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
基本信息
- 批准号:7801300
- 负责人:
- 金额:$ 26.33万
- 依托单位:
- 依托单位国家:美国
- 项目类别:
- 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:
- 关键词:AdenovirusesAnimalsAtherosclerosisBiological AssayBioreactorsCapsidCell LineCellsCholesterolClinicClinical TrialsCulture MediaCyclic GMPCytolysisDefectDependovirusDevelopmentDiseaseDyslipidemiasEvaluationFamilial HypercholesterolemiaGene Transduction AgentGenerationsGenesGenomeGoalsGovernmentGrantHarvestHeatingHemophilia AHepatitis BHepatocyteHumanHuman Cell LineHybridsInfectionInheritedLaboratoriesLeadLegal patentLicensingLiverLow Density Lipoprotein ReceptorMetabolicMethodsModelingModificationMusOrganPersonsPharmaceutical PreparationsPhaseProcessProductionProductivityPublic HealthRecombinantsRefractoryRelative (related person)RunningSamplingSatellite VirusesSerotypingSerumStagingSuspension CultureSuspension substanceSuspensionsSystemTechnologyTherapeuticTissuesTransfectionTransgenesTranslational ResearchVirionbasecellular transductioncommercial applicationflasksgene therapylarge scale productionnovelnovel therapeuticsparticlepremature atherosclerosispreventpromoterpublic health relevancereplicaseresearch studyscale upvector
项目摘要
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):本申请的总体目标是开发一种可扩展的生产系统,用于生产携带人低密度脂蛋白受体(hLDLR)转基因的基于腺相关病毒(AAV)血清型8的基因治疗载体,用于治疗家族性高胆固醇血症(FH).重组AAV 8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载体相比具有上级组织转导能力。AAV 8载体有效地靶向肝脏,因此是治疗血脂异常如FH的有前景的治疗剂。这种常染色体显性遗传疾病是由于低密度脂蛋白受体缺乏,传统的药物治疗是难治的。在动脉粥样硬化的鼠模型中,AAV 8-hLDLR载体能够使高达85%的肝细胞存活,纠正潜在的代谢缺陷并预防所产生的动脉粥样硬化。将该研究转化为临床的主要障碍是能够产生足够量的AAV 8载体。本文提出的用于生产AAV 8-hLDLR载体的可扩展方法将基于最初为AAV 2开发的腺病毒-AAV杂合(Ad杂合)系统。该系统完全依赖于腺病毒-AAV杂合感染以将重组AAV基因组递送至含有AAV衣壳基因的包装细胞系,因此适合与大规模悬浮培养物一起使用。我们的目标是产生包括AAV 8包装细胞系和hLDLR Ad杂合体的Ad杂合体系统组件,并使用它们以可接受的产率和产品效力生产AAV 8-hLDLR载体。还将研究一种新的修饰,其中双重腺病毒/Ad杂交感染过程被单一感染取代。将专门开发用于表征载体产率和效价的高通量精确测定法,以与Ad杂交系统一起使用。在I期的最后阶段,新衍生的包装线将适应于无血清条件下的悬浮培养,并在hLDLR Ad杂合感染后评估载体产率和效价。在第I阶段结束时,我们的目标是建立用于生产AAV 8-hLDLR的高产悬浮培养系统,其既适合于规模扩大又可为监管机构所接受。这些成就将带领我们进入第二阶段,在第二阶段,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优化上游工艺,开发可扩展的下游工艺,并纳入几种新的过程中和QC检测。
公共卫生相关性:靶向肝脏的基因疗法有可能治疗许多严重的公共卫生问题,包括B肝炎和丙型肝炎,以及遗传性疾病,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过早的动脉粥样硬化。该项目的总体目标是开发一个大规模的生产系统,用于家族性高胆固醇血症新型治疗药物的商业化生产。
项目成果
期刊论文数量(0)
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Karen Kozarsky其他文献
Karen Kozarsky的其他文献
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