# Patent

## Title
Chondroitin sulfate synthesis promoter

## Abstract

The invention provides a chondroitin sulfate synthesis promoter useful for the treatment of diseases such as articular disease and discopathy. The chondroitin sulfate synthesis promoter contains, as an active ingredient, chondroitin sulfate glucuronyltransferase protein and/or chondroitin sulfate N-acetylgalactosaminyltransferase-1 protein, or a gene encoding the enzyme protein(s).

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a divisional of U.S. application Ser. No. 11/466,260 filed on Aug. 22, 2006, now abandoned which claims priority to Japanese application number 2006-045813, filed on Feb. 22, 2006 and Japanese application number 2006-224252, filed on Aug. 21, 2006, all of which are incorporated herein by reference.


BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a chondroitin sulfate synthesis promoter, and to a treatment agent for articular disease and discopathy employing the promoter.
2. Background Art
Cartilage contains a macromolecular substance, called aggrecan, which is predominantly formed of chondroitin sulfate proteoglycan. Aggrecan is a type of proteoglycan in which a polysaccharide sulfate is covalently bound to a core protein, and is known to play an important role in organisms. In cartilage, aggrecan, having a hyaluronic acid-binding domain in a core protein portion, forms a macromolecular complex with a link protein and hyaluronic acid. Aggrecan, having 100 or more chondroitin sulfate chains bound to a core protein portion thereof, retains a large number of water molecules. Thus, aggrecan is present in cartilage in the form of highly hydrated gel and is believed to play important roles such as absorbing shock and reducing friction (lubrication).
Meanwhile, several glycosyltransferases have recently been reported to be involved in synthesis of chondroitin sulfate chains: chondroitin synthase-1 (CSS-1, J. Biol. Chem., October 2001; 276: 38721-38726), chondroitin synthase-2 (CSS-2, J. Biol. Chem., August 2003; 278: 30235-30247, WO 03/102194), chondroitin synthase-3 (CSS-3, J. Biol. Chem., October 2003; 278: 39711-39725, WO 03/102193), chondroitin sulfate glucuronyltransferase (CSGlcAT, J. Biol. Chem., October 2002; 277: No. 41: 38179-38188), chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1, J. Biol. Chem., March 2002; 277: No. 11: 8841-8846), and chondroitin sulfate N-acetylgalactosaminyltransferase-2 (CSGalNAcT-2, J. Biol. Chem., January 2003; 278: 3063-3071, Japanese Patent Application Laid-Open (kokai) No. 2003-289883).
Although general functions of these enzymes are already known, the roles of these enzymes in organisms have not yet been elucidated. In addition, an enzyme involved in biosynthesis of chondroitin sulfate (a part of aggrecan biosynthesis) has not been identified. Therefore, whether or not these enzymes increase the amount of chondroitin sulfate in organisms remains unknown.
SUMMARY OF THE INVENTION
Degradation of cartilage is accelerated by aging or overload. For example, in recent aging society, an increased number of elderly people suffer knee joint pain caused by wear of knee cartilage, leading to keen demand for means of repairing cartilage functions. In cartilage of elderly people, shock absorption performance and friction reduction performance (lubricating action) are considerably impaired. Loss of functions intrinsic to cartilage may predominantly be attributable to a decrease in the amount of chondroitin sulfate chains contained by proteoglycan such as aggrecan. Under such circumstances, the present inventors have attempted to repair and improve functions of cartilage through promoting biosynthesis of chondroitin sulfate chains of aggrecan.
In order to solve the aforementioned object, the present inventors have conducted extensive studies, and have found that among chondroitin sulfate synthesis-related enzymes, chondroitin sulfate glucuronyltransferase (hereinafter sometimes abbreviated as CSGlcAT) and chondroitin sulfate N-acetylgalactosaminyltransferase-1 (hereinafter sometimes referred to as CSGalNAcT-1) are expressed in mouse embryonic cartilage and mouse cartilage differentiated cells in an expression pattern which is considerably correlated with a known aggrecan expression pattern. The inventors have also found that forced over-expression of CSGlcAT and CSGalNAcT-1 in chondroid cells remarkably promotes synthesis of chondroitin sulfate. The inventors have further found that introduction of CSGalNAcT-1 Gene to mouse intervertebral discs increases the levels of chondroitin sulfate in the intervertebral discs, through in vivo experiment.
The present invention has been accomplished on the basis of these findings.
Accordingly, in one aspect of the present invention, there is provided a chondroitin sulfate synthesis promoter comprising, as an active ingredient, a gene encoding chondroitin sulfate glucuronyltransferase and/or a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1.
Preferably, the gene encoding chondroitin sulfate glucuronyltransferase is a nucleic acid molecule selected from at least one of the following:
(a): a nucleic acid molecule including a nucleotide sequence defined by SEQ ID NO: 1,
(b): a nucleic acid molecule which can be hybridized with a complement of a nucleic acid of SEQ ID NO: 1 under stringent conditions, or
(c): a nucleic acid molecule including a nucleotide sequence identical to that defined by SEQ ID NO: 1, except that one or more nucleotide(s) have been substituted, deleted, inserted, or transposed, wherein the nucleic acid molecule encodes a protein having activity of transferring glucuronate from a glucuronate donor to a non-reducing end N-acetylgalactosamine residue present in the chondroitin skeleton.
Preferably, the chondroitin sulfate synthesis promoter comprises, as an active ingredient, a nucleic acid molecule including a nucleotide sequence encoding an amino acid sequence defined by SEQ ID NO: 2.
Preferably, the gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 is a nucleic acid molecule selected from at least one of the following:
(d): a nucleic acid molecule including a nucleotide sequence defined by SEQ ID NO: 3,
(e): a nucleic acid molecule which can be hybridized with a complement of a nucleic acid of SEQ ID NO: 3 under stringent conditions, or
(f): a nucleic acid molecule including a nucleotide sequence identical to that defined by SEQ ID NO: 3, except that one or more nucleotide(s) have been substituted, deleted, inserted, or transposed, wherein the nucleic acid molecule encodes a protein having activity of transferring an N-acetylgalactosamine residue from an N-acetylgalactosamine donor to a non-reducing end D-glucuronate residue of an N-acetylgalactosamine acceptor substrate containing a sugar chain represented by formula;

GlcUA-Gal-Gal-Xyl

(wherein GlcUA represents a D-glucuronate residue, Gal represents a D-galactose residue, Xyl represents a D-xylose residue, and “-” represents a glycosidic linkage).

Preferably, the chondroitin sulfate synthesis promoter comprises, as an active ingredient, a nucleic acid molecule including a nucleotide sequence encoding an amino acid sequence defined by SEQ ID NO: 4.
In another aspect of the present invention, there is provided a chondroitin sulfate synthesis promoter comprising, as an active ingredient, chondroitin sulfate glucuronyltransferase and/or chondroitin sulfate N-acetylgalactosaminyltransferase-1.
Preferably, the chondroitin sulfate glucuronyltransferase is an enzyme selected from at least one of the following:
(A): an enzyme comprising a protein which includes an amino acid sequence defined by SEQ ID NO: 2, or
(B): an enzyme comprising a protein which includes an amino acid sequence identical to that defined by SEQ ID NO: 2, except that one or more amino acid residue(s) have been substituted, deleted, inserted, or transposed, wherein the protein has activity of transferring glucuronate from a glucuronate donor to a non-reducing end N-acetylgalactosamine residue present in the chondroitin skeleton.
Preferably, the chondroitin sulfate N-acetylgalactosaminyltransferase-1 is an enzyme selected from at least one of the following:
(C): an enzyme comprising a protein which includes an amino acid sequence defined by SEQ ID NO: 4, or
(D): an enzyme comprising a protein which includes an amino acid sequence identical to that defined by SEQ ID NO: 4, except that one or more amino acid residue(s) have been substituted, deleted, inserted, or transposed, wherein the protein has activity of transferring an N-acetylgalactosamine residue from an N-acetylgalactosamine donor to a non-reducing end D-glucuronate residue of an N-acetylgalactosamine acceptor substrate containing a sugar chain represented by formula;

GlcUA-Gal-Gal-Xyl

(wherein GlcUA represents a D-glucuronate residue, Gal represents a D-galactose residue, Xyl represents a D-xylose residue, and “-” represents a glycosidic linkage).

Preferably, the gene encoding chondroitin sulfate glucuronyltransferase and a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 are incorporated into an expression vector or a host cell.
In yet another aspect of the present invention, there is provided a treatment agent for articular disease and discopathy employing, as an active ingredient, any of the chondroitin sulfate synthesis promoters.
The chondroitin sulfate synthesis promoter according to the present invention can promote synthesis of chondroitin sulfate contained in proteoglycan such as aggrecan. Therefore, the synthesis promoter is suitable for improving cartilage functions and treating articular disease and discopathy.
Accordingly, the present invention provides a method for promoting chondroitin sulfate synthesis in a living subject wherein the living subject includes a joint, a cartilage, an intervertebral disc or a cell, the method comprising the steps of:

    
    
        providing a gene comprising a gene encoding chondroitin sulfate glucuronyltransferase or a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1; and
        introducing the gene into the living subject.
    
    


Preferably, the step of introducing the gene into the living subject is performed with a gene gun or via injection.
Further, the present invention provides a method for promoting a chondroitin sulfate synthesis in a living subject wherein the living subject includes a joint, a cartilage, an intervertebral disc or a cell, the method comprising the steps of:

    
    
        providing a chondroitin sulfate glucuronyltransferase or chondroitin sulfate N-acetylgalactosaminyltransferase-1; and
        introducing a chondroitin sulfate glucuronyltransferase or chondroitin sulfate N-acetylgalactosaminyltransferase-1 into the living subject.
    
    


Preferably, the step of introducing a chondroitin sulfate glucuronyltransferase or chondroitin sulfate N-acetylgalactosaminyltransferase-1 into the living subject is performed via injection.
Further, the present invention provides a method for treating an articular disease or discopathy, the method comprising the steps of:

    
    
        applying a therapeutically effective amount of an active ingredient comprising a chondroitin sulfate glucuronyltransferase, a gene encoding chondroitin sulfate glucuronyltransferase, a chondroitin sulfate N-acetylgalactosaminyltransferase-1, and/or a gene encoding a chondroitin sulfate N-acetylgalactosaminyltransferase-1 with the affected joint or intervertebral disc; and
        promoting a chondroitin sulfate synthesis in the affected joint or intervertebral disc.
    
    


These and other objects/aspects of the present invention will become apparent in light of the present specification, claims and drawings appended hereto.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows in situ hybridization results to confirm expression of chondroitin sulfate glycosyltransferases in cartilage (photographs);
FIG. 2 shows RT-PCR results representing expression patterns of chondroitin sulfate glycosyltransferases during differentiation of ATDC5 cells;
FIG. 3 shows RT-PCR results representing expression patterns of chondroitin sulfate glycosyltransferases during differentiation of N1511 cells;
FIG. 4 shows expression levels of chondroitin sulfate glycosyltransferases in ATDC5 cells, N1511 cells, and mouse embryonic fibroblasts (MEFs);
FIG. 5 shows expression levels of CSGlcAT and CSGalNAcT-1 in transfected cells;
FIG. 6 shows results of Alcian Blue staining and immunostaining (employing anti-chondroitin 4-sulfate (C4S) antibody or anti-aggrecan antibody) of control (Mock) and sample of transfected cells (photographs);
FIG. 7 shows chondroitin sulfate synthesis activities of control (Mock) and sample of transfected cells as determined by labeled chondroitin sulfate levels in culture media and cell lysates, wherein “-” denotes no treatment, “HSase” denotes heparitinase mixture treatment, and “CHase” denotes chondroitinase ABC treatment (FIGS. 7A-7F);
FIG. 8 shows results of disaccharide analysis of GAG digested products formed through chondroitinase ABC treatment of control (Mock) and sample of transfected cells, the disaccharide composition being determined in culture media and cell lysates (FIGS. 8A-8F);
FIG. 9 shows results of Alcian Blue staining of intervertebral disc in transgenic mice for CSGalNAcT-1 (photographs), wherein the photographs of a, c and e are for the mock, the photographs of b, d and f are for the transgenic mice, a and b are the overall view for the intervertebral disc, c and d are closeups for the nucleus pulposus cells and e and f are closeups for the chondrocytes; and
FIG. 10 shows levels for alcian blue stain of intervertebral disc in transgenic mice for CSGalNAcT-1.



BEST MODE FOR PRACTICING THE INVENTION
While this invention is susceptible of embodiment in many different forms, there is shown in the drawings and will be described in detail, several specific embodiments with the understanding that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the invention to the embodiments illustrated.
In a first embodiment of the invention, the chondroitin sulfate synthesis promoter contains, as an active ingredient, a gene encoding chondroitin sulfate glucuronyltransferase and/or a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1.
Examples of the gene coding for chondroitin sulfate glucuronyltransferase (CSGlcAT) include a nucleic acid molecule including a nucleotide sequence defined by SEQ ID NO: 1 and encoding human CSGlcAT. The nucleic acid molecule may be obtained through, for example, isolating RNA from human bone culture cells or similar cells and performing RT-PCR by use of a primer design based on SEQ ID NO: 1.
The gene encoding CSGlcAT is not limited to a nucleic acid including a nucleotide sequence defined by SEQ ID NO: 1. So long as the nucleic acid molecule encodes a protein having CSGlcAT activity, there may be employed a nucleic acid molecule which can be hybridized with a complement of a nucleic acid of SEQ ID NO: 1 under stringent conditions. The stringent conditions include routine washing conditions employed in southern blotting; e.g., 65° C., 0.1×SSC, 0.1% SDS solution.
So long as the nucleic acid molecule encodes a protein having CSGlcAT activity, the gene encoding CSGlcAT may be a nucleic acid molecule including a nucleotide sequence identical to that defined by SEQ ID NO: 1, except that one or more nucleotide(s) have been substituted, deleted, inserted, or transposed. The number of such nucleotides is preferably 2 to 20, more preferably 2 to 10, particularly preferably 2 to 5.
The gene encoding CSGlcAT also includes a nucleic acid which has a high sequence identity (including as preferable examples, 75-100%, 80-100%, or 90-100% sequence identity, or 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity; higher sequence identities are more preferable among the above examples) to a nucleotide sequence defined by SEQ ID NO: 1.
The aforementioned homologue genes include naturally occurring species-dependent variants and variants produced through treatment such as UV irradiation or treatment with a variation-inducing agent. The gene encoding CSGlcAT may be a gene derived from a non-human organism. However, when the product is employed as a drug for human use, a human-derived gene is preferred. Thus, the gene encoding CSGlcAT may be a nucleic acid molecule including a nucleotide sequence encoding an amino acid sequence defined by SEQ ID NO: 2.
As used herein, the term “CSGlcAT activity” refers to activity of transferring glucuronate from a glucuronate donor to a non-reducing end N-acetylgalactosamine residue present in the chondroitin skeleton. The activity may be determined through a method disclosed in J. Biol. Chem. 2002 Oct. 11; 277 (41): 38179-88.
Examples of the gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 (CSGalNAcT-1) include a nucleic acid molecule including a nucleotide sequence defined by SEQ ID NO: 3 and encoding human CSGalNAcT-1. The nucleic acid molecule may be obtained through, for example, isolating RNA from human bone culture cells or similar cells and performing RT-PCR by use of a primer design on the basis of SEQ ID NO: 3.
The gene encoding CSGalNAcT-1 is not limited to a nucleic acid including a nucleotide sequence defined by SEQ ID NO: 3. So long as the nucleic acid molecule encodes a protein exhibiting CSGalNAcT-1 activity, a nucleic acid molecule which can be hybridized with a complement of a nucleic acid of SEQ ID NO: 3 under stringent conditions may be employed. The stringent conditions include routine washing conditions employed in southern blotting; e.g., 65° C., 0.1×SSC, 0.1% SDS solution.
So long as the nucleic acid molecule encodes a protein exhibiting CSGalNAcT-1 activity, the gene encoding CSGalNAcT-1 may be a nucleic acid molecule including a nucleotide sequence identical to that defined by SEQ ID NO: 3, except that one or more nucleotide(s) have been substituted, deleted, inserted, or transposed. The number of such nucleotides is preferably 2 to 20, more preferably 2 to 10, particularly preferably 2 to 5.
The gene encoding CSGalNAcT-1 also includes a nucleic acid which has a high sequence identity (including as preferable examples, 75-100%, 80-100%, or 90-100% sequence identity, or 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity; higher sequence identities are more preferable among the above examples) to a nucleotide sequence defined by SEQ ID NO: 3.
The aforementioned homologue genes include naturally occurring species-dependent variants and variants produced through treatment such as UV irradiation or treatment with a variation-inducing agent. The gene encoding CSGalNAcT-1 may be a gene derived from a non-human organism. However, when the product is employed as a drug for human use, a human-derived gene is preferred. Thus, the gene encoding CSGalNAcT-1 may be a nucleic acid molecule including a nucleotide sequence encoding an amino acid sequence defined by SEQ ID NO: 4.
As used herein, the term “CSGalNAcT-1 activity” refers to activity of transferring an N-acetylgalactosamine residue from an N-acetylgalactosamine donor to a non-reducing end D-glucuronate residue of an N-acetylgalactosamine acceptor substrate containing a sugar chain represented by formula:
GlcUA-Gal-Gal-Xyl
(wherein GlcUA represents a D-glucuronate residue, Gal represents a D-galactose residue, Xyl represents a D-xylose residue, and “-” represents a glycosidic linkage).
The CSGalNAcT-1 activity may be determined through a method disclosed in J. Biol. Chem. 2002 Mar. 15; 277 (11): 8841-6.
When the aforementioned gene encoding chondroitin sulfate glucuronyltransferase and/or gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 is formulated as an active ingredient of the chondroitin sulfate synthesis promoter, the gene(s) may be formulated through a method for preparing a genetic drug generally employed in a genetic therapy.
Preferably, the gene encoding chondroitin sulfate glucuronyltransferase and a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 are incorporated into an expression vector.
In the above expression vector, regions relating to the gene expression (promoter region, enhancer region, operator region, etc.) may be appropriately arranged so that the gene is expressed. For example, the expression vector may include vectors such as pcDNA3.1 (+), pCDM8, or pcDNA3 expression vector (product of Invitrogen).
The gene can be incorporated into an expression vector using techniques well known to the skilled artisan.
Preferably, the gene encoding chondroitin sulfate glucuronyltransferase and a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 are incorporated into a host cell.
Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the polypeptides of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells, fungal cells, insect cells, and plant or animal cells, in particular mammalian cells. Other suitable host cells can be found in Goeddel (1991) Gene Expression Technology Methods in Enzymology 185, Academic Press, San Diego, Calif. A method for incorporating the gene into the host cell is not particularly limited. Methods for introducing exogenous genetic material into host cells are well known to the skilled artisan, and the gene can be incorporated into the host cell using such techniques. Preferably, a gene which is incorporated into an expression vector may be incorporated into the host cell.
Specific examples of methods for incorporating the gene into the vector, and introducing genes to target tissue are shown below.
For example, the CSGlcAT gene or the CSGalNAcT-1 gene is ligated to a promoter which can be expressed in target tissue, to thereby produce a gene construct, and the gene construct per se or a vector containing the gene construct is introduced into target tissue. Although the promoter may be a constitutionally expressed promoter such as a CMV promoter, the promoter is preferably a tissue-specific promoter. Examples of the target tissue include articular cartilage and intervertebral disk tissue. In the case where the target is chondrocytes or vertebral pulp cells, a promoter which functions in chondrocytes or vertebral pulp cells; e.g., a collagen gene promoter, may be employed. Examples of employable vectors include virus vectors such as an adenovirus vector and a retrovirus vector, and plasmid vectors. In one possible mode, a CSGlcAT gene or a CSGalNAcT-1 gene is ligated to a type-2 collagen promoter, to thereby form a gene construct, and the gene construct is introduced to an adenovirus vector. Through incorporation of the product, the gene of the present invention can be introduced to target tissue (e.g., intervertebral disk tissue).
Other than the above gene construct, a nucleotide sequence defined by SEQ ID NO: 1 or a nucleotide sequence defined by SEQ ID NO: 3 may be ligated with a tag sequence generally employed in a recombination technique, to thereby form a gene construct. For example, a gene construct in which a V5 epitope tag is ligated to the nucleotide sequence may be produced. Of course, a gene construct in which both of a promoter and a tag sequence are ligated to the nucleotide sequence may be produced. Through employment of a general recombination technique, a gene construct in which a different sequence is ligated to the nucleotide sequence is expressed, whereby a fusion protein can be expressed.
The gene construct containing a CSGlcAT gene or a CSGalNAcT-1 gene or the vector containing the gene construct may be introduced in combination with a pharmacologically acceptable carrier. Examples of the pharmacologically acceptable carrier include physiological saline, a stabilizer, a nuclease inhibitor, and a complexing agent such as EDTA. Alternatively, the gene construct containing a CSGlcAT gene or a CSGalNAcT-1 or the vector containing the gene construct may be introduced in the form of a complex with ribosome. Still alternatively, cells such as chondrocytes transfected with the gene construct containing a CSGlcAT gene or a CSGalNAcT-1 gene or the vector containing the gene construct may serve as a chondroitin sulfate synthesis promoter and be introduced to tissue such as cartilage.
The CSGlcAT gene or the CSGalNAcT-1 gene of the present invention may be applied to a genetic therapy employing a gene gun (e.g., a gene gun or a particle gun). The gene gun is a device for introducing an extraneous gene into a cell or tissue preferably through implanting a carrier harboring an extraneous gene or a composition containing an extraneous gene by pressure or other means. In other words, the present invention may employ a gene gun for introducing the gene of the present invention through implanting a gene introduction carrier harboring the gene of the present invention into cartilage, a vertebral pulp cell, etc., which gene gun has a tube for accelerating a gas such as helium, a mechanism for holding the gene introduction carrier harboring the gene, and other members and which has dimensions suitable for introduction of the gene to the target tissue. The carrier which is employed in the gene gun method preferably has little affect on cells and tissue. Although other carriers can be used, one preferred example of a carrier is gold particles.
In one possible mode, the CSGlcAT gene or the CSGalNAcT-1 gene of the invention is chemically or physically adsorbed onto spermidine-coated gold particles, to thereby provide a gene-harboring carrier, and the carrier is implanted into the target tissue such as cartilage or cells by means of a gene gun, whereby the gene of the present invention is introduced to the target tissue or cells.
Although no particular limitation is imposed on the administration method, the genetic drug is preferably administered topically to the target tissue. The dose of the genetic drug may be appropriately adjusted in accordance with the expression efficiency of a nucleic acid molecule and gravity of the target disease.
In a second embodiment of the present invention, the chondroitin sulfate synthesis promoter contains, as an active ingredient, a chondroitin sulfate glucuronyltransferase or chondroitin sulfate N-acetylgalactosaminyltransferase-1.
Examples of the CSGlcAT protein include human CSGlcAT having an amino acid sequence defined by SEQ ID NO: 2. The protein may be obtained through extraction from an organism, followed by purification, or through a genetic engineering technique. For example, the protein is obtained through introducing a gene having a nucleotide sequence as defined by SEQ ID NO: 1 into E. coli, animal cells, or a non-human transgenic animal, to thereby express a recombinant protein, followed by purification. Examples of the vector for introducing the gene into E. coli include pET vector (product of Novagen) and pGEX vector (product of Amersham Pharmacia), and examples of the vector for introducing the gene into animal cells include pcDNA vector (product of Invitrogen).
The CSGlcAT protein is believed to readily undergo variation such as substitution of amino acids depending on species. However, if an amino acid is substituted by another amino acid having similar characteristics (conservative substitution) or a certain amino acid not essential for exerting transfer activity is deleted, CSGlcAT activity is believed not to be affected. Therefore, so long as the nucleic acid molecule encodes a protein exhibiting CSGlcAT activity, in an amino acid sequence defined by SEQ ID NO: 2, one or more amino acid(s) may be substituted, deleted, inserted, or transposed. The number of such nucleotides is preferably 2 to 20, more preferably 2 to 10, particularly preferably 2 to 5.
The CSGlcAT protein may be derived from a non-human organism. However, when the product is employed as a drug for human use, a human-derived protein (this includes human-derived protein obtained by means of genetic engineering using a human-derived gene, for example) is preferred.
Examples of the CSGalNAcT-1 protein include human CSGalNAcT-1 protein having an amino acid sequence defined by SEQ ID NO: 4. The protein may be obtained through extraction from an organism, followed by purification, or through a genetic engineering method. For example, the protein is obtained through introducing a gene having a nucleotide sequence as defined by SEQ ID NO: 3 into E. coli, animal cells, or a non-human transgenic animal, to thereby express a recombinant protein, followed by purification.
The CSGalNAcT-1 protein is believed to readily undergo variation such as substitution of amino acids depending on species. However, if an amino acid is substituted by another amino acid having similar characteristics (conservative substitution) or a certain amino acid not essential for exerting transfer activity is depleted, CSGalNAcT-1 activity is believed not to be affected. Therefore, so long as the nucleic acid molecule encodes a protein exhibiting CSGalNAcT-1 activity, in an amino acid sequence defined by SEQ ID NO: 4, one or more amino acid(s) may be substituted, deleted, inserted, or transposed. The number of such nucleotides is preferably 2 to 20, more preferably 2 to 10, particularly preferably 2 to 5.
The CSGalNAcT-1 protein may be derived from a non-human organism. However, when the product is employed as a drug for human use, a human-derived protein (this includes human-derived protein obtained by means of genetic engineering using a human-derived gene, for example) is preferred.
It is generally known that enzymes exist which have glycochains. Also, it is possible to express enzymes which have glycochains through genetic engineering. A chondroitin sulfate glucuronyltransferase or chondroitin sulfate N-acetylgalactosaminyltransferase-1 in the present invention may have parts such as glycochains in addition to their protein.
The CSGlcAT protein or the CSGalNAcT-1 protein per se may be employed as a chondroitin sulfate synthesis promoter. Alternatively, the above protein may be blended with a pharmacologically acceptable carrier, and the mixture may be employed as the synthesis promoter. Examples of the carrier include a vehicle, a stabilizer, a tonicity agent, a surfactant, and a buffer. No particular limitation is imposed on the drug form of CSGlcAT protein or CSGalNAcT-1 protein, and examples include injection, ointment, cream, fomentation, and liniment.
Although no particular limitation is imposed on the administration method, the protein drug is preferably administered topically to the target tissue. The dose of the protein drug may be appropriately adjusted in response to gravity of the target disease or other factors.
No particular limitation is imposed on the diseases and disorders to which the chondroitin sulfate synthesis promoter of the present invention is applied, so long as they can be treated by increasing the chondroitin sulfate level. However, the chondroitin sulfate synthesis promoter is preferably employed for improving functions of articular cartilage and intervertebral disk tissue. For example, the chondroitin sulfate synthesis promoter may serve as a treatment agent for a decrease in cartilage; articular diseases such as osteoarthritis and traumatic articular disorders; or discopathy such as intervertebral disk degradation or herniated disk, which are caused by aging, disease, injury, etc.
The present inventors have elucidated that each of CSGlcAT and CSGalNAcT-1 promotes synthesis of chondroitin sulfate in chondroid cells. Therefore, a compound which increases expression of a CSGlcAT gene or a CSGalNAcT-1 gene may be a candidate for a low-molecular drug for promoting synthesis chondroitin sulfate. In one screening procedure for the chondroitin sulfate synthesis promoter, each test compound is added to cartilage culture cells, and the protein level or mRNA level in relation to CSGlcAT or CSGalNAcT-1 is determined. Among test compounds, a compound which increases the protein level or mRNA level in relation to CSGlcAT or CSGalNAcT-1 is selected.
Alternatively, a CSGlcAT gene and/or a CSGalNAcT-1 gene is expressed in vitro in cultured chondrocytes, to thereby form cells exhibiting enhanced chondroitin sulfate synthesis performance, which serve as transplant chondrocytes. The cultured chondrocytes are preferably human-derived. By use of a virus vector or a plasmid vector containing a CSGlcAT gene and/or a CSGalNAcT-1 gene, these genes are expressed in cultured chondrocytes, and promotion of chondroitin sulfate synthesis is confirmed. Thereafter, the thus-gene-introduced cultured chondrocytes are transplanted into a disordered site.
Further the present invention provides a method for promoting chondroitin sulfate synthesis in a living subject wherein the living subject includes a joint, a cartilage, an intervertebral disc or a cell (including a chondrocyte), the method comprising the steps of:

    
    
        providing a gene comprising a gene encoding chondroitin sulfate glucuronyltransferase or a gene encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1; and
        introducing the gene into the living subject.
    
    


Preferably the step of introducing the gene into the living subject is performed with a gene gun or via injection. Please refer to the above said explanations about the gene gun or gene therapy.
Preferably a living subject is a living subject derived from mammal such as human, domestic animal (including for example, a horse, cattle, and pig), or pet (including for example, dog and cat), for example.
Further the present invention provides a method for promoting a chondroitin sulfate synthesis in a living subject wherein the living subject includes a joint, a cartilage, an intervertebral disc or a cell (including a chondrocyte), the method comprising the steps of:

    
    
        providing a chondroitin sulfate glucuronyltransferase or chondroitin sulfate N-acetylgalactosaminyltransferase-1; and
        introducing a chondroitin sulfate glucuronyltransferase or chondroitin sulfate N-acetylgalactosaminyltransferase-1 into the living subject.
    
    


Preferably a living subject is a living subject derived from mammal such as human, domestic animal (including for example, a horse, cattle, and pig), or pet (including for example, dog and cat), for example.
Preferably the step of introducing a chondroitin sulfate glucuronyltransferase or chondroitin sulfate N-acetylgalactosaminyltransferase-1 into the living subject can be performed via injection, for example.
Further the present invention provides a method for treating an articular disease or discopathy, the method comprising the steps of:

    
    
        applying a therapeutically effective amount of an active ingredient comprising a chondroitin sulfate glucuronyltransferase, a gene encoding chondroitin sulfate glucuronyltransferase, a chondroitin sulfate N-acetylgalactosaminyltransferase-1, or a gene encoding a chondroitin sulfate N-acetylgalactosaminyltransferase-1 with the affected joint or intervertebral disc; and
        promoting a chondroitin sulfate synthesis in the affected joint or intervertebral disc.
    
    


The method to apply the active ingredient with the affected joint or intervertebral disc is not particularly limited. When the active ingredient is selected from a gene encoding chondroitin sulfate glucuronyltransferase and a gene encoding a chondroitin sulfate N-acetylgalactosaminyltransferase-1, gene gun or injection can be used preferably. When the active ingredient is selected from a chondroitin sulfate glucuronyltransferase and a chondroitin sulfate N-acetylgalactosaminyltransferase-1, injection can be used preferably.
A therapeutically effective amount is a concentration or amount of the active ingredient which is effective for achieving a therapeutic effect. This amount may also be determined empirically.
EXAMPLES
The present invention will next be described in detail by way of examples, which should not be construed as limiting the invention thereto.
Example 1
Expression of Chondroitin Sulfate Glycosyltransferase in Cartilage
Humeri were obtained from mouse E16.5 embryos, and in situ hybridization was performed in order to confirm expression of chondroitin sulfate glycosyltransferase in cartilage.
Digoxigenin (DIG)-11-UTP-labeled single-strand antisense RNA probes corresponding to mouse CSS-1, CSS-2, CSS-3, CSGlcAT, CSGalNAcT-1, CSGalNAcT-2, aggrecan, versican, and type-10 collagen α1 chain (Col10a1) were prepared by use of a DIG RNA labeling kit (product of Boehringer Mannheim) according to the manufacturer's instructions. Each humerus sample was collected from a mouse E16.5 embryo, and a paraffin section thereof was prepared. After removal of paraffin, the section was fixed with 4% paraformaldehyde for 10 minutes and treated with 20-ng/mL protein kinase K (product of Roche) at 37° C. for 7 minutes. Another fixation was performed with 4% paraformaldehyde for 10 minutes, followed by treating with 0.2-mol/L hydrochloric acid for 10 minutes and 0.25-mol/L acetic anhydride/0.1-mol/L triethylamine (TEA) for 10 minutes. The thus-treated section was dehydrated with ethanol and dried under air. The dried section was hybridized with 1.0-μg/mL RNA probe at 50° C. for 20 hours. The product was washed with 2×SSC (buffer: 0.3-mol/L sodium citrate and 3-mol/L sodium chloride)/50% formamide. Subsequently, the washed product was treated with 20 μg/mL RNaseA/TNE buffer (10-mmol/L Tris-HCl (pH: 8.0), 0.5-mol/L sodium chloride, and 1-mmol/L dihydrogen sodium ethylenediaminetetraacetate (EDTA)) at 37° C. for 20 minutes, and washed with 2×SSC at 50° C. for 10 minutes and a 20×SSC at 50° C. for 20 minutes. The product was blocked with a 1.5% blocking reagent (product of Applied Biosystems) at 37° C. for one hour, reacted with a 1:500 anti-DIG antibody (product of Roche) at 37° C. for one hour, and colored with an NBT/BIP (nitroblue tetrazolium hydrochloride/bromochloroindolylphosphoric acid, product of Roche) at room temperature for 56 hours. Hematoxylin staining and alcian blue staining were also performed.
The results are shown in FIG. 1.
Among six chondroitin sulfate glycosyltransferases, considerable expression of mRNA was confirmed in cartilage for four glycosyltransferases: CSS-1, CSS-2, CSGlcAT, and CSGalNAcT-1. CSS-3 and CSGalNAcT-2 were not found to be expressed. In articular cartilage covering the articular plate and growth cartilage, the above four glycosyltransferases were colocalized with core proteins of aggrecan and versican, confirming expression of the four glycosyltransferases.
Example 2
Expression Patterns of Chondroitin Sulfate Glycosyltransferases During Chondrocyte Differentiation
Chondrogenic ATDC5 cells were cultured in Dulbecco's modified Eagle medium (DMEM)/F-12 medium containing 5% fetal bovine serum (FBS), penicillin and streptomycin. In order to induce differentiation of chondrocytes, the cells at confluency were treated with 10-μg/mL bovine insulin, 10-μg/mL human transferrin, and 3×10−8-mol/L sodium selenite.
On days 1, 3, 5, 7, 14, and 21 after induction of differentiation (day 0: at about 80% confluency (growing phase) before induction of differentiation), the expression amount of mRNA (a gene transcription product) corresponding to mouse CSS-1, CSS-2, CSS-3, CSGlcAT, CSGalNAcT-1, CSGalNAcT-2, or aggrecan was determined through real time RT-PCR.
mRNA was extracted by use of Micro-FastTrack (product of Invitrogen), and cDNA was reverse transcripted therefrom by use of SuperScript First-Strand (product of Invitrogen). The primers and fluorescence-labeled probes corresponding to the genes are shown in Table 1, and were designed by use of Primer Express 1.0 software.







TABLE 1





Gene (GenBank


Accession No.)

















aggrecan
Forward primer
ctgcccttgccccgtaa


derived from mouse

(SEQ ID NO: 5)


(NM_007424)
Reverse primer
gacaggtcaaagatgggctttg




(SEQ ID NO: 6)



Probe
ccctgggcagcgtgatcctcac




(SEQ ID NO: 7)


 


CSS-1
Forward primer
ctggacctgctgctcctgtat


derived from mouse

(SEQ ID NO: 8)


(XM_194358)
Reverse primer
tcttcagggaattggacaggaa




(SEQ ID NO: 9)



Probe
cagcagaccttcagcaagatgcagtttgt




(SEQ ID NO: 10)


 


CSS-2
Forward primer
gggctttggagtcttgctctct


derived from mouse

(SEQ ID NO: 11)


(XM_129886)
Reverse primer
ggcgagcactgacgatgtc




(SEQ ID NO: 12)



Probe
acagcaactgcgcccccacct




(SEQ ID NO: 13)


 


CSS-3
Forward primer
ggaaactgggttttggagagacta


derived from mouse

(SEQ ID NO: 14)


(XM_128873)
Reverse primer
ccgtaagccagataggatgacttta




(SEQ ID NO: 15)



Probe
acggaatccaatgcatttacaaaaacgatc




(SEQ ID NO: 16)


 


CSGlcAT
Forward primer
tggccgtcgcggttaa


derived from mouse

(SEQ ID NO: 17)


(NM_133913)
Reverse primer
tccatgagacaccacctgcat




(SEQ ID NO: 18)



Probe
cgtacagtagcacatcacttccctcggttact




(SEQ ID NO: 19)


 


CSGaLNAcT-1
Forward primer
tgagctggtagaagccatcga


derived from mouse

(SEQ ID NO: 20)


(BC057630)
Reverse primer
gttcggtaaatcccttctatgaagtc




(SEQ ID NO: 21)



Probe
cggccctggagagtctaaacagccct




(SEQ ID NO: 22)


 


CSGaLNAcT-2
Forward primer
gctgagccaggtaaaaaggtgtt


derived from mouse

(SEQ ID NO: 23)


(NM_030165)
Reverse primer
aaaccagagtcctttttatgaacca




(SEQ ID NO: 24)



Probe
caggacgtgccgccccctg




(SEQ ID NO: 25)









PCR was performed by use of 5-μL cDNA, 25-μL TaqMan Universal PCR Master Mix (product of Applied Biosystems), 100-nmol/L probe, and 100-nmol/L primer (total 50 μL), and determination was performed on a 96-well plate by means of an ABI Prism 7700 (product of Applied Biosystems).
The aforementioned mRNA levels were standardized by an amount of a gene transcription product (mRNA level) of glycerinaldehyde triphosphate dehydroganase (GAPDH) determined through the same method. The thus-standardized mRNA expression levels were plotted as fold numbers against day 0 (before induction of differentiation (FIG. 2)).
Example 3
Expression Patterns of Chondroitin Sulfate Glycosyltransferases During Chondrocyte Differentiation
Chondrogenic N1511 cells were cultured in a minimum essential medium α (α-MEM medium) supplemented with 10% FBS, penicillin, and streptomycin at 37° C. under 5% CO2. The cells at confluency were treated with 1×10−6-mol/L dexamethasone solution and 1×10−7-mol/L rat parathyroid hormone (PTH) solution, to thereby induce differentiation. In a manner similar to that of Example 2, on days 1, 3, 5, 7, 14, and 21 after induction of differentiation (day 0: at about 80% confluency (growing phase) before induction of differentiation), the level of mRNA (a gene transcription product) corresponding to CSS-1, CSS-2, CSS-3, CSGlcAT, CSGalNAcT-1, CSGalNAcT-2, or aggrecan was determined. In a manner similarly to that of Example 2, the aforementioned mRNA levels were standardized by an mRNA level of GAPDH. The thus-standardized mRNA expression levels were plotted as fold numbers with respect to those at day 0 (before induction of differentiation) (FIG. 3).
As is clear from FIGS. 2 and 3, in both the case where differentiation of ATDC5 cells was induced and the case where differentiation of N1511 cells was induced, CSGlcAT transcriptional level and CSGalNAcT-1 transcriptional level increased remarkably, as compared with other enzymes. Meanwhile, it has already been known that aggrecan synthesis is promoted in chondrogenic cells such as ATDC5 cells and N1511 cells during an induced differentiation step. For example, in the case of ATDC5 cells, the aggrecan core protein transcription product (mRNA) level reaches the peak 7 to 10 days after induction of differentiation. In the above experiments, the aggrecan transcriptional level reached the peak approximately at day 7, indicating that transcriptional levels of CSGlcAT and CSGalNAcT-1 are correlated to the change in gene transcriptional level with respect to aggrecan core protein. Therefore, CSGlcAT and CSGalNAcT-1 are believed to be involved in chondroitin sulfate chain synthesis in cartilage. Note that a CSS-3 transcription product (mRNA) was not detected over the period of the experiment.
Example 4
Expression Patterns of Chondroitin Sulfate Glycosyltransferases in Mouse Embryonic Fibroblasts
Mouse embryonic fibroblasts (MEFs, product of BD Bioscience) were cultured in a DMEM medium supplemented with 10% FBS, penicillin, and streptomycin at 37° C. under 5% CO2.
At a growing phase (about 80% confluency) and one week after confluency, the level of mRNA (a gene transcription product) corresponding to CSS-1, CSS-2, CSGlcAT, CSGalNAcT-1, CSGalNAcT-2, or aggrecan was determined through the same method as employed in Example 2. Similar to Example 2, the mRNA levels were standardized by an mRNA level of GAPDH.
Expression levels (mRNA levels) of the genes at the growing phase (day 0) before induction of differentiation and at one week after induction of differentiation in Examples 2 and 3, and expression levels (mRNA levels) of the genes at the growing phase and at one week after confluency in Example 4 were plotted as fold numbers with respect to those at the growing phase of ATDC5 cells determined in Example 2 (FIG. 4).
mRNA expression levels of CSGlcAT, CSGalNAcT-1, and aggrecan were remarkably increased one week after induction of differentiation in chondrogenic cells such as ATDC5 cells and N1511 cells, and the increase was found to be correlated with increase in aggrecan. In contrast, mRNA expression level of CSGlcAT was virtually unobserved in mouse embryonic fibroblasts (MEFs), which are not differentiated to chondrocytes, and mRNA expression level of CSGalNAcT-1 was not considerably changed as compared with chondrogenic cells.
In Examples 2, 3, and 4, expression of CSGlcAT and CSGalNAcT-1 correlated highly with chondrocyte differentiation in chondrogenic cells.
Similar to Example 1, mRNA expression level of CSGalNAcT-2 was greater in fibroblasts and was virtually unobserved in chondrogenic cells.
Example 5
Establishment of CSGlcAT-Transfected Cells and CSGalNAcT-1-Transfected Cells
Rat chondrosarcoma cells (LTC cells) were transfected with human CSGlcAT and CSGalNAcT-1 expression vectors, respectively, followed by culturing. The transfected cells were selected by use of 1-mg/mL G418 disulfate (product of Nacalai Tesque).
Each of the above expression vectors was produced by incorporating cDNA of human CSGlcAT or cDNA of CSGalNAcT-1 into a pcDNA3.1 (+) vector (product of Invitrogen) (the expression vectors were donated by Dr. Goto of Advanced Industrial Science and Technology, Research Center for Glycoscience).
Rat chondrosarcoma cells (LTC cells) were cultured in a DMEM medium supplemented with 10% FBS, penicillin, and streptomycin at 37° C. under 5% CO2. Each expression vector was cut with a restriction enzyme PvuI (product of New England Biolabs) and linearized, and the LTC cells were transfected with the vector using FuGENE6 (product of Roche) in accordance with the manufacturer's instructions. On the day following transfection, 1-mg/mL G418 was added to the above culture, and culturing was further performed for 10 days, whereby transfected cells were selected. As a control, Mock-transfected cells were established in a similar manner through transfection with pcDNA3.1 (+) (product of Invitrogen) instead of the above enzymes.
Example 6
Analysis of Transfected Cells
The transfected cells established in Example 5 were analyzed as follows.
(1) Determination of Expression Levels of Enzymes:
CSGlcAT, CSGalNAcT-1, and Mock were analyzed in terms of expression levels of the corresponding exogenous enzymes (human-derived) and endogenous enzymes (rat-derived) according to the method employed in Example 2 through real time RT-PCR. The primers and fluorescence-labeled probes corresponding to the genes are shown in Table 2, and were designed by use of Primer Express 1.0 software.







TABLE 2





Gene (GenBank 


Accession No.)

















CSGlcAT
Forward primer
ctagaccaaagtgatgaagacttcaaac


derived from human

(SEQ ID NO: 26)


(AB037823)
Reverse primer
tgtaccgagtcctgagcacctt




(SEQ ID NO: 27)



Probe
ctacagggaccccaacaagccctacaag




(SEQ ID NO: 28)


 


CSGalNAcT-1
Forward primer
agcagcaccgcaactacgt


derived from human

(SEQ ID NO: 29)


(AB08 1516)
Reverse primer
ctggcttggtactgcccatt




(SEQ ID NO: 30)



Probe
ctgaagcggcagatcgcacagct




(SEQ ID NO: 31)


 


CSGlcAT
Forward primer
tggccgtcgctgttaa


derived from rat

(SEQ ID NO: 32)


(XM_216063)
Reverse primer
tccatgagacaccacctgcat




(SEQ ID NO: 33)



Probe
cgtacagtggcacatcacttccctcggttact




(SEQ ID NO: 34)


 


CSGalNAcT-1
Forward primer
tgagctagtggaagctatcga


derived from rat

(SEQ ID NO: 35)


(XM_224757)
Reverse primer
gttcggtagatcccttctatgaagtc




(SEQ ID NO: 36)



Probe
cagccctggagagtctaaacagccct




(SEQ ID NO: 37)









Reference to the respective expression vectors as controls, copy numbers (transcriptional levels) of human CSGlcAT and CSGalNAcT-1 and rat CSGlcAT and CSGalNAcT-1 are shown in FIG. 5.
(2) Alcian Blue Staining and Immunostaining:
In Alcian Blue staining, each transfected cell was seeded onto a DMEM medium supplemented with 10% FBS, penicillin, and streptomycin placed in a 6-well plate, followed by culturing at 37° C. under 5% CO2. One day after, the culture was fixed in an 80%-confluency state with 4% paraformaldehyde and treated for 30 minutes with 0.1% Alcian Blue dissolved in 0.1-mol/L hydrochloric acid (FIG. 6, upper).
Immunostaining was performed by use of a mouse monoclonal antibody reacting with chondroitin-4-sulfate (C4S) (Ab-Chondroitin-4-Sulfate/chicken (LY111), product of SEIKAGAKU CORPORATION) and a rabbit polyclonal antibody against aggrecan (FIG. 6, lower) (the rabbit polyclonal antibody against aggrecan was donated by Dr. Yada of Institute for Molecular Science of Medicine, Aichi Medical University).
(3) Determination of Chondroitin Sulfate Synthesis Activity:
LTC cells transfected with a CSGlcAT gene and those transfected with a CSGalNAcT-1 gene were cultured in cell culture dishes (3003, product of Falcon) containing a DMEM medium supplemented with 10% FBS, penicillin, and streptomycin, at 37° C. under 5% CO2. On the following day, 100-μCi/mL3 5 S sulfate was added to each culture, followed by culturing for 24 hours, so as to label the cells. Subsequently, cells and a culture medium were separately collected and extracted with 0.1-mol/L sodium hydride solution at 4° C. for 16 hours. Subsequently, the system was neutralized with 4-mol/L acetic acid. Under 20-mmol/L Tris-HCl (pH: 8.0) buffered conditions, 5-μL protein kinase K (20 mg/mL, product of Roche) was added to the system, followed by treatment at 37° C. for 12 hours. Through heating to 100° C., protein kinase K was inactivated. Then, 10-μL DNaseI (10 mg/mL, product of Boehringer Mannheim) and 10-μL RNaseA (10 mg/mL, product of Wako) were added to the above system, followed by treatment at 37° C. for two hours. Glycosaminoglycan (GAG) chains were collected from the reaction mixture. The thus-collected GAG chains were applied to a DEAE-Sepharose column. The column was washed with a 0.2-mol/L aqueous sodium chloride solution, and GAG fractions were eluted with an aqueous 2-mol/L sodium chloride solution. The thus-obtained GAG was precipitated with a 1.3% aqueous potassium acetate solution and 95% ethanol, and the thus-obtained precipitate was suspended in a buffer (50-mmol/L Tris-HCl (pH: 7.5)-0.2-mol/L sodium chloride). The GAG suspension was divided into three portions, and the portions were treated with a heparitinase mixture containing heparitinase I, heparitinase II, and heparinase (all enzymes are products of SEIKAGAKU CORPORATION); treated with a chondroitinase ABC (product of SEIKAGAKU CORPORATION); and non-treated, respectively. Subsequently, each of the thus-treated GAG samples was applied to a Superrose 6 (product of Amersham Biosciences) column which had been equilibrated with an aqueous solution (50-mmol/L Tris-HCl (pH: 7.5)-0.2-mol/L sodium chloride), and eluted with the aqueous solution (50-mmol/L Tris-HCl (pH: 7.5)-0.2-mol/L sodium chloride), whereby 0.5-mL fractions were collected. Radioactivity of labeled GAG contained in each fraction was measured by means of a liquid scintillation counter, and an elution curve was obtained through the data of scintillation counting (FIG. 7, lower).
The graph in FIG. 7 shows scintillation counts (transfected cell lysate and culture medium) of digested chondroitin sulfate fractions (19.5 mL to 21 mL) obtained from chondroitinase ABC-treated samples (graphs in FIGS. 7A-7F elution curve graphs). Each scintillation count is a sum of the count attributed to the respective transfected cells and that attributed to the respective culture medium.
(4) Disaccharide Analysis of Chondroitinase-ABC Digests of GAG Samples:
With reference to a method disclosed in Analytical Biochemistry, 177, 327-332 (1989), chondroitinase-ABC-digested products of glycosaminoglycan chains synthesized by the transfected cells were analyzed in terms of disaccharide composition.
LTC cells transfected with a CSGlcAT gene and those transfected with a CSGalNAcT-1 gene were cultured in cell culture dishes (3003, product of Falcon) containing a DMEM medium supplemented with 10% FBS, penicillin, and streptomycin, at 37° C. under 5% CO2 for 24 hours. Subsequently, cells and a culture medium were separately collected and extracted 0.1-mol/L sodium hydride solution at 4° C. for 16 hours. Subsequently, the system was neutralized with 4-mol/L acetic acid. Under 20-mmol/L Tris-HCl (pH: 8.0) buffered conditions, 5-μL protein kinase K (20 mg/mL, product of Roche) was added to the system, followed by treating at 37° C. for 12 hours. Through heating to 100° C., protein kinase K was inactivated. Then, 10-μL DNaseI (10 mg/mL, product of Boehringer Mannheim) and 10-μL RNaseA (10 mg/mL, product of Wako) were added to the above system, followed by treating at 37° C. for two hours. Glycosaminoglycan (GAG) chains were collected from the reaction mixture. The thus-collected GAG chains were applied to a DEAE-Sepharose column. The column was washed with a 0.2-mol/L aqueous sodium chloride solution, and GAG fractions were eluted with an aqueous 2-mol/L sodium chloride solution. The thus-obtained GAG was precipitated with a 1.3% aqueous potassium acetate solution and 95% ethanol, and the thus-obtained precipitate was suspended in a buffer (50-mmol/L Tris-HCl (pH: 7.5)-0.2-mol/L sodium chloride). The GAG suspension was treated with a chondroitinase ABC (product of SEIKAGAKU CORPORATION).
GAG-digested products formed through chondroitinase ABC treatment were analyzed through fluorometric postcolumn HPLC so as to confirm presence of disaccharide (FIG. 8).
The graphs in FIG. 8 shows total peak area of the respective sugar with respect to Mock, CSGlcAT, and CSGalNAcT-1 obtained in disaccharide analysis (see FIGS. 8A-8F). Each bar shows the sum of peak areas (0S, 4S, and Di(4,6)S). The values were standardized on the basis of a peak area obtained from a disaccharide standard sample (10 μmol/L) through the same method, and were reduced to amount of GAG/dish.
As used herein, “Di(4,6)S” denotes an unsaturated disaccharide which forms a disaccharide unit of chondroitin sulfate and which has a 4-sulfate group and 6-sulfate group (instead of 4-hydroxyl group and 6-hydroxyl group) in N-acetylgalactosamine; “4S” denotes an unsaturated disaccharide which forms the disaccharide unit and which has only a 4-sulfate group in N-acetylgalactosamine; and “0S” denotes an unsaturated disaccharide having no sulfate unit in the disaccharide unit.
In analysis (1) above, CSGlcAT-transfected cells and CSGalNAcT-1-transfected cells exhibited a remarkably high expression of the introduced genes, as compared with the control.
In immunostaining analysis (2) above, as compared with the control (Mock), CSGlcAT-transfected cells and CSGalNAcT-1-transfected cells have higher chondroitin 4-sulfate contents, and have an aggrecan core protein content almost equivalent to that of the control. The LTC cells are maturation chondrocytes, and cell characteristics remain during continuous culturing. In other words, the LTC cells constantly synthesize predetermined aggrecan molecules. Consequently, the core protein amount of aggrecan was found to be constant.
In analysis (3) above, transfected cells overexpressing CSGlcAT and CSGalNAcT-1 exhibited chondroitin sulfate synthesis activity of 1.6 fold and 2.2 fold, respectively, as compared with the control. As shown in the graph in FIG. 7 (middle, right), chondroitin sulfate elution curves of Mock, CSGlcAT, and CSGalNAcT-1-transfected cells, which had been treated with the heparitinase mixture, exhibited a common peak profile corresponding to fractions. Therefore, the synthesized chondroitin sulfate chains released to the culture medium had no variation in size among samples.
The above analyses revealed that, in CSGlcAT-transfected cells and CSGalNAcT-1-transfected cells, the amount of aggrecan core protein was not changed, but chondroitin sulfate chains of the same length were synthesized 1.6 fold and 2.2 fold in amount, respectively, indicating that the number of chondroitin sulfate chains in one aggrecan molecule increased 1.6 fold and 2.2 fold, respectively.
In analysis (4), chondroitin sulfate synthesized in each transfected cells exhibited a large amount of disaccharide essential units having a 4-sulfate structure. As compared with CSGlcAT-transfected cells, CSGalNAcT-1-transfected cells synthesized chondroitin sulfate in a larger amount.
Examples 2, 3, and 4 have revealed that, among chondroitin sulfate glycosyltransferases believed to be related to biosynthesis of chondroitin sulfate, CSGlcAT and CSGalNAcT-1 are closely related to biosynthesis of chondroitin sulfate in cartilage. In particular, CSGalNAcT-1 is closely related to the biosynthesis. The Examples have also revealed that an increase in expression levels of CSGlcAT and CSGalNAcT-1 leads to an increase in amounts of chondroitin sulfate chains bound to the core protein of one aggrecan molecule, as compared with the control.
Accordingly, when expression or activity of these enzymes is promoted, or these enzymes are exogenously administered, synthesis of chondroitin sulfate chains is expected to be promoted in cartilage, whereby the number of chondroitin sulfate chains which forms parts of aggrecan can be increased.
Example 7
Biosynthesis of Chondroitin Sulfate (CS) in Mouse Intervertebral Discs by In Vivo Gene Delivery of CSGalNAcT-1
cDNA encoding hCSGalNAcT-1 (CSGalNAcT-1 of human origin) was prepared by PCR using primers (5′-CACCATGATGATGGTTCGCCG-3′ SEQ ID NO: 38, 5′-TGTTTTTTTGCTACTTGTCTTCTG-3′ SEQ ID NO: 39) and a plasmid (hCSGalNAcT-1/pcDNA3.1) as a template, and the prepared cDNA was subcloned into adenoviral expression vectors (pAd/CMV/V5-DEST, manufactured by Invitrogen, Co.) through entry-vectors (pENT/D-TOPO, manufactured by Invitrogen, Co.) to obtain adenoviral vectors for expression of hCSGa1NAcT-1. Next, the adenoviral vectors for expression of hCSGalNAcT-1 were transduced into 293A cells (manufactured by Invitrogen, Co.) using an ViraPower Adenovirus Expression System (manufactured by Invitrogen, Co.) according to the manufacturer's instructions to obtain adenoviral particles for expression of hCSGalNAcT-1.
The particles' titer was checked according to the above manufacturer's instructions. 1.5×107 plaque-forming unit (pfu) ADENOVIRAL particles were dissolved in 30 μl phosphate buffer saline and injected into mouse intervertebral discs of 4 month old ICR mice at a dosage of 1.5×107 plaque-forming unit (pfu). In addition, adenoviral particles were prepared with pAd/CMV/V5-GW/Lac-Z (manufactured by Invitrogen, Co.) according to the above-described same procedures and used as a negative control (“mock”).
At day 7 after the injection the mice were dissected and the intervertebral discs were stained with alcian blue. Quantitative imaging by NIH-image (produced by National Institute of Health) were then performed on the results of alcian blue stain and the alcian blue stain were figured in quantitative data (FIG. 10).
Histological analysis showed intense alcian blue staining in the pericellular zone of the nucleus pulposus cells in the disc and chondrocytes in the vertebral endplate of the mice injected with the CSGalNAcT-1 gene, compared to that of mice injected with the mock (FIG. 9). This result teaches that the volume or biosynthesis of CS in mouse intervertebral discs is increased by in vivo gene delivery of CSGalNAcT-1.
And upon the above, it is asserted that genetic therapy with delivery of the gene of CSGalNAcT-1 into a joint increases the volume of CS in that joint toward attacking and/or curing diseases or symptoms associated with the decrease of the volume of CS.
Results in the present invention teach that the introduction of CSGalNAcT-1 gene and/or CSGlcAT gene, or exogenous administration of CSGalNAcT-1 protein and/or CSGlcAT protein promotes expression or activity of these enzymes in joints such as cartilage or intervertebral disc. Then the synthesis of chondroitin sulfate chain can be promoted, resulting in an increase of chondroitin sulfate chains of aggrecan.
In addition, it is believed that improved water retention of aggrecan by virtue of an increase in chondroitin sulfate chains, improves functions of cartilage and imparts an increase in shock absorbing and friction reducing (lubrication) performance there to. When expression or activity of these enzymes is promoted, or these enzymes are exogenously administered, formation of cartilage is expected to be promoted. For example, a cartilage formation promoter and a cartilage repairing agent for repairing damaged cartilage can be provided. Through employment of a drug or drug treatment comprising CSGalNAcT-1 or CSGlcAT as an active ingredient, or a genetic therapy employing a gene encoding CSGalNAcT-1 or CSGlcAT, a cartilage repairing agent or a treatment agent for articular disease and discopathy can be effectively provided.

## Claims

1. A method for promoting chondroitin sulfate synthesis in a living subject wherein the living subject is a joint, a cartilage, or an intervertebral disc, the method comprising the steps of:
providing a nucleic acid encoding chondroitin sulfate N-acetylgalactosaminyltransferase-1 and having a nucleotide sequence defined by SEQ. ID NO: 3; and
introducing the nucleic acid into the living subject.

2. A method for promoting a chondroitin sulfate synthesis in a living subject as described in claim 1, wherein the step of introducing the nucleic acid into the living subject is performed with a gene gun or via injection.

3. A method for promoting a chondroitin sulfate synthesis in a living subject as described in claim 1, wherein the nucleic acid encodes a protein having activity of transferring an N-acetylgalactosamine residue from an N-acetylgalactosamine donor to a non-reducing end D-glucuronate residue of an N-acetylgalactosamine acceptor substrate containing a sugar chain represented by formula:


GlcUA-Gal-Gal-Xyl


(wherein GlcUA represents a D-glucuronate residue, Gal represents a D-galactose residue, Xyl represents a D-xylose residue, and “-” represents a glycosidic linkage).

4. The method of claim 1 wherein the nucleic acid is introduced into an intervertebral disc of the living subject.