Targeted gene therapy toward astrocytoma using a Cre/loxP-based adenovirus system.
The aim of this study was to establish a novel adenovirus-based gene therapy system targeting astrocytoma. For this purpose, the Cre recombinase (Cre)/loxP system together with the astrocytoma-specific promoter for GFAP were used. We constructed an adenovirus (Ad) vector that expressed Cre under the control of the GFAP promoter (AxGFAPNCre), as well as another Ad vector containing a switching unit. The latter vector contained a stuffer sequence encoding GFP (AxCALGLTK) with a functional polyadenylation signal between two loxP sites, followed by the herpes simplex virus thymidine kinase (HSV-TK) gene under the control of the CAG promoter. In this system, gene expression of either the stuffer sequence (GFP) or the downstream gene (HSV-TK) was switched on by co-expression of Cre recombinase. Western blot analysis demonstrated specific expression of high levels of TK protein in C6 glioma cells after co-infection of AxGFAPNCre and AxCALGLTK. In vivo, AxGFAPNCre/AxCALGLTK injection into C6 gliomas in the subcutaneous tissue of nude mice followed by intraperitoneal ganciclovir (GCV) treatment significantly suppressed tumor growth compared with control mice. Co-infection of AxGFAPNCre and AxCALNLLacZ resulted in LacZ expression in C6 glioma cells and some reactive astrocytes, whereas GFP was expressed in other cell types surrounding the injected site. Furthermore, a combination of AxGFAPNCre/AxCALGLTK and intraperitoneal GCV injection significantly regressed intracranial C6 gliomas in the rat striatum and prolonged the survival time compared with control rats. The present results indicate that this cell-type-specific gene therapy using a Cre/loxP adenovirus system is both operational and effective, at least against astrocytoma.
Maeda
Mitsuyo
M
Department of Anatomy, Graduate School of Medicine, Osaka City University, 1-4-3, Asahimachi, Abeno-ku, Osaka 545-8585, Japan. maedam@med.osaka-u.ac.jp
Namikawa
Kazuhiko
K
Kobayashi
Ikue
I
Ohba
Norihiro
N
Takahara
Yuki
Y
Kadono
Chiho
C
Tanaka
Akemi
A
Kiyama
Hiroshi
H
D000256:Adenoviridae / Q000235:genetics; D000818:Animals; D001254:Astrocytoma / Q000473:pathology / Q000628:therapy*; D015153:Blotting, Western / Q000379:methods; D001932:Brain Neoplasms / Q000473:pathology / Q000628:therapy / Q000821:virology; D004305:Dose-Response Relationship, Drug; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use*; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods; D019426:Integrases / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use*; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D009368:Neoplasm Transplantation / Q000379:methods; D008249:Protein-Lysine 6-Oxidase / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use*; D051381:Rats; D017208:Rats, Wistar; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use*
D016326:Extracellular Matrix Proteins; D014764:Viral Proteins; C077112:Lox protein, mouse; D008249:Protein-Lysine 6-Oxidase; C045073:Cre recombinase; D019426:Integrases