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）：我们正在申请题为“基于 FPLC 的重组腺相关病毒 (rAAV) 纯化”的 S10 共享仪器拨款，以开发新的病毒纯化方法，以支持两项计划项目拨款和其他主要研究者 (PI) ）在坦普尔大学医学院需要 rAAV 血清型进行体内研究 该提案的主要目的是资助购买通用电气 AKTA 纯快速蛋白液体。该仪器将取代目前使用的超过 10 年的 AKTA-FPLC，但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。 ，用于基因替换/增强以增加衰竭后心脏中的蛋白质水平的动物研究。产生的绝大多数 rAAV 用于小型啮齿动物模型，但最近，犬和猪模型已使用。我们目前使用三步和四步纯化方法，包括粗细胞裂解液的碘克沙醇梯度，然后进行离子交换 FPLC，然后进行三步法透析。例如，FPLC 后和透析前的氯化铯密度梯度用于大型动物研究的病毒。 25 公斤的猪比 25 克的小鼠重 1000 倍。我们之前已经生成了用于将 1 x 1014 颗粒注射到羊体内的 rAAV6 和 rAAV9（REF），目前该病毒载体核心为两个 PPG 提供了 6 名研究人员和超过 6 名额外的研究人员。天普大学医学院和其他机构的几位研究人员资助了本申请中描述的研究项目。 目前的仪器 AKTA-FPLC 无法扩展以满足当前 AAV 纯化的需求，它是为小动物模型和体外 AAV 纯化而购买的，研究人员现在使用纯化的 AAV 在猪和狗动物模型中进行转导。 ，数量级比啮齿动物大。我们现在每周执行更多数量的 FPLC 运行，以维持我们的生产计划。在此应用中，我们建议用新的主力 FPLC 替换旧的主力仪器，以持续纯化。重组 AAV，以及天普大学医学院 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该仪器将服务于两个目标： 目标 1：生成用于体内研究的高度纯化的 rAAV。坦普尔大学医学院由 NIH 资助的小组目标 1A：将开始大型动物研究的病毒生产和完成生产之间的时间从几个月缩短到几周：目标 1B：开发。目标 2：开发腺病毒和慢病毒以及其他 AAV 血清型的新柱纯化方法。