Selective gene transfer into neurons via Na,K-ATPase beta1. Targeting gene transfer with monoclonal antibody and adenovirus vector.
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 beta-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 beta-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 beta-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 beta1 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.
Ishii
Keiji
K
Department of Molecular Medicine, Sapporo Medical University School of Medicine, Sapporo, Japan.
Nakamura
Kiminori
K
Kawaguchi
Satoshi
S
Li
Rong
R
Hirai
Sachie
S
Sakuragi
Naoya
N
Wada
Takuro
T
Kato
Kazunori
K
Yamashita
Toshihiko
T
Hamada
Hirofumi
H
D000256:Adenoviridae; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics*; D006825:Hybridomas / Q000166:cytology; D007150:Immunohistochemistry; D013058:Mass Spectrometry; D051379:Mice; D009422:Nervous System Diseases / Q000628:therapy*; D009474:Neurons / Q000378:metabolism*; D016716:PC12 Cells; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013116:Spinal Cord / Q000166:cytology*
D000911:Antibodies, Monoclonal; D000254:Sodium-Potassium-Exchanging ATPase