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.

摘要 病毒作为用于基因和细胞治疗的载体以及用于免疫治疗的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 发展目前的方法大多基于过滤和色谱法，这两种方法的规模都很小 并且需要清洁的进料流以良好地运行。病毒颗粒难以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。大量的昂贵的，高质量的 通常需要核酸酶（Benzonase）来充分降低裂解物的粘度，以有效地裂解物。 膜和柱操作。基于亲和性的载体纯化已经被开发用于仅一种类型的微生物。 载体（一些腺相关病毒株），也非常昂贵。总之，病毒纯化是 有挑战性的和大量的有价值的产品在低效的过滤器中损失。 我们开发了一种新的基于DNA的亲合力试剂，称为DeNAno，它可以提供显著的 在商业病毒的开发和制造方面取得了进展，适用于各种病毒 向量。DeNAno使用大量的亲合力而不是亲和力来捕获生物靶标，如病毒。德纳诺 颗粒由单链DNA多联体组成，其中含有数百个拷贝的 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， 然后使用DeNAno作为病毒纯化的捕获试剂。DeNAno库是通过滚动 含有随机序列的环状寡核苷酸模板的环状复制，和特异性病毒 通过生物淘选方法回收结合剂。DeNAno粒子可以通过以下方式从目标中释放出来 通过去除二价阳离子破坏它们的二级结构;它们也可以用纳米标记。 颗粒磁铁矿或涂覆在微米级磁铁矿上以允许它们用作磁性捕获试剂。 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 与目标病毒结合该项目的具体目标是：1）表征结合到 模型病毒（腺病毒）和2）通过将它们附着到磁铁矿上来证明它们作为纯化工具用途 这样它们就可以用于磁性捕获。病毒在被二价阳离子捕获后会被释放 去除并再次通过磁体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污染和宿主细胞的存在 蛋白质在最后的准备。如果成功，该项目将展示DeNAno的潜力 简化病毒生产的技术，并为其他基于DeNANO的应用程序铺平道路 针对其他商业上重要的病毒