Production, Purification and Characterization of Adeno-Associated Vectors
The use of recombinant adeno-associated viral vectors (rAAVs) as gene transfer tools has increased dramatically during the past several years, establishing AAV as the vector of choice for many therapeutic applications. With the steady advance of the field toward clinical studies, and the isolation and engineering of several novel AAV serotypes, efficient, scalable, and versatile production and purification methods are continuously under development. Here, we review the current state of the art in the various production and purification methods for rAAVs. Classical parameters and methodologies to characterize rAAV stocks will be also discussed.
Keywords: Gene therapy, helper viruses, empty capsids, GMP, gradient vs. column purification, Parvoviridae, AAV serotype 2, chromosome 19, ITR, p5, p19, Rep 78, Rep 68, p5 promoter, p19 promoter, cap gene, VP1, VP2, VP3, adenoviruses (Ad), herpes simplex virus (HSV), vaccinia virus, and cytomegalovirus (CMV), AAV7, AAV8, AAV9, baculovirus vectors, virus-associated (VA) RNA, E2A, E4, Hela cells, HEK 293 cells, E1B genes, HeLa, A549, HEK293 cell lines, RPE65 deficiency, polyethylene glycol (PEG), Cesium chloride (CsCl) isopycnic gradient centrifugation, Column chromatography, AVB Sepharose High performance medium, Nhydroxysuccinimide (NHS)-activated Sepharose High Performance, human factor IX transgene, CD8 T cell, benzonase, anion-exchange chromatography, Vector Titration, ELISA, Transduction Titer, polyacrylamide gel electrophoresis (PAGE)
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