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

描述（由申请人提供）：本申请的总体目标是开发一种可扩展的生产系统，用于生产基于8血清型腺相关病毒（AAV）的基因治疗载体，该载体携带人类低密度脂蛋白受体（hLDLR）转基因，用于治疗家族性高胆固醇血症（FH）。重组AAV8载体是ReGenX独家授权的一组新一代载体之一，与目前可用的AAV载体相比，具有优越的组织转导能力。AAV8载体有效地靶向肝脏，因此有希望治疗血脂异常，如FH。这种常染色体显性疾病是由低密度脂蛋白受体缺乏引起的，对传统的药物治疗是难治的。在小鼠动脉粥样硬化模型中，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预防由此产生的动脉粥样硬化。将这项研究转化为临床的一个主要障碍是能否产生足够数量的AAV8载体。本文提出的用于生产AAV8-hLDLR载体的可扩展过程将基于最初为AAV2开发的腺病毒- aav杂交（Ad hybrid）系统。该系统完全依赖于腺病毒-AAV杂交感染，将重组AAV基因组传递到含有AAV衣壳基因的包装细胞系中，因此适合大规模悬浮培养。我们的目标是产生Ad杂交系统组件，包括AAV8包装细胞系和hLDLR Ad杂交，并使用它们来生产具有可接受产量和产品效力的AAV8-hLDLR载体。本文还将研究一种新的修饰方法，即用单一感染过程代替双重腺病毒/Ad杂交感染过程。将专门开发用于Ad杂交系统的高通量精确测定方法来表征载体产量和效力。在第一阶段的最后阶段，新衍生的包装线将适用于无血清条件下的悬浮培养，并在hLDLR和杂交感染后评估媒介产量和效力。在第一阶段结束时，我们的目标是建立一个高产的悬浮培养系统，用于生产AAV8-hLDLR，该系统既可以扩大规模，又可以被监管机构接受。这些成就将引导我们进入第二阶段，届时将在cGMP设施中重新获得最佳系统组件。在第二阶段，我们设想基于生物反应器的上游工艺优化，开发可扩展的下游工艺，并纳入几个新的过程中和QC分析。

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.