Novel adeno-associated virus vector production system development

新型腺相关病毒载体生产系统的开发

• 批准号: 8311963
• 负责人: WEIDONG XIAO
• 金额: $ 45.07万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-02-15 至 2017-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8311963
• 关键词: Abbreviations; Adenovirus Vector; Adenoviruses; Adopted; Animal Experiments; Baculoviruses; Biomedical Engineering; Bioreactors; Cell Culture Techniques; Cell Line; Cell Nucleus; Cells; Centrifugation; Clinical; Clinical Trials; Cyclic GMP; DNA; Data; Dependovirus; Development; Disease; Dose; Engineering; Face; Factor IX; Future; Gene Expression; Gene Transduction Agent; Generations; Genes; Genetic Recombination; Genome; Goals; HeLa S3; Helper Viruses; Hemophilia A; Hemophilia B; Hereditary Disease; Herpesviridae; Human; Inverted Terminal Repeat; Leber' s amaurosis; Mediating; Mus; Patients; Plasmids; Procedures; Process; Production; Protocols documentation; Quality of life; RPE65 protein; Recombinant adeno-associated virus (rAAV); Recombinants; Reporting; Research; Research Project Grants; Role; Safety; Serotyping; Single Stranded DNA Virus; Suspension substance; Suspensions; System; Systems Development; TCF3 gene; Technology; Therapeutic; Transfection; Transgenes; Vaccinia; Vaccinia virus; Vaccinium; Viral; Virion; Virus Replication; Vision; adeno-associated viral vector; base; bioprocess; cost effective; flexibility; follow-up; gene therapy; human subject; improved; innovation; large scale production; manufacturing process; meetings; novel; particle; promoter; response; tool; transgene expression; vector

DESCRIPTION (provided by applicant): AAV is a single stranded DNA virus which has shown great promise as a gene therapy vector. Recombinant AAV vectors delivering RPE65 gene have been demonstrated to cure Leber's congenital amaurosis (LCA) disease and patients are able to regain normal vision after receiving the vectors. In hemophilia B clinical trial, patient receiving AAV vectors were able to express therapeutic level of factor IX gene expression. Systemic delivery of rAAV into human subjects would require a lot more vectors. We anticipate the demand for high quality rAAV vector for human clinical trials will exceed the current capability in our field. To face this challenge, we propose to improve rAAV vector production utilizing a new concept to eliminate wild type replication rcAAV particles and engineer scalable manufacturing processes. We would also investigate the complimentary helper function of adenovirus over cytoplasmic carrier helper in facilitating rAAV replication and packaging. In order to further improve the quality of recombinant AAV production for human gene therapy, we hypothesize that unwanted replication competent AAV particle formation could be eliminated by sequestering the helper function and vector sequences into different cellular compartments. The production and process system will eliminate plasmid transfection and other procedures which are not feasible for large scale production. Hence, our three specific aims are: 1. To study enhancement of AAV encapsidation by adenovirus in a cytoplasmic helper packaging system; 2. To develop a rAAV production system utilizing a cytoplasmic rep/cap carrier; 3. To engineer a protocol for scalable production of rAAV vectors free of contaminants. Completion of these specific aims will greatly enhance the way of rAAV vectors to be used in the field of human gene therapy. PUBLIC HEALTH RELEVANCE: The completion of this project will improve AAV production technology and allow a high quality rAAV vector to be produced economically in large scale. It has an immediate impact on human gene therapy and has the potential to improve the quality of life of patients with genetic diseases, especially for hemophilia which requires systemic delivery of vectors.

描述（由申请人提供）：AAV 是一种单链 DNA 病毒，作为基因治疗载体已显示出巨大的前景。携带RPE65基因的重组AAV载体已被证明可以治愈莱伯氏先天性黑蒙（LCA）疾病，患者在接受载体后能够恢复正常视力。在血友病 B 临床试验中，接受 AAV 载体的患者能够表达治疗水平的因子 IX 基因表达。将 rAAV 系统性递送至人类受试者体内需要更多的载体。我们预计人体临床试验对高质量 rAAV 载体的需求将超过我们领域目前的能力。为了应对这一挑战，我们建议利用新概念来改进 rAAV 载体的生产，以消除野生型复制 rcAAV 颗粒并设计可扩展的制造工艺。我们还将研究腺病毒相对于细胞质载体辅助病毒在促进 rAAV 复制和包装方面的互补辅助功能。为了进一步提高用于人类基因治疗的重组 AAV 生产的质量，我们假设可以通过将辅助功能和载体序列隔离到不同的细胞区室中来消除不需要的具有复制能力的 AAV 颗粒形成。该生产和工艺系统将消除质粒转染和其他不适合大规模生产的程序。因此，我们的三个具体目标是： 1. 研究细胞质辅助包装系统中腺病毒对AAV衣壳化的增强作用； 2. 开发利用细胞质rep/cap载体的rAAV生产系统； 3. 设计一个可规模化生产无污染物的 rAAV 载体的方案。这些具体目标的完成将大大增强rAAV载体在人类基因治疗领域的应用方式。 公共卫生相关性：该项目的完成将改进 AAV 生产技术，并允许大规模、经济地生产高质量的 rAAV 载体。它对人类基因治疗有立竿见影的影响，并有可能改善遗传性疾病患者的生活质量，特别是对于需要全身递送载体的血友病。