Original Article

Gene Therapy (2010) 17, 503–510; doi:10.1038/gt.2009.157; published online 3 December 2009

High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency

E Ayuso1,2, F Mingozzi3, J Montane1, X Leon1,2, X M Anguela1,2, V Haurigot3,4, S A Edmonson3,4, L Africa3, S Zhou3, K A High3,4, F Bosch1,2 and J F Wright3,5

  1. 1Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy, School of Veterinary Medicine, Universitat Autònoma de Barcelona, Bellaterra, Spain
  2. 2CIBER de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), Barcelona, Spain
  3. 3Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, Philadelphia, PA, USA
  4. 4Howard Hughes Medical Institute, Philadelphia, PA, USA
  5. 5Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

Correspondence: Dr JF Wright, Center for Cellular and Molecular Therapeutics, Children's Hospital of Philadelphia, ARC 1216C, 3615 Civic Center Blvd., Philadelphia, PA 19104, USA. E-mail: wrightf@email.chop.edu

Received 31 July 2009; Revised 15 October 2009; Accepted 26 October 2009; Published online 3 December 2009.

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Abstract

The purity of adeno-associated virus (AAV) vector preparations has important implications for both safety and efficacy of clinical gene transfer. Early-stage screening of candidates for AAV-based therapeutics ideally requires a purification method that is flexible 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 flexibility for purification of different serotypes; however, a commonly used first-generation CsCl-based protocol was found to result in AAV vectors containing large amounts of protein and DNA impurities and low transduction efficiency 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 efficiency observed with several AAV serotypes in multiple tissues and species. Vectors purified by the optimized CsCl protocol were found to be comparable in purity and functional activity to those prepared by more scalable, but less flexible serotype-specific purification processes developed for manufacture of clinical vectors, and are therefore ideally suited for pre-clinical studies supporting translational research.

Keywords:

AAV; vector purity; transduction efficiency