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(由申请人提供)：我们正在申请一项名为“基于FPLC的重组腺相关病毒(RAAV)纯化”的S10共享仪器资助，以开发新的病毒纯化方法，以支持坦普尔大学医学院的两个计划项目拨款和额外的主要研究人员(PI)，他们需要rAAV血清型来进行活体研究。这项提议的主要目的是为购买通用电气阿克塔纯快速蛋白质液相色谱(FPLC)仪器提供资金。这款仪器将取代已有10年历史的Akta-FPLC，目前正在使用，但已被通用电气淘汰。转化医学中心和心血管研究中心内的许多研究目前正在使用在载体核心中纯化的rAAV进行动物研究，以在失败后增加心脏中的蛋白质水平的基因替换/增强。绝大多数生产的rAAV用于小型啮齿动物模型，然而最近，狗和猪的模型已被用于使用rAAV将转基因运送到心脏。我们目前使用的是三步和四步纯化方法，包括碘二醇梯度粗细胞裂解液，然后离子交换FPLC，然后透析为三步法。四步法包括FPLC后和透析前的氯化铯密度梯度。四步法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rAAV物理滴度；一头25公斤的猪的重量是一只25克小鼠的1000倍。我们之前已经产生了rAAV6和rAAV9，用于将1x1014颗粒注射到绵羊(参考文献)。目前，病毒载体核心为两名PPG提供服务，其中包括坦普尔大学医学院的6名调查人员和另外6名以上的调查人员，以及其他机构的几名调查人员。美国国立卫生研究院资助了本申请书中描述的研究项目 所有这些都需要纯化的rAAV。目前的仪器Akta-FPLC不能适应当前AAV纯化的需求，它是为小动物模型和体外AAV纯化而购买的。研究人员现在正在使用纯化的AAV在猪和狗的动物模型中进行转导，这些动物模型比啮齿动物大几个数量级。我们现在每周执行更多的FPLC运行，以保持我们的生产计划。在这项应用中，我们建议用新的主力FPLC取代陈旧的主力仪器，以继续纯化重组AAV，并潜在地柱纯化坦普尔大学医学院PI所需的其他重组病毒。在病毒载体核心中建立该仪器将有两个目标：目标1：为坦普尔大学医学院NIH资助小组的活体研究产生高纯度的rAAV。目标1A：最大限度地减少从开始大型动物研究的病毒生产到完成生产的时间，从几个月到几个星期。目标1B：开发每个血清型的标记基因库，并将此资源提供给坦普尔大学医学院的PI。目的2：建立新的腺病毒和慢病毒以及其他AAV血清型的柱纯化方法。