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Selective gene transfer into neurons via Na,K‐ATPase β1. Targeting gene transfer with monoclonal antibody and adenovirus vector
Author(s) -
Ishii Keiji,
Nakamura Kiminori,
Kawaguchi Satoshi,
Li Rong,
Hirai Sachie,
Sakuragi Naoya,
Wada Takuro,
Kato Kazunori,
Yamashita Toshihiko,
Hamada Hirofumi
Publication year - 2008
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1164
Subject(s) - monoclonal antibody , microbiology and biotechnology , immunocytochemistry , biology , transfection , antigen , flow cytometry , genetic enhancement , reporter gene , viral vector , gene delivery , antibody , virology , gene , gene expression , immunology , recombinant dna , biochemistry , endocrinology
Background Neuron‐selective gene transfer is an attractive therapeutic strategy for neurological disorders. However, optimal targets and gene delivery systems remain to be determined. Methods Following immunization of mice with PC12 cells, hybridomas were screened by β‐Gal reporter gene assay using FZ33 fiber‐modified adenovirus vectors. Subsequently, the efficacy and specificity of monoclonal antibody (mAb)‐mediated gene transfer via FZ33 and FdZ adenovirus vectors were evaluated by flow cytometry, chemiluminescent β‐Gal reporter gene assay, and immunocytochemistry. Finally, the antigen recognized by the mAb was identified by mass spectrometry and transfection analysis. Results A hybridoma clone 6E3 producing monoclonal antibody, mAb6E3, was screened. Flow cytometry, chemiluminescent β‐Gal reporter gene assay, and immunocytochemistry with mAb6E3 and the fiber mutant adenovirus demonstrated efficient gene transfer into the PC12 cells. Treatment of neuron–glia cocultures with mAb6E3 and FdZ adenovirus resulted in neuron‐selective gene transfer. Immunohistochemical images of rat spinal cord tissue showed that mAb6E3 reacts specifically with neurons. Finally, Na,K‐ATPase β1 was identified as the antigen of mAb6E3. Conclusions Hybridoma screening using FZ33 fiber‐modified adenovirus vectors serves as an efficient approach to detect antigens in mAb‐targeted gene transfer. Neuronal tropism in the central nervous system through mAb6E3 represents an important initial step towards neuron‐selective gene transfer in the treatment of local neurological disorders, such as spinal cord injury. Copyright © 2008 John Wiley & Sons, Ltd.