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

用于更有效地制造病毒载体系统的新型生产细胞系

基本信息

批准号：
10597799
负责人：
Elaine Hamm
金额：
$ 33.05万
依托单位：
CLAIRIGENE, LLC
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-01-12 至 2025-01-11
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10597799
关键词：
Adenoviruses Apoptosis Apoptotic Biological Products Biological Response Modifier Therapy Biological Sciences Biotechnology Businesses CRISPR/Cas technology Cell Cycle Arrest Cell Line Cell Proliferation Cell Therapy Cells Certification Chemicals Clinical Clinical Trials Clustered Regularly Interspaced Short Palindromic Repeats Collaborations Data Dependovirus Development Dideoxy Chain Termination DNA Sequencing Dose Duchenne muscular dystrophy Embryo Engineering Enzymes Family Fluorescence-Activated Cell Sorting Foundations Future Gene Delivery Gene Transfer Genes Genetic Transcription Genome Genotype Growth Guide RNA HDAC8 gene Heterochromatin Histone Deacetylase Histone Deacetylase Inhibitor Human In Vitro Insertional Mutagenesis Integrase Kidney Laboratories Lentivirus Lentivirus Vector Life Cycle Stages Mediating Methods Microscopy Molecular Morphology Neurons Oncogenic Open Reading Frames Patients Phase Phase I/II Clinical Trial Phenotype Play Production Proteins Protocols documentation Qualifying RNA Recombinant adeno-associated virus (rAAV)Regulation Reporter Risk Risk Reduction Role Running Safety Serious Adverse Event Signal Transduction Simplexvirus Small Business Technology Transfer Research Solid Structure System Testing Therapeutic Time Tissues Toxic effect Transfection Treatment Efficacy Universities Viral Viral Genes Viral Packaging Viral Vector Virion Virus Western Blotting Work adeno-associated viral vector commercialization delivery vehicle exome sequencing experience gene therapy immunogenicity improved in vivo interest loss of function manufacture manufacturing process manufacturing technology novel postmitotic programs prototype rational design research and development screening small molecule success targeted delivery transcriptome sequencing transduction efficiency tumorigenesis vector

项目摘要

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.

抽象的特定靶向载体的特定靶向量和此类治疗效率提高的重大进展已经建立了基因传递的载体，刺激了对发展的主要兴趣和侧重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针对广泛应用程序范围，包括CRISPR/CAS9系统。在开发基因治疗产品中，我们专注于重组腺相关病毒（AAV）的载体和整合酶缺陷型慢病毒载体（IDLV），因为它们显示出基因治疗适应症的最大潜力。两个系统在瞬态“撞击”模式下运行，与最小的集成水平相关，这大大降低了因此，插入诱变，肿瘤发生和其他相关毒性的风险很高对于基因治疗应用有利。此外，我们最近进化了这些非整合向量进入高效且安全的送货车，以将CRISPR/CAS9组件转移到各种基因转移到各种原代细胞和组织，包括体外和体内的有丝分裂后神经元。尽管如此优点，偶发向量，包括IDLV和AAV，以相对较低的水平表示为基因组被包装到异染色质结构中，在很大程度上通过转录机制无法访问。一贯我们先前证明了在载体生产步骤中添加的组蛋白脱乙酰基酶抑制剂（HDACSI）可以大大提高其滴度和翻译效率。然而，大多数基于小分子的HDAC 抑制剂是非选择性的，显然表现出高度不良的脱离目标活动，包括细胞周期停滞，细胞分化的变化和凋亡信号传导。因此，更有选择性和需要采取旨在下调HDAC兴趣表达的目标策略。当前的建议是根据我们的初步数据，HDAC家族HDAC8的I类可能发挥重要作用在包括AAV和IDLV在内的各个媒介生命周期的调节中的作用。我们已经表明矢量 - 生产者细胞，人类胚胎肾脏HEK293T敲除HDAC8支持的改进包装和向量的滴度。在当前的建议中，我们旨在创建（i）一个稳定的单克隆生产商HDAC8 - / - HEK293细胞系，以获取更多信息 AAV和IDLV向量的有效生产。遵循表征和优化，我们旨在证明使用病毒向量作为输送系统的一系列基因治疗应用的产品。评估是否可以普遍使用开发的细胞来生产其他非整合系统，我们将旨在将来测试它们用于制造腺病毒（AD），单纯疱疹病毒I型（HSV-1）和非 - 人慢病毒载体。细胞和基因疗法的渠道不断增长导致大幅增加对高质量基因输送车的需求；在这里，我们认为改进的生产商细胞将创建一个我们支持这一需求的巨大商机。