Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock

Davidoff, Andrew M.; Ng, Catherine Y.; Sleep, Susan; Gray, John T.; Azam, Selina A.; Zhao, Yuan; McIntosh, Jenny; Karimipoor, Morteza; Nathwani, Amit C.

doi:10.1016/j.jviromet.2004.07.001

Cited by 109 publications

(92 citation statements)

References 18 publications

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“…[1][2][3][4][5][6][7] An early challenge for AAV production and purification was to obtain sufficient amounts of high-titer vectors to support research studies in animal models. With steady advances in the field into clinical studies, and the isolation of several novel AAV serotypes, 8 more efficient and versatile production and purification methods have been required.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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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, secondgeneration 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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Section: Introductionmentioning

confidence: 99%

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

et al. 2009

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“…[23][24][25] These techniques are valuable and demonstrate success but may require special equipment (HPLC instrument and ultracentri- Purification of adenovirus and adeno-associated virus AM Duffy et al fuges) and often are lengthy procedures. In contrast, ion exchange on a membrane matrix appears to lend itself to both time efficiency and generation of comparable yields of virus to that purified by alternative more laborious techniques 26 without the need for specialized and expensive equipment as has been demonstrated here. A critical factor in the generation of rAAV is the efficiency of plasmid transfection into the virus-producing HEK 293T cells, depending on the mode of transfection, viability and state of the cells, quality of DNA and length of transfection time.…”

Section: Adeno-associated Virusmentioning

confidence: 81%

Purification of adenovirus and adeno-associated virus: comparison of novel membrane-based technology to conventional techniques

et al. 2005

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Adenovirus (Ad) and Adeno-associated virus (AAV) are efficient gene delivery systems; manipulation of the wild-type genome allows their use as vectors for the overexpression of desirable transgenes. Generation and purification of such viral vectors can be labour intensive, costly and require specialized equipment, but a new generation of membranemediated ion exchange kits for purification of recombinant virus may facilitate this process. Here, we examine the yields, transgene expression and purity of preparations of Ad and AAV purified using commercially available kits in comparison to other established techniques for purification of recombinant viral vectors. We demonstrate comparable results for Ad and AAV respectively in all parameters investigated, with a substantial reduction in purification time for the kit-based technology. Such approaches are attractive methods for small-scale purification of recombinant Ad and AAV viral vectors. Gene Therapy (2005) 12, S62-S72.

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“…Vector producer cells were harvested, subjected to five cycles of freeze thaw to release vector particles, and cellular debris was removed by centrifugation (2000 g). The supernatant containing vector particles was then filtered through 0.45 lm filters (Millipore) and diluted with 20 mM bis-tris propane buffer prior to chromatography (Davidoff et al, 2004).…”

Section: Vector Productionmentioning

confidence: 99%

“…Significant progress has recently been made in large-scale production and robust purification of AAV to support clinical development (Brument et al, 2002;Davidoff et al, 2004;Shiau et al, 2005;Okada et al, 2009;Lock et al, 2012). However, production of high-titer AAV is still a significant challenge, NIBSC/Medicines and Healthcare Products Regulatory Agency, Hertfordshire EN6 3QG, United Kingdom.…”

Section: Introductionmentioning

confidence: 99%

“…/hum.2014 as in the case of the first marketed product Glybera, which is required to be administered to patients as a dose of 3x10 12 vg/ kg via 40 or 60 multiple injections (Bryant et al, 2013). To improve AAV production, in terms of high-titer and better quality production of AAV vectors, significant efforts have been made, including the generation of partial stable producer cell lines with the integration of AAV plasmids to allow a two-step and controlled production process (Gao et al, 1998;Martin et al, 2013) and the reduction of immunoreactivity of AAV vectors (Wu, 2001;Kaludov et al, 2002;Davidoff et al, 2004;Qu et al, 2007).…”

Section: Introductionmentioning

confidence: 99%

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Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

et al. 2014

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Adeno-associated viral (AAV) vectors show great promise because of their excellent safety profile; however, preexisting immune responses have necessitated the administration of high titer AAV, posing a significant challenge to the advancement of gene therapy involving AAV vectors. Recombinant AAV vectors contain minimum viral proteins necessary for their assembly and gene delivery functions. During the process of AAV assembly and production, AAV vectors acquire, inherently and submissively, various cellular proteins, but the identity of these proteins is poorly characterized. We reason that by identifying host cell proteins inherently associated with AAV vectors we may better understand the contribution of cellular components to AAV vector assembly and, ultimately, may improve the production of AAV vectors for gene therapy. In this study, three serotypes of recombinant AAV, namely AAV2, AAV5, and AAV8, were investigated. We used liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) methods to identify protein composition in purified AAV vectors, confirmed protein identities using western blotting, and explored the potential function of selected proteins in AAV vector production using small hairpin (shRNA) methods. Using LC-MS/MS, we identified 44 AAVassociated cellular proteins including Y-box binding protein (YB1). We showed for the first time that the establishment of a novel producer cell line by introducing an shRNA sequence down-regulating YB1 resulted in up to 45-and 9-fold increase in physical vector genome titers of AAV2 and AAV8, respectively, and up to 7-fold increase in AAV2 transduction vector genome titers. Our results revealed that YB1 gene knockdown promoted AAV2 rep expression and vector DNA production and reduced the number of empty particles in AAV2 products, suggesting that YB1 plays an important role in AAV vector assembly by competition with adenovirus E2A and AAV capsid proteins for binding to the inverted terminal repeat (ITR) sequence. The significance and implications of our findings in future improvement of AAV production are discussed.

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Purification of recombinant adeno-associated virus type 8 vectors by ion exchange chromatography generates clinical grade vector stock

Cited by 109 publications

References 18 publications

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

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

Purification of adenovirus and adeno-associated virus: comparison of novel membrane-based technology to conventional techniques

Establishment of a Novel Cell Line for the Enhanced Production of Recombinant Adeno-Associated Virus Vectors for Gene Therapy

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