Development of a Production Method for Scaling Up Lentiviral Vector Manufacture

描述（由申请人提供）：本研究项目的长期目标是开发一种高效的大规模商业系统，用于在悬浮细胞中制造慢病毒基因治疗载体。本提案是根据PA-06-013， “医疗、牙科和生物技术的制造工艺”提交的。开发稳健、可扩展、封闭、符合cgmp的生产系统，以生成和生产适合临床和商业用途的慢病毒基因治疗载体，这是一个关键的未满足需求。慢病毒载体正在开发用于许多治疗应用，临床研究正在进行中。不幸的是，由于缺乏稳定的包装细胞系，并且由于使用瞬态转染的标准方法效率低下，缺乏鲁棒性和有限的可扩展性，慢病毒载体难以大量生产，瞬态转染是制造过程中的关键步骤。MaxCyte开发了一种细胞转染和生产系统，该系统是一种高效，可扩展的工程细胞功能过程，并使基于细胞的制造能够用于临床应用。在该项目的第一阶段，我们证明了该系统能够在小规模和大规模转染中在贴壁和悬浮细胞中生产高滴度的慢载体。下一个挑战是优化和扩大无菌生产系统，并在cGMP生产设施中演示大规模慢载体生产技术。这一成就将使fda批准的细胞装载技术首次应用于无血清培养基中培养的非贴壁细胞中，通过瞬时转染可大规模生产慢病毒载体。因此，本二期项目的具体目标是：1)优化培养适合载体生产的悬浮细胞的培养条件；2)实现MaxCyte细胞处理仪器与Wave细胞生产技术的无菌整合；3)优化病毒生产能力，最大限度地提高转染细胞产生和收集的感染性病毒颗粒的数量；4)在小型商业规模上论证完全集成的病毒生产系统的可行性；5)在cGMP生产设施中演示大规模慢矢量生产。印第安纳大学载体生产设施（IUVPF）和SAFC制药公司对MaxCyte细胞装载技术的应用表达了兴趣，强调了该项目的重要性。这些设施为基因治疗临床试验提供慢病毒载体（和许多其他病毒载体）的合同制造服务。IUVPF将为Aim 5进行cGMP生产，从而展示将MaxCyte技术转移到大规模商业生产的能力。

DESCRIPTION (provided by applicant): The long-term goal of this research program is to develop an efficient large-scale commercial system for manufacture of lentiviral gene therapy vectors in suspension cells. This proposal is submitted in response to PA-06-013, "Manufacturing Processes of Medical, Dental and Biological Technologies." There is a critical unmet need to develop robust, scalable, closed, cGMP-compliant manufacturing systems for generating and producing lentivirus gene therapy vectors that are suitable for clinical and commercial use. Lentiviral vectors are being developed for a number of therapeutic applications and clinical studies are underway. Unfortunately, lentiviral vectors are difficult to produce in large numbers due to the lack of stable packaging cell lines and due to inefficiencies, lack of robustness, and limited scalabilities associated with standard methods using transient transfection, which is a key step in the manufacturing process. MaxCyte has developed a cell transfection and production system that is an efficient, scalable process for engineering cell function and enabling cell-based manufacturing for clinical applications. In Phase 1 of this project, we demonstrated that this system enables high titer lentivector production in adherent and suspension-adapted cells in both small and large scale transfections. The next challenge is to optimize and scale up an aseptic production system and demonstrate large scale lentivector production technology in a cGMP manufacturing facility. This achievement will enable the first application of an FDA-approved cell loading technology for scalable production of lentiviral vectors by transient transfection in non-adherent cells cultured in serum-free medium. Therefore, the Specific Aims of this Phase 2 project are to: 1) optimize culture conditions for culturing suspension-adapted cells for vector production; 2) achieve sterile integration of the MaxCyte cell processing instrumentation with the Wave cell production technology; 3) optimize viral production capacity by maximizing the number of infectious viral particles that can be produced by and collected from transfected cells; 4) demonstrate feasibility of the completely integrated viral production system at a small commercial scale; and 5) demonstrate large scale lentivector production in a cGMP manufacturing facility. The interest expressed in this application of the MaxCyte cell loading technology by Indiana University Vector Production Facility (IUVPF) and SAFC Pharma emphasizes the importance of the project. These facilities provide contract manufacturing services for lentiviral vectors (and a number of other viral vectors) for gene therapy clinical trials. IUVPF will perform cGMP manufacturing for Aim 5 and thereby demonstrate the ability to transfer the MaxCyte technology to large scale commercial manufacturing. Public Health Relevance: There is a critical unmet need to develop robust, scalable, closed, cGMP-compliant manufacturing systems for producing lentiviral gene therapy vectors that are suitable for clinical and commercial use. The goal of this Phase 2 SBIR project is to optimize and validate a proprietary cell transfection and production system that provides an efficient, large-scale commercial process for developing and manufacturing lentiviral gene therapy vectors for clinical applications. The benefits of this production system will translate into safer, better, cost effective gene therapies on the market more rapidly to benefit patients in many disease areas with unmet need today, such as: Parkinson's disease, age-related macular degeneration, beta-thalassemia, sickle-cell anemia, muscular dystrophy, and a variety of lympho-hematopoetic pathologies including HIV infection, leukemias, and lymphomas.