title: AAV2/8 vectors purified from culture medium with a simple and rapid protocol transduce murine liver, muscle, and retina efficiently.
abstract: During the production of some adeno-associated virus (AAV) serotypes, a large amount of vectors is found in the medium of producing cells. For their purification, previous protocols used tangential flow filtration (TFF) of the medium followed by iodixanol gradient centrifugation. Taking advantage of the higher purity of the medium than the cell-derived material as the source of AAV, we tested a simple method that combines production of large culture medium volumes containing AAV from cell stacks with medium clarification+TFF without further time-consuming and nonscalable centrifugation. To test this, we selected AAV2/8, which is emerging as a favored serotype for transduction of liver, muscle, and retina and abundantly found in the extracellular medium. We show that yields and in vitro infectivity of AAV2/8 vectors produced from the culture medium using this method are higher than those of vectors purified from the same cell lysate using a conventional CsCl2 gradient ultracentrifugation-based method, although purity appears inferior. In addition, we found that the transduction efficiency of AAV2/8 purified from medium was similar to that of AAV2/8 purified from the same cell lysate in the murine liver, muscle, and retina. Considering that the purification protocol from the medium we describe requires 3 hr as opposed to the 63 hr of a conventional two-round CsCl2-gradient ultracentrifugation+desalting, we conclude that TFF of the medium containing AAV2/8 represents a quick and scalable method to purify research-grade vectors for use in animal models.
authors.affiliation: 1 Telethon Institute of Genetics and Medicine (TIGEM) , Naples 80131, Italy .
authors.forename: Monica
authors.initials: M
authors.lastname: Doria
authors.forename: Antonella
authors.initials: A
authors.lastname: Ferrara
authors.forename: Alberto
authors.initials: A
authors.lastname: Auricchio
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D005591:Chemical Fractionation / Q000379:methods*; D003470:Culture Media; D000229:Dependovirus / Q000235:genetics / Q000302:isolation & purification*; D015316:Genetic Therapy / Q000379:methods; D005822:Genetic Vectors / Q000008:administration & dosage / Q000235:genetics / Q000302:isolation & purification*; D057809:HEK293 Cells; D006801:Humans; D008099:Liver / Q000378:metabolism; D051379:Mice; D018482:Muscle, Skeletal / Q000378:metabolism; D009928:Organ Specificity; D012160:Retina / Q000378:metabolism; D014161:Transduction, Genetic; D014162:Transfection
chemical_list: D003470:Culture Media