GE Healthcare Fast Protein Liquid Chromatography FPLC product number 13470501.

GE Healthcare 快速蛋白液相色谱 FPLC 产品编号 13470501。

• 批准号: 7792259
• 负责人: JASTI S. RAO
• 金额: $ 9.47万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-03-25 至 2011-03-24
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7792259
• 关键词: Affinity; Affinity Chromatography; Alzheimer' s Disease; Anions; Binding Sites; Biocompatible Materials; Biological; Cations; Cause of Death; Charge; Chromatography; Dementia; Elderly; Engineering; Funding; Gene Expression; Goals; Healthcare; Heparin; Human; Ion-Exchange Chromatography Procedure; Ionic Strengths; Ligands; Liquid substance; Malignant Neoplasms; Methods; Modality; Phase; Preparation; Production; Recombinant adeno-associated virus (rAAV); Research; Research Personnel; Research Project Grants; Sepharose; Serotyping; Services; Social Welfare; Sodium Chloride; Solutions; System; Tail; Testing; Tissues; United States National Institutes of Health; Virus; cancer therapy; cost; fast protein liquid chromatography; gene therapy; high standard; immunogenic; interest; meetings; novel; public health relevance; therapeutic gene; tool; vector

DESCRIPTION (provided by applicant): Independent and productive investigators have NIH funded projects. These investigators all have keen well-established interests in developing novel gene therapy modalities for cancer and Alzheimer's disease (AD) using recombinant adeno-associated viruses (rAAVs) as a vehicle to deliver therapeutic genes. Despite recent rapid progress in cancer therapy, cancer is expected to be the leading cause of death worldwide in the year 2010. AD is the most common cause of dementia in the elderly. To date, however, no satisfactory treatments are available for most cancers and AD. Thus, further studies in cancer as well as AD treatment are essential for human welfare. Due to limited means to produce highly purified rAAVs with relatively large quantities, the progress of the research projects which require rAAVs have been slow. Therefore, the long term objectives are to meet the specific needs of investigators and facilitate their research projects by providing and maintaining high quality service for production of highly purified rAAVs. We will utilize fast protein liquid chromatography (FPLC) to purify rAAVs. A major strength of FPLC is that a biological material from a large number of impurities can be highly purified with a relatively large quantity. To achieve these goals, the specific aims are: (1) To assist in establishing highly efficient methods for purification of specific serotypes of rAAVs. (2) To provide investigators with highly purified rAAVs in a relatively large quantify for their research projects. (3) To maintain the highest standards in continuously providing purified rAAVs. The AKTAExplorer 100A FPLC system will enable us not only to meet the needs of investigators but also to facilitate their research projects by producing highly purified rAAVs. The AKTA FPLC system will be used to perform both affinity and ion exchange chromatography. Chromatography is commonly used in the purification of biological materials. Specifically, affinity chromatography involves the use of packing which has been chemically modified by attaching a compound with a specific affinity for the desired molecules, primarily biological compounds (ex. heparin for AAV2 and AAV6). The packing material used, called the affinity matrix, must be inert and easily modified. Agarose is the most common substance used, in spite of its cost. The ligands, or "affinity tails", that are inserted into the matrix can be genetically engineered to possess a specific affinity. The desired molecules adsorb to the ligands on the matrix until a solution of high salt concentration is passed through the column. This causes desorption of the molecules from the ligands, and they elute from the column. Ion exchange chromatography is commonly used in the purification of biological materials. There are two types of exchange: cation exchange in which the stationary phase carries a negative charge, and anion exchange in which the stationary phase carries a positive charge. Charged molecules in the liquid phase pass through the column until a binding site in the stationary phase appears. The molecule will not elute from the column until a solution of varying pH or ionic strength is passed through it. Separation by this method is highly selective. PUBLIC HEALTH RELEVANCE: Among the many viruses currently being developed as vectors for human gene therapy, AAV is one of the most promising tools because AAV is nonpathogenic, nontoxic, low immunogenic and allows longterm gene expression in many tissues. However, preparation of highly pure rAAV with a relatively large quantity has been problematic and challenging. The AKTA FPLC system will enable us to prepare highly pure rAAV from a large number of impurities, which are essential to test novel gene therapy modalities for cancer and Alzheimer's disease (AD). To date, however, no satisfactory treatments are available for either AD or many cancers making further research vitally important for human welfare.

描述（由申请人提供）：独立和富有成效的研究人员有NIH资助的项目。这些研究人员都有强烈的兴趣，在开发新的基因治疗模式，癌症和阿尔茨海默病（AD）使用重组腺相关病毒（rAAV）作为载体，提供治疗基因。尽管最近癌症治疗进展迅速，但预计癌症将成为2010年全球死亡的主要原因。AD是老年人痴呆症最常见的原因。然而，迄今为止，大多数癌症和AD都没有令人满意的治疗方法。因此，进一步研究癌症以及AD治疗对人类福利至关重要。由于生产相对大量的高纯度rAAV的手段有限，需要rAAV的研究项目进展缓慢。因此，长期目标是满足研究者的特定需求，并通过提供和保持高质量的服务来促进他们的研究项目，以生产高度纯化的rAAV。我们将利用快速蛋白液相色谱法（FPLC）纯化rAAV。FPLC的主要优点是可以相对大量地高度纯化来自大量杂质的生物材料。为了实现这些目标，具体目标是：（1）协助建立用于纯化特定血清型rAAV的高效方法。(2)为研究者的研究项目提供相对大量的高纯度rAAV。(3)在持续提供纯化rAAV方面保持最高标准。AKTAExplorer 100A FPLC系统不仅可以满足研究人员的需求，还可以通过生产高纯度的rAAV来促进他们的研究项目。AKTA FPLC系统将用于进行亲和色谱和离子交换色谱。色谱法通常用于生物材料的纯化。具体地，亲和色谱法涉及使用已经通过连接对所需分子（主要是生物化合物（例如，AAV2和AAV6用肝素）。所使用的填充材料，称为亲和基质，必须是惰性的，并且容易改性。琼脂糖是最常用的物质，尽管它的成本。插入基质中的配体或“亲和尾”可经遗传工程改造以具有特异性亲和性。所需的分子吸附到基质上的配体上，直到高盐浓度的溶液通过柱。这导致分子从配位体上解吸，并且它们从柱上脱离。离子交换色谱法通常用于生物材料的纯化。有两种类型的交换：阳离子交换，其中固定相携带负电荷，和阴离子交换，其中固定相携带正电荷。液相中的带电分子通过色谱柱，直到固定相中出现结合位点。只有当不同pH值或离子强度的溶液通过柱时，分子才能从柱上分离出来。用这种方法分离是高度选择性的。 公共卫生关系：在目前被开发作为人类基因治疗载体的许多病毒中，AAV是最有前途的工具之一，因为AAV是非致病性的，无毒的，低免疫原性的，并且允许在许多组织中长期基因表达。然而，制备相对大量的高纯度rAAV是有问题和挑战性的。AKTA FPLC系统将使我们能够从大量杂质中制备高纯度的rAAV，这些杂质对于测试癌症和阿尔茨海默病（AD）的新型基因治疗方式至关重要。然而，迄今为止，对于AD或许多癌症都没有令人满意的治疗方法，使得进一步的研究对人类福利至关重要。