132. An Optimized CsCl Gradient -Based Method to Generate High Concentration AAV Vector Stocks of High Purity and In Vivo Potency

Abstract

Top of pageAbstract Gradient ultracentrifugation using cesium chloride is a commonly used method for purification of AAV vectors, and remains an important technique to support research of novel vector serotypes. While more recently developed column chromatographic methods have been reported that facilitate large scale vector purification, such methods currently have two important limitations: 1) chromatography resins and conditions need to be developed and optimized for individual AAV serotypes because chromatography depends on interaction of vector surface features that vary significantly between serotypes; and 2) most column chromatography methods result in co-purification of AAV empty capsids, increasing the total particle to infectious unit ratio of the resulting purified product. In this study we report an optimized cesium chloride gradient method that enables purification of various AAV serotypes using a single procedure, and results in highly purified vector essentially free of empty capsids and associated impurities in high yield. This process provides a method to enable rapid production and purification of novel vector constructs, key to the ability of our vector core facility to efficiently support research on various vector constructs and serotypes. Major steps of this flexible and high yielding process include 1) transient transfection using separate plasmids for transgene, AAV packaging and Ad helper functions; 2) highly efficient nuclease digestion of transfected cell lysates to reduce nucleic acids impurities; 3) optimized polyethylene glycol differential precipitation to achieve significant purification and concentration of vector prior to gradient ultracentrifugation; 4) modified cesium chloride gradient centrifugation and fractionation methods in which separation of the vector product from cellular impurities and empty capsids has been optimized; and 5) a final vector concentration, buffer exchange and formulation process which enables generation of stable, highly concentrated vector (exceeding 5|[times]|1013 vg/mL) without aggregation. Visualization of vector bands and those corresponding to impurities (including emtpy capsids and dense particles) following ultracentrifugation was performed using halogen beam illumination, facilitating enhanced yield and purity. Vectors (including serotypes 1, 2, 6 and 8) generated using this optimized and flexible protocol demonstrate high protein purity as measured by SDS-PAGE analysis and densitometric quantification of capsid proteins VP1, 2 and 3, and high biological activity as determined by in vivo transduction.