Creating an optimized and scalable production platform in suspension adapted serum-free HEK293 cells for rAAV gene delivery using a Design-of-Experime

使用实验设计在悬浮适应的无血清 HEK293 细胞中创建优化且可扩展的生产平台,用于 rAAV 基因递送

基本信息

  • 批准号:
    2776049
  • 负责人:
  • 金额:
    --
  • 依托单位:
  • 依托单位国家:
    英国
  • 项目类别:
    Studentship
  • 财政年份:
    2023
  • 资助国家:
    英国
  • 起止时间:
    2023 至 无数据
  • 项目状态:
    未结题

项目摘要

The scope of the collaboration between the University College London and Sartorius is to establish a human embryonic kidney (HEK)293 cell line suspension platform for recombinant adeno-associated vector-based gene therapy manufacturing. Recombinant adeno-associated virus (rAAV) vectors are a leading gene delivery platform, and as June 2022, there are two rAAV-based gene therapies approved (Luxturna, in 2017, a rAAV2-based gene therapy and Zolgesma, in 2019, a rAAV9-base gene therapy) and more than seven-hundred active clinical trials. The conventional process for the manufacture of rAAV vectors is the usage of HEK293 cell line, which is transiently transfected with the genetic material required to produce the viral vector (either for research, pre-clinical and early clinical studies purposes) (Escandell et al., 2022). The widespread use of the HEK/transient transfection platform is due to recent developments supporting the design of the molecular constructs and the introduction of the plasmid DNA (pDNA) into HEK293 cells with chemical transfection reagents. However, vector production is a complex process that requires an efficient and optimized method of introducing the necessary virus genes into the cells (Dismuke & Kotin, 2017), suitable analytics and properly defined critical quality attributes (CQAs) for processes monitoring to ensure robustness and quality of the final product (Escandell et al., 2022). Despite advances in the clinic, rAAV vector manufacturing remains a challenging strategy for therapeutic applications requiring high systemic doses due to the high cost of materials (pDNA as GMP starting material), as well as a lack of process scalability and robustness. The recent development of HEK293 cell lines optimized for transfection in suspension culture has already improved the scale of production (Chahal et al., 2014). Also, stable producer cell lines represent a potential alternative to transient manufacturing systems, being more robust and easier to scale-up (Escandell et al., 2022). Although many strategies have been developed, to introduce the virus genes into HEK293 cells and to create serum-free, suspension-adapted cell lines, no rAAV production platform has been fully optimized or standardized (Dismuke & Kotin, 2017). Efficient and scalable production of rAAV is critical to enable lower cost of goods and easier drug commercialization for gene therapy applications. Therefore, the aim of the project is to optimize a transient and stable platform for suspension adapted HEK293 for rAAV production using Design of Experiments (DoE) to study the interaction of multiple factors and its impact on CQAs affecting the product quality. The process conditions and factors that impact rAAV vector production such as temperature, agitation, pH, production cell line, cell density, culture medium, harvest time, and plasmid concentrations (Zhao et al., 2020) will be investigated using rAAV2 as a representative vector to optimize virus production. Another rAAV serotype relevant to specific target tissue will be evaluated and further optimized in a data-driven approach. Sartorius transient and stable producer cell lines will serve as basis for the project. The transient cell was successfully adapted to growth in suspension culture using a proprietary serum-free, chemically defined media by direct adaptation. The PhD thesis will comprise selection of different cell clones based on cell growth and virus titer and the evaluation of different proprietary and benchmarking media and feed supplement / strategy. Analytical methods to measure metabolites, infectious titers, AAV protein identity, vector genome quantification, AAV capsid/titer, empty to full ratio and/or other product qualities were developed in-house, and samples will be retained for analysis (when performed by Sartorius).
伦敦大学学院和Sartorius之间的合作范围是建立一个人胚肾(HEK)293细胞系悬浮平台,用于基于重组腺相关载体的基因治疗生产。重组腺相关病毒(rAAV)载体是一种领先的基因递送平台,截至2022年6月,已有两种基于rAAV的基因疗法获得批准(Luxturna,2017年,基于rAAV 2的基因疗法和Zolgesma,2019年,基于rAAV 9的基因疗法)和700多项活跃的临床试验。用于制造rAAV载体的常规方法是使用HEK 293细胞系,其用产生病毒载体所需的遗传物质瞬时转染(用于研究、临床前和早期临床研究目的)(Escandell等人,2022年)。HEK/瞬时转染平台的广泛使用是由于最近的发展支持分子构建体的设计和用化学转染试剂将质粒DNA(pDNA)引入HEK 293细胞。然而,载体生产是一个复杂的过程,需要将必要的病毒基因引入细胞的有效和优化的方法(Dismuke & Kotin,2017),合适的分析和适当定义的关键质量属性(CQA)用于过程监测,以确保最终产品的稳健性和质量(Escandell et al.,2022年)。尽管在临床上取得了进展,但由于材料的高成本(pDNA作为GMP起始材料)以及缺乏工艺可扩展性和稳健性,rAAV载体制造对于需要高全身剂量的治疗应用仍然是一种具有挑战性的策略。最近开发的优化用于悬浮培养中转染的HEK 293细胞系已经提高了生产规模(Chahal等人,2014年)。此外,稳定的生产细胞系代表了瞬时制造系统的潜在替代方案,其更稳健且更容易扩大规模(Escandell等人,2022年)。虽然已经开发了许多策略,将病毒基因引入HEK 293细胞并创建无血清,悬浮适应的细胞系,但没有rAAV生产平台已经完全优化或标准化(Dismuke & Kotin,2017)。rAAV的高效和可规模化生产对于实现更低的商品成本和更容易的基因治疗应用的药物商业化至关重要。因此,该项目的目的是使用实验设计(DoE)优化用于rAAV生产的悬浮适应HEK 293的瞬时和稳定平台,以研究多个因素的相互作用及其对影响产品质量的CQA的影响。影响rAAV载体生产的工艺条件和因素如温度、搅拌、pH、生产细胞系、细胞密度、培养基、收获时间和质粒浓度(Zhao et al.,2020)将使用rAAV 2作为代表性载体进行研究以优化病毒生产。将评价与特定靶组织相关的另一种rAAV血清型,并以数据驱动的方法进一步优化。Sartorius瞬时和稳定生产细胞系将作为该项目的基础。通过直接适应,使用专有的无血清、化学成分确定的培养基,使瞬时细胞成功适应悬浮培养生长。博士论文将包括根据细胞生长和病毒滴度选择不同的细胞克隆,以及评估不同的专有和基准培养基和饲料补充剂/策略。内部开发了测量代谢物、感染滴度、AAV蛋白鉴别、载体基因组定量、AAV衣壳/滴度、空/满比和/或其他产品质量的分析方法,并将保留样品用于分析(当由Sartorius进行时)。

项目成果

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其他文献

吉治仁志 他: "トランスジェニックマウスによるTIMP-1の線維化促進機序"最新医学. 55. 1781-1787 (2000)
Hitoshi Yoshiji 等:“转基因小鼠中 TIMP-1 的促纤维化机制”现代医学 55. 1781-1787 (2000)。
  • DOI:
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    0
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LiDAR Implementations for Autonomous Vehicle Applications
  • DOI:
  • 发表时间:
    2021
  • 期刊:
  • 影响因子:
    0
  • 作者:
  • 通讯作者:
生命分子工学・海洋生命工学研究室
生物分子工程/海洋生物技术实验室
  • DOI:
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    0
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吉治仁志 他: "イラスト医学&サイエンスシリーズ血管の分子医学"羊土社(渋谷正史編). 125 (2000)
Hitoshi Yoshiji 等人:“血管医学与科学系列分子医学图解”Yodosha(涉谷正志编辑)125(2000)。
  • DOI:
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Effect of manidipine hydrochloride,a calcium antagonist,on isoproterenol-induced left ventricular hypertrophy: "Yoshiyama,M.,Takeuchi,K.,Kim,S.,Hanatani,A.,Omura,T.,Toda,I.,Akioka,K.,Teragaki,M.,Iwao,H.and Yoshikawa,J." Jpn Circ J. 62(1). 47-52 (1998)
钙拮抗剂盐酸马尼地平对异丙肾上腺素引起的左心室肥厚的影响:“Yoshiyama,M.,Takeuchi,K.,Kim,S.,Hanatani,A.,Omura,T.,Toda,I.,Akioka,
  • DOI:
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的其他文献

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{{ truncateString('', 18)}}的其他基金

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评估用于航空航天应用的新型抗疲劳钛合金
  • 批准号:
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  • 财政年份:
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