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  Pharmaceutical Patents  

 

Title:  Method of expressing foreign gene in kidney
United States Patent: 
7,381,709
Issued: 
June 3, 2008

Inventors: 
Maruyama; Hiroki (Niigata, JP), Miyazaki; Jun-ichi (Osaka, JP)
Assignee:  Japan Science and Technology Corporation (Kawaguchi-Shi, JP)
Appl. No.: 
10/478,537
Filed: 
May 24, 2002
PCT Filed: 
May 24, 2002
PCT No.: 
PCT/JP02/05067
371(c)(1),(2),(4) Date: 
November 24, 2003
PCT Pub. No.: 
WO02/097087
PCT Pub. Date: 

December 05, 2002


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

The invention of this application provides a method of expressing a gene, which comprises transferring a plasmid vector recombined with an exogenous gene into the kidney by administration via the renal vein so that the exogenous gene is expressed mainly in renal interstitial fibroblasts. The method of this invention can transfer the exogenous gene into the kidneys safely and efficiently and express the transgene over a long period of time.

Description of the Invention

This application is a U.S. national stage of International Application No. PCT/JP02/05067 filed May 24, 2002.

TECHNICAL FIELD

The invention of this application relates to a method of expressing an exogenous gene in the kidney. More specifically, the invention of this application relates to a novel method of expressing a gene, in which transfer of an exogenous gene and long-term expression thereof are permitted in order to in vivo or ex vivo gene therapy of renal diseases and the like.

BACKGROUND ART

Kidney-targeted gene transfer has the potential to be one of the most important tools for broadening the understanding of renal disease processes of genetic renal diseases, glomerulonephritis, diabetic nephropathy, acute and chronic renal failure, renal graft and the like, or for revolutionizing treatments of these renal diseases.

Although various methods for kidney-targeted transfer of a gene and an expression thereof have been so far reported, the amount of expressed protein and duration of the expression have been insufficient for a therapeutic use (Gene Ther. 4, 426-431, 1997; J. Clin. Invest. 101, 1320-1325, 1998; Kidney Int. 57, 1973-1980, 2000; Hum. Gene Ther. 8, 1243-1251, 1997; Gene Ther. 7, 279-285, 2000; Biochem. Biophys. Res. Commun. 186, 129-34, 1992; J. Clin. Invest. 92, 2597-2601, 1993; Biochem. Biophys. Res. Commun. 206, 525-532, 1995).

Further, as a vector for transferring an exogenous gene (therapeutic gene), viral vectors (adenovirus vector, herpesvirus vector and the like) are mainly used in view of the transfer efficiency. The use of these viral vectors are however still problematic in securing safety, excluding antigenicity and the like.

Moreover, regarding a sort of nonviral vector, methods of improving the transfer efficiency using synthetic polymers, such as a complex with a cationic liposome (lipoplex) and a complex with a cationic polymer (polyplex), have been also proposed. However, in case of using these synthetic polymers as a vector, it must be concerned with a problem such as disorder of an immune system resulted from the adjuvant function thereof.

Meanwhile, a method has been proposed, in which a recombinant plasmid vector is directly injected into the skeletal muscle, skin or cardiac muscle of mammals and a protein or a peptide is produced in vivo by expression of the gene that is encoding thereof and incorporated in the plasmid vector. This method is based on interesting characteristics of plasmid DNA (naked DNA) reported by Wolff et al. That is, Wolff et al. have reported that; when a plasmid vector recombined with genes encoding various enzymes (.beta.-galactosidase, luciferase and the like) was directly injected into the muscle of mammals, the recombinant plasmid DNA kept its existence as an episome (extrachromosomal element) in the muscle cells over a long period of time (for several months) without being replicated or incorporated into a host genome, and it was observed in the meantime that those enzymes encoded by insert genes was continuously expressed (Science, 247:1465-1468, 1990). Since then, the gene transfer and expression method by intramuscular injection of this plasmid vector has been expected to be a main stream of future gene therapy that replaces the recombinant viral vector or the like, and its application range has been variously studied. For example, a method had been developed, in which an antigen is expressed in muscle cells by means of intramuscularly injection of a plasmid vector with an incorporated DNA fragment encoding the antigenic protein, and immunological resistance of host is acquired stimulation of the antigen-specific immune response therein. This method is named "DNA vaccine", and excellent in cost and safety in comparison with ordinary inactivated or attenuated virus vaccines. Thus, it attracts much interest as a vaccine in the next generation. In addition to the use as a DNA vaccine for acquiring an immunological resistance, a method of directly transferring the recombinant plasmid vector was shown to be effective for controlling a systemic function with a physiologically active substance or the like. For example, it was reported that a recombinant plasmid vector encoding cytokines (IL-2, IL-4, TGF.beta.1) was intramuscularly injected into mice, whereby the cytokines expressed in the muscle cells was subjected to systemic circulation and function systemically (Pro. Natl. Acad. Sci. USA, 93:10876-10880, 1996). Incidentally, one of the inventors of this application invented a method using electroporation as an effective approach to transfer a recombinant plasmid vector into cells, and the invention has already been applied for patent (official gazette of JP-A-2000-004715).

As mentioned above, the gene transfer using the recombinant plasmid vector (naked DNA) has been so far employed as a method of directly transferring a vector into tissue cells such as muscle cells, and has never been taken advantage of as a vector to transfer an exogenous gene into in vivo organs.

This invention aims to provide a novel method that enables long-term expression of a transgene using a recombinant plasmid vector as a kidney-targeted gene transfer vector.

Further, it was known that peritubular capillaries (PTC) of the kidney, which comprise a network of interstitial vessels that connects veins at each cortical level, play a major role in maintaining renal function and hemodynamics [The kidney Vol. 1, 6th edn. (ed. Brenner, B. M.) 277-318, W. B. Saunders, Philadelphia, USA, 2000]. Progressive tubulointerstitial fibrosis, which is accompanied by the loss of PTC or tubules typifies, was known as common symptom in all progressive renal diseases (J. Am. Soc. Nephrol. 7, 2495-2508, 1996). The severity of chronic tubulointerstitial changes, rather than that of glomerular damage, was strongly related with decline in renal function and long-term prognosis (Lancet. 2, 363-366, 1968; Hum. Pathol. 1, 631-641, 1970; Nephrol. Dial. Transplant, 5, 889-893, 1990; Kidney Blood Press. Res. 19, 191-195, 1996; J. Am. Soc. Nephrol. 9, 231-242, 1998; J. Am. Soc. Nephrol. 11, 47-56, 2000). Moreover, it was known that an endothelium of the PCT is one of the main targets of acute and chronic renal transplant rejection (17. J. Am. Soc. Nephrol. 10, 2208-2214, 1999; Lab. Invest. 80, 815-830, 2000; J. Am. Soc. Nephrol. 12, 574-582, 2001).

Accordingly, the invention of this application more specifically aims to express an exogenous gene in fibroblasts adjacent to the endothelium of PTC over a long period of time.

DISCLOSURE OF INVENTION

As an invention for solving the foregoing problems, this application provides a method of expressing a gene, which comprises transferring a plasmid vector recombined with an exogenous gene into the kidney by administration via the renal vein so that the exogenous gene is expressed mainly in renal interstitial fibroblasts.

In the method of this invention, the preferable embodiment is that the exogenous gene is a gene for therapy of renal diseases. Another preferable embodiment is that the administration via the renal vein is performed through a percutaneous vein catheter.

In the method of this invention, still another preferable embodiment is that the recombinant plasmid is transferred into the kidney nephrectomized from renal cells or human body in a transplantable state.

BEST MODE FOR CARRYING OUT THE INVENTION

In the method of this invention, an exogenous gene for being incorporated into a plasmid vector includes a gene (polynucleotides such as DNA and RNA), for example, which encodes various cytokines and hormones that influence the kidney function. Further, a therapeutic gene for a renal diseases is also available in accordance with purpose of the method of this invention. Moreover, in the method of this invention, therapeutic genes for other non-renal diseases (for example, .alpha.-galactosidase A gene on Fabry disease, and Epo gene on .beta.-thalassemia) can be used. That is, in the method of this invention, a transgene can be expressed in the kidney over a long period of time also by transferring a plasmid vector in a small amount of, for example, 2 .mu.g. Therefore, a treatment of various diseases with supplying a secretory protein, in which the kidney fills a role of gene expression factory (biofactory), will be put into practice.

With respect to such a gene (polynucleotide), a genomic gene can be used as such, or mRNA transcribed from a gene and cDNA synthesized from mRNA can also be used. In case of using mRNA or cDNA, a promoter/enhancer sequence or the like which controls gene expression in the kidney is ligated with a polynucleotide.

The plasmid vector is not particularly limited, and plasmid DNA ordinarily used for animal cell expression can properly be used. The incorporation of an exogenous gene polynucleotide into a plasmid DNA can also be performed by a known method.

The thus-constructed recombinant plasmid vector is retrogradely administered into the kidney via the renal vein. The targeted kidney is a human or non-human mammal kidney. When the human kidney is targeted, an effect as gene therapy is expected. Further, in case of the non-human animal, for example, a renal disease model animal is targeted, whereby the effect of the therapeutic gene transferred can be confirmed at the animal individual level. Alternatively, a novel renal disease animal model can be provided by in vivo expression of a gene, which is responsible for a renal disease.

The transfer of a recombinant plasmid into the kidney can be performed by meas of injecting plasmid solution into temporarily clamped the renal vein, in which the plasmid solution is prepared by mixing an injection liquid with the recombinant plasmid. Or, it is also possible to inject plasmid solution through the catheter that is percutaneous vein catheter inserted in the femoral vein, jugular vein, subclavian vein, cubital vein or the like. The concentration of the plasmid in the injection liquid or the dose of the injection liquid varies according to sort of a targeted animal. In case of humans, the plasmid may be administered at a dose of from 1 .mu.g to 20 mg.

In the kidney, to which the recombinant plasmid vector has been administered by the foregoing method, an exogenous gene incorporated in the vector is expressed for at least 6 months. Accordingly, an improvement of the renal function or a therapeutic effect for a renal disease with a gene is maintained for a long period of time.

The method of this invention can also be applied to a renal tissue nephrectomized from the human body in a transplantable state. In this case as well, the kidney is nephrectomized along with the vein, and the plasmid solution is administered into the kidney through this vein. And the kidney having injected therein the recombinant plasmid vector is transplanted into the human body, whereby the transgene is expressed within the kidney to attain the improvement of the renal function and the effect for renal disease therapy over a long period of time.
 

Claim 1 of 2 Claims

1. A method of expressing an exogeneous gene, which comprises administering into a renal vein through a percutaneous vein catheter a plasmid vector comprising an exogenous gene operably linked to a promoter that results in expression of said exogenous gene in renal cells, wherein said administering results in delivery of vector to the peritubular papillaries (PCT) of kidney and expression of the exogenous gene product in the renal cells adjacent to the PCT.
 

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If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.

 

 

     
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