GE Healthcare Fast Protein Liquid Chromatography FPLC product number 13470501.

描述(由申请人提供)：独立和富有成效的调查人员有国家卫生研究院资助的项目。这些研究人员都热衷于开发利用重组腺相关病毒(RAAVs)作为载体传递治疗性基因的癌症和阿尔茨海默病(AD)的新型基因治疗模式。尽管癌症治疗最近取得了快速进展，但预计2010年癌症仍将成为全球主要的死亡原因。AD是导致老年人痴呆症的最常见原因。然而，到目前为止，还没有令人满意的治疗方法可用于大多数癌症和AD。因此，对癌症和阿尔茨海默病治疗的进一步研究对人类福祉至关重要。由于大量生产高纯度rAAVs的手段有限，需要rAAVs的研究项目进展缓慢。因此，长期目标是通过为生产高纯度rAAVs提供和保持高质量的服务来满足研究人员的特定需求并促进他们的研究项目。我们将利用快速蛋白质液相色谱(FPLC)纯化rAAVs。FPLC的一个主要优点是，从大量杂质中提取的生物材料可以以相对较大的数量高度纯化。为了实现这些目标，具体目标是：(1)协助建立高效的rAAVs血清型纯化方法。(2)为科研人员提供较大数量的高纯度rAAVs。(3)保持最高标准，持续提供纯化的rAAVs。AKTAExplorer 100A FPLC系统将使我们不仅能够满足研究人员的需求，还将通过生产高纯度的rAAVs来促进他们的研究项目。Akta FPLC系统将用于进行亲和层析和离子交换层析。层析法通常用于生物材料的提纯。具体地说，亲和层析涉及使用经过化学修饰的填料，通过附着对所需分子具有特定亲和力的化合物，主要是生物化合物(例如。AAV2和AAV6的肝素)。所使用的包装材料称为亲和力基质，必须是惰性的且易于修改。琼脂糖是最常用的物质，尽管它的成本很高。插入到基质中的配体或“亲和尾巴”可以通过基因工程获得特定的亲和力。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色谱柱。这会导致分子从配体上解吸，然后它们从柱子上洗脱。离子交换层析是一种常用的生物材料纯化方法。交换有两种类型：固定相带负电荷的阳离子交换和固定相带正电荷的阴离子交换。液相中的带电分子通过色谱柱，直到固定相中出现结合部位。在不同pH或离子强度的溶液通过色谱柱之前，分子不会从色谱柱中洗脱。用这种方法分离具有很高的选择性。 公共卫生相关性：在目前被开发为人类基因治疗载体的许多病毒中，AAV是最有前景的工具之一，因为AAV是非致病性、无毒、低免疫原性的，并允许在许多组织中长期表达基因。然而，制备相对大量的高纯度rAAV一直是一个难题和挑战。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)的新基因治疗方式至关重要。然而，到目前为止，无论是AD还是许多癌症，都没有令人满意的治疗方法，这使得进一步的研究对人类福祉至关重要。

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.