1111. Removal of Empty Particles from Type 1 Adeno-Associated Virus Vector Stocks by Ion Exchange Chromatography Potentiates Transgene Expression
Authors: Masashi Urabe, Ke-Qin Xin, Yoko Obara, Takayo Nakakura, Hiroaki Mizukami, Akihiro Kume, Kenji Okuda, Keiya Ozawa
Journal: Molecular Therapy (2006)

Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), a virus protein shell without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell surface receptors and when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric points of the vector may differ from that of the VLP. In an attempt to separate type 1 rAAV particles from VLPs by ion exchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)4N] 2SO4, effectively separated rAAV1 particles from VLPs, which was confirmed by electron microscopic observation of samples from pooled fractions. The rAAV1-GFP or -SEAP (secreted form of alkaline phosphatase) chromatographically separated from VLPs induced stronger transgene expression in HEK293 cells than rAAV1 contaminated with VLPs. The transduction of mouse muscles with VLP-free rAAV1-SEAP also showed higher serum SEAP levels than rAAV1-SEAP contaminated with VLPs. These results suggested that chromatographic separation of rAAV1 from empty capsids was possible and increased the efficacy of rAAV1. It will be useful for the purification of large quantities of rAAV1 for large-animal or human applications. Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), a virus protein shell without the vector genome. The contaminating VLPs would interfere with transduction by competing for cell surface receptors and when administered in vivo, contribute to antigen load, which may elicit a stronger immune response. Density-gradient ultracentrifugation provides a means to separate VLPs from rAAV particles, but is not feasible for large scale preparations of vectors. Since the compositions of the VLP and vector differ by the single-stranded DNA genome, we hypothesized that the isoelectric points of the vector may differ from that of the VLP. In an attempt to separate type 1 rAAV particles from VLPs by ion exchange chromatography, we tested a number of buffer systems and found that trimethylammonium sulfate, or [(CH3)4N] 2SO4, effectively separated rAAV1 particles from VLPs, which was confirmed by electron microscopic observation of samples from pooled fractions. The rAAV1-GFP or -SEAP (secreted form of alkaline phosphatase) chromatographically separated from VLPs induced stronger transgene expression in HEK293 cells than rAAV1 contaminated with VLPs. The transduction of mouse muscles with VLP-free rAAV1-SEAP also showed higher serum SEAP levels than rAAV1-SEAP contaminated with VLPs. These results suggested that chromatographic separation of rAAV1 from empty capsids was possible and increased the efficacy of rAAV1. It will be useful for the purification of large quantities of rAAV1 for large-animal or human applications.