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.
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Acknowledgements
We thank M Molas, M Moya, A Tai, and Y Zhao for expert technical assistance, C Mann, D Callejas, A Andaluz, F García for their assistance with dog experiments, and B Hauck and O Zelenaia for assistance with vector production, characterization, and helpful scientific discussion. This work was supported by grants from the Ministerio de Ciencia e Innovación, SAF2005-01262 and SAF2008-00962, the CIBER de Diabetes y Enfermedades Metabólicas Asociadas, Spain, European Community (CLINIGENE, LSHB-CT-2006–018933), the National Institute of Health grant 2PO1 HL64190 to KAH, and the Howard Hughes Medical Institute.
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JFW has consulted for Genzyme Corporation and Tacere Therapeutics, and is an inventor on patents on scalable AAV manufacturing methods, but not the optimized purification protocol described in this manuscript. FM has consulted for Arthrogen, BV, and holds a patent on prevention of immune responses to AAV vectors. KAH has consulted for Tacere Therapeutics and holds patents on prevention of immune responses to AAV vectors and other AAV technologies. All other authors declare no conflict of interest.
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Ayuso, E., Mingozzi, F., Montane, J. et al. High AAV vector purity results in serotype- and tissue-independent enhancement of transduction efficiency. Gene Ther 17, 503–510 (2010). https://doi.org/10.1038/gt.2009.157
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DOI: https://doi.org/10.1038/gt.2009.157
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