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）：我们正在申请一项题为“基于FPLC的重组腺相关病毒（rAV）纯化”的S10共享仪器资助，以开发新的病毒纯化方法，以支持两项计划项目赠款和其他主要研究者（PI）天普大学医学院需要rAV血清型进行体内研究。本提案的主要目的是为购买通用电气AKTA纯快速蛋白质液相色谱仪提供资金。该仪器将取代目前使用超过10年的AKTA-FPLC，但已被GE停产。转化医学中心和心血管研究中心的许多研究目前正在使用在载体核心中纯化的rAAV，用于基因置换/增强的动物研究，以增加衰竭后心脏中的蛋白质水平。所产生的绝大多数rAAV用于小型啮齿动物模型，然而，最近，犬和猪模型已用于使用rAAV将转基因递送至心脏。我们目前使用三步和四步纯化方法，包括粗细胞裂解物的碘克沙醇梯度，然后是离子交换FPLC，然后是三步法的透析。四步法包括在FPLC之后和透析之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较大的rAAV物理滴度; 25 kg的猪比25 g的小鼠重1000倍。我们之前已经产生了rAAV 6和rAAV 9，用于将1 × 1014个颗粒注射到绵羊体内（REF）。目前，病毒载体核心为两个PPG提供服务，其中有6名研究人员，坦普尔大学医学院的另外6名研究人员和其他机构的几名研究人员。美国国立卫生研究院资助的研究项目在此申请中描述 都需要纯化的rAAV。目前的仪器AKTA-FPLC不能按比例缩放以适应当前的AAV纯化需求，其被购买用于小动物模型和体外AAV纯化。研究人员现在正在使用纯化的AAV在猪和狗动物模型中进行转导，数量级大于啮齿动物。我们现在每周进行更多的FPLC运行，以维持我们的生产计划。在本申请中，我们建议用新的主力FPLC替换老化的主力仪器，用于重组AAV的继续纯化，以及坦普尔大学医学院PI所需的其他重组病毒的潜在柱纯化。在病毒载体核心中建立这种仪器将服务于两个目的：目的1：由美国国立卫生研究院资助的坦普尔大学医学院的研究组产生高度纯化的rAAV。 目标1A：最大限度地缩短大型动物研究开始病毒生产和完成生产之间的时间，从数月到数周。 目标1B：为每种血清型开发标记基因库，并将此资源提供给坦普尔大学医学院的PI。目的2：开发腺病毒和慢病毒以及其他血清型AAV的新柱纯化方法。