Development of a Production Method for Scaling Up Lentiviral Vector Manufacture

描述(申请人提供)：该研究计划的长期目标是开发一种高效的大规模商业系统，用于在悬浮细胞中生产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。迫切需要开发健壮的、可扩展的、封闭的、符合cGMP的制造系统，以产生和生产适合临床和商业使用的慢病毒基因治疗载体。慢病毒载体正在被开发用于一些治疗应用，临床研究正在进行中。遗憾的是，由于缺乏稳定的包装细胞系，以及与使用瞬时转染法的标准方法相关的低效、缺乏稳健性和有限的可扩展性，慢病毒载体难以大量生产，而瞬时转染法是制造过程中的关键步骤。Maxcell已经开发了一种细胞转染和生产系统，这是一种高效、可扩展的过程，用于设计细胞功能并使基于细胞的制造能够用于临床应用。在这个项目的第一阶段，我们证明了这个系统能够在贴壁和悬浮适应的细胞中以小规模和大规模的转染法生产高滴度的慢病毒载体。下一个挑战是优化和扩大无菌生产系统，并在cGMP制造设施中演示大规模慢病毒载体生产技术。这一成果将使FDA批准的细胞装载技术首次应用于可规模化生产慢病毒载体，方法是在无血清培养的非贴壁细胞中瞬时转染。因此，这一二期项目的具体目标是：1)优化悬浮适应细胞的培养条件，用于载体生产；2)实现MaxCyte细胞处理仪器与Wave细胞生产技术的无菌集成；3)通过最大限度地提高转基因细胞能够生产和收集的传染性病毒颗粒的数量，优化病毒生产能力；4)证明完全集成的病毒生产系统在小型商业规模上的可行性；以及5)在cGMP制造设施中进行大规模慢病毒载体的生产。印第安纳大学载体生产设施(IUVPF)和SAFC Pharma对MaxCyte细胞装载技术的这一应用表示的兴趣强调了该项目的重要性。这些工厂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制造，从而展示将MaxCyte技术转化为大规模商业制造的能力。 与公共卫生相关：开发健壮的、可扩展的、封闭的、符合cGMP的制造系统，以生产适合临床和商业使用的慢病毒基因治疗载体，这是一个关键的未得到满足的需求。该二期SBIR项目的目标是优化和验证专利细胞转染和生产系统，该系统为临床应用的慢病毒基因治疗载体的开发和制造提供了一个高效、大规模的商业流程。这一生产系统的好处将更快地转化为市场上更安全、更好、更具成本效益的基因疗法，使许多疾病领域的患者受益，这些疾病目前尚未得到满足，例如：帕金森氏病、老年性黄斑变性、β-地中海贫血、镰状细胞性贫血、肌肉营养不良以及各种淋巴造血疾病，包括艾滋病毒感染、白血病和淋巴瘤。

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.