Removal of empty capsids from type 1 adeno-associated virus vector stocks by anion-exchange chromatography potentiates transgene expression.
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.
Division of Genetic Therapeutics, Jichi Medical School, 3311-1 Yakushiji, Tochigi 329-0498, Japan. murabe@jichi.ac.jp
Masashi
M
Urabe
Ke-Qin
KQ
Xin
Yoko
Y
Obara
Takayo
T
Nakakura
Hiroaki
H
Mizukami
Akihiro
A
Kume
Kenji
K
Okuda
Keiya
K
Ozawa
D002213:Capsid / Q000378:metabolism*; D002852:Chromatography, Ion Exchange; D000229:Dependovirus / Q000145:classification / Q000235:genetics*; D015870:Gene Expression; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000378:metabolism; D014161:Transduction, Genetic; D019076:Transgenes
D049452:Green Fluorescent Proteins