Gene therapy is a novel therapeutic method for the treatment of acquired, refractory and fatal diseases in addition to inheritable gene deficiency diseases. [1][2][3][4][5][6][7][8] Moreover, its application range is spreading to acute diseases or traumas. 9,10) The gene delivery systems in vivo can be categorized as viral and nonviral approaches. [11][12][13] Safety in usage of viral vectors for clinical gene therapy is not yet sufficient, 14,15) whereas plasmid DNA (pDNA), which is a typical nonviral vector, has advantages in safety compared with a viral vector. Recently, gene transfection efficiency using nonviral vectors has improved due to the development of various gene carriers such as cationic liposome. [16][17][18][19][20] However, transfection utilizing naked pDNA is the simplest and safest nonviral gene delivery system since naked pDNA can be used without consideration of cytotoxicity by gene carrier. When the genes were administered by the vasculature route, they were distributed to the whole body via the bloodstream, leading to inadequate organ-selective or diseased site-selective gene delivery, and were rapidly degraded by reticuloendothelial cells (liver Kupffer cell, etc.) and nuclease in blood. 21) Although it was previously reported that the organ-selective gene transfection using naked pDNA had been achieved by direct injection, electroporation, gene gun and so on, [22][23][24][25] there is great concern about safety because physical forces against the organs are required; consequently, the continuous administration of pDNA is limited.We previously developed a method of application of drugs to the surface of intraperitoneal organs such as kidney, [26][27][28] liver, 29,30) stomach 31) and intestine, 32) and found it to be a useful method for site-selective drug delivery to these organs. Furthermore, we reported on liver-and lobe-selective gene expression following the instillation of naked pDNA to the liver surface in mice. 33,34) Kidney-targeted gene therapy is expected to treat fatal diseases, including renal cell carcinoma, chronic renal fibrosis and glomerulonephritis. [35][36][37] Therefore, the present study was undertaken to elucidate the unilateral kidney-selective gene transfer following the administration of naked pDNA to the surface of mouse kidney.
MATERIALS AND METHODS
MaterialsAll chemicals were of the highest purity available.Construction and Preparation of pDNA pCMV-luciferase was constructed by subcloning the HindIII/XbaI firefly luciferase cDNA fragment from pGL3-control vector (Promega, Madison, WI, U.S.A.) into the polylinker of pcDNA3 vector (Invitrogen, Carlsbad, CA, U.S.A.). pDNA was amplified in Escherichia coli strain DH5a, isolated, and purified using an EndoFree ® Plasmid Giga Kit (QIAGEN GmbH, Hilden, Germany). pDNA was dissolved in 5% dextrose solution and was stored at Ϫ20°C until experiments were performed.In Vivo Gene Transfer Experiments All animal procedures in the present study conformed to the Guidelines for Animal Experimentation in Nagasaki University. Fiv...
