AKTA Pure L

AKTA 纯 L

• 批准号: 9075312
• 负责人: JOSEPH E RABINOWITZ
• 金额: $ 5.26万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9075312
• 关键词: 10 year old; Adenoviruses; Animal Model; Animals; Canis familiaris; Cardiovascular system; Cells; Dialysis procedure; Failure; Family suidae; Funding; Genes; Grant; Heart; In Vitro; Injection of therapeutic agent; Institution; Ion Exchange; Methods; Modeling; Mus; Production; Program Research Project Grants; Proteins; Recombinant adeno-associated virus (rAAV); Recombinants; Research; Research Personnel; Research Project Grants; Resources; Rodent; Rodent Model; Running; Schedule; Serotyping; Sheep; Subfamily lentivirinae; Time; Transgenes; United States National Institutes of Health; Universities; Viral Vector; Virus; aged; base; cesium chloride; density; fast protein liquid chromatography; gene replacement; in vivo; instrument; instrumentation; iodixanol; medical schools; particle; recombinant virus; translational medicine; vector

 DESCRIPTION (provided by applicant): We are applying for an S10 Shared Instrumentation Grant entitled "FPLC based purification of recombinant Adeno-associated virus (rAAV)" to develop new methods of virus purification to support two Program Project Grants and additional Principle Investigators (PIs) at the Temple University School of Medicine who require rAAV serotypes for in vivo studies. The primary purpose of this proposal is to fund the purchase of a General Electric AKTA pure fast protein liquid chromatography (FPLC) instrument. This instrument will replace the >10 years old AKTA-FPLC currently being used, but discontinued by GE. Numerous studies within the Center for Translational Medicine and the Cardiovascular Research Center are currently using rAAV, purified in the vector core, for animal studies of gene replacement/enhancement to augment protein levels in the heart after failure. The vast majority of rAAV produced is used in small rodent models, recently however, Canine and Porcine models have been used to deliver transgenes to the heart using rAAV. We are currently using three and four step purification methods consisting of Iodixanol gradient of crude cell lysate, followed by ion exchange FPLC then dialysis for the three step method. The four-step method includes a cesium chloride density gradient after FPLC and before dialysis. The four-step method is used on viruses intended for large animal studies. The larger animal models require larger physical titers of rAAV, for example; a 25 kg pig weighs 1000 times more than a 25 g mouse. We have previously generated rAAV6 and rAAV9 for injection of 1 x 1014 particles into sheep (REF). Currently the viral vector core serves two PPG with 6 investigators and greater than 6 additional investigators at Temple University School of Medicine and several investigators at other institutions. The NIH funded research projects described in this application all require purified rAAV. The current instrument AKTA-FPLC cannot scale to accommodate the current demand for AAV purification, it was purchased for small animal model and in vitro AAV purification. Investigators are now using the purified AAV for transduction in pig and dog animal models, orders of magnitude larger than rodents. We are now performing greater numbers of FPLC runs/week to maintain our production schedule. In this application we propose to replace an aged workhorse instrument with a new workhorse FPLC for the continued purification of recombinant AAV, and potentially column purification of other recombinant viruses required by the PIs of the Temple University School of Medicine. The establishment of this instrument in the Viral Vector Core will serve two Aims: Aim 1: Generate highly purified rAAV for in vivo research by NIH-funded groups at Temple University School of Medicine. Aim 1A: Minimize the time between starting virus production for a large animal study and completing production from months to several weeks. Aim 1B: Develop stocks of marker genes for each serotype and make this resource available to PIs at Temple University School of Medicine. Aim 2: Develop new column purification methods for Adenovirus and Lentiviruses as well as additional serotypes of AAV.

 描述（由应用程序提供）：我们正在申请S10共享仪器赠款，标题为“基于FPLC的重组腺相关病毒（RAAV）的纯化”，以开发新的病毒纯化方法，以支持两种计划项目赠款和其他主要研究人员（PIS）在Temple University of Medicine of Medicine of Medicine of Raav SerotypeS for Invivo In Invivo研究。该提案的主要目的是资助购买通用Akta纯快速蛋白质液相色谱（FPLC）仪器的购买。该仪器将取代目前使用的> 10年历史的Akta-FPLC，但被GE停用。当前，在转化医学中心和心血管研究中心的大量研究中，正在使用纯化的Raav，用于矢量核心，用于衰竭后心脏增强蛋白质水平的基因替代/增强的动物研究。产生的绝大多数RAAV用于小型啮齿动物模型，但是最近，犬和猪模型已用于使用RAAV将翻译为心脏。我们目前正在使用三种和四个步骤纯化方法，这些方法由粗细细胞裂解物的碘糖梯度组成，然后是离子交换FPLC，然后是三个步骤方法的透析。四步方法包括FPLC后和透析之前的氯化邻苯康属密度梯度。四步方法用于用于大型动物研究的病毒。例如，较大的动物模型需要更大的物理滴度。 25千克猪的重量比25克小鼠高1000倍。我们以前已经生成了RAAV6和RAAV9，用于将1 x 1014颗粒注入绵羊（参考）。目前，病毒媒介核心为两名PPG提供了6名调查人员的服务，并在Temple University医学院和其他机构的几位调查人员提供了更多的调查员。 NIH资助的研究项目中描述的 所有需要纯化的Raav。当前的仪器AKTA-FPLC无法扩展以适应当前对AAV纯化的需求，它是用于小型动物模型和体外AAV纯化的。研究人员现在使用纯化的AAV在猪和狗动物模型中翻译，比啮齿动物大的数量级。现在，我们每周进行更多的FPLC运行，以维持我们的生产计划。在此应用程序中，我们建议用新的主力FPLC代替一种老年人的主力仪器，以持续纯化重组AAV，并有可能对坦普尔大学医学院PIS所需的其他重组病毒纯化。在病毒媒介核心中建立该乐器将达到两个目的：目标1：在Temple University医学院的NIH资助小组为体内研究生成高度纯化的RAAV。 AIM 1A：最大程度地减少大型动物研究的病毒生产与完成几个月至数周之间的生产之间的时间。 AIM 1B：为每种血清型开发标记基因的库存，并在Temple University医学院提供此资源。 AIM 2：为腺病毒和慢病毒以及AAV的其他血清型开发新的色谱柱纯化方法。