Coelectrotransfer to skeletal muscle of three plasmids coding for antiangiogenic factors and regulatory factors of the tetracycline-inducible system: tightly regulated expression, inhibition of transplanted tumor growth, and antimetastatic effect.
We describe an approach employing intramuscular plasmid electrotransfer to deliver secretable forms of K1-5 and K1-3-HSA (a fusion of K1-3 with human serum albumin), which span, respectively, five and three of the five kringle domains of plasminogen. A tetracycline-inducible system (Tet-On) composed of three plasmids coding, respectively, for the transgene, the tetracycline transcriptional activator rtTA, and the silencer tTS was employed. K1-3-HSA and K1-5, produced from C2C12 muscle cells, were found to inhibit endothelial cell (HMEC-1) proliferation by 30 and 51%, respectively. In vivo, the expression of the transgene upon doxycycline stimulation was rapid, stable, and tightly regulated (no background expression) and could be maintained for at least 3 months. Blood half-lives of 2.1 and 3.7 days were found for K1-5 and K1-3-HSA, respectively. The K1-5 protein was secreted from muscle into blood at a level of 45 ng/ml, which was sufficient to inhibit MDA-MB-231 tumor growth by 81% in nude mice and B16-F10 melanoma cell lung invasion in C57BL/6 mice by 73%. PECAM-1 immunostaining studies revealed modest tumor vasculature in mice expressing K1-5. In contrast, K1-3-HSA, although secreted into blood at much higher level (250 ng/ml) than K1-5, had no effect on tumor growth.
Martel-Renoir
Dominique
D
Vectorologie et Transfert de Gènes, UMR 8121, Institut Gustave Roussy, 39 Rue Camille Desmoulins, 94805, Villejuif, France. renoir@igr.fr
Trochon-Joseph
Véronique
V
Galaup
Ariane
A
Bouquet
Céline
C
Griscelli
Franck
F
Opolon
Paule
P
Opolon
David
D
Connault
Elisabeth
E
Mir
Lluis
L
Perricaudet
Michel
M
D020533:Angiogenesis Inhibitors / Q000235:genetics* / Q000378:metabolism; D018274:Electroporation; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D018482:Muscle, Skeletal / Q000378:metabolism*; D009362:Neoplasm Metastasis / Q000628:therapy; D009369:Neoplasms / Q000628:therapy*; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D010958:Plasminogen / Q000235:genetics / Q000378:metabolism; D013752:Tetracycline / Q000378:metabolism; D013997:Time Factors
D020533:Angiogenesis Inhibitors; D010455:Peptides; D010958:Plasminogen; D013752:Tetracycline