Scalable Production of New Generation Adeno-Associated Virus Gene Therapy Vectors

新一代腺相关病毒基因治疗载体的规模化生产

• 批准号: 7801300
• 负责人: Karen Kozarsky
• 金额: $ 26.33万
• 依托单位: REGENX BIOSCIENCES, LLC
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7801300
• 关键词: Adenoviruses; Animals; Atherosclerosis; Biological Assay; Bioreactors; Capsid; Cell Line; Cells; Cholesterol; Clinic; Clinical Trials; Culture Media; Cyclic GMP; Cytolysis; Defect; Dependovirus; Development; Disease; Dyslipidemias; Evaluation; Familial Hypercholesterolemia; Gene Transduction Agent; Generations; Genes; Genome; Goals; Government; Grant; Harvest; Heating; Hemophilia A; Hepatitis B; Hepatocyte; Human; Human Cell Line; Hybrids; Infection; Inherited; Laboratories; Lead; Legal patent; Licensing; Liver; Low Density Lipoprotein Receptor; Metabolic; Methods; Modeling; Modification; Mus; Organ; Persons; Pharmaceutical Preparations; Phase; Process; Production; Productivity; Public Health; Recombinants; Refractory; Relative (related person); Running; Sampling; Satellite Viruses; Serotyping; Serum; Staging; Suspension Culture; Suspension substance; Suspensions; System; Technology; Therapeutic; Tissues; Transfection; Transgenes; Translational Research; Virion; base; cellular transduction; commercial application; flasks; gene therapy; large scale production; novel; novel therapeutics; particle; premature atherosclerosis; prevent; promoter; public health relevance; replicase; research study; scale up; vector

DESCRIPTION (provided by applicant): The overall goal of this application is to develop a scalable production system for the manufacture of an adeno- associated virus (AAV) serotype 8-based gene therapy vector bearing a human low density lipoprotein receptor (hLDLR) transgene for the treatment of familial hypercholesterolemia (FH). Recombinant AAV8 vector is one of a group of new generation vectors exclusively licensed to ReGenX with superior tissue transduction capabilities compared to currently available AAV vectors. AAV8 vectors efficiently target the liver and are therefore promising therapeutics for the treatment of dyslipidemias such as FH. This autosomal dominant disorder results from a deficiency of LDL receptor and is refractory to traditional pharmacologic therapy. In murine models of atherosclerosis, AAV8-hLDLR vectors are able to transduce up to 85% of hepatocytes, correct the underlying metabolic defect and prevent the resulting atherosclerosis. A major barrier to the translation of this research to the clinic has been the ability to produce AAV8 vector in sufficient quantities. The scalable process proposed here for the production of an AAV8-hLDLR vector will be based on the adenovirus-AAV hybrid (Ad hybrid) system initially developed for AAV2. The system relies entirely on adenovirus-AAV hybrid infection to deliver the recombinant AAV genome to packaging cell lines which contain the AAV capsid gene and is therefore suitable for use with large scale suspension cultures. We aim to generate Ad hybrid system components including AAV8 packaging cell lines and hLDLR Ad hybrids and use them to produce AAV8-hLDLR vector with acceptable yields and product potency. A novel modification in which the dual adenovirus/Ad hybrid infection process is replaced with a single infection will also be investigated. High throughput precise assays to characterize vector yield and potency will be specifically developed for use with the Ad hybrid system. In the final stages of Phase I the newly derived packaging lines will be adapted to suspension culture under serum free conditions and vector yields and potency assessed following hLDLR Ad hybrid infection. By the conclusion of phase I we aim to have established a high yielding suspension culture system for the production of AAV8-hLDLR which is both amenable to scale up and acceptable to regulatory agencies. These accomplishments will lead us to Phase II where optimal system components will be re-derived at a cGMP facility. In Phase II we envisage bioreactor-based optimization of the upstream process, development of a scalable downstream process and incorporation of several new in-process and QC assays. PUBLIC HEALTH RELEVANCE: Gene therapy targeted to the liver has the potential to treat many diseases of serious public health concern, including Hepatitis B and C, and inherited diseases such as hemophilia and familial hypercholesterolemia, the latter of which is characterized by high cholesterol levels and premature atherosclerosis. The overall goal of this project is to develop a large-scale production system for the commercial manufacture of a novel therapeutic for familial hypercholesterolemia.

描述（由申请人提供）：该应用程序的总体目标是开发一种可扩展的生产系统，用于生产腺相关病毒（AAV）基于8的血清型基因治疗载体，该基因治疗载体带有人类低密度脂蛋白受体（HLDLR）转基因，以治疗家族性高胆固醇血症（FH）。与当前可用的AAV矢量相比，重组AAV8矢量是一组新一代向量之一，具有优质组织转导能力的较高的组织转导能力。 AAV8载体有效地靶向肝脏，因此是治疗血脂异常（例如FH）的有前途的治疗剂。这种常染色体显性疾病是由LDL受体缺乏引起的，对传统的药物治疗是难治性的。在动脉粥样硬化的鼠模型中，AAV8-HLDLR载体能够转导高达85％的肝细胞，纠正潜在的代谢缺陷并防止导致的动脉粥样硬化。这项研究向诊所转换的主要障碍是能够以足够数量生产AAV8载体。此处提出的用于生产AAV8-HLDLR矢量的可扩展过程将基于最初为AAV2开发的腺病毒-AAV混合动力车（AD混合）系统。该系统完全依靠腺病毒-AAV杂交感染来将重组AAV基因组传递到包装细胞系中，该细胞系包含AAV CAPSID基因，因此适合与大规模悬浮培养物一起使用。我们旨在生成AD混合系统组件，包括AAV8包装细胞系和HLDLR AD混合动力，并使用它们来生产具有可接受的产量和产品效力的AAV8-HLDLR载体。还将研究一种新型的修饰，其中还将研究双重腺病毒/AD杂种感染过程被单一感染所取代。高吞吐量精确的测定法以表征向量产量和效力，以与AD混合系统一起使用。在第一阶段的最后阶段，新得出的包装线将适用于无血清条件下的悬浮培养，并在HLDLR AD混合感染后评估的载体产量和效力。通过第一阶段的结论，我们的目标是建立一个高产的悬浮培养系统来生产AAV8-HLDLR，这既可以扩大规模且可以接受监管机构。这些成就将使我们进入II阶段，其中最佳系统组件将在CGMP设施中重新介绍。在第二阶段，我们设想基于生物反应器的上游过程的优化，开发可扩展的下游过程以及纳入了几种新的过程中和QC分析。 公共卫生相关性：针对肝脏的基因治疗有可能治疗许多严重的公共卫生问题，包括乙型肝炎和C，以及遗传性疾病，例如血友病和家族性高胆固醇血症，后者的特征是高胆固醇水平和早产性动脉粥样硬化。该项目的总体目标是开发一种大规模生产系统，用于为家族性高胆固醇血症的新型治疗性商业制造。