A Novel Strategy for Recombinant Adeno-Associated Virus Vector Production

描述(申请人提供)：重组腺相关病毒(RAAV)载体在治疗各种遗传性和获得性疾病方面具有很大的前景。RAAV载体已在多种动物模型和临床试验中显示出治疗效果，可将基因输送到各种组织，如肝脏、骨骼肌、中枢神经系统和视网膜。RAAV载体对于基因治疗方法很有吸引力，因为载体可以提纯到高滴度，用于感染分裂或未分裂的细胞，并且通常保持上体。 极大地降低了插入突变的风险。目前制备重组腺病毒载体的方法包括转染腺病毒(Ad)、疱疹病毒或杆状病毒载体。这些方法中的每一种都对rAAV生产有用，但每一种方法在大规模rAAV载体生产方面也都受到一个或多个显着限制。因此，有必要开发一种高效的、容易扩展的替代方法来生产临床级rAAV载体。RAAV载体的生产在一定程度上依赖于AAV Rep和Cap蛋白的表达能力。先前在Ad载体中保持和表达Rep基因的尝试被证明是非常困难的，因为由于对Ad复制的未知毒性，Rep基因不稳定。我们利用计算工程技术在重组的Ad载体中成功构建了一个Rep基因，并在连续传代过程中保持稳定。这一观察结果将使我们能够开发利用Ad高效生产rAAV载体的方法，这是该领域的一项突破。这项建议的第一个目的是开发一种以时间协调的方式在适当的表达水平表达AAV2 Rep和Cap蛋白的Ad载体，以指导最佳的rAAV生产。该建议的第二个目的是评估使用Ad载体大规模生产rAAV的能力，并比较基于Ad的系统与使用疱疹病毒和杆状病毒方法生产rAAV的效率。这些方法将为高效、大规模生产用于基因治疗的rAAV载体奠定基础。

DESCRIPTION (provided by applicant): Recombinant Adeno-Associated Virus (rAAV) vectors hold great promise for therapeutic treatment of a variety of inherited and acquired diseases. rAAV vectors have shown therapeutic efficacy in a variety of animal models and in clinical trials for gene delivery to various tissues such as liver, skeletal muscle, central nervous system, and the retina. rAAV vectors are attractive for gene therapy approaches since the vectors may be purified to high titers, used to infect dividing or non-dividing cells, and usually remain episomal in transduced cells greatly reducing the risk of insertional mutagenesis. Current approaches to produce rAAV vectors include transfection of producer cell lines and infection using adenovirus (Ad), herpesvirus, or baculovirus vectors. Each of these approaches is useful for rAAV production but each approach also suffers from one or more significant limitations for large-scale rAAV vector production. Thus, there is a significant need to develop an efficient, readily scalable, alternative approach to produce clinical grade rAAV vectors. rAAV vector production relies, in part, on the ability to express the AAV Rep and Cap proteins. Prior attempts to maintain and express the Rep gene in an Ad vector proved very difficult since the Rep gene was not stable due to an unknown toxicity to Ad replication. We used computational engineering to generate a Rep gene that is stably maintained over serial passage in a recombinant Ad vector. This observation will allow us to develop approaches to utilize Ad for the efficient production of rAAV vectors, a breakthrough in the field. The first aim of this proposal is to develop an Ad vector expresses the AAV2 Rep and Cap proteins in a temporally coordinated manner and at the appropriate expression levels to direct optimal rAAV production. The second aim of this proposal is to evaluate the ability to produce rAAV on a large-scale using Ad vectors and to compare the efficiency of the Ad-based system to rAAV production using the herpesvirus and baculovirus approaches. These approaches will provide the foundation for the efficient and large-scale production of rAAV vectors for use in gene therapy.