Rapid and simplified purification of recombinant adeno-associated virus
Highlights
► The optimal condition for aqueous two-phase partitioning to purify AAV, i.e. 10%PEG8000–13.2 (NH4)2SO4 at pH 8.0 in HEPES buffer, yielded a purity even higher than conventional CsCl gradient density centrifugation methods, and a higher efficiency of infection in vivo. ► In addition, the infection in embryonic mouse organs demonstrated that the PEG8000/(NH4)2SO4 aqueous two-phase partitioning purified AAV is not toxic to the embryos, and is sufficiently pure for in vivo studies in mouse embryos. ► The method does not require ultrahigh speed gradient centrifugation nor chromatography.
Introduction
Recombinant adeno-associated virus (AAV) is a non-pathogenic gene therapy method that has been used in clinical trials previously. AAV is a good candidate for clinical use as it provides long-term transgene expression in animal models, it is associated with little toxicity, and has good overall safety profiles in both pre-clinical animal studies and in clinical trials. Many investigators have published the need for a highly purified AAV in large quantity in order to perform pre-clinical and clinical trials (Ayuso et al., 2010, Lock et al., 2010).
Currently, the principal methods for purifying AAV include using CsCl ultrahigh speed density gradient centrifugation or chromatography, with the former being adopted by many investigators. This method, however, is time consuming (Ayuso et al., 2010). Iodixanol (OptiPrep) ultrahigh speed density gradient centrifugation is a similar method developed for AAV purification, with a higher viral recovery compared to the CsCl method, but the Iodixanol reagent is expensive and the method still remains relatively time-consuming; the latter drawback prevents its use for assessing quickly gene expression effects of AAV in in vivo studies (Klein et al., 2008, Zolotukhin et al., 1999).
The majority of chromatographic purification methods use either ion exchange or affinity based techniques to purify AAV. The latter method uses an antibody against AAV capsid, which therefore recognizes only assembled (serotype specific) particles, which has limited its popularity among investigators. Heparin-based affinity chromatography has also been used to purify AAV, a technique based on the property that AAV binds heparin sulfate proteoglycan with high efficiency during AAV infection; however, heparin affinity is known to be serotype specific (AAV 2). During heparin-based chromatography, other proteins present in the cell lysate that have affinity for heparin sulfate co-elute with AAV, and in the case of production of AAV in insect cells, baculovirus may also bind to heparin. This contamination would necessitate at least one additional step to purify further AAV from the eluate of heparin-based chromatography. Thus, both the CsCl or iodixanol ultrahigh speed density gradient centrifugation, as well as chromatography are time consuming and serotype-restricted techniques.
AAV has been shown to have more than ten serotypes that show tropism in vivo in a tissue dependent manner (Wu et al., 2006). Commercial purification kits are available only for AAV serotype 2. Specifically, a serotype 2 AAV purification kit is the certain commercially available kit on the market. Current chromatography purification methods are not fit for a variety of AAV serotypes, and ultrahigh speed density gradient centrifugation is time consuming and needs expensive equipment (ultrahigh speed centrifuge) that are not always available.
The technique of chemical partitioning in aqueous two-phase systems (ATPs) has been shown to be a powerful method for separating and purifying mixtures of soluble proteins. ATPs can remove undesirable by-products in crude supernatants. These systems are composed of aqueous solutions of either two water-soluble polymers, usually polyethylene glycol (PEG) and dextrin, or else a polymer and a salt, usually PEG and a phosphate or sulfate. Compared with other commonly used separation and purification techniques, ATPs have a number of advantages, such as ease of scale-up, the ability to handle particulate materials, especially for the virus, and the ease of processing. No one has previously reported the use of ATPs to remove soluble bulk proteins from AAV–protein mixtures to further purify AAV.
AAV serotype 8 has been used widely in recent years to transduce multiple organs in animals, but a purification kit is not available commercially. A simple method described in this study can be used in a typical laboratory with only a standard desktop centrifuge, avoiding the need for ultrahigh speed centrifuge or chromatography, to purify AAV serotype 8 and also conventional AAV serotype 2. The method involves PEG8000 precipitation, chloroform treatment, PEG/(NH4)2SO4 aqueous two phase extraction and final dialysis. The whole purification process can be achieved in one week, starting with transfecting HEK293 cells in culture, through to harvesting cells to purify the virus for use in in vivo studies. This rapid and simple method should be applicable for other serotype AAV purification and can be used to assess quickly the function of a gene or shRNA carried by the AAV vector. The purity of this method is high enough for use in in vivo studies.
Section snippets
Chemical reagents and equipment
Polyethylenimine (PEI) was purchased from Polysciences (Warrington, PA). NaCt (sodium citrate), (NH4)2SO4, Na2CO3, 40% PEG1450, 40% PEG4000 and 40% PEG8000 (w/w) solution were purchased from Sigma–Aldrich (St. Louis, MO). Eppendorf 5810R and 5415R desktop centrifuges with cooling system, 37 °C water bath, sterile cell culture hood and cell culture incubator were used in this study.
Plasmids
Plasmids used to co-transfect in this study are (1) Vector, pAAV-CMV-ZsGreen plasmid, briefly pAAV-GFP carrying the
Results
AAV serotype 8-ZsGreen virus, which carries the green fluorescent protein (GFP) transgene (AAV8-GFP) was produced by triple plasmid co-transfection with the PEI chemical transfection reagent method (Lock et al., 2010). The treatments were combined with benzonase, RNase A and 0.5% sodium deoxycholate in a 37 °C water bath for 1 h. Benzonase and RNase were used in low concentration (50 U/ml and10 μg/ml respectively) for nuclease treatment of AAV8-GFP virus released from cells. Sodium deoxycholate,
Discussion
AAV serotype 8 has been reported to infect efficiently brain, liver, skeletal muscle, cardiac muscle and pancreas (Wang et al., 2006, Zincarelli et al., 2008). Our aqueous two-phase extraction method has the ability to extract AAV serotype 8 from lysed cells as a single process, delivering a near-purified AAV. Thus, this technology is potentially an attractive alternative to traditional AAV purification with CsCl density gradient ultrahigh speed centrifugation and chromatography. The
Conclusion
The optimal condition for aqueous two-phase partitioning to purify the AAV8-GFP in these experiments, i.e. 10%PEG8000–13.2 (NH4)2SO4 at pH8.0 in HEPES buffer, yielded a purity even higher than conventional CsCl gradient density centrifugation methods (Fig. 3A), and a higher efficiency of infection in vivo (Fig. 5). In addition, the infection in embryonic mouse organs demonstrated that the PEG8000/(NH4)2SO4 aqueous two-phase partitioning purified AAV is not toxic to the embryos, and is pure
Author contributions
P.G. designed and performed the experiments, analyzed data and wrote the manuscript. G.G. viewed and finalized the manuscript. J.P. performed embryonic intra cardiac microinjection. K.P. and Y.G. viewed and corrected the manuscript.
Competing financial interests
The authors declare no competing financial interests.
Acknowledgments
We would like to thank Sean-Paul Williams and Jessica Thomas for their assistant with animal work.
References (11)
- et al.
Preclinical in vivo evaluation of pseudotyped adeno-associated virus vectors for liver gene therapy
Blood
(2003) - et al.
AAV8, 9, Rh10, Rh43 vector gene transfer in the rat brain: effects of serotype, promoter and purification method
Molecular Therapy
(2008) - et al.
Embryonic mouse blood flow and oxygen correlate with early pancreatic differentiation
Developmental Biology
(2011) - et al.
Adeno-associated virus serotypes: vector toolkit for human gene therapy
Molecular Therapy
(2006) - et al.
Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection
Molecular Therapy
(2008)
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2023, Biochemical Engineering Journal