A Novel Strategy for Recombinant Adeno-Associated Virus Vector Production

重组腺相关病毒载体生产的新策略

• 批准号: 8601421
• 负责人: PATRICK HEARING
• 金额: $ 7.89万
• 依托单位: STATE UNIVERSITY NEW YORK STONY BROOK
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-01-01 至 2015-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8601421
• 关键词: Address; Adenovirus Vector; Adenoviruses; Amino Acid Sequence; Animal Model; Baculoviruses; Capsid; Cell Line; Cells; Clinical; Clinical Trials; Code; Codon Nucleotides; DNA biosynthesis; Disease; Dose; Engineering; Enhancers; Excision; Foundations; Gene Delivery; Gene Expression; Genes; Herpesviridae; Human; Infection; Inherited; Insertional Mutagenesis; Interphase Cell; Leber' s amaurosis; Liver; Measurable; Methods; Neuraxis; Patients; Production; Proteins; RPE65 protein; Recombinant adeno-associated virus (rAAV); Recombinants; Retina; Risk; Serial Passage; Serotyping; Side; Site; Skeletal Muscle; Structural Protein; System; Terminal Repeat Sequences; Therapeutic; Tissue-Specific Gene Expression; Tissues; Toxic effect; Transfection; Treatment Efficacy; Virion; Virus; Virus Replication; Vision; adeno-associated viral vector; base; cellular transduction; cis acting element; gene therapy; innovation; large scale production; nonhuman primate; novel; novel strategies; promoter; public health relevance; recombinase; response; success; vector

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.

描述（由申请人提供）：重组腺相关病毒（rAAV）载体对于各种遗传性和获得性疾病的治疗性治疗具有很大的希望。rAAV载体已经在多种动物模型和临床试验中显示出治疗功效，用于将基因递送至各种组织，例如肝脏、骨骼肌、中枢神经系统和视网膜。rAAV载体对于基因治疗方法是有吸引力的，因为载体可以被纯化至高滴度，用于感染分裂或非分裂细胞，并且通常保持游离型 在转导的细胞中，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感染。这些方法中的每一种都可用于rAAV生产，但每一种方法也遭受大规模rAAV载体生产的一个或多个显著限制。因此，非常需要开发一种有效的、易于扩展的替代方法来产生临床级rAAV载体。rAAV载体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。我们使用计算工程来产生Rep基因，其在重组Ad载体中连续传代稳定维持。这一观察将使我们能够开发利用Ad有效生产rAAV载体的方法，这是该领域的一个突破。该提议的第一个目的是开发一种Ad载体，其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。这些方法将为高效和大规模生产用于基因治疗的rAAV载体提供基础。