Novel Materials for Viral Purification

用于病毒纯化的新型材料

• 批准号: 9408588
• 负责人: BRADLEY T MESSMER
• 金额: $ 22.42万
• 依托单位: ABREOS BIOSCIENCES, INC.
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-01 至 2019-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9408588
• 关键词: Adenoviruses; Affinity; Area; Avidity; Binding; Cancer Vaccines; Capsid Proteins; Cell Culture Techniques; Cell Therapy; Cells; Chromatography; Column Chromatography; Communicable Diseases; Concatenated DNA; Coupling; DNA; DNA Library; Dependovirus; Development; Disease; Divalent Cations; Excision; Exhibits; Filtration; Future; Gene Transduction Agent; Goals; Immobilization; Individual; Label; Libraries; Magnetism; Malignant Neoplasms; Membrane; Methods; Modeling; Nucleic Acids; Oligonucleotides; Oncolytic; Preparation; Process; Production; Proteins; Protocols documentation; Reagent; Recovery; Residual state; Sepharose; Single-Stranded DNA; Source; Specificity; Stream; Structure; Subfamily lentivirinae; Techniques; Technology; Testing; Viral; Viral Vector; Virus; Viscosity; Work; base; bioprocess; cancer therapy; commercial application; cost; feeding; gene therapy; interest; magnetic beads; magnetite ferrosoferric oxide; nanoparticulate; novel; nuclease; operation; particle; tool; vector

Abstract Viruses have significant commercial application as vectors for gene and cell therapy, as well as vaccines for cancer treatment and infectious diseases. The purification of commercial viruses is a major area of bioprocess development. Current methods are mostly based on filtration and chromatography, both of which scale poorly and require clean feed streams to perform well. Viral particles are difficult to recover from culture supernatants or cell lysates because they are similar in size to cell debris. A large amount of expensive, high-quality nuclease (Benzonase) is typically needed to reduce the viscosity of the lysate sufficiently for efficient membrane and column operations. Affinity based vector purification has been developed for only one type of vector (some strains of adeno-associated virus), and is also very expensive. In summary, virus purification is challenging and significant amounts of valuable product are lost in inefficient filters. We have developed a novel DNA-based avidity reagent, termed DeNAno, that can provide a significant advance in the development and manufacturing of commercial viruses, with applicability to a variety of viral vectors. DeNAno uses massive avidity rather than affinity to capture biologic targets such as viruses. DeNAno particles are composed of a single-stranded DNA concatemer, which contains several hundred copies of a template oligonucleotide. The goal of this project is to develop DeNAno particles that bind to a target virus, and then use the DeNAno as a capture reagent for virus purification. DeNAno libraries are produced by rolling circle replication of a circular oligonucleotide template containing a random sequence, and specific viral binders are recovered by a biopanning process. DeNAno particles can be released from their targets by disrupting their secondary structure through divalent cation removal; they can also be labeled with nano- particulate magnetite or coated onto micron-sized magnetite to allow their use as magnetic capture reagents. We have already performed a selection on adenovirus-coated beads and obtained a pool of DeNAno particles that bind the target virus. The specific aims of this project are to: 1) characterize DeNAno particles that bind to the model virus (adenovirus) and 2) demonstrate their use as a purification tool by attaching them to magnetite so that they can be used in magnetic capture. The virus will be released following capture by divalent cation removal and the magnetic DeNAno cleared from the viral preparation, again by magnet. We will assess the capture efficiency, virus yield and infectivity, residual DeNAno contamination, and the presence of host cell proteins in the final preparation. If successful, this project will demonstrate the potential of the DeNAno technology to streamline virus production and pave the way for additional DeNAno-based applications targeting other commercially important viruses.

抽象的 病毒作为基因和细胞治疗的载体以及疫苗具有重要的商业应用 癌症治疗和传染病。商业病毒的纯化是生物过程的一个主要领域 发展。目前的方法主要基于过滤和色谱法，这两种方法的扩展性都很差 并且需要清洁的进料流才能良好运行。病毒颗粒很难从培养上清液中回收 或细胞裂解物，因为它们的大小与细胞碎片相似。量大价高，品质优良 通常需要核酸酶（Benzonase）来充分降低裂解物的粘度，以有效地 膜和柱操作。基于亲和力的载体纯化仅针对一种类型的 载体（某些腺相关病毒株），而且也非常昂贵。综上所述，病毒纯化是 具有挑战性的且大量有价值的产品在低效的过滤器中丢失。 我们开发了一种新型的基于 DNA 的亲和力试剂，称为 DeNAno，它可以提供显着的亲和力。 商业病毒的开发和制造取得进展，适用于多种病毒 向量。 DeNAno 使用大量的亲和力而不是亲和力来捕获病毒等生物目标。德纳诺 颗粒由单链DNA串联体组成，其中包含数百个拷贝 模板寡核苷酸。该项目的目标是开发与目标病毒结合的 DeNAno 颗粒，以及 然后使用 DeNAno 作为病毒纯化的捕获试剂。 DeNAno 文库是通过滚动生成的 含有随机序列和特定病毒的环状寡核苷酸模板的环状复制 通过生物淘选过程回收粘合剂。 DeNAno 粒子可以通过以下方式从其目标释放： 通过去除二价阳离子破坏其二级结构；它们也可以用纳米标记 颗粒状磁铁矿或涂覆在微米尺寸的磁铁矿上，以使其用作磁性捕获试剂。 我们已经对腺病毒包被的珠子进行了选择，并获得了 DeNAno 颗粒池 结合目标病毒。该项目的具体目标是：1）表征结合到的 DeNAno 粒子 模型病毒（腺病毒）和 2) 通过将它们附着在磁铁矿上来展示它们作为纯化工具的用途 以便它们可以用于磁捕获。病毒将被二价阳离子捕获后释放 去除并再次通过磁铁从病毒制剂中清除磁性 DeNAno。我们将评估 捕获效率、病毒产量和感染性、残留的 DeNAno 污染以及宿主细胞的存在 最终制剂中的蛋白质。如果成功，该项目将展示 DeNAno 的潜力 简化病毒生产并为其他基于 DeNAno 的应用铺平道路的技术 针对其他商业上重要的病毒。