A novel producer cell line for more efficient manufacturing of viral vector systems

摘要 靶向递送载体及其治疗效果提高方面的重大进展 用于基因传递的载体已经被制造出来，激发了人们对发展和 专注于基因治疗适应症的治疗产品的商业化。CLAIRIgene LLC专注于 各种病毒基因到细胞转移传递平台的开发和优化具有广泛的针对性 应用范围广泛，包括CRISPR/CAS9系统。在开发中的基因治疗产品中，我们 重组腺相关病毒载体和整合酶缺陷型慢病毒 载体(IDLV)，因为它们在基因治疗适应症中显示出最大的交付潜力。两个系统 工作在与最低集成度相关的瞬时“肇事逃逸”模式下，这将大大降低 因此，插入突变、致癌和其他相关毒性的风险很高。 有利于基因治疗的应用。此外，我们最近进化了这些非积分向量 为CRISPR/Cas9组分的基因到细胞转移提供高效和安全的载体 原代细胞和组织，包括体外和体内的有丝分裂后神经元。尽管如此， 优势，包括IDLV和AAV在内的附体载体在相对较低的水平表达，因为它们的基因组 被包装成异染色质结构，在很大程度上无法被转录机器访问。始终如一， 我们先前证明了在载体生产步骤中添加组蛋白脱乙酰酶抑制物(HDACsi) 可显著提高其效价和转导效率。然而，大多数以小分子为基础的HDAC 抑制剂是非选择性的，并且明显表现出高水平的不受欢迎的非靶标活性，包括 细胞周期停滞，细胞分化和凋亡信号的变化。因此，更具选择性和 有必要采取有针对性的战略，以期降低人类发展援助中心感兴趣的表达。目前的建议是 根据我们的初步数据表明，HDAC家族的I类HDAC8可以发挥重要的作用 在包括AAV和IDLV在内的外体载体的生命周期调节中的作用。我们已经证明了向量- 生产细胞，人类胚胎肾脏HEK293T敲除HDAC8支持改进的包装 以及载体的滴度。 在目前的方案中，我们的目标是创建(I)稳定的、克隆的产生HDAC8-/-HEK293细胞系，以获得更多 高效生产AAV和IDLV载体。在进行表征和优化之后，我们的目标是认证 该产品适用于使用病毒载体作为递送系统的一系列基因治疗应用。评估 所开发的电池能否普遍用于其他非集成系统的生产，我们将 目的在未来检测它们制造腺病毒(Ad)、I型单纯疱疹病毒(HSV-1)和非 人类慢病毒载体。不断增长的细胞和基因治疗管道导致了 对高质量基因输送工具的需求；在这里，我们相信改进的生产细胞将创造一个 这为我们提供了支持这一需求的巨大商机。

ABSTRACT Significant advances in the specific targeting of delivery vectors and the increased therapeutic efficacy of such vectors for gene delivery have been made, stimulating major interest in the development and commercialization of therapeutic products focused on gene therapy indications. CLAIRIgene LLC focused on the development and optimization of various viral gene-to-cell transfer delivery platforms targeted a broad range of applications, including CRISPR/Cas9 systems. Of the gene therapy products in development, we focused on recombinant Adeno-Associated Virus (AAV)-based vectors and Integrase-Deficient Lentiviral Vectors (IDLVs), as they show the greatest potential for delivery in gene therapy indications. Both systems operate at transient “hit-and-run” mode, associated with minimal integration levels, which substantially reduces the risk of insertional mutagenesis, oncogenesis and other associated toxicities, therefore is highly advantageous for gene therapy applications. Furthermore, we recently evolved these non-integrating vectors onto efficient and safe delivery vehicles for gene-to-cell transfer of CRISPR/Cas9 components into various primary cells and tissues, including post-mitotic neurons in vitro and in vivo. Notwithstanding these advantages, episomal vectors, including IDLV and AAV expressed at relatively low levels, as their genomes are packaged into heterochromatin structures, largely inaccessible by transcription machinery. Consistently, we previously demonstrated that histone deacetylase inhibitors (HDACsi) added during vector production step can dramatically increase its titer and transduction efficiency. Nevertheless, most small-molecule-based HDAC inhibitors are non-selective, and evidently demonstrate high-level of undesirable off-target activities, including cell cycle arrest, changes in cellular differentiation and apoptotic signaling. Therefore, more selective and targeted strategies aimed to downregulate expression of HDAC-of-interest are needed. The current proposal is based on our preliminary data demonstrated that class I of the HDAC family HDAC8 could play an important role in the regulation of life cycle of episomal vectors, including AAV and IDLV. We have shown that vector- producer cells, human embryonic kidney HEK293T knockouted for HDAC8 support the improved packaging and titer of the vectors. In the current proposal, we aim to create (i) a stable, monoclonal producer HDAC8-/- HEK293 cell line for more efficient production of AAV and IDLV vectors. Following characterization and optimization, we aim to certify the product for a range of gene therapy applications using viral vectors as delivery systems. To evaluate whether the developed cells could be used universally for production of other non-integrating systems, we will aim in the future testing them for manufacturing adenovirus (Ad), herpes simplex virus type I (HSV-1), and non- human lentiviral vectors. The growing pipeline of cell and gene therapies has led to a substantial increase in demand to high quality gene delivery vehicles; here, we believe that the improved producer cells will create a significant business opportunity for us to support this demand.