Use Of Decorine For Increasing Muscle Mass

Abstract

The invention concerns decorin for increasing muscle mass, particularly in the treatment of muscular dystrophies.

Description

TECHNICAL DOMAIN

[0001] The aim of this invention is to increase muscle mass in humans or animals.

[0002] More specifically, it advocates the use of decorin to develop muscle mass, particularly for treating pathological conditions associated with muscular wasting, such as muscular dystrophy.

PRIOR STATE OF THE ART

[0003] Neuromuscular diseases include various conditions that are generally associated with temporary or permanent loss of muscular strength. This loss of strength is usually accompanied by muscular wasting, also known as amyotrophia.

[0004] Myopathies, which involve damage to the actual muscle fibres, are an important group of these muscular diseases, and among them, progressive muscular dystrophies are characterised by a decrease in muscular strength, generally with atrophy of the muscles, as well as abnormalities in the muscle biopsy showing modifications of the tissue. This group notably includes Duchenne muscular dystrophy (or DMD), Becker muscular dystrophy (or BMD) and the limb girdle muscular dystrophies.

[0005] Associated genetic abnormalities have been identified for some of these diseases. Duchenne or Becker muscular dystrophies are related to alterations in the gene encoding dystrophin, type 2A limb girdle muscular dystrophy (LGMD 2A or calpainopathy) to alterations in the calpain 3 gene, while the sarcoglycanopathies or the dystrophy types LGMD 2C, LGMD 2D, LGMD 2E, LGMD 2F are related to defects in the .gamma.-, .alpha.-, .beta.- and .delta.-sarcoglycan genes respectively (McNally E M, Pytel P, Muscle diseases: the muscular dystrophies. Annu Rev Pathol. 2007; Vol 2: 87-109).

[0006] In these particular cases, different gene therapy strategies are being developed but are difficult to put into practice.

[0007] Nevertheless and more generally in all cases of muscular wasting, there is a clear need to develop technical solutions to increase muscle mass and/or volume.

[0008] The document WO 2005/094446 identified antibodies against an epitope located between residues 40 and 64 of mature human myostatin which could increase muscle mass. However, this strategy based on the recognition of myostatin by an antibody is not free of problems. Alternative solutions therefore need to be found.

[0009] The present invention is based on the discovery by the inventors of this property of decorin.

[0010] Decorin belongs to the SLRP (Small Leucine-Rich Proteoglycan) family of proteins and includes an LRR (Leucine-Rich Repeat) segment. Decorin is a member of class I of the SLRPs. The members of this family are secreted with a propeptide which, in some cases, is cleaved. Decorin also has a glycosaminoglycan (GAG) chain.

[0011] Decorin is a protein of the extracellular matrix, with a similar structure to that of the protein biglycan. It plays a role in assembling the matrix and interacts with various partners, such as type I, II, III and IV collagen, or TGF-beta (Ameye L, Young M F, Mice deficient in small leucine-rich proteoglycans: novel in vivo models for osteoporosis, osteoarthritis, Ehlers-Danlos syndrome, muscular dystrophy, and corneal diseases. Glycobiology 2002; Vol 12:107 R-116R; Reed C C, Iozzo R V, The role of decorin in collagen fibrillogenesis and skin homeostasis. Glycoconj J. 2002; Vol 19(4-5): 249-55).

[0012] On the basis of its interaction with TGF-beta, WO 96/25178 proposed the use of decorin to treat diseases associated with tissue fibrosis, i.e. excessive production of extracellular matrix, without relating this to the muscle mass problem.

DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention thus concerns the use of decorin to counter muscle wasting and even to increase muscle mass.

[0014] For this invention, the term "muscle mass" could be replaced either by muscle weight or volume.

[0015] More precisely the invention also concerns: [0016] a composition containing decorin to treat diseases associated with muscle wasting; [0017] the use of decorin in the preparation of a medicinal product for treating diseases associated with muscle wasting; [0018] a composition containing decorin to increase muscle mass; [0019] the use of decorin to increase muscle mass.

[0020] There are a number of conditions in which muscle wasting occurs.

[0021] Firstly, it may result from pathological conditions, particularly in the case of neuromuscular diseases. Duchenne muscular dystrophy is a disease particularly targeted, but all forms of neuromuscular diseases, especially muscular dystrophies, can be treated.

[0022] In addition, cachexia or marasmus is also a medical condition targeted by this invention. This state is characterized by extreme thinness, especially of the muscles, caused by prolonged illness or inadequate calorie or protein intake.

[0023] This condition is particularly seen in cases of chronic diseases such as cancer or AIDS or in individuals with either heart failure, where there is atrophy of skeletal muscles in 68% of patients, or urinary incontinence.

[0024] Although not actually considered as pathological, some situations are associated with muscle wasting: ageing, prolonged immobilisation etc. Here again, therefore, there is a reason for increasing the muscle mass.

[0025] The invention also offers the possibility particularly in the area of food production of increasing animal meat production. The use of decorin is therefore of particular interest in animals.

[0026] This invention is based therefore on detection of the stimulating properties of decorin, particularly related to muscle volume.

[0027] In the invention, "decorin" is used generically to mean the protein described by Krusius et al. (Krusius T., Ruoslahti E., Primary structure of an extracellular matrix proteoglycan core protein deduced from cloned cDNA. Proc Natl Acad Sci USA 1986; Vol 83(20): 7683-87). The human protein described in this document has the sequence SEQ ID NO: 1. It is in the form of a preproprotein of 359 amino acids. Both native proteins and those deprived of their propeptide and/or their signal sequence (329 aa), are covered by this invention.

[0028] Although decorin naturally has a glycosaminoglycan (GAG) chain, a decorin without GAG (GAG-) can also be used in the context of this invention. This can, for example, be obtained by enzyme treatment.

[0029] The decorin can be obtained from any organism, but in this invention, decorin of human origin is preferred. More generally and advantageously, the protein comes from the same organism as the organism into which it will be administered. Preferably therefore, for therapeutic indications in humans, human decorin is used to advantage.

[0030] One of the primary benefits indeed of the solution proposed in this invention is that decorin is a protein naturally present in mammals, especially humans, and therefore a priori is not likely to cause side effects or immune responses.

[0031] It has also been shown for human decorin that transcriptional variants exist (variants b, c, d and e), resulting in protein isoforms, of sequence SEQ ID NO: 2, 3, 4 and 5, respectively, included in this submission.

[0032] In the context of the invention, the term "decorin" thus has a wide meaning and covers: [0033] the native protein, particularly the sequence SEQ ID NO: 1; [0034] the protein with or without the GAG chain (GAG+ or GAG-, respectively); [0035] the protein lacking the propeptide and/or the signal sequence; [0036] variants of these proteins, especially embodied in the sequences SEQ ID NOS: 2 to 5; [0037] more generally active fragments of these proteins, [0038] or active derivatives or functional equivalents.

[0039] As far as the fragments or derivatives are concerned, they are, of course, active fragments or derivatives. The activity in question which these fragments or derivatives must possess concerns the ability to increase muscle mass, which is easily assessed by using the test described in this submission.

[0040] In practice, they are to advantage 60% identical to one of sequences SEQ ID NO: 1 to 7, even more advantageously 70%, 80% or 90% identical.

[0041] Thus, by way of example for the derivatives, it could be sequence SEQ ID NO: 6 corresponding to the murine protein of 354 aa, which is 80% identical to the human sequence SEQ ID NO: 1.

[0042] According to a preferred embodiment of the invention, the decorin is in the form of an active fragment. To advantage by "fragment" we mean a peptide containing less than 100 amino acids, to even greater advantage, less than 50 amino acids.

[0043] The use of a peptide instead of the protein has certain advantages, particularly in terms of its production but also concerning the possible risk of undesirable interference in vivo.

[0044] It has been shown as part of this application that a 41 residue fragment of the N-terminal part of murine decorin (SEQ ID NO: 7) corresponding to residues 31-71 of the sequence SEQ ID NO: 6, reported to fix zinc (Yang V W, LaBrenz S R, Rosenberg L C, McQuillan D, Hook M. Decorin is a Zn2+metalloprotein. J Biol Chem. 1999, 274(18): 12454-60), had the required activity. It has also been shown that an even smaller fragment of 30 residues (residues 42 to 71 of the sequence SEQ ID NO: 6 corresponding to SEQ ID NO: 15) was also active.

[0045] The corresponding domain, present in human decorin, can be easily determined by the methodology described in this document. Such a fragment may for example have the sequence SEQ ID NO: 16.

[0046] More generally, the invention therefore concerns the use of a fragment of decorin including the zinc binding domain, in practice the residues 31 to 71, possibly 42 to 71 of the murine sequence. In a particular embodiment, the sequence of the fragment in question corresponds to sequence SEQ ID NO: 7 or SEQ ID NO: 15. In addition, fragments which are to advantage 50% identical to SEQ ID NO: 7 or 15, or even more advantageously 60%, 70%, 80% or 90% identical to them, and which retain their ability to bind zinc, are also covered by this invention.

[0047] In addition, decorin, its fragments and active derivatives may also be in the form of fusion proteins or chimeric proteins with another protein fragment at their N- or C-terminal ends, which can, for example, but without being limited to this, increase the residence time of the protein in the organism. A preferred example is the chimera consisting of the constant region of mammalian IgGs, attached via a hinge sequence to decorin or one of its fragments. Another example is human or mammalian albumin, also attached to decorin or to a protein fragment of decorin. Such combinations can be obtained both from a recombinant cDNA and by chemical bonding of the 2 proteins.

[0048] The present invention is therefore based on an exogenous supply of decorin. In fact, the composition covered by the invention consists of either the protein as such or a system producing the protein.

[0049] As far as the protein itself is concerned, it could be either native decorin, purified from an organism naturally producing this protein, or a recombinant protein produced by any of the synthesis systems available and known to those working in the field.

[0050] Alternatively, a nucleic acid sequence encoding decorin is put into an expression system, to advantage under the control of a promoter in a vector. After introduction into the body, the decorin is produced in vivo. The transfer of the nucleic acids (DNA or RNA) can be done either with viral approaches to gene transfer (e.g. adeno-associated virus or AAV) or with non-viral approaches (e.g. by simple intramuscular injection of a plasmid). Genomic DNA may be of interest since in some cases, the presence of introns stabilises the prespliced mRNA and improves its stability in the nucleus and its export, which leads to better protein expression.

[0051] Decorin, its derivatives or fragments, can thus be provided in the form of nucleic acids, particularly DNA or RNA, and may for example be in the form of transcripts occurring naturally in humans or the mouse. The following sequences are preferred: [0052] Sequence SEQ ID NO: 8, corresponding to the A1 variant (Accession Number NM.sub.--001920.3), which is the longest transcript and encodes the isoform a of the human decorin sequence SEQ ID NO: 1 (Accession Number NP.sub.--001911); [0053] Sequence SEQ ID NO: 9, corresponding to the A2 variant (Accession Number NM.sub.--133503.2), which uses an alternative exon at the 5'UTR compared with the variant A1 and encodes the same protein sequence SEQ ID NO: 1 (Accession Number NP.sub.--598010.1); [0054] Sequence SEQ ID NO: 10, corresponding to the B variant (Accession Number NM.sub.--133504.2), which lacks exons 3 and 4 in the coding region, compared with the A1 variant. This causes no change in reading frame but codes for an isoform b of the protein, which lacks an internal fragment of 109 aa, and has the sequence SEQ ID NO: 2 (Accession Number NP.sub.--598011.1); [0055] Sequence SEQ ID NO: 11, corresponding to the C variant (Accession Number NM.sub.--133505.2), which lacks exons 3, 4 and 5 in the coding region, compared with the A1 variant. This causes a change of internal reading frame and the isoform c encoded of SEQ ID NO: 3 (Accession Number NP.sub.--598012.1) is shorter than isoform a by 147 amino acids; [0056] Sequence SEQ ID NO: 12, corresponding to the D variant (Accession Number NM.sub.--133506.2), which lacks exons 4, 5, 6 and 7 in the coding region, compared with the A1 variant. This causes no change in reading frame but codes for an isoform d of the protein, which lacks an internal fragment of 187 aa, and has the sequence SEQ ID NO: 4 (Accession Number NP.sub.--598013.1); [0057] Sequence SEQ ID NO: 13, corresponding to the E variant (Accession Number NM.sub.--133507.2), which lacks exons 3, 4, 5, 6 and 7 in the coding region, compared with the A1 variant. This causes a change of internal reading frame and the isoform e encoded of SEQ ID NO: 5 (Accession Number NP.sub.--598014.1) is shorter than isoform a by 284 amino acids; [0058] Sequence SEQ ID NO: 14, encoding the murine protein sequence SEQ ID NO: 6 (Accession Number P28654).

[0059] As already mentioned, decorin is known to be a zinc metalloprotein. Owing to this and in order to potentiate its activity, one could choose to provide additional zinc to that naturally available in the organism to which the decorin is administered. Thus, according to this embodiment, the composition containing the decorin also includes zinc, e.g. as zinc chloride, preferably at a concentration between 1 and 50 .mu.M, even equal to 15 .mu.M.

[0060] A composition containing decorin according to the invention for the treatment of diseases associated with muscle wasting or intended to increase muscle mass may also contain any acceptable compound or excipient, particularly a pharmaceutical compound or excipient. The route of administration may be intramuscular or intravenous, or even subcutaneous, intraperitoneal or oral.

[0061] To promote the engraftment of precursor cells or stem cells, it may be advantageous to combine the administration of decorin with the cell grafts (myoblasts, stem cells etc.). This administration can be simultaneous or separated in time.

[0062] It can also be advantageous to combine gene therapy for the treatment of a neuromuscular disease with administration of decorin. In a preferred embodiment, a therapeutic gene is associated with decorin treatment. Administration of the two treatments can be simultaneous or separated in time.

[0063] The beneficial effects of decorin result in an increase in muscle volume (either mass or weight), due to an increase in the area of fibres possibly associated with an increase in the number of fibres. These positive effects can be observed in the various different skeletal muscles, both in an organism with a disease affecting its muscle mass and in a healthy individual. In principle, there are no side effects and no immune reaction.

EXAMPLES OF EMBODIMENTS

[0064] The invention and the advantages resulting from it are better illustrated by the following examples of embodiments and the attached figures. These are in no way limiting.

[0065] The invention is further illustrated by means of recombinant mouse decorin injected intramuscularly into mdx mice with a gene encoding an altered dystrophin and serving as an experimental model of Duchenne muscular dystrophy, and gamma-sarcoglycan-/-mice (mouse model of sarcoglycanopathies on a pure C57/B16 background).

LEGENDS TO THE FIGURES

[0066] FIG. 1 is a view of the tibialis anterior muscle taken from mdx mouse 7 that had received (on the left) or not (on the right) an intramuscular injection of decorin.

[0067] FIG. 2 is a view of the tibialis anterior muscle taken from mdx mouse 8 that had received (on the left) or not (on the right) an intramuscular injection of decorin.

[0068] FIG. 3 is a view of the tibialis anterior muscle taken from gamma-sarcoglycan-/-mouse 4 at D18 that had received (on the left) or not (on the right) an intramuscular injection of decorin.

[0069] FIG. 4 is a view of a cross-section of the tibialis anterior muscle taken from gamma-sarcoglycan-/-mouse 4 at D18 that had received (LTA4 on the right of the figure) or not (RTA4 on the left of the figure) an intramuscular injection of decorin.

I) MATERIALS AND METHODS

[0070] Preparing the mDecorin Solution

[0071] The protein used was recombinant mouse decorin (mDecorin) of sequence SEQ ID NO: 6, provided by R&D Systems.

[0072] Twenty-four to forty hours before the injection, 100 .mu.l of 150 mM sterile NaCl and 6 .mu.l of 250 .mu.M ZnCl.sub.2 were added to 100 .mu.g of mDecorin powder. The final volume was 106 .mu.l with a final concentration of approximately 1 .mu.g/.mu.l. For the injections into the control muscles a mixture was also prepared of 100 .mu.l of 150 mM NaCl and 6 .mu.l of 250 .mu.M ZnCl.sub.2. All these solutions, after being vortexed, were stored at 4.degree. C.

In Vivo Injection

[0073] All the mice were treated according to EU directives on human health and the use of experimental animals.

[0074] mdx dystrophic (S-linked muscular dystrophy) or gamma-sarcoglycan-/-mice were used that were at least 6 weeks old. 20 .mu.g of mDecorin, i.e. 22 .mu.l of the solution described above (20 .mu.g Decorin+15 .mu.M ZnCl.sub.2/22 .mu.l NaCl), were injected into the left tibialis anterior (LTA), the muscle treated. 22 .mu.l of the control solution described above (15 .mu.M ZnCl.sub.2/22 .mu.l NaCl) were administered into the control muscle, the right tibialis anterior (RTA). A specific number of days after injection, the mice were sacrificed and the RTA and LTA were removed, weighed then frozen for further histological study.

Preparation and Injection of the Solution Containing the Peptide mDCN 31-71:

[0075] The peptide with the sequence SEQ ID NO: 7 was synthesised by the company NeoMPS with purity >65%. It was dissolved at 2 mg/ml in 150 mM NaCl and stored at -80.degree. C.

[0076] For injections, the preparation protocol was identical to that used for the protein, i.e. 24-40 hours before injection, the desired amount of peptide was removed from the stock solution and mixed with a solution of zinc chloride (ZnCl.sub.2) and 150 mM NaCl, to produce a final zinc concentration of 15 .mu.M. The injection protocol was identical to that used for the protein.

Histological Analyses

[0077] Laminin Labelling:

[0078] Cryostat sections (8 .mu.m) were made of treated and control muscles using standard techniques. The slides were fixed with Dakopen (DAKO.RTM., ref.: S 2002) for 10 minutes open to the air and then blocked with a solution of PBS/10% goat serum for 30 min at room temperature in a humidity chamber. The rabbit anti-laminin antibody (DAKO.RTM., ref.: Z0097) was applied to the slides at a dilution of 1:1000 for 12 hours in the humidity chamber. The slides were then rinsed in PSB (5 minutes) while being agitated and the secondary antibody (Envision HRP rabbit kit) was applied to the slides in a humidity chamber for 30 min at room temperature. After rinsing the slides in PBS (5 minutes) while being agitated, the DAB (DAKO.RTM., ref.: K 3466) was applied to the sections for 2 to 5 minutes at room temperature in a humidity chamber. The slides were rinsed constantly and were mounted in the fume cupboard. The results were analysed using ELLIX software.

[0079] HPS Staining:

[0080] Cryostat sections (8 .mu.m) were made of treated and control muscles using standard techniques. The slides were immersed in Harris haematoxylin for 3 minutes and then rinsed with running water. The slides were then put into acid alcohol, rinsed and soaked in Scott's tap water substitute for one minute. After rinsing, the slides were immersed in phloxine for 30 seconds, rinsed with running water and soaked in absolute alcohol for one minute. After exposure to the saffron for 3 minutes, the slides were rinsed with absolute alcohol and mounted with Eukitt resin, the solvent for which is xylene. The results were analysed using the CARTHOGRAPH program.

II) RESULTS

[0081] 1/ Weight of Muscles at Different Times after Injection into Dystrophic mdx Mice:

[0082] The RTA and LTA muscles were collected 7 (D7), 14 (D14) or 21 (D21) days after the injection and weighed. The experiment was repeated on three separate mice each time. The results are summarised in the following tables:

Day 7:

TABLE-US-00001 [0083] Growth in % Mouse Muscles Weight (g) (100*LTA/RTA) - 100 Mouse 1 RTA 1 0.0661 3.18 LTA 1 0.0682 Mouse 2 RTA 2 0.0774 0.90 LTA 2 0.0781 Mouse 3 RTA 3 0.0749 2.94 LTA 3 0.0771

Day 14:

TABLE-US-00002 [0084] Mouse Muscles Weight Growth Mouse 4 RTA 4 0.0707 58.98 LTA 4 0.1124 Mouse 5 RTA 5 0.0694 48.41 LTA 5 0.103 Mouse 6 RTA 6 0.0854 6.67 LTA 6 0.0911

Day 21:

TABLE-US-00003 [0085] Mouse Muscles Weight Growth Mouse 7 RTA 7 0.068 53.09 LTA 7 0.1041 Mouse 8 RTA 8 0.0567 66.31 LTA 8 0.0943 Mouse 9 RTA 9 0.0731 37.21 LTA 9 0.1003

[0086] The difference in muscle mass at day 21 between an mdx mouse that had received or had not received an intramuscular injection of decorin can be seen in FIGS. 1 and 2 for mice 7 and 8, respectively. There is a clear increase in muscle mass (+53.09% and +66.31%, respectively).

2/ Weight of Muscles at D18 after Injection into Dystrophic Gamma-Sarcoglycan-/-Mice:

[0087] A second series of experiments was performed on four gamma-sarcoglycan-/-mice. The protocol was identical to that described for mdx mice. The mice were sacrificed on D18. The results, shown in FIGS. 3 and 4, are presented in the following table:

TABLE-US-00004 Mouse Muscles Weight (g) Growth Mouse 1 RTA 1 0.0456 10.75 LTA 1 0.0505 Mouse 2 RTA 2 0.0413 17.43 LTA 2 0.0485 Mouse 3 RTA 3 0.0528 12.31 LTA 3 0.0593 Mouse 4 RTA 4 0.0444 24.10 LTA 4 0.0551

3/ Injection of the Peptide 31-71 Derived from the N-Terminal Part of Murine Decorin in mdx Mice:

[0088] To verify whether the N-terminal part of decorin is sufficient to produce observable increases in muscle mass, similar experiments were performed in the presence of the mDCN 31-71 peptide (SEQ ID NO: 7) corresponding to residues 31-71 of murine decorin (SEQ ID NO:6). This peptide has been described as being sufficient and necessary for binding zinc (Yang V W, LaBrenz S R, Rosenberg L C, McQuillan D, Hook M. Decorin is a Zn2+ metalloprotein. J Biol Chem. 1999, 274(18): 12454-60.).

[0089] mdx mice were injected intramuscularly into the TA with the following formulations:

LTA 1: 65 .mu.g peptide 41 DCN+15 .mu.M ZnCl2/33 .mu.l NaCl;

RTA 2: 15 .mu.M ZnCl2/33 .mu.l NaCl.

[0090] At D18, the mice were sacrificed and the weight of the RTA and LTA muscles was measured. The results are given in the following table:

TABLE-US-00005 Muscle Weight (mg) Growth Mouse 4 RTA 4 53.6 8.77 LTA 4 58.3 Mouse 5 RTA 5 39.2 19.39 LTA 5 46.8 Mouse 6 RTA 6 40.1 24.69 LTA 6 50

[0091] These results show that an effect on muscle growth is indeed maintained in the presence of just this part of decorin.

[0092] Similar results were obtained with an even shorter peptide of 30 amino acids, with the sequence SEQ ID NO:15.

Sequence CWU 1

1

161359PRTartificial sequencehuman DCN (variant A) 1Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala1 5 10 15Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro65 70 75 80Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr Glu Ile 85 90 95Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu Ile Leu 100 105 110Val Asn Asn Lys Ile Ser Lys Val Ser Pro Gly Ala Phe Thr Pro Leu 115 120 125Val Lys Leu Glu Arg Leu Tyr Leu Ser Lys Asn Gln Leu Lys Glu Leu 130 135 140Pro Glu Lys Met Pro Lys Thr Leu Gln Glu Leu Arg Ala His Glu Asn145 150 155 160Glu Ile Thr Lys Val Arg Lys Val Thr Phe Asn Gly Leu Asn Gln Met 165 170 175Ile Val Ile Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser Gly Ile Glu 180 185 190Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile Arg Ile Ala 195 200 205Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro Ser Leu Thr 210 215 220Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser225 230 235 240Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser 245 250 255Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg 260 265 270Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly Leu 275 280 285Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile 290 295 300Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr Lys305 310 315 320Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln Tyr 325 330 335Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala 340 345 350Ile Gln Leu Gly Asn Tyr Lys 3552250PRTartificial sequencehuman DCN (variant B) 2Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala1 5 10 15Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60Val Gln Cys Ser Asp Leu Glu Leu Gly Thr Asn Pro Leu Lys Ser Ser65 70 75 80Gly Ile Glu Asn Gly Ala Phe Gln Gly Met Lys Lys Leu Ser Tyr Ile 85 90 95Arg Ile Ala Asp Thr Asn Ile Thr Ser Ile Pro Gln Gly Leu Pro Pro 100 105 110Ser Leu Thr Glu Leu His Leu Asp Gly Asn Lys Ile Ser Arg Val Asp 115 120 125Ala Ala Ser Leu Lys Gly Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser 130 135 140Phe Asn Ser Ile Ser Ala Val Asp Asn Gly Ser Leu Ala Asn Thr Pro145 150 155 160His Leu Arg Glu Leu His Leu Asp Asn Asn Lys Leu Thr Arg Val Pro 165 170 175Gly Gly Leu Ala Glu His Lys Tyr Ile Gln Val Val Tyr Leu His Asn 180 185 190Asn Asn Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro Gly His 195 200 205Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro 210 215 220Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val Tyr Val225 230 235 240Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 245 2503212PRTartificial sequencehuman DCN (variant C) 3Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala1 5 10 15Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60Val Gln Cys Ser Asp Leu Gly Leu Pro Pro Ser Leu Thr Glu Leu His65 70 75 80Leu Asp Gly Asn Lys Ile Ser Arg Val Asp Ala Ala Ser Leu Lys Gly 85 90 95Leu Asn Asn Leu Ala Lys Leu Gly Leu Ser Phe Asn Ser Ile Ser Ala 100 105 110Val Asp Asn Gly Ser Leu Ala Asn Thr Pro His Leu Arg Glu Leu His 115 120 125Leu Asp Asn Asn Lys Leu Thr Arg Val Pro Gly Gly Leu Ala Glu His 130 135 140Lys Tyr Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile Ser Val Val145 150 155 160Gly Ser Ser Asp Phe Cys Pro Pro Gly His Asn Thr Lys Lys Ala Ser 165 170 175Tyr Ser Gly Val Ser Leu Phe Ser Asn Pro Val Gln Tyr Trp Glu Ile 180 185 190Gln Pro Ser Thr Phe Arg Cys Val Tyr Val Arg Ser Ala Ile Gln Leu 195 200 205Gly Asn Tyr Lys 2104172PRTartificial sequencehuman DCN (variant D) 4Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala1 5 10 15Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro Lys Asp Leu Pro65 70 75 80Pro Asp Thr Thr Leu Leu Asp Leu Gln Asn Asn Lys Ile Thr Glu Ile 85 90 95Lys Asp Gly Asp Phe Lys Asn Leu Lys Asn Leu His Val Val Tyr Leu 100 105 110His Asn Asn Asn Ile Ser Val Val Gly Ser Ser Asp Phe Cys Pro Pro 115 120 125Gly His Asn Thr Lys Lys Ala Ser Tyr Ser Gly Val Ser Leu Phe Ser 130 135 140Asn Pro Val Gln Tyr Trp Glu Ile Gln Pro Ser Thr Phe Arg Cys Val145 150 155 160Tyr Val Arg Ser Ala Ile Gln Leu Gly Asn Tyr Lys 165 170575PRTartificial sequencehuman DCN (variant E) 5Met Lys Ala Thr Ile Ile Leu Leu Leu Leu Ala Gln Val Ser Trp Ala1 5 10 15Gly Pro Phe Gln Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30Ala Ser Gly Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro 35 40 45Ser Leu Gly Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val 50 55 60Val Gln Cys Ser Asp Leu Gly Cys Leu Pro Ser65 70 756354PRTartificial sequencemurine DCN 6Met Lys Ala Thr Leu Ile Phe Phe Leu Leu Ala Gln Val Ser Trp Ala1 5 10 15Gly Pro Phe Glu Gln Arg Gly Leu Phe Asp Phe Met Leu Glu Asp Glu 20 25 30Ala Ser Gly Ile Ile Pro Tyr Asp Pro Asp Asn Pro Leu Ile Ser Met 35 40 45Cys Pro Tyr Arg Cys Gln Cys His Leu Arg Val Val Gln Cys Ser Asp 50 55 60Leu Gly Leu Asp Lys Val Pro Trp Asp Phe Pro Pro Asp Thr Thr Leu65 70 75 80Leu Asp Leu Gln Asn Asn Lys Ile Thr Glu Ile Lys Glu Gly Ala Phe 85 90 95Lys Asn Leu Lys Asp Leu His Thr Leu Ile Leu Val Asn Asn Lys Ile 100 105 110Ser Lys Ile Ser Pro Glu Ala Phe Lys Pro Leu Val Lys Leu Glu Arg 115 120 125Leu Tyr Leu Ser Lys Asn Gln Leu Lys Glu Leu Pro Glu Lys Met Pro 130 135 140Arg Thr Leu Gln Glu Leu Arg Val His Glu Asn Glu Ile Thr Lys Leu145 150 155 160Arg Lys Ser Asp Phe Asn Gly Leu Asn Asn Val Leu Val Ile Glu Leu 165 170 175Gly Gly Asn Pro Leu Lys Asn Ser Gly Ile Glu Asn Gly Ala Phe Gln 180 185 190Gly Leu Lys Ser Leu Ser Tyr Ile Arg Ile Ser Asp Thr Asn Ile Thr 195 200 205Ala Ile Pro Gln Gly Leu Pro Thr Ser Leu Thr Glu Val His Leu Asp 210 215 220Gly Asn Lys Ile Thr Lys Val Asp Ala Pro Ser Leu Lys Gly Leu Ile225 230 235 240Asn Leu Ser Lys Leu Gly Leu Ser Phe Asn Ser Ile Thr Val Met Glu 245 250 255Asn Gly Ser Leu Ala Asn Val Pro His Leu Arg Glu Leu His Leu Asp 260 265 270Asn Asn Lys Leu Leu Arg Val Pro Ala Gly Leu Ala Gln His Lys Tyr 275 280 285Ile Gln Val Val Tyr Leu His Asn Asn Asn Ile Ser Ala Val Gly Gln 290 295 300Asn Asp Phe Cys Arg Ala Gly His Pro Ser Arg Lys Ala Ser Tyr Ser305 310 315 320Ala Val Ser Leu Tyr Gly Asn Pro Val Arg Tyr Trp Glu Ile Phe Pro 325 330 335Asn Thr Phe Arg Cys Val Tyr Val Arg Ser Ala Ile Gln Leu Gly Asn 340 345 350Tyr Lys 741PRTartificial sequencemurine 31-71 DCN 7Asp Glu Ala Ser Gly Ile Ile Pro Tyr Asp Pro Asp Asn Pro Leu Ile1 5 10 15Ser Met Cys Pro Tyr Arg Cys Gln Cys His Leu Arg Val Val Gln Cys 20 25 30Ser Asp Leu Gly Leu Asp Lys Val Pro 35 4082305DNAartificial sequencevariant A1 human DCN 8gaatctacaa taagacaaat ttcaaatcaa gttgctccac tatactgcat aagcagttta 60gaatcttaag cagatgcaaa aagaataaag caaatgggag gaaaaaaaag gccgataaag 120tttctggcta caatacaaga gacatatcat taccatatga tctaatgtgg gtgtcagccg 180gattgtgttc attgagggaa accttatttt ttaactgtgc tatggagtag aagcaggagg 240ttttcaacct agtcacagag cagcacctac cccctcctcc tttccacacc tgcaaactct 300tttacttggg ctgaatattt agtgtaatta catctcagct ttgagggctc ctgtggcaaa 360ttcccggatt aaaaggttcc ctggttgtga aaatacatga gataaatcat gaaggccact 420atcatcctcc ttctgcttgc acaagtttcc tgggctggac cgtttcaaca gagaggctta 480tttgacttta tgctagaaga tgaggcttct gggataggcc cagaagttcc tgatgaccgc 540gacttcgagc cctccctagg cccagtgtgc cccttccgct gtcaatgcca tcttcgagtg 600gtccagtgtt ctgatttggg tctggacaaa gtgccaaagg atcttccccc tgacacaact 660ctgctagacc tgcaaaacaa caaaataacc gaaatcaaag atggagactt taagaacctg 720aagaaccttc acgcattgat tcttgtcaac aataaaatta gcaaagttag tcctggagca 780tttacacctt tggtgaagtt ggaacgactt tatctgtcca agaatcagct gaaggaattg 840ccagaaaaaa tgcccaaaac tcttcaggag ctgcgtgccc atgagaatga gatcaccaaa 900gtgcgaaaag ttactttcaa tggactgaac cagatgattg tcatagaact gggcaccaat 960ccgctgaaga gctcaggaat tgaaaatggg gctttccagg gaatgaagaa gctctcctac 1020atccgcattg ctgataccaa tatcaccagc attcctcaag gtcttcctcc ttcccttacg 1080gaattacatc ttgatggcaa caaaatcagc agagttgatg cagctagcct gaaaggactg 1140aataatttgg ctaagttggg attgagtttc aacagcatct ctgctgttga caatggctct 1200ctggccaaca cgcctcatct gagggagctt cacttggaca acaacaagct taccagagta 1260cctggtgggc tggcagagca taagtacatc caggttgtct accttcataa caacaatatc 1320tctgtagttg gatcaagtga cttctgccca cctggacaca acaccaaaaa ggcttcttat 1380tcgggtgtga gtcttttcag caacccggtc cagtactggg agatacagcc atccaccttc 1440agatgtgtct acgtgcgctc tgccattcaa ctcggaaact ataagtaatt ctcaagaaag 1500ccctcatttt tataacctgg caaaatcttg ttaatgtcat tgctaaaaaa taaataaaag 1560ctagatactg gaaacctaac tgcaatgtgg atgttttacc cacatgactt attatgcata 1620aagccaaatt tccagtttaa gtaattgcct acaataaaaa gaaattttgc ctgccatttt 1680cagaatcatc ttttgaagct ttctgttgat gttaactgag ctactagaga tattcttatt 1740tcactaaatg taaaatttgg agtaaatata tatgtcaata tttagtaaag cttttctttt 1800ttaatttcca ggaaaaaata aaaagagtat gagtcttctg taattcattg agcagttagc 1860tcatttgaga taaagtcaaa tgccaaacac tagctctgta ttaatcccca tcattactgg 1920taaagcctca tttgaatgtg tgaattcaat acaggctatg taaaattttt actaatgtca 1980ttattttgaa aaaataaatt taaaaataca ttcaaaatta ctattgtata caagcttaat 2040tgttaatatt ccctaaacac aattttatga agggagaaga cattggtttg ttgacaataa 2100cagtacatct tttcaagttc tcagctattt cttctacctc tccctatctt acatttgagt 2160atggtaactt atgtcatcta tgttgaatgt aagcttataa agcacaaagc atacatttcc 2220tgactggtct agagaactga tgtttcaatt tacccctctg ctaaataaat attaaaacta 2280tcatgtgaaa aaaaaaaaaa aaaaa 230592151DNAartificial sequencevariant A2 human CN 9ggaataataa gacacgccct gaaggagtac atcgtctagt gagggacaga ccaagcacgc 60aaaacaaatt gcaatataat gtgataagtt ctttaaaaga ggtaagagca acgtgctttg 120ggagcagaga agagggagaa agcagcatct tgcctggatg agccagggga cacagaagag 180aagcccacta tctcatttaa tctttacaac tctcttgcaa ggttccctgg ttgtgaaaat 240acatgagata aatcatgaag gccactatca tcctccttct gcttgcacaa gtttcctggg 300ctggaccgtt tcaacagaga ggcttatttg actttatgct agaagatgag gcttctggga 360taggcccaga agttcctgat gaccgcgact tcgagccctc cctaggccca gtgtgcccct 420tccgctgtca atgccatctt cgagtggtcc agtgttctga tttgggtctg gacaaagtgc 480caaaggatct tccccctgac acaactctgc tagacctgca aaacaacaaa ataaccgaaa 540tcaaagatgg agactttaag aacctgaaga accttcacgc attgattctt gtcaacaata 600aaattagcaa agttagtcct ggagcattta cacctttggt gaagttggaa cgactttatc 660tgtccaagaa tcagctgaag gaattgccag aaaaaatgcc caaaactctt caggagctgc 720gtgcccatga gaatgagatc accaaagtgc gaaaagttac tttcaatgga ctgaaccaga 780tgattgtcat agaactgggc accaatccgc tgaagagctc aggaattgaa aatggggctt 840tccagggaat gaagaagctc tcctacatcc gcattgctga taccaatatc accagcattc 900ctcaaggtct tcctccttcc cttacggaat tacatcttga tggcaacaaa atcagcagag 960ttgatgcagc tagcctgaaa ggactgaata atttggctaa gttgggattg agtttcaaca 1020gcatctctgc tgttgacaat ggctctctgg ccaacacgcc tcatctgagg gagcttcact 1080tggacaacaa caagcttacc agagtacctg gtgggctggc agagcataag tacatccagg 1140ttgtctacct tcataacaac aatatctctg tagttggatc aagtgacttc tgcccacctg 1200gacacaacac caaaaaggct tcttattcgg gtgtgagtct tttcagcaac ccggtccagt 1260actgggagat acagccatcc accttcagat gtgtctacgt gcgctctgcc attcaactcg 1320gaaactataa gtaattctca agaaagccct catttttata acctggcaaa atcttgttaa 1380tgtcattgct aaaaaataaa taaaagctag atactggaaa cctaactgca atgtggatgt 1440tttacccaca tgacttatta tgcataaagc caaatttcca gtttaagtaa ttgcctacaa 1500taaaaagaaa ttttgcctgc cattttcaga atcatctttt gaagctttct gttgatgtta 1560actgagctac tagagatatt cttatttcac taaatgtaaa atttggagta aatatatatg 1620tcaatattta gtaaagcttt tcttttttaa tttccaggaa aaaataaaaa gagtatgagt 1680cttctgtaat tcattgagca gttagctcat ttgagataaa gtcaaatgcc aaacactagc 1740tctgtattaa tccccatcat tactggtaaa gcctcatttg aatgtgtgaa ttcaatacag 1800gctatgtaaa atttttacta atgtcattat tttgaaaaaa taaatttaaa aatacattca 1860aaattactat tgtatacaag cttaattgtt aatattccct aaacacaatt ttatgaaggg 1920agaagacatt ggtttgttga caataacagt acatcttttc aagttctcag ctatttcttc 1980tacctctccc tatcttacat ttgagtatgg taacttatgt catctatgtt gaatgtaagc 2040ttataaagca caaagcatac atttcctgac tggtctagag aactgatgtt tcaatttacc 2100cctctgctaa ataaatatta aaactatcat gtgaaaaaaa aaaaaaaaaa a 2151101570DNAartificial sequenceVariant B human DCN 10atgaaggcca ctatcatcct ccttctgctt gcacaagttt cctgggctgg accgtttcaa 60cagagaggct tatttgactt tatgctagaa gatgaggctt ctgggatagg cccagaagtt 120cctgatgacc gcgacttcga gccctcccta ggcccagtgt gccccttccg ctgtcaatgc 180catcttcgag tggtccagtg ttctgatttg gaactgggca ccaatccgct gaagagctca 240ggaattgaaa atggggcttt ccagggaatg aagaagctct cctacatccg cattgctgat 300accaatatca ccagcattcc tcaaggtctt cctccttccc ttacggaatt acatcttgat 360ggcaacaaaa tcagcagagt tgatgcagct agcctgaaag gactgaataa tttggctaag 420ttgggattga gtttcaacag catctctgct gttgacaatg gctctctggc caacacgcct 480catctgaggg agcttcactt ggacaacaac aagcttacca gagtacctgg tgggctggca 540gagcataagt acatccaggt tgtctacctt cataacaaca atatctctgt agttggatca 600agtgacttct gcccacctgg acacaacacc aaaaaggctt cttattcggg tgtgagtctt 660ttcagcaacc cggtccagta ctgggagata cagccatcca ccttcagatg tgtctacgtg 720cgctctgcca ttcaactcgg aaactataag taattctcaa gaaagccctc atttttataa 780cctggcaaaa tcttgttaat gtcattgcta aaaaataaat aaaagctaga tactggaaac 840ctaactgcaa tgtggatgtt ttacccacat gacttattat gcataaagcc aaatttccag 900tttaagtaat tgcctacaat aaaaagaaat tttgcctgcc attttcagaa tcatcttttg 960aagctttctg ttgatgttaa ctgagctact agagatattc ttatttcact aaatgtaaaa 1020tttggagtaa atatatatgt caatatttag taaagctttt cttttttaat ttccaggaaa 1080aaataaaaag agtatgagtc ttctgtaatt cattgagcag ttagctcatt tgagataaag 1140tcaaatgcca aacactagct ctgtattaat ccccatcatt actggtaaag cctcatttga 1200atgtgtgaat tcaatacagg ctatgtaaaa tttttactaa tgtcattatt ttgaaaaaat 1260aaatttaaaa atacattcaa aattactatt gtatacaagc

ttaattgtta atattcccta 1320aacacaattt tatgaaggga gaagacattg gtttgttgac aataacagta catcttttca 1380agttctcagc tatttcttct acctctccct atcttacatt tgagtatggt aacttatgtc 1440atctatgttg aatgtaagct tataaagcac aaagcataca tttcctgact ggtctagaga 1500actgatgttt caatttaccc ctctgctaaa taaatattaa aactatcatg tgaaaaaaaa 1560aaaaaaaaaa 1570111456DNAartificial sequencevariant C human DCN 11atgaaggcca ctatcatcct ccttctgctt gcacaagttt cctgggctgg accgtttcaa 60cagagaggct tatttgactt tatgctagaa gatgaggctt ctgggatagg cccagaagtt 120cctgatgacc gcgacttcga gccctcccta ggcccagtgt gccccttccg ctgtcaatgc 180catcttcgag tggtccagtg ttctgatttg ggtcttcctc cttcccttac ggaattacat 240cttgatggca acaaaatcag cagagttgat gcagctagcc tgaaaggact gaataatttg 300gctaagttgg gattgagttt caacagcatc tctgctgttg acaatggctc tctggccaac 360acgcctcatc tgagggagct tcacttggac aacaacaagc ttaccagagt acctggtggg 420ctggcagagc ataagtacat ccaggttgtc taccttcata acaacaatat ctctgtagtt 480ggatcaagtg acttctgccc acctggacac aacaccaaaa aggcttctta ttcgggtgtg 540agtcttttca gcaacccggt ccagtactgg gagatacagc catccacctt cagatgtgtc 600tacgtgcgct ctgccattca actcggaaac tataagtaat tctcaagaaa gccctcattt 660ttataacctg gcaaaatctt gttaatgtca ttgctaaaaa ataaataaaa gctagatact 720ggaaacctaa ctgcaatgtg gatgttttac ccacatgact tattatgcat aaagccaaat 780ttccagttta agtaattgcc tacaataaaa agaaattttg cctgccattt tcagaatcat 840cttttgaagc tttctgttga tgttaactga gctactagag atattcttat ttcactaaat 900gtaaaatttg gagtaaatat atatgtcaat atttagtaaa gcttttcttt tttaatttcc 960aggaaaaaat aaaaagagta tgagtcttct gtaattcatt gagcagttag ctcatttgag 1020ataaagtcaa atgccaaaca ctagctctgt attaatcccc atcattactg gtaaagcctc 1080atttgaatgt gtgaattcaa tacaggctat gtaaaatttt tactaatgtc attattttga 1140aaaaataaat ttaaaaatac attcaaaatt actattgtat acaagcttaa ttgttaatat 1200tccctaaaca caattttatg aagggagaag acattggttt gttgacaata acagtacatc 1260ttttcaagtt ctcagctatt tcttctacct ctccctatct tacatttgag tatggtaact 1320tatgtcatct atgttgaatg taagcttata aagcacaaag catacatttc ctgactggtc 1380tagagaactg atgtttcaat ttacccctct gctaaataaa tattaaaact atcatgtgaa 1440aaaaaaaaaa aaaaaa 1456121336DNAArtificial sequencevariant D human DCN 12atgaaggcca ctatcatcct ccttctgctt gcacaagttt cctgggctgg accgtttcaa 60cagagaggct tatttgactt tatgctagaa gatgaggctt ctgggatagg cccagaagtt 120cctgatgacc gcgacttcga gccctcccta ggcccagtgt gccccttccg ctgtcaatgc 180catcttcgag tggtccagtg ttctgatttg ggtctggaca aagtgccaaa ggatcttccc 240cctgacacaa ctctgctaga cctgcaaaac aacaaaataa ccgaaatcaa agatggagac 300tttaagaacc tgaagaacct tcacgttgtc taccttcata acaacaatat ctctgtagtt 360ggatcaagtg acttctgccc acctggacac aacaccaaaa aggcttctta ttcgggtgtg 420agtcttttca gcaacccggt ccagtactgg gagatacagc catccacctt cagatgtgtc 480tacgtgcgct ctgccattca actcggaaac tataagtaat tctcaagaaa gccctcattt 540ttataacctg gcaaaatctt gttaatgtca ttgctaaaaa ataaataaaa gctagatact 600ggaaacctaa ctgcaatgtg gatgttttac ccacatgact tattatgcat aaagccaaat 660ttccagttta agtaattgcc tacaataaaa agaaattttg cctgccattt tcagaatcat 720cttttgaagc tttctgttga tgttaactga gctactagag atattcttat ttcactaaat 780gtaaaatttg gagtaaatat atatgtcaat atttagtaaa gcttttcttt tttaatttcc 840aggaaaaaat aaaaagagta tgagtcttct gtaattcatt gagcagttag ctcatttgag 900ataaagtcaa atgccaaaca ctagctctgt attaatcccc atcattactg gtaaagcctc 960atttgaatgt gtgaattcaa tacaggctat gtaaaatttt tactaatgtc attattttga 1020aaaaataaat ttaaaaatac attcaaaatt actattgtat acaagcttaa ttgttaatat 1080tccctaaaca caattttatg aagggagaag acattggttt gttgacaata acagtacatc 1140ttttcaagtt ctcagctatt tcttctacct ctccctatct tacatttgag tatggtaact 1200tatgtcatct atgttgaatg taagcttata aagcacaaag catacatttc ctgactggtc 1260tagagaactg atgtttcaat ttacccctct gctaaataaa tattaaaact atcatgtgaa 1320aaaaaaaaaa aaaaaa 1336131223DNAArtificial sequenceVariant E human DCN 13atgaaggcca ctatcatcct ccttctgctt gcacaagttt cctgggctgg accgtttcaa 60cagagaggct tatttgactt tatgctagaa gatgaggctt ctgggatagg cccagaagtt 120cctgatgacc gcgacttcga gccctcccta ggcccagtgt gccccttccg ctgtcaatgc 180catcttcgag tggtccagtg ttctgatttg ggttgtctac cttcataaca acaatatctc 240tgtagttgga tcaagtgact tctgcccacc tggacacaac accaaaaagg cttcttattc 300gggtgtgagt cttttcagca acccggtcca gtactgggag atacagccat ccaccttcag 360atgtgtctac gtgcgctctg ccattcaact cggaaactat aagtaattct caagaaagcc 420ctcattttta taacctggca aaatcttgtt aatgtcattg ctaaaaaata aataaaagct 480agatactgga aacctaactg caatgtggat gttttaccca catgacttat tatgcataaa 540gccaaatttc cagtttaagt aattgcctac aataaaaaga aattttgcct gccattttca 600gaatcatctt ttgaagcttt ctgttgatgt taactgagct actagagata ttcttatttc 660actaaatgta aaatttggag taaatatata tgtcaatatt tagtaaagct tttctttttt 720aatttccagg aaaaaataaa aagagtatga gtcttctgta attcattgag cagttagctc 780atttgagata aagtcaaatg ccaaacacta gctctgtatt aatccccatc attactggta 840aagcctcatt tgaatgtgtg aattcaatac aggctatgta aaatttttac taatgtcatt 900attttgaaaa aataaattta aaaatacatt caaaattact attgtataca agcttaattg 960ttaatattcc ctaaacacaa ttttatgaag ggagaagaca ttggtttgtt gacaataaca 1020gtacatcttt tcaagttctc agctatttct tctacctctc cctatcttac atttgagtat 1080ggtaacttat gtcatctatg ttgaatgtaa gcttataaag cacaaagcat acatttcctg 1140actggtctag agaactgatg tttcaattta cccctctgct aaataaatat taaaactatc 1200atgtgaaaaa aaaaaaaaaa aaa 1223141065DNAArtificial sequenceMurine DCN 14atgaaggcaa ctctcatctt cttccttctg gcacaagtct cttgggctgg accatttgaa 60cagagaggct tatttgactt catgctagaa gatgaggctt ctggcataat cccttatgac 120cctgacaatc ccctgatatc tatgtgcccc taccgatgcc agtgtcatct tcgagtggtg 180cagtgttctg atctgggttt ggacaaagtg ccctgggatt ttccacccga cacaaccttg 240ctagacctgc aaaacaacaa aattacagag atcaaagaag gggccttcaa gaacctgaag 300gacttgcata ccttgatcct tgtcaacaac aagatcagca aaatcagtcc agaggcattc 360aaacctctcg tgaagttgga aaggctttac ctgtctaaga accaactaaa ggaactgcct 420gaaaaaatgc ccagaactct ccaggaactt cgtgtccatg agaatgagat caccaagctg 480cggaaatccg acttcaatgg actgaacaat gtgcttgtca tagaactggg cggcaaccca 540ctgaaaaact ctgggattga aaacggagcc ttccagggac tgaagagtct ctcatacatt 600cgcatctcag acaccaacat aactgcgatc cctcaaggtc tgcctacttc tctcactgaa 660gtgcatctag atggcaacaa gatcaccaag gttgatgcac ccagcctgaa aggactgatt 720aatttgtcta aactgggatt gagcttcaac agcatcaccg ttatggagaa tggcagtctg 780gccaatgttc ctcatctgag ggaactccac ttggacaaca acaaactcct cagggtgcct 840gctgggctgg cacagcataa gtatatccag gtcgtctacc ttcacaacaa caacatctcc 900gcagttgggc aaaatgactt ctgccgagct ggacacccct ctcgaaaggc ttcctactcg 960gctgtgagtc tttacggcaa ccctgtccgg tattgggaaa tctttccaaa caccttcaga 1020tgtgtctatg tgcgttctgc cattcaactt ggaaactaca agtaa 10651530PRTartificial sequencemurine 42-71 DCN 15Asp Asn Pro Leu Ile Ser Met Cys Pro Tyr Arg Cys Gln Cys His Leu1 5 10 15Arg Val Val Gln Cys Ser Asp Leu Gly Leu Asp Lys Val Pro 20 25 301641PRTartificial sequencehuman DCN fragment 16Ile Gly Pro Glu Val Pro Asp Asp Arg Asp Phe Glu Pro Ser Leu Gly1 5 10 15Pro Val Cys Pro Phe Arg Cys Gln Cys His Leu Arg Val Val Gln Cys 20 25 30Ser Asp Leu Gly Leu Asp Lys Val Pro 35 40

Claims

1. Composition containing a fragment of decorin able to bind zinc to treat diseases associated with muscle wasting.

2. Composition according to claim 1, characterised in that the diseases are selected from the group of neuromuscular diseases, to advantage muscular dystrophies such as Duchenne muscular dystrophy, and the cachexia.

3. Composition according to claim 1 or 2, characterised in that the fragment comprises the sequence SEQ ID NO: 7 or 15.

4. Composition according to claim 1 or 2, characterised in that the fragment has the sequence SEQ ID NO: 7 or 15.

5. Composition containing decorin to increase muscle mass.

6. Composition according to claim 5, characterised in that the aim of increasing muscle mass is to compensate for wasting resulting from immobilisation or old age.

7. Composition according to claim 5 or 6, characterised in that it is for use in animals.

8. Composition according to one of claim 5 or 6, characterised in that decorin is in the form of an active fragment.

9. Composition according to claim 8, characterised in that the active fragment is able to bind zinc.

10. Composition according to claim 9, characterised in that the fragment comprises the sequence SEQ ID NO: 7 or 15.

11. Composition according to claim 9, characterised by the fragment has the sequence SEQ ID NO: 7 or 15.

12. Composition according to claim 1 or 5, characterised in that the decorin is in the form of an active fragment, a recombinant protein, a fusion protein or a nucleic acid encoding such a protein or fragment.

13. Composition according to claim 1 or 5, characterised in that the decorin is for intramuscular, intraperitoneal or intravenous injection.

14. Composition according to claim 1 or 5, characterised in that the decorin is associated with other treatments, particularly gene therapy and cell grafting.