AKTA Pure L

点击翻译按钮获取中文摘要

 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.