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

摘要 在递送载体的特异性靶向和这种递送载体的增加的治疗功效方面的显著进展是值得注意的。 已经制造了用于基因递送的载体，激发了对开发和 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递送平台的开发和优化针对广泛的 包括CRISPR/Cas9系统。在开发中的基因治疗产品中，我们 专注于基于重组腺相关病毒（AAV）的载体和整合酶缺陷型慢病毒 载体（IDLV），因为它们显示出在基因治疗适应症中递送的最大潜力。两个系统 在瞬时“打了就跑”模式下运行，与最小集成水平相关联，这大大降低了 因此，插入诱变、肿瘤发生和其它相关毒性风险是高度 对于基因治疗应用是有利的。此外，我们最近进化了这些非整合载体， 用于将CRISPR/Cas9组分的基因到细胞转移到各种细胞中的高效和安全的递送载体上。 原代细胞和组织，包括体外和体内的有丝分裂后神经元。尽管有这些 优点是，附加型载体，包括以相对低水平表达的IDLV和AAV，作为它们的基因组， 被包装成异染色质结构，很大程度上不能被转录机器所接近。我们一贯认为， 我们先前证明了在载体生产步骤中加入的组蛋白去乙酰化酶抑制剂（HDACsi） 可以显著增加其滴度和转导效率。然而，大多数基于小分子的HDAC 抑制剂是非选择性的，并且明显表现出高水平的不期望的脱靶活性，包括 细胞周期停滞、细胞分化和凋亡信号传导的变化。因此，更有选择性， 需要旨在下调目的HDAC表达的靶向策略。当前的提议是 基于我们的初步数据表明，HDAC家族的I类HDAC 8可以发挥重要作用， 在调节附加型载体（包括AAV和IDLV）的生命周期中的作用。我们已经证明了这个矢量- 生产细胞，敲除HDAC 8的人胚肾HEK 293 T支持改进的包装 和载体的滴度。 在目前的提议中，我们的目标是（i）建立一个稳定的单克隆生产HDAC 8-/-HEK 293细胞系，用于更多的研究。 AAV和IDLV载体的高效生产。经过表征和优化，我们的目标是证明 该产品用于一系列使用病毒载体作为递送系统的基因治疗应用。评价 无论开发的细胞可以普遍用于生产其他非整合系统，我们将 目的是在未来测试他们的生产腺病毒（Ad），单纯疱疹病毒I型（HSV-1），和非- 人慢病毒载体。不断增长的细胞和基因治疗管道导致了 高质量的基因载体的需求;在这里，我们相信，改进的生产细胞将创造一个 这对我们来说是一个巨大的商机，以支持这一需求。

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.