1. ¹Division of Genetic Therapeutics, Jichi Medical School, 3311-1 Yakushiji, Tochigi 329-0498, Japan
2. ²Department of Molecular Biodefense Research, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Yokohama 236-0004, Japan

Correspondence: Masashi Urabe, Fax: +81 285 44 8675. E-mail: murabe@jichi.ac.jp

Received 25 September 2005; Revised 25 November 2005; Accepted 28 November 2005.

Abstract

Production of recombinant adeno-associated virus (rAAV) results in substantial quantities of empty capsids or virus-like particles (VLPs), virus protein shells 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 point 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. The rAAV1-GFP chromatographically separated from VLPs induced stronger GFP expression in HEK293 cells than rAAV1-GFP contaminated with VLPs. The transduction of mouse muscles with rAAV1-SEAP (secreted form of alkaline phosphatase) isolated from VLPs also showed higher serum SEAP levels than rAAV1-SEAP with VLPs. These results suggest that chromatographic separation of rAAV1 from empty capsids increased the efficacy of rAAV1.