Flexible Platform for End-to-end Manufacturing of Gene Therapies to Advance Development of Treatments for Ultra-rare Diseases

摘要/摘要 FDA已经证明了基因疗法在解决遗传性疾病方面的成功 批准基于AAV的LUXTURNA®和ZOLGENSMA®。有很大的希望基因 治疗方法也将能够解决更多的10,000+单基因疾病，这些疾病已经 到目前为止已经确定了。基因疗法研发人员面临成本和生产渠道方面的挑战 生产基因治疗产品的能力。超罕见疾病的治疗进展 遗传疾病的发病率不到百万分之一，尤其处于不利地位。 因为这些疗法通常对生物制药制造商没有商业利益。 迫切需要创新，以满足基因疗法开发商的需求 超罕见病获得GMP生产的小剂量药品。我们 提出一种灵活、经济高效的端到端制造平台，用于生产小型 甲型肝炎病毒基因治疗产品的剂量。我们在HEK中使用了三重转染法 细胞，可以通过切换疾病特异性来适应多种遗传性疾病 基因组插入到其中一个质粒中，并通过切换适应多种AAV血清型 甲型肝炎病毒特异的质粒。我们易于使用的微生物反应器平台将细胞培养到高密度 在灌流中。我们将优化高密度细胞培养的三重转染工艺，以 提高生物反应器的生产率，并产生用于治疗少量患者的材料。 此外，我们的平台满足了AAV下游纯化方面的创新需求 产品。缺乏目的基因的空衣壳可以构成所产生的AAV的10%-90% 在细胞培养中。目前的媒介纯化方法需要专门的设备，这些设备可能 在标准实验室中不易获得。我们将在下游开发一种新的 一种基于电动分离的提纯方法，大大简化了对 空空的AAV衣壳。最后，由于提供了三次转染所需的GMP级质粒 流程也可能是瓶颈，也可能是一笔巨大的费用，我们将证明我们的 Platform能够整合质粒制造，包括生产和纯化， 为AAV疗法的产生提供端到端的解决方案。这个平台，将(1)加快 通过增加AAV的体积产率来治疗超罕见疾病的可用性 上游流程，最大限度地降低成本；(2)减少相关时间、成本和劳动力 与下游AAV的纯化；以及(3)结合质粒生产，确保及时 以及这种关键原材料的低成本供应。这一平台将使对病毒的访问大众化 为超罕见疾病的基因疗法的开发者提供载体。

SUMMARY / ABSTRACT The success of gene therapies for addressing inherited disorders has been demonstrated by FDA approval of AAV-based LUXTURNA® and ZOLGENSMA®. There is great hope that gene therapies will also be able to address more of the 10,000+ monogenic diseases that have been identified to date. Gene therapy developers face challenges in cost and access to manufacturing capacity to produce the gene therapy product. The development of treatments for ultra-rare genetic diseases, which occur in fewer than one person in a million, are especially disadvantaged as these therapies are not typically of commercial interest to biopharmaceutical manufacturers. There is an urgent need for innovation in meeting the need for developers of gene therapies for ultra-rare diseases to gain access to GMP manufacturing of small doses of drug product. We propose a flexible, cost-efficient, end-to-end manufacturing platform for production of a small number of doses of AAV gene therapy products. We use the method of triple transfection in HEK cells, which can be adapted to multiple genetic diseases by switching the disease-specific genomic insert in one of the plasmids, as well as adapted to multiple AAV serotypes by switching the AAV-specific plasmids. Our easy-to-use microbioreactor platform grows cells to high-density in perfusion. We will optimize the the triple-transfection process for high-density cell cultures to increase bioreactor productivity and generate material for treating small numbers of patients. Additionally, our platform addresses the need for innovation in downstream purification of AAV products. Empty capsids lacking the gene of interest can compose 10% - 90% of AAV produced in cell cultures. Current vector purification approaches require specialized equipment which may not be readily available in the standard laboratory. We will develop a novel downstream purification method based on electrokinetic separation which greatly simplifies elimination of empty AAV capsids. Finally, as the supply of GMP-grade plasmid needed for the triple transfection process can also be a bottleneck, as well as significant expense, we will demonstrate that our platform is capable of incorporating plasmid manufacturing, including production and purification, to provide an end-to-end solution to generation of AAV therapies. This platform, will (1) expedite the availability of AAV treatments for ultra-rare diseases by increasing the volumetric yields of upstream process with minimization of the cost; (2) reduce the time, cost and labor associated with the downstream AAV purification; and (3) incorporate plasmid production to ensure timely and low-cost supply of this critical raw material. This platform will democratize access to viral vector for developers of gene therapies for ultra-rare diseases.