目状态：
已结题

项目摘要

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.

描述（由申请人提供）：基因疗法具有治疗和治愈多种疾病的巨大潜力。然而，在大多数此类疾病得到治疗之前，基因传递技术需要在安全性、效率和表达稳定性方面显着提高。基于腺相关病毒（AAV）的载体已被证明在实验室和临床中都非常有前途，但它们仍然存在一些缺点。特别是，大多数人群都曾接触过 AAV 血清型 2 以及其他血清型，因此免疫系统已做好中和 AAV 的准备。 AAV 载体的抗体中和是一个既定问题，细胞免疫反应也可能是一个挑战。我们将尝试解决前一个问题，并进一步研究后者涉及的基本机制。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。具体来说，生成了编码病毒外壳蛋白的衣壳基因中具有随机点突变的大型病毒文库，并使用高通量筛选选择具有新特性的变体。我们利用这种方法来生成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，这些抗体极大地抑制野生型衣壳或外壳蛋白的 AAV 基因传递。我们将研究人类抗体逃避变体介导治疗基因促红细胞生成素高效基因递送至携带抗 AAV 抗体的动物的肌肉和肝脏的潜力。此外，虽然抗体中和 AAV 是一个既定问题，但人们对 AAV 与免疫系统其他组成部分的相互作用知之甚少。因此，补体[和T细胞]免疫中和该病毒的基本机制将在体外和体内进行研究。总之，病毒自然进化是为了它们自己的目的，并不总是满足人类治疗的需要。因此，这项工作中开发的将病毒重新进化为增强型人类疗法的新方法将对增强型病毒基因传递载体（包括替代 AAV 血清型以及其他载体）的分子工程产生广泛而普遍的影响。此外，它将深入了解其他免疫系统成分对 AAV 的反应。