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

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Abstract

Recombinant vectors based on the recently isolated AAV serotype 8 (rAAV-8) shows great promise for gene therapy, particularly for disorders affecting the liver. Transition of this vector system to the clinic, however, is limited by the lack of an efficient scaleable purification method. In this report, we describe a simple method for purification of rAAV-8 vector particles based on ion exchange chromatography that generates vector stocks with greater than 90% purity. The average yield of purified rAAV-8 from five different vector preparation was 41%. Electron microscopy of these purified stocks revealed typical icosohedral virions with less than 10% empty particles. Liver targeted delivery of ion-exchange purified rAAV-8 vector encoding the human factor IX (hFIX) gene, resulted in plasma hFIX levels approaching 30% of normal in immunocompetent mice, which is 20-fold higher than observed with an equivalent number of rAAV-5 ion exchange purified vector particles. The method takes less then 5 h to process and purify rAAV-8 vector from producer cells and represents a significant advance on the CsCl density centrifugation technique in current use for purification of rAAV-8 vector systems and will likely facilitate the transition of the rAAV-8 vector system to the clinic.

Introduction

Recombinant adeno-associated viral (rAAV) vectors are a promising system for gene therapy of disorders that affect the liver such as haemophilia B. There are currently eight immunologically distinct serotypes of AAV, however, vectors based on serotype 2 (AAV-2) have been most extensively evaluated in preclinical studies as this was the first serotype to be fully characterized (Samulski et al., 1983). Based on highly encouraging efficacy data in animal models, AAV-2 based vectors are currently being evaluated in a Phase I/II study of liver targeted gene transfer in patients with severe haemophilia (Nathwani et al., 2003, High et al., 2003). Vectors based on the alternative serotypes of AAV are gaining in popularity because their unique tropism may result in more efficient transduction of the target tissue than currently possible with rAAV-2 based vectors (Grimm et al., 2003, Rutledge et al., 1998, Zabner et al., 2000, Davidson et al., 2000). Indeed, the most recent isolate, AAV-8, mediates between 10- to 100-fold higher transduction of murine liver then observed with equivalent numbers of AAV-2 particles (Gao et al., 2002, Sarkar et al., 2004). Consequently, fewer vector particles will be required to achieve transgene expression at therapeutic levels which will ease pressure on vector production whilst adding a measure of safety as biodistribution of rAAV to non-target sites, including the gonads, is directly proportional to the dose of rAAV administered (Nathwani et al., 2001). An equally important advantage afforded by AAV-8 vector system is an ability to circumvent pre-existing immunity to AAV-2 that is prevalent in over 70% of humans as a consequence of infection with wild type virus. In animal model, neutralizing anti-AAV-2 antibodies block transduction with rAAV-2 based vectors, but not with vectors based on rAAV-8 (Gao et al., 2002). Hence, vectors based on alternative serotypes may prove to be a valuable resource for gene therapy.

A major limitation to the clinical use of rAAV-8 vectors is the lack of efficient methods for generating clinical grade vector particles. Key to the transition of the rAAV-2 vector system to the clinic was the establishment of heparin affinity chromatography purification method based on the finding that heparan sulphate proteoglycans served as a natural receptor for AAV-2 (Summerford and Samulski, 1998, Clark et al., 1999).

As the cellular receptor for AAV-8 has not been described, it is not amenable to purification using affinity chromatography. Currently, rAAV-8 vectors are purified by density gradient centrifugation using CsCl, the traditional approach for isolating rAAV vectors. Density centrifugation is time consuming, difficult to scale-up and yields vector stocks that are heavily contaminated with cellular proteins, thus, rendering them unsuitable for clinical use. In addition, CsCl has been shown to denature rAAV vector particles leading to a substantial reduction of the therapeutic potency (Auricchio et al., 2001a). As an alternative to affinity purification methods, many investigators have shown that ion exchange chromatography can be used to purify rAAV-2 and 5 based vectors without compromising potency (Kaludov et al., 2002, Zolotukhin et al., 2002, Smith et al., 2003, Brument et al., 2002). An ion exchange method for purification of rAAV-5 vector using membrane immobilised resin that generates vector stock with purity and potency that is comparable to that achieved with mucin affinity chromatography has recently been described (Sleep et al., 2003). However, to date, a scaleable chromatographic method for purification of rAAV-8 vectors has not been described.

In this report, a simple but versatile method for purification of rAAV-8 vectors based on ion exchange chromatography is described. The purity of the resulting vector stocks is in excess of 80% as assessed by SDS PAGE electrophoresis. Additionally, the ion-exchange purified rAAV-8 particles are highly potent in murine models after liver targeted delivery of vector. This novel method is amenable to scale-up under good manufacturing practice (GMP) conditions and will be valuable in the development of AAV-8 based gene transfer vectors for clinical applications.

Section snippets

Production of pseudotyped vectors

Pseudotyped AAV vector stocks in which an AAV-2 genome was cross-packaged with AAV-8 capsid proteins were prepared according to the method described previously (Gao et al., 2002). In brief, subconfluent 293T cells were co-transfected using calcium phosphate with pAV-2 vector plasmids together with adenoviral helper plasmid [80-XX6 (Nathwani et al., 2000)] and a chimeric packaging plasmid [pAAV8-2 (Gao et al., 2002)] kindly provided by Dr James Wilson (Philadelphia, PA). Vector plasmid pAV-2

Results and Discussion

Preliminary experiments indicated that rAAV-8 vectors did not bind to either heparin or mucin matrixes that have been used effectively to purify rAAV-2 and 5 vectors, respectively (Summerford and Samulski, 1998, Auricchio et al., 2001b) (data not shown). To establish if ion-exchange chromatography could be used to purify rAAV-8 vector particles we used our previously described two-step ion exchange chromatography method for purification of rAAV-5 (Sleep et al., 2003). This method utilizes the

Acknowledgements

We wish to thank Dr K. G. Murti for his help with electron microscopy of rAAV vector particles. In addition, support from Drs Peter Savory and Ajay Lajmi of Pall Corporation, in Europe and USA is greatly appreciated.

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This work was supported by The ASSISI Foundation of Memphis, the American Lebanese Syrian Associated Charities (ALSAC), The Katharine Dormandy Trust, UK and The National Blood Service (R&D Research grant BS02/1/RB28).

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