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

总结/摘要 基因疗法在解决遗传性疾病方面的成功已被FDA证实 基于AAV的LUXTURNA®和ZOLGENSMA®的批准。有很大的希望基因 治疗也将能够解决更多的10，000+单基因疾病， 识别日期。基因治疗开发人员面临成本和制造业准入的挑战 生产基因治疗产品的能力。超罕见病治疗的发展 遗传性疾病的发病率不到百万分之一， 因为这些疗法通常对生物制药商没有商业意义。 迫切需要创新，以满足基因疗法开发者的需求， 超罕见疾病，以获得小剂量药品的GMP生产。我们 提出一个灵活的，具有成本效益的，端到端的制造平台，用于生产小型 AAV基因治疗产品的剂量数。我们采用三重转染的方法 细胞，它可以适应多种遗传疾病，通过切换疾病特异性 基因组插入质粒之一，以及适应多种AAV血清型通过转换 AAV特异性质粒。我们易于使用的微型生物反应器平台可高密度培养细胞 灌注。我们将优化高密度细胞培养的三重转染过程， 增加生物反应器生产率并产生用于治疗少量患者的材料。 此外，我们的平台还满足了AAV下游纯化创新的需求。 产品.缺乏目的基因的空衣壳可占所产生的AAV的10% -90 在细胞培养中。目前的载体纯化方法需要专门的设备， 在标准实验室中不容易获得。我们将在下游开发一种新的 一种基于电动分离的纯化方法， 空AAV衣壳。最后，作为三重转染所需的GMP级质粒的供应， 过程也可能是一个瓶颈，以及显着的费用，我们将证明，我们的 平台能够整合质粒制造，包括生产和纯化， 以提供端到端的解决方案来产生AAV疗法。该平台将（1）加快 通过增加AAV的体积产量， 上游过程的成本最小化;（2）减少相关的时间，成本和劳动力 与下游AAV纯化;和（3）并入质粒生产以确保及时 和低成本供应这一关键原材料。这个平台将使病毒式的 为超罕见疾病的基因疗法开发者提供载体。

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.