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Gene Therapy (2010) 17, 503510
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ORIGINAL ARTICLE
High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efciency
E Ayuso1,2, F Mingozzi3, J Montane1, X Leon1,2, XM Anguela1,2, V Haurigot3,4, SA Edmonson3,4, L Africa3, S Zhou3, KA High3,4, F Bosch1,2 and JF Wright3,5
1Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy, School of Veterinary Medicine, Universitat Autnoma de Barcelona, Bellaterra, Spain; 2CIBER de Diabetes y Enfermedades Metablicas Asociadas (CIBERDEM), Barcelona, Spain; 3Center for Cellular and Molecular Therapeutics, Childrens Hospital of Philadelphia, Philadelphia, PA, USA; 4Howard Hughes Medical Institute, Philadelphia, PA, USA and 5Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA
The purity of adeno-associated virus (AAV) vector preparations has important implications for both safety and efcacy of clinical gene transfer. Early-stage screening of candidates for AAV-based therapeutics ideally requires a purication method that is exible and also provides vectors comparable in purity and potency to the prospective investigational product manufactured for clinical studies. The use of cesium chloride (CsCl) gradient-based protocols provides the exibility for purication of different serotypes; however, a commonly used rst-generation CsCl-based protocol was found to result in AAV vectors containing large amounts of protein and DNA impurities and low transduction efciency in vitro and in vivo. Here, we describe and characterize an optimized, second-
generation CsCl protocol that incorporates differential precipitation of AAV particles by polyethylene glycol, resulting in higher yield and markedly higher vector purity that correlated with better transduction efciency observed with several AAV serotypes in multiple tissues and species. Vectors puried by the optimized CsCl protocol were found to be comparable in purity and functional activity to those prepared by more scalable, but less exible serotype-specic purication processes developed for manufacture of clinical vectors, and are therefore ideally suited for pre-clinical studies supporting translational research.
Gene Therapy (2010) 17, 503510; doi:http://dx.doi.org/10.1038/gt.2009.157
Web End =10.1038/gt.2009.157 ; published online 3 December 2009
Keywords: AAV; vector purity; transduction efciency
Introduction
Over the past several years, many protocols for the purication of recombinant adeno-associated virus (AAV) vectors have been established.17 An early
challenge for AAV production and purication was to obtain sufcient amounts of...