Development of a Production Method for Scaling Up Lentiviral Vector Manufacture

描述（由申请方提供）：本研究项目的长期目标是开发一种有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。“有一个关键的未满足的需求，开发强大的，可扩展的，封闭的，符合cGMP的生产系统，用于生成和生产慢病毒基因治疗载体，适用于临床和商业用途。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以及由于效率低、缺乏稳健性和与使用瞬时转染的标准方法相关的有限的可扩展性，慢病毒载体难以大量生产，瞬时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，这是一种高效、可扩展的工艺，用于工程化细胞功能，并使基于细胞的制造能够用于临床应用。在该项目的第1阶段，我们证明了该系统能够在小规模和大规模转染中在粘附和悬浮适应细胞中产生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生产设施中展示大规模慢病毒载体生产技术。这一成就将使FDA批准的细胞加载技术首次应用于通过在无血清培养基中培养的非粘附细胞中瞬时转染可规模化生产慢病毒载体。因此，该II期项目的具体目的是：1）优化培养悬浮适应细胞用于载体生产的培养条件; 2）实现MaxCyte细胞处理仪器与Wave细胞生产技术的无菌整合; 3）通过最大化转染细胞可产生和收集的感染性病毒颗粒数量来优化病毒生产能力; 4）证明完全整合的病毒生产系统在小商业规模下的可行性;和5）证明在cGMP生产设施中的大规模慢病毒载体生产。 印第安纳州大学载体生产设施（IUVPF）和SAFC Pharma对MaxCyte细胞加载技术的应用表示出的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将进行Aim 5的cGMP生产，从而证明将MaxCyte技术转移到大规模商业生产的能力。 公共卫生相关性：存在关键的未满足的需求，以开发用于生产适合于临床和商业用途的慢病毒基因治疗载体的稳健的、可扩展的、封闭的、符合cGMP的制造系统。该SBIR项目的第2阶段目标是优化和验证专有的细胞转染和生产系统，该系统为开发和生产用于临床应用的慢病毒基因治疗载体提供了一种有效的大规模商业化工艺。这种生产系统的好处将转化为更安全，更好，更经济的基因疗法更快地进入市场，使许多疾病领域的患者受益，这些疾病领域目前尚未满足需求，例如：帕金森病，年龄相关性黄斑变性，β地中海贫血，镰状细胞贫血，肌肉萎缩症，以及各种淋巴造血病理学，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.