A Novel Strategy for Recombinant Adeno-Associated Virus Vector Production

描述（由申请人提供）：重组腺相关病毒（rAAV）载体对于治疗多种遗传性和获得性疾病具有广阔的前景。 rAAV 载体已在多种动物模型和将基因递送至肝脏、骨骼肌、中枢神经系统和视网膜等多种组织的临床试验中显示出治疗功效。 rAAV 载体对于基因治疗方法很有吸引力，因为载体可以纯化至高滴度，用于感染分裂或非分裂细胞，并且通常保持附加型 在转导细胞中，大大降低了插入突变的风险。目前生产 rAAV 载体的方法包括转染生产细胞系和使用腺病毒 (Ad)、疱疹病毒或杆状病毒载体进行感染。这些方法中的每一种对于 rAAV 生产都是有用的，但每种方法对于大规模 rAAV 载体生产也都存在一个或多个显着的限制。因此，迫切需要开发一种有效的、易于扩展的替代方法来生产临床级 rAAV 载体。 rAAV 载体的生产部分依赖于表达 AAV Rep 和 Cap 蛋白的能力。先前在Ad载体中维持和表达Rep基因的尝试被证明是非常困难的，因为Rep基因由于对Ad复制的未知毒性而不稳定。我们使用计算工程生成了 Rep 基因，该基因在重组 Ad 载体中连续传代时稳定维持。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，这是该领域的突破。该提案的第一个目标是开发一种 Ad 载体，以时间协调的方式和适当的表达水平表达 AAV2 Rep 和 Cap 蛋白，以指导最佳的 rAAV 生产。该提案的第二个目的是评估使用 Ad 载体大规模生产 rAAV 的能力，并将基于 Ad 的系统与使用疱疹病毒和杆状病毒方法生产 rAAV 的效率进行比较。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.