WO1997004090A1

WO1997004090A1 - SEQUENCE AND EXPRESSION OF Sox-18

Info

Publication number: WO1997004090A1
Application number: PCT/AU1996/000455
Authority: WO
Inventors: Timothy L. Dunn; George E. Muscat; Lesley Mynett-Johnson; Peter Koopman
Original assignee: University of Queensland UQ
Current assignee: University of Queensland UQ
Priority date: 1995-07-18
Filing date: 1996-07-18
Publication date: 1997-02-06
Anticipated expiration: 1998-01-18
Also published as: AU6509096A

Abstract

A gene designated 'Sox-18' encodes a transcription factor. A subsequence of the Sox-18 nucleotide sequence has been identified that encodes an activation domain of the Sox-18 transcription factor. Polypeptides comprising all or a part of the Sox-18 expression product can be used to regulate myogenesis, proliferation of skeletal and cardiac muscle, and differentiation of smooth muscle, for example, in the context of treating atherosclerosis, restenosis and pulmonary disease.

Description

TITLE SEQUENCE AND EXPRESSION OF Sox-18

BACKGROUND OF THE INVENTION

The present invention relates to Sox-18 , a new HMG-box transcription factor.

The number of known members of the Sox gene family is rapidly increasing. Sox genes are characterized by a conserved DNA sequence encoding an approximately 80-amino acid domain responsible for sequence-specific DNA binding. This domain has homology with the HMG (high mobility group) box DNA-binding domain, originally identified in the transcription factor UBF (Jantzen et al . , 1992, Genes Dev. 6 1950-1963). The HMG-box binding domain contains highly conserved proline, aromatic and basic residues, as described in Laudet et al . , 1993, Nucleic Acids Res. 21 2493-2500. More than 60 different HMG-box proteins have been reported and/or entered into sequence databases (Laudet et al . , 1993). These fall into two broad categories, those with sequence-specific DNA-binding activity (Sry, TCF-1, LEF-1 and the Sox proteins) , and those which bind DNA in a sequence- independent manner (HMG protein, UBF). All known members of the sequence-specific group bind to variations of the WCAA G motif (Giese et al . , 1991, Genes Dev. 5 2567- 2578; Ferrari et al . , 1992, EMBO J. 11 4497-4506 and Van de etering and Clevers, EMBO J. 11 3039-3044), where W = A or T.

The Sox (£ry like HMG box gene) family takes its name from the first member isolated, the mammalian Y- linked testis determining gene, Sry, whose expression in early embryogenesis is sufficient to cause the male development of a chromosomally female mouse (Koopman et al . , 1991, Nature 351 117-121). In the course of cloning murine Sry, four other HMG-box genes expressed during embryogenesis were identified (Sox-1-4; Gubbay et al . , 1990, Nature 351 245-250). Subsequent PCR cloning has now led to the identification of 18 different Sox genes in the mouse, most of which are as yet uncharacterized, and many of which have orthologues across the plant and animal kingdoms (Laudet et al . , 1993 and references therein) . As used in this specification, orthologues are nucleotide sequences with sequence similarity that derives from common ancestry.

A full-length cDNA sequence has been reported for human Sry (Sinclair et al . , 1990, Nature 346 240-244), mouse Sox-A and 5 (Denny et al . , 1992, Nucleic Acids Res 20 2887; Van de Wetering et al . , 1993, EMBO J. 12 3847- 3854), human and marsupial Sox-3 (Foster and Graves, 1994, Proc. Natl. Acad. Sci. U.S.A. 91 1927193 1) and human TCF- 1 (Oosterwegel et al . , 199 1, J. Exp. Med. 173 1133-1142). Several members of the Sox family show tissue-specific expression during embryogenesis, suggesting a developmental role, including Sry (genital ridge and testis; Koopman et al . , 1991, Nature 351 117- 121) and Sox-1 , 2 and 3 (nervous system; J. Collignon and R.Lovell-Badge, data not shown).

TCF- 1 (Van de Wetering, 1992, J. Biol. Chem. 267 8530-8536)., LEF-1 (Travis et al . , 1991, Genes Dev. 5 880-94) and Sox-4 (Van de Wetering et al . , 1993) have been implicated in the regulation of lymphoid differentiation and have been shown to be restricted in their expression, Sox-5 is expressed in post-meiotic round spermatids, suggesting a role in spermatogenesis (Denny et al . , 1992 EMBO J. 1 1 3705-3712 and Connor et al . , 1994, Nucleic Acids Res. 22 33393346) and Soχ-9 is expressed during chondrogenesis in mouse embryos (Wright et al . , 1995, Nature Genet. 9 15-20). In Wright et al . , 1993, Nucleic Acids Res. 21 744,

Sox genes encoding the HMG-box domain were identified in embryonic mouse tissues using a reverse transcriptase- mediated PCR. Seven distinct subgroups have been identified within the murine Sox gene family according to the amino acid identity within the HMG-box region (Van de Wetering et al . , 1993 and Wright et al . , 1993). These subgroups were identified using degenerate primers which span a highly conserved 230-base pair region in Syr and the known Sox genes, encoding the HMG-box domain.

While various Sox genes have been identified, a need exists for the identification and characterization of Sox genes present in muscle tissue. Identification and characterization of such genes and their corresponding polypeptide sequences would provide valuable insight into regulation of tissue development and therapies for treatment of diseases that affect muscle tissue.

The inventors have recently identified a Sox gene designated Sox-18 from a mouse cardiac and skeletal muscle cDNA library. The full length nucleotide and deduced amino acid sequences relating to this gene were published on the GenBank database on 7 March 1995. However, this sequence information did not disclose various biological fragments of Sox-18 , or therapeutic potential of Sox-18 or methods for treatment of various disease conditions using Sox-18 which are described hereinafter.

SUMMARY OF THE INVENTION

It is therefore an object of the invention to provide methods for treatment of diseases involving muscle cells, wherein a Sox nucleotide sequence or polypeptide according to the invention is used as a therapeutic agent to treat such diseases.

A subsequence of the Sox-18 nucleotide sequence has been identified that encodes an activation domain of this transcription factor. Polypeptides comprising all or a part of the Sox-18 expression product can be used to regulate myogenesis, proliferation of skeletal and cardiac muscle, and differentiation of smooth muscle, for example, in the context of treating atherosclerosis, restenosis and pulmonary disease.

The invention further provides an isolated nucleotide sequence comprising nucleotide 566 through nucleotide 778 of Figure 2, and an isolated nucleotide sequence encoding amino acids 160 to 255 of the amino acid sequence of Figure 2.

It is also an object of the invention to provide an isolated polypeptide comprising amino acids 160 to 255 of Figure 2.

It is a further object of the invention to provide a homolog of the abovementioned nucleotide sequences. The invention further comprises such homologs obtained from muscle tissue, lung tissue or cardiac tissue. The invention further comprises a homolog wherein said homolog is obtained from a mammal selected from the group consisting of human, mouse, rat, chicken, guinea pig and rabbit. By "obtained from", is meant that something is isolated from, or derived from, a particular source.

The nucleotide sequence in accordance with the invention may be obtained from tissue isolated directly from a mammalian source. A homolog of the invention may also be obtained from a DNA library derived from mammalian tissue (such as muscle, heart or lung tissue). Such a library may be a mouse heart cDNA library.

It is also an object of the invention to provide a polypeptide homolog of the abovementioned polypeptide comprising amino acids 160 to 255. The invention further comprises such polypeptide homologs obtained from muscle tissue, lung tissue or cardiac tissue.

The invention further comprises a polypeptide homolog of the abovementioned polypeptide comprising amino acids 160 to 255 wherein said homolog is obtained from a mammal selected from the group consisting of human, mouse, rat, chicken, guinea pig and rabbit.

The invention also provides a method for inducing transcription of a gene, comprising the steps of: (a) transforming a mammalian cell with a plasmid comprising a Sox-18 nucleotide sequence, wherein the expression of said gene is under the control of a constitutive promoter; and

(b) transforming the cell of step (a) with a plasmid comprising said gene, which is operably linked to a DNA sequence comprising the sequence AACAAAG.

The invention also contemplates a method for inducing myogenesis in a mammalian cell, comprising transforming said cell with a plasmid comprising a Sox-18 nucleotide sequence, wherein said sequence is operably linked, in the sense orientation, to a constitutive promoter.

In this regard, the term Sox-18 as used with reference to methods of use thereof includes within its scope the full length Sox-18 DNA sequence comprising the sequence of nucleotides shown in Figure 2 as well as biological fragments thereof.

The invention further comprises a method for inhibiting myogenesis in a mammalian cell, comprising transforming said cell with a plasmid comprising a Sox-18 nucleotide, sequence, wherein said sequence is operably linked, in the antisense orientation, to a constitutive promoter.

The invention also comprises a method for inhibiting the proliferation of skeletal muscle proliferation in a mammalian cell, comprising transforming said cell with a plasmid comprising a Sox-18 nucleotide sequence, whereinsaid sequence is operably linked, in the sense orientation, to a constitutive promoter.

In yet another embodiment of the invention, there is provided a method for inhibiting the proliferation of cardiac muscle proliferation in a mammalian cell, comprising transforming said cell with a plasmid comprising a Sox- 18 nucleotide sequence, wherein said sequence is operably linked, in the sense orientation, to a constitutive promoter.

The invention also provides a method for inducing differentiation of smooth muscle in a mammalian smooth muscle cell, comprising transforming said cell with a plasmid comprising a Sox-18 nucleotide sequence, wherein said sequence is operably linked, in the sense orientation, to a constitutive promoter.

It is another object of the invention to provide a pharmaceutical composition comprising a nucleotide sequence, encoding a Sox-18 polypeptide. Methods for treatment of atherosclerosis, restenosis, and pulmonary disease (including pulmonary thrombosis and pulmonary fibrosis), comprising administering to a patient in need of such treatment a pharmaceutically effective amount of said pharmaceutical composition, are also provided. The invention also provides a method for stimulation of vascularisation in skeletal muscle tissue in vivo, a method for stimulation of vascular endothelial cell proliferation in vivo, a method for inducing endothelialisation of vascular grafts in vivo , comprising administering to a patient in need of the aforementioned treatments, a pharmaceutically effective amount of the composition of claim 19.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA shows an amino-acid sequence comparison of the HMG-box region of Sox-7 , Sox- 17 , Sox-18 and the Sox HMG-box consensus (Wright et al . , 1993). Identities are represented by upper case, gaps are shown with dashes (-). The sequences chosen for degenerate PCR primers are underlined. GenBank Accession Nos. (HMG-box region only): Sox-17, L29085; Sox- 18, L29086. Figure IB shows the nucleotide sequence for the Sox- 17 HMG-box.

Figure 2 shows the full nucleotide sequence of mouse Sox-18 (GenBank Accession No. L35032) and deduced amino acid sequence. The HMG-box is shown in bold type; a potential body (A) signal is underlined.

Figure 3 shows Northern analysis of Sox-18 expression in various mouse tissues. The source from which RNA was isolated are marked, skeletal muscle from the hind limb is marked as "muscle".

Figure 4A shows EMSA of ³P kinase radiolabelled SoCM ( Sox Consensus Motif (SoCM = AACAAAG) ) (0.5 ng) and affinity purified recombinant GST-Sox-lS fusion protein (5 μg) performed as described in Downes et al . , Cell Growth Diff . 4:901 (1993). Competition with unlabelled double stranded oligo was carried out at 25- and 100-fold molar excess. C denotes the control binding reaction in the absence of ^' unlabelled competitor. The unlabelled competitors are SoCM, non-hormone response elements (SRE, MEF-I and MEF-11) and the characterized steroid hormone response elements (MCRBPI [RARE] and AMHC [TRE]).

Figure 4B shows transcriptional activation of Gal chimeric constructs in transient transfection assays into COS-l cells using DOTAP (BoehringerMannheim) . 5 μg of the pG5ElbCAT reporter (containing five Gal4 binding motifs) was co-transfected with 3 μg of each of the Gal chimers indicated. GalO contains amino acid 1-147 of the yeast Gal4 transcription factor responsible for the DNA binding activity. Gal-Spl and Gal-MyoD contain the complete ORF of the Spl and MyoD transcription factors respectively.

Figure 5A shows Gal-Sox-lS expression constructs used in co-transfection assays to map the activation domain of Sox-18 (performed as in FIG. 4). The respective level of activation of the pG5ElbCAT reporter is shown. The G refers to the plasmid GALO into which the various fragments were cloned, the numbers in brackets correspond to the amino acid positions in murine Sox-18 .

Figure 5B shows co-transfection assays of the eukaryotic expression plasmid pSG5-Sox-18 and TK-Sox (Denny et al . , 1992, EMBO J. 11 3705-3712) CAT containing four Sox consensus motifs cloned into TK-CAT. 5 μg of CAT reporter was co-transfected with increasing amounts of SG5Sox-18 .

Figure 5C shows co-transfection of 5 μg of TK-CAT, TK-Sox (Berta et al . , 1990, Nature 348 448-450) (containing one Sox motif) or TK-Sox (Denny et al . , 1992) (containing four Sox motifs) with the 2 μg of control vector (SG5) or the Sox-18 expression vector (SG5-Sox- 18 ) . Figure 5D shows the effect of phosphorylation reagents 8-Bromo cAMP(M) and Okadaic Acid (M) on the transcriptional activity of the Sox-18 activation domain. CAT assays were performed on COS-l cells cotransfected with either GalO or G-Sox-18 (160-255) and treated with either 8-Bromo cAMP or Okadaic Acid. Results shown are the mean CAT activity and the standard deviations derived from experiments done at least in triplicate.

Figure 6A shows chromosomal localisation of Sox- 18 . Haplotype analysis of 45 mice from the EUC1B interspecific backcross for the markers D2Nds3, Sox-18 and Acra4. Each filled box represents a mouse scored for the both Mus spretus and Mus musculus alleles, but not both. Numbers beneath each column indicate the number of mice in each recombinant class. Only mice typed for all three markers are shown. Allele sizes were 7.5 kb and 4.5 kb for Mus spretus and 6.0 kb and 4.5 kb for Mus musculus . The marker D2Mit 74 is not included due to the low number of mice typed with it, though it too can be placed proximal to Acra4 due to its typing in one of the mice showing a recombination between Sox-18 and Acra4 .

Figure 6B shows linkage distance (in centimorgans) between Sox-18 and the markers Acra4 and D2Nds3, including standard errors . The co-segregating marker D2Mlt74 is shown on the left hand side. Linkage distances are not represented to scale.

Figure 7 shows wholemount in situ hybridisations showing expression of Sox-18 in 9.5 days post coitumJ d . p . c . ) in developing mouse embryos, a) Sideview showing Sox-18 expression in the intersomitic region and the vasculature with an anti-sense probe. As a negative control, staining with a sense probe is also shown; b) Posterior view of hindlimb region, showing clear expression in the inter-somitic vasculature/arteries .

Figure 8 shows Northern blot analysis demonstrating the effect of Sox- 18 Gene expression on the steady state levels of myogenin and p21 mRNAs.

Figure 9 shows the CAT assay results of co¬ transfection analysis demonstrating the trans-activation of myogenin gene transcription by Sox-18 sense gene expression.

DETAILED DESCRIPTION OF THE INVENTION

As background to the present invention, Sox-18 is a member of the newly identified Sox gene family (Sry- like HMG box gene) . The Sox-18 gene was isolated from a murine heart (cardiac) cDNA library. Sox-18 mRNA expression is restricted to heart, lung and skeletal muscle in the adult mouse. Very abundant expression was observed in the lung, which is comprised of smooth muscle and endothelial cell tissue.

It also has been demonstrated above that Sox-18 binds DNA efficiently in a sequence specific manner and trans-activates gene expression in a dose dependent manner. This protein specifically recognizes nucleic acids containing the sequence motif 5 ' AACAAAG 3 ' . Sox- 18 was discovered to function as a transcription factor. The full-length Sox-18 polypeptide binds specifically to the Sox consensus motif AACAAAG and is able to activate transcription via this site. The trans¬ activation domain of the Sox-18 protein was mapped to a region comprising amino acids 160 to 255 of Figure 2.

Nucleotide Sequences of the Invention

The invention provides the nucleotide sequence encoding the Sox-18 transactivation domain (nucleotides 317 through 554 of Figure IA) . A "nucleotide sequence" designates mRNA, RNA, cRNA, cDNA or DNA. One of skill in the art will appreciate that cDNA is complementary DNA produced from a RNA template, usually by the action of RNA-dependent DNA polymerase (reverse transcriptase). If the RNA template has not been processed to remove the introns, the cDNA will not be identical to the gene from which the RNA was transcribed.

The invention also provides homologs of the Sox-18 nucleotide sequences of the invention as described above. Such "Sox-18 " homologs, "as used in this specification include all nucleotide sequences encoding subsequences of this polypeptide, such as the transactivation domain. In this regard, codon sequence redundancy means that changes can be made to a nucleotide sequence without affecting the corresponding polypeptide sequence.

The homologs of the invention further include nucleotide sequences encoding polypeptides that have the same functional characteristics as the Sox-18 polypeptides of the invention. One of skill in the art will appreciate that conservative amino acid substitutions can be made in a Sox-18 polypeptide according to the invention and that such substituted polypeptides will retain the functional characteristics of a Sox-18 polypeptide according to the invention. The homologs of the invention also cover alternately spliced variants which may be obtained by using a nucleotide sequence in accordance with the invention as a probe using a mouse heart cDNA library.

The homologs of the invention further comprise nucleotide sequences that hybridize with a Sox nucleotide sequence of the invention under stringent conditions . Suitable hybridization conditions are discussed below.

A homolog within the invention does not include the Sox-1 7 full length gene. In addition, a homolog of the invention does not include the Sox-1 , 2 , 3 , 4 , 5 , 6, 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , or 16 ( "Sox genes 1- 16"). Furthermore, a homolog of the invention does not include the Syr gene.

The invention also includes all nucleotide sequences encoding the Sox-1 7 HMG-box polypeptide of Figure 1 and a Sox-17 HMG-box nucleotide sequence (Figure IB) .

Sox-18 homologs of the invention may be prepared according to the following procedure:

(i) designing primers which are preferably degenerate which span at least a fragment of a DNA sequence of the invention; and

(ii) using such primers to amplify, via PCR techniques, said at least a fragment from cDNA derived from total RNA or poly A+ RNA obtained from a mammalian source selected from the group consisting of human, rat, chicken, guinea pig and rabbit. The invention contemplates those sequences isolated from muscle tissue.

"Hybridization" is used here to denote the pairing of complementary nucleotide sequences to produce a DNA- DNA hybrid or a DNA-RNA hybrid. Complementary base sequences are those sequences that are related by the base-pairing rules. In DNA, A pairs with T and C pairs with G. In RNA U pairs with A and C pairs with G.

Typically, nucleotide sequences to be compared by means of hybridization are analyzed using dot blotting, slot blotting, or Southern blotting. Southern blotting is used to determine the complementarity of DNA sequences. Northern blotting determines complementarity of DNA and RNA sequences. Dot and Slot blotting can be used to analyze DNA/DNA or DNA/RNA complementarity. These techniques are well known by those of skill in the art. Typical procedures are described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, et al . , eds.) (John Wiley & Sons, Inc. 1995) at pages 2.9.1 through 2.9.20. Briefly, for Southern blotting, DNA samples are separated by size using gel electrophoresis. The size- separated DNA samples are transferred to and immobilized on a membrane (typically, nitrocellulose) and the DNA samples are probed with a radioactive, complementary nucleic acid. In dot blotting, DNA samples are directly spotted onto a membrane (nitrocellulose or nylon) . In slot blotting, the spotted DNA samples are elongated. The membrane is then probed with a radioactive complementary nucleic acid.

A probe is a biochemical labeled with a radioactive isotope or tagged in other ways for ease in identification. A probe is used to identify a gene, a gene product or a protein. Thus a nucleotide sequence probe can be used to identify complementary nucleotide sequences. An mRNA probe will hybridize with its corresponding DNA gene. Typically, the following general procedure can be used to determine hybridization under stringent conditions. A nucleotide according to the invention (such as Sox-18 or a subsequence thereof) will be immobilized on a membrane using one of the above- described procedures for blotting. A sample nucleotide sequence will be labeled and used as a "probe." Using procedures well known to those skilled in the art for blotting described above, the ability of the probe to hybridize with a nucleotide sequence according to the invention can be analyzed.

One of skill in the art will recognize that various factors can influence the amount and detectability of the probe bound to the immobilized DNA. The specific activity of the probe must be sufficiently high to permit detection. Typically, a specific activity of at least IO⁸ dpm/ug is necessary to avoid weak or undetectable hybridization signals when using a radioactive hybridization probe. A probe with a specific activity of 10⁸ to 10^s dpm/ug can detect approximately 0.5 pg of DNA. It is well known in the art that sufficient DNA must be immobilized on the membrane to permit detection. It is desirable to have excess immobilized DNA and spotting lOug of DNA is generally an acceptable amount that will permit optimum detection in most circumstances. Adding an inert polymer such as 10% (w/v) dextran sulfate (mol. wt. 500,000) or PEG 6000 to the hybridization solution can also increase the sensitivity of the hybridization. Adding these polymers has been known to increase the hybridization signal. See Ausubel, supra , at p 2.10.10.

To achieve meaningful results from hybridization between a first nucleotide sequence immobilized on a membrane and a second nucleotide sequence to be used as a hybridization probe, (1) sufficient probe must bind to the immobilized DNA to produce a detectable signal (sensitivity) and (2) following the washing procedure, the probe must be attached only to those immobilized sequences with the desired degree of complementarity to the probe sequence (specificity).

"Stringency," as used in this specification, means the condition with regard to temperature, ionic strength and the presence of certain organic solvents, under which nucleic acid hybridizations are carried out. The higher the stringency used, the higher degree of complementarity between the probe and the immobilized DNA.

"Stringent conditions" designates those conditions under which only a nucleotide sequences that have a high frequency of complementary base sequences will hybridize with each other. Exemplary stringent conditions are (1) 0.75 M dibasic sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1% sarkosyl at about 42°C for at least about 30 minutes, (2) 6.0M urea/0.4% sodium laurel sulfate/0.1% SSX at about 42° C for at least about 30 minutes, (3) 0. IX SSC/0.1% SDS at about 68°C for at least about 20 minutes, (4) IX SSC/0.1% SDS at about 55°C for about one hour, (5) IX SSC/0.1% SDS at about 62°C for about one hour, (6) IX SSC/0.1% SDS at about 68°C for about one hour, (7) 0.2X SSC/0.1% SDS at about 55°C for about one hour, (8) 0.2X SSC/0.1% SDS at about 62°C for about one hour, and (9) 0.2X SSC/0.1% SDS at about 68°C for about one hour. See, e . g. CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (Ausubel, et al. , eds.) (John Wiley & Sons, Inc. 1995), pages 2.10.1-2.10.16 of which are hereby incorporated by reference and Sambrook, et al . , MOLECULAR CLONING. A LABORATORY MANUAL (Cold Spring Harbor Press, 1989) at §§1.101-1.104.

Stringent washes are typically carried out for a total of about 20 minutes to about 60 minutes. In certain instances, more than one stringent wash will be required to remove sequences that are not highly similar to Sox-18 or a subsequence thereof. Typically, two washes of equal duration, such as two 15 or 30 minute washes, are used. One of skill in the art will appreciate that other longer or shorter times may be employed for stringent washes to ensure identification of sequences similar to Sox-18 .

While stringent washes are typically carried out at temperatures from about 42°C to about 68°C, one of skill in the art will appreciate that other temperatures may be suitable for stringent conditions. Maximum hybridization typically occurs at about 20 to about 25°C below the T_m for DNA-DNA hybrids. It is well known in the art that T_m is the melting temperature, or temperature at which two nucleotide sequences dissociate. Methods for estimating T-, are well known in the art. See, e.g. Ausubel, supra , at page 2.10.8. Maximum hybridization typically occurs at about 10 to about 15°C below the T_m for DNA-RNA hybrids.

Other typical stringent conditions are well-known in the art. One of skill in the art will recognize that various factors can be manipulated to optimize the specificity of the hybridization. Optimization of the stringency of the final washes can serve to ensure a high degree of hybridization between the Sox- 18 gene (or subsequence thereof) and other similar nucleotide sequences.

In a typical hybridization procedure, DNA is first immbolized on a membrane such as a nitrocellulose membrane or a nylon membrane. Procedures for DNA immobilization on such membranes are well known in the art. See, e.g., Ausubel, supra at pages 2.9.1-2.9.20. The membrane is prehybridized at 42°C for 30-60 minutes in 0.75 M dibasic sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1% sarkosyl. Membranes are then hybridized at 42°C in ACES hybridization solution (Life Technologies, Inc., Gaithersburg, Md. ) containing labeled probe for one hour. Next, membranes are subjected to two high stringency 10 minute washes at 42°C in 0.75 M dibasic sodium phosphate/0.5 M monobasic sodium phosphate/1 mM disodium EDTA/1% sarkosyl. Following this, the membranes are washed with 2X SSC at room temperature, to remove unbound probe.

In another typical hybridization procedure, DNA immobilized on a membrane is hybridized overnight at 42°C in prehybridization solution. Following hybridization, blots are washed with two stringent washes, such as 6.0M urea/0.4% sodium laurel sulfate/0.1% SSX at 42° C. Following this, the membranes are washed with 2X SSC at room temperature. Autoradiogrpahic techniques for detecting radioactively labeled probes bound to membranes are well known in the art. Polypeptides of the Invention

As used in this specification, a "Sox-18 polypeptide" includes, but is not limited to, a full- length Sox- 18 polypeptide (amino acids 1 through 378 of Figure 2), a polypeptide comprising the amino acid sequence encoded by amino acids 160 to 255 of Figure 2, and a polypeptide comprising the amino acid sequence of the Sox- 18 HMG box of Figure IA.

The invention also includes within its scope a polypeptide encoded by the human Sox-18 gene as well as mammalian homologs of the Sox-18 polypeptide. As used in this specification, a "Sox-18 polypeptide homolog" is a polypeptide encoded by a "Sox-18 homolog," which is defined above. Thus, the invention contemplates polypeptides which are functionally similar to the Sox-18 polypeptide. Such polypeptides may contain conservative amino acid substitutions compared to the Sox-18 polypeptide of Figure 2.

A Sox-18 polypeptide of the invention may be prepared by a procedure including the steps of:

(a) ligating a DNA sequence encoding a recombinant Sox-18 polypeptide or biological fragment thereof into a suitable expression vector to form an expression construct; (b) transfecting the expression construct into a suitable host cell;

(c) expressing the recombinant protein; and

(d) isolating the recombinant protein.

As used in this specification, an expression construct is a nucleotide sequence comprising a first nucleotide sequence encoding a polypeptide, wherein said first sequence is operably linked to regulatory nucleotide sequences (such as a promoter and a termination sequence) that will induce expression of said first sequence. Both constitutive and inducible promoters may be useful adjucts for expression of a Sox- 18 polypeptide or Sox-18 polypeptide homolog according to the invention. An expression construct according to the invention may be a vector, such as a plasmid cloning vectors . A vector according the the invention may be a prokaryotic or a eukaryotic expression vector, which are well known to those of skill in the art.

Suitable host cells for expression may be prokaryotic or eukaryotic. One preferred host cell for expression of a polypeptide according to the invntion is a bacterium. The bacterium used may be Escherichia coli . The recombinant protein may be conveniently prepared by a person skilled in the art using standard protocols as for example described in Sambrook et al . (1989, supra , in particular Sections 16 and 17). Myogenesis and Sox-18 It has been discovered that transforming mammalian cells with the Sox-18 gene in the sense orientation causes induction of myogenesis. Conversely, it has been discovered that mammalian cells transformed with the Sox- 18 gene in the antisense orientation inhibits myogenesis. Accordingly the present invention contemplates methods utilizing the Sox-18 gene for induction and inhibition of myogenesis.

Myogenesis involves two processes, determination and differentiation. Determination is the process whereby pluripotential precursor cellε commit to the myogenic lineage and become myoblasts. During differentiation, proliferating myoblasts permanently exit the cell cycle and fuse to become post-mitotic, multinucleated myotubes with a contractile phenotype, that express myogenic markers (reviewed in (Olson, E.N., 1992, Dev. Biol . , 154, 261-272; Olson, E.N. 1993, Mol . Endocrinol . , 7, 1369-1378). Insights into this process have been provided by the identification of a group of basic helix loop helix (bHLH) proteins encoded by the myoD gene family (myoD, myf-5, myogenin, and MRF-4/myf- 6/herculin) . The protein products of the myoD gene family are muscle-specific trans-activators that can direct the fate of mesodermal cell lineages, repress proliferation, activate differentiation and the contractile phenotype and function at the nexus of command circuits that control the mutually exclusive events of division and differentiation (reviewed in Olson, 1992, supra ; Olson, 1993, supra ; Muscat et al . , 1995a). Gene targeting studies have suggested that while myoD and myf-5 are required for determination (Rudnicki et al . , 1993, Cell , 75, 1351-1359), myogenin is specifically required for differentiation (Hasty et al . , 1993, Nature , 364, 501-506).

MyoD forms heterodimers with ubiquitously expressed members of the helix-loop-helix (HLH) protein family, such as E12 and E47 (the alternatively spliced products of the E2A gene). MyoD-E2A heterodimers bind to a consensus DNA binding sequence, the E-box motif (CANNTG), present in muscle-specific enhancers (reviewed in (Olson, 1992, supra; Olson, 1993, supra ) . The MyoD proteins also act in concert with a variety of other ubiquitous (eg Spl, CTF, SRF) and tissue specific (e.g., MEF-2) transcription factors to regulate myogenic promoters (reviewed in (Olson, 1992, supra ; Olson, 1993, supra ) .

Direct interaction of MyoD and myogenin with the nuclear retinoblasto a phosphoprotein (RB) has been observed (Gu et al . , 1993, Cell ,72, 309-324) and the binding of RB to MyoD is necessary to stabilise the DNA- bound (MyoD/E2A-protein) heteromeric complex.

It is now becoming apparent that control of myoblast fate is regulated by factors that ultimately affect the formation/stability of this transcriptionally active multi-protein complex (Gu et al . , 1993, supra ; Schneider et al . , 1994, Science , 264, 1467-1471).

RB activity is controlled by cell division kinase (cdk) complexes with the D-cyclins (see Sherr, 1994, Trends Cell Biol . , 4, 15-18. for review). The activity of cdks are regulated at the level of synthesis of the subunit partners (eg cyclins) of the complex, post¬ translational modification and by binding of inhibitors including _p2l^ciP1/Wafl (Sherr, 1994, Cell , 79, 551-555; Sherr, 1995, Genes Dev. , 9, 1149-1163). In C2 cells in culture, serum withdrawal induces differentiation, repression of cyclin Dl and induction of p21 mRNA/protein (Gu et al . , 1993, supra ; Guo et al . , 1995, Mol . Cell . Biol . , 15, 3823-3829; Halevy et al . 1995, Science , 2647, 1018-1021; Parker et al . , 1995, Science , 2647, 1024-1027; Skapek et al . , 1995, Science 267, 1022-1024).

The critical role of these cell cycle regulators in myogenesis has been demonstrated by (i) the inhibition of myogenesis by forced expression of cyclin Dl results in phosphorylation and inhibition of myoD function and (ii) the ectopic expression of p21 in growing myoblasts results in cell cycle arrest (Rao et al . , 1994, Mol . Cell . Biol . , 14, 5259-5267.; Guo et al . , 1995, supra ; Skapek et al . , 1995, supra ) Other Methods of the Invention

In yet another aspect, the invention provides a method of promotion of differentiation of endothelial cells, smooth muscle cells, and striated muscle cells.

The invention also provides a method for suppression of proliferation of endothelial cells, smooth muscle cells and striated muscle cells by administration of a nucleotide sequence or polypeptide of the invention.

Preferably, the DNA molecule or protein is injected directly into the endothelial and/or smooth/striated muscle. Therefore, the DNA molecule or protein of the invention may be utilized as a differentiation-inducing therapeutic agent in regard to treatment of, for example, restonosis or re-blockage of arteries after angioplasty wherein promotion of differentiation and suppression of proliferation is required to promote circulatory blood flow. In still yet another aspect, the invention provides a method for stimulation of endothelial cell, smooth muscle cell, and striated muscle cell proliferation by administration of an antisense DNA or RNA molecule according to the invention to a subject requiring such treatment. The invention further provides a method for suppression or inhibition of endothelial cell, smooth muscle cell and striated muscle cell differentiation by administration of such an antisense DNA or RNA molecule.

Suitably, the antisense DNA or RNA molecule is injected directly into the endothelial and/or smooth/striated muscle. Accordingly, the antisense DNA or RNA molecules in accordance with the invention may be used as a proliferative therapeutic agent in regard to treatment of, for example, ischemic heart injury and atherosclerotic plaques wherein promotion of smooth muscle cell proliferation is required to accelerate neovascularization. The therapeutic agents of the invention may also be utilized as part of a suitable drug delivery system to a particular tissue that may be targeted.

In a broader sense, the potential uses for the Sox-18 gene or its protein product fall into two broad categories, viz. (1) the promotion of endothelial and/or smooth/striated muscle differentiation and/or proliferation, and (2) the suppression of endothelial and/or smooth/striated muscle differentiation and/or proliferation. As such, the gene or its protein product (or any part or combination of parts of either), can be described as a therapeutic agent. Thus, the therapeutic agent may be a Sox-18 nucleotide sequence, including a subsequence of the full-length Sox- 18 gene, which may be used alone or in combination with any other molecule, SOX-18 polypeptide, or polypeptide fragments alone or in combination with any other molecule, antibodies to SOX-18 alone or in combination with any other molecule, sense or anti-sense oligonucleotides corresponding to the sequence of Sox-18 (alone or in combination with any other molecule) .

The method of administration of the therapeutic agent will differ depending on the intended use and or species, and will involve non-viral and viral vectors, cationic liposomes, retroviruses and adenoviruses such as, for example, described in Mulligan, R.C, (1993 Science , 260, 926-932) which is hereby incorporated by reference. Such methods may include:

(i) Local application of the therapeutic agent by injection (Wolff et al . , 1990, Science , 247, 1465-1468, which is hereby incorporated by reference), surgical implantation, instillation or any other means. This method may be useful where effects are to be restricted to specific endothelial and/or smooth/striated muscles. This method may also be used in combination with local application by injection, surgical implantation, instillation or any other means, of cells responsive to the therapeutic agent so as to increase the effectiveness of that treatment. This method may also be used in combination with local application by injection, surgical implantation, installation or any other means, of another factor or factors required for the activity of the therapeutic agent.

(ii) General systematic delivery by injection of DNA, oligonucleotides (Calabretta et al . , 1993, Cancer Treat . Rev. , 19, 169-179, which is hereby incorporated by reference), RNA or protein, alone or in combination with liposomes (Zhu et al . , 1993, Science, 261, 209-212, which is hereby incorporated by reference), viral capsids or nanoparticles (Bertling et al . , 1991, Biotech . Appl . Biochem . , 13, 390-405, which is hereby incorporated by reference) or any other mediator of delivery. This method may be advantageous for all intended uses whether or not the effect is intended to be targeted to specific tissues or parts of the body, and regardless of whether the intended result is the stimulation or inhibition or suppression of Soχ-18 gene or protein activity. Where specific targeting is required, this might be achieved by linking the agent to a targeting molecule (the so-called "magic bullet" approach employing for example, an antibody), or by local application by injection, surgical implantation or any other means, of another factor or factors required for the activity of the therapeutic agent, or of cells responsive to the therapeutic agent. (iii) Injection or implantation or delivery by any means, of cells that have been modified ex vivo by transfection (for example, in the presence of calcium phosphate: Chen et al . , 1987, Mole . Cell Biochem. , 7, 2745-2752, or of cationic lipids and polyamines: Rose et al . , 1991, BioTech . , 10, 520-525, which articles are hereby incorporated by reference), infection, injection, electroporation (Shigekawa et al . , 1988, BioTech . , 6, 742-751, which is hereby incorporated by reference) or any other way so as to increase the expression or activity of Sox-18 (gene or protein) in those cells. The modification may be mediated by plasmid, bacteriophage, cosmid, viral (such as adenoviral or retroviral; Mulligan, 1993, Science , 260, 926-932; Miller, 1992, Nature , 357, 455-460; Salmons et al . , 1993, Hum. Gen . Ther. , 4, 129-141, which articles are hereby incorporated by reference) or other vectors, or other agents of modification such as liposomes (Zhu et al . , 1993, Science, 261, 209-212, which is hereby incorporated by reference), viral capsids or nanoparticles (Bertling et al . , 1991, Biotech . Appl . Biochem. , 13, 390-405, which is hereby incorporated by reference), or any other mediator of modification. The use of cells as a delivery vehicle for genes or gene products has been described by Barr et al . , 1991, Science , 254, 1507-1512 and by Dhawan et al . , 1991, Science , 254, 1509-1512, which articles are hereby incorporated by reference. Treated cells may be delivered in combination with any nutrient, growth factor, matrix or other agent that will promote their survival in the treated subject.

The following examples are provided to illustrate various embodiments of the present invention, but do not limit the scope of the present invention.

EXAMPLE 1

(a) Isolation of HMG-box regions

A procedure similar to that described by Wright et al . , 1993, supra , which is hereby incorporated by reference, was used to isolate HMG-box regions of novel Sox genes. The procedure used by Wright et al . was modified by using adult mouse (Swiss random outbred) cardiac and skeletal muscle total RNA as a template for reverse transcriptase PCR. For amplification, the degenerate primers shown in Figure IA were used, which span a highly conserved 230-base pair region in Syr and the other known Sox genes. Using this approach, a DNA fragment encoding the HMG-box of a novel gene was amplified, and this gene was designated Sox-1 7 (Figure IB).

The Sox-17 gene HMG box has a high level of amino¬ acid identity (91 %) with the HMG-box of Sox-7 (Wright et al., 1993), as shown in Figure IA. The HMG-box region of Sox-n shows significantly higher amino acid identity with that of Sox-1 than other known Sox genes and therefore belongs to the Sox-1 subgrouping.

(b) Cloning and sequencing of Sox-18

The gene for Sox- 18 was isolated from a murine heart cDNA library using the procedure described in Wright et al . , 1993, supra , which is hereby incorporated by reference.

A 41-base pair region of the amplified Sox-17 HMG- box with the greatest level of sequence divergence with respect to other members of the Sox gene family was concatenated by ligation, radiolabelled, and used to screen an λZAPII murine heart cDNA library (Stratagene). The 41-base pair region used is the Hindl/MJboII fragment of the amplified DNA fragment of part (a) above and comprises the sequence: 5 ' TGACCTTGGCAGAGAAGCGGCCCTTCGTGGAAGAGGCCGAGC 3 ' pBluescript clones were isolated, restriction mapped and the HMG-box region sequenced. Sequencing was performed using the dideoxy chain-termination method with sequence being read at least once from each DNA strand. Several cDNA clones isolated and sequenced contained a related but distinct HMG-box designated Sox-18 .

Sox-18 has 88% amino acid identity with Sox-17. Sox-18 also shows higher amino acid identity to Sox-7 than any of the other known Sox genes, placing it in the same sub-grouping as Sox-17. An alignment of the Sox-1 , 17 and 18 HMG-box amino acid sequence is given in FIG. 1.

The full-length cDNA sequence of Sox-18 is presented in FIG. 2. This sequence was determined using three over-lapping clones and is 1588-bp long, encompassing a reading frame of 1134 nucleotides. The deduced protein sequence is 378 amino acids, with the HMG-box DNA-binding domain spanning amino acids 76-155. The sequence shows that the Sox-18 protein has a serine rich tail — 9 of the 27 C-terminal amino acid are Ser residues. A Ser-rich domain has also been identified in Sox-4, which has been implicated in trans-activation (Van de Wetering et al . , 1993). (b) Northern analysis To determine whether Sox-18 expression is restricted to muscle, a Northern blot was prepared using total RNA isolated from adult mouse lung, hind limb muscle, heart, spleen, kidney, liver and brain tissue. Total RNA was isolated using the method of Chomezynski, et al . , Anal . Biochem. 162:156 (1987). 10 μg of RNA was electrophoresed in a 1.2% (w/v) agarose gel containing 1% (v/v) formaldehyde and transferred to Nylon membrane. An antisense ³²-labelled RNA probe was transcribed from the 3' end of the Sox-18 cDNA not containing the HMG-box, using T7 polymerase and a Pstl -cut Pstl-Xbal sub-clone in pBluescript (Stratagene) as a template. Filters were hybridized in 5 x SSPE/60% formamide at 42°C, washed at high stringency (0. 1 x SSC/65°C) and autoradiographed. SSC is 0.15M NaCl/0.015 M Na3 citrate pH 7.6. SSPE is 0.15 M NaCl/0.2 M Na-phosphate/0.02 M EDTA pH 7.7. As shown in Figure 3, the Sox-18 probe specifically hybridized to a 1.6 kb transcript in RNA from lung, heart and skeletal muscle tissue, demonstrating that Sox-18 mRNA is expressed in smooth and striated muscle of the adult mouse. (c) Expression of Sox-18 in Other Tissues

It was demonstrated that the Sox- 18 gene is expressed in primary cultures of endothelial cell and smooth muscle.

In situ studies in mouse embryos have demonstrated that Sox-18 is expressed in the pre-somitic mesoderm from 8 days post coitum (dpc) and significantly expressed in the inter-somitic region between 9 and 11 dpc. This region includes the progenitors of endothelial cells and smooth muscle. Furthermore, Sox-18 is abundantly expressed in the vasculature of the early mouse embryo.

EXAMPLE 2

(a) Trans-activation and DNA-binding properties of Sox-18 Recombinant Sox-18 was expressed in E. coli as a glutathione-S-transferase (GST) fusion protein. Procedures for preparing GST fusion proteins are well- known in the art. To prepare the Sox-18-GST fusion protein, a procedure in Hosking Nucl. Acid Res 23:2626 (1995), which is hereby incorporated by reference. This recombinant Sox-18-GST interacts with the Sox

Consensus Motif (SoCM = AACAAAG) in a dose dependent manner. EMSA competition assays indicated that only the unlabelled SoCM specifically competed with the Sox- 18 :SoCM complex, and none of the non-HMG-box binding sites had any effect (FIG. 4A) . Several other Sox-like DNA motifs (specifically AACAATC, GACAAAG, TACAATC, AACAAAC and CACAATTG) were also unable to effectively compete the Sox-18 :SoCM complex. Thus, Sox-18 binds DNA in a highly sequence-specific manner.

It is well-known in the art that transcription factors can be analyzed using the Gal4 hybrid assay system. In this system, chimeric genes are constructed using the Gal4 DNA-binding domain and protein domains of choice. The Gal4 system can be used to (1) determine whether a chosen protein is capable of regulating transcription and (2) identify modular domains. Gal4 assay procedures, which are well-known in the art, are described in Van de Wetering et al . , EMBO J. 12 : 3487 (1993), Dubin et al . , 1994, Mol . Endo . 8 1182, Lee et al . , Proc . Natl . Acad. Sci . U. S . A . 90 6145 (1993) and Weintraub et al . , Genes Dev. 5 1377-1386 (1991).

A Gal-Sox-J8 fusion was constructed containing the full open reading frame of Sox-18. The Gal-Sox-18 fusion plasmid was co-transfected into COS-l cells with plasmid pG5ElbCAT. pG5ElbCAT contains a CAT reporter gene linked to 5 Gal4 binding (or target) sites and is described in Lillie et al . , 1989, Nature 338 31-44, hereby incorporated by reference. As shown in Figure 4B, Sox- 18 is able to activate transcription 6-fold above the negative controls (GALO and Gal-Sox-18 antisense). Sox- 18 also activated transcription at levels similar that activated by known transcription factors (muscle specific MyoD and the ubiquitous Spl transcription factors) (Figure 4B) . (b) Mapping the Sox-18 activation domain Using the Gal4 hybrid assay system, the activation domain of Sox- 18 was mapped to a 95 amino acid segment immediately downstream of the HMG box. This segment encodes amino acids 160-255 of the Sox-18 protein. This domain was identified using the expression constructs shown in figure 5A.

The 95 amino acid domain increased CAT activity, using the Gal4 assay, over 100-fold above background wherein the full length Sox-18 protein activates only around 10-fold, suggesting that the additional DNA binding domain may suppress trans-activation. The activation domain of the related Sry protein was shown to be confined to a glutamine/histidine rich region, just downstream of the HMG box (Dubin et al . , Mol . Endocr in . 8: 1182 (1994). A serine rich region in the carboxy terminus of Sox-4 was shown to have trans-activation activity. Van de Wetering, et al . , supra . The activation domain of Sox-18 does not show significant homology, at either the nucleotide or amino acid level, to any previously characterized protein in nucleotide or amino acid sequence databases.

(c) Sox- 18 -mediated regulation of transcription via SoCM

To determine whether the Sox-18 gene product could regulate transcription through the SoCM ( Sox consensus motif), Sox-18 cDNA was cloned into the SV40 expression vector, pSG5 (Stratagene) to make SG5-Sox-18. This was cotransfected with increasing amounts of one of the following reporters: (1) TK-CAT (basal reporter) or (2) TK-Sox(4) (Denny et al . , EMBO J. 11: 3705-12 (1992) reporter (TK-CAT containing four SoCMs) into COS-l cells. A dose dependent specific increase in CAT expression from TK-Sox (4) was demonstrated with the increasing amounts of SG5-Sox-18 (FIG. 5B) . These results indicate that Sox- 18 can specifically trans-activate gene expression via the AACAAAG SoCA motif.

However, when a plasmid containing a single Sox motif (TK-Sox (1)) was co-transfected with SG5-Sox-18, no significant trans-activation was detected. (FIG. 5C).

(d) Phosphorylation and Sox-18 The role of phosphorylation in the transcriptional regulation of Sox-18 was examined. The reagents 8-Br- cAMP (stimulates cAMP-dependent protein kinase A pathways) and okadaic acid (inhibits serine/threonine protein phosphatases) were used to globally increase the level of protein phosphorylation. The basal vector (GALO) or the Gal-Sox-18 activation domain construct G- Sox-18 (encoding amino acids 160-255) were co-transfected with the pGSElbCAT reporter, in the presence or absence of either 8-Br-cAMP or okadaic acid.

Both reagents increased the expression of CAT from the basal vector, GALO, by approximately 2- and 3-fold respectively, as shown in Figure 5D. However, in the presence of the chimeric, G-Sox-18 (160-255) plasmid, 8- Br-cAMP increased CAT expression approximately 3.5-fold, whereas okadaic acid increased expression only 2-fold. These results, with respect to the effect of these phosphorylating agents on basal transcription, indicate that 8-Br-cAMP increases the activity of Sox-18 , suggesting that cAMP dependent protein kinase cascades target the Sox-18 activation domain and regulate the activity of this protein.

EXAMPLE 3 Chromosomal localisation of Sox 18 A similar approach to that described in an article by Wright et al. (Nature Genetics, 9, 15-20) which article is hereby incorporated by reference, was used to map Sox-18 to distal mouse chromosome 2.

The chromosomal localization of Sox-18 was completed using the EUCIB interspecific backcross method as described in an article by the European Backcross Collaborative Group (1994, Human Mol . Genetics , 3, 621- 627) which is hereby incorporated by reference. Briefly, a 1550 bp EcoRI cDNA probe containing most of the Sox-18 transcript was used to identify a Mus spretus/Mus musculus restriction fragment variant in Tagl-digested genomic DNA (see legend to FIG. 6 for allele sizes). Single hybridizing fragments were observed for several enzymes indicating that no other Sox genes are detected by this probe. Linkage analysis suggests a localization to distal chromosome 2. Sox-18 co-segregated with the anonymous marker D2Mit74 in 34 animals typed. Linkage data for two other distal chromosome markets, Acra4 and D2Nds3, are given in FIGS. 6A and 6B, along with the haplotype analysis which places Sox-18 proximal to Acra4.

Two mouse mutations, ragged(Ra) and wasted(wst), have been recently mapped to distal mouse chromosome 2 (Abbot et al . , 1994, Genomics 20, 94-980. The wst locus maps distal to Acra 4, excluding Sox-18 as a candidate for wst. However, the Ra locus co-segregated with D2Mit74 in the same study, and hence it remains a candidate for the Ra gene defect, which in Ra/Ra homozygotes results in nakedness, edema and death after weaning.

EXAMPLE 4

Expression of Sox-18 during mouse embryo development Analysis by whole mount in situ hybridization

Expression of Sox-18 during mouse embryo development was studied by wholemount in situ hybridisation as described in an article by Wright et al .

(1995, Nature Genetics 9, 15-20). Briefly, antisense and sense RNA probes were prepared from subclones of the Sox-

18 cDNA 3' to the HMG box but not containing any HMG box or polyA+ sequences. Hybridisations were carried out essentially as described in Wilkinson et al . (1993, entitled "Detection of messenger RNA by in situ hybridisation to tissue sections in whole mounts."

Methods in Enzymology 225, 361-373). Specimens were photographed on an Olympus stereomicroscope using Kodak

Extachrome Film. By 8 - 9.5 days post coitum (dpc), Sox-18 expression is prominent in the dorsal aorta and the so¬ called intersegmental arteries forming between the nascent somites (i.e., the inter-somitic region that includes the progenitors of endothelial cells and smooth muscle) (Figure 7). At this stage (9.5 dpc), blood vessels are devoid of smooth muscle and consist entirely of endothelial cells. The Sox-18 expression in the dorsal aorta is transient, disappearing by 9.5 dpc. Between 8.5 and 11.5 dpc, Sox-18 expression is seen in developing vasculature throughout the embryo, most prominently associated with nascent vessels such as the capillaries of the trunk, limbs and head.

At 12.5 dpc and beyond, expression of Sox-18 appears to subside, possibly correlating with a cessation of angiogenesis and vasculogenesis in the most visible surface components of the embryo.

In addition to expression in the embryo proper, Sox-18 expression is also seen in the developing yolk sac and allantois.

These studies strongly suggest that Sox-18 has a role in the developing vascular system. As blood vessels at these stages of embryogenesis consist entirely of endothelial cells and are devoid of smooth muscle, these studies strongly suggest that Sox-18 expression is associated with the endothelial cell lineage.

The timing, sites and transient nature of Sox-18 expression are consistent with Sox-18 acting aε a switch required for differentiation of endothelial cells. The pattern of expression shown by Sox-18 is remarkable similar to that of flk-l(fetal liver kinase), a receptor tyrosine kinase that is specific to endothelial cells and their precursors. Both genes are expressed in blood vessels of the mouse embryo from approximately 8 dpc (Yamaguchi, T.P. et al . , 1993, Development , 118, 489-498; Shalabay. F. et al . , 1995 Nature, 376, 62-66) in extraembryonic tissues such as the yolk sac and allantois, and in adult tissues such as the lung, heart, and skeletal muscle (Dunn et al . , 1995, Gene, 161: 223-225; Larsson-Blo berg and Dzierzak, 1994, FEBS Lett . , 348, 119-125). These results raise the . possibility of a regulatory relationship between Sox-18 and regulatory receptor molecules such as flk-1. The requirement for proper vasculogenesis was demonstrated by experiments in which this gene was inactivated by homologous recombination in embryonic stem cells; mice lacking Flk-1 failed to develop endothelial cells and died in utero by 10.5 dpc (Shalaby et al . , 1995, supra ) .

It is also possible that Sox-18 may be involved in the regulation of genes encoding other molecules known to play a role in blood vessel development, including fltl(Fong et al . , 1995, Nature, 376, 66-70), Tek and Tiel (Dumont et al . , 1994, Genes & Development , 8, 1897-1909; Puri et al . , 1995, EMBO J. , 14, 5884-5891; Sato et al . , 1995, Nature, 376:70-74).

EXAMPLE 5 Expression of Sox 18 in myogenic cells

The requirement of Sox-18 for myogenesis was investigated by studying whether Sox- 18 is involved in the modulation of expression of p21 and myogenin which are respectively the critical regulators of the cell cycle and muscle differentiation. This investigation was carried out by studying the 'loss of function' of Sox-18 in myogenic C2C12 cells by constitutive over-expression of pSG5-Sox-18 AS (an expression vector comprising Sox-18 in the anti-sense orientation under the control of the SV40 promoter in the vector pSG5 described above).

Wild-type myogenic C2C12 cells were stably tranfected with pSG5-Sox-18 sense [denoted as Sox-18 sense] and pSG5-Sox-18 antisense [denoted as Sox-18 antisense]. Total RNA was isolated from wild type cells, Sox-18 sense and Sox-18 antisense stably transfected cells as confluent myoblasts (CMB) in growth medium (20% FCS in DMEM) and myotubes cells after 24 h and 72 h of serum withdrawal in differentiation medium (2% Horse Serum in DMEM) . These yotube samples harvested after 24 and 72 hours, were denoted as MT-1 and MT-3, respectively. RNA (2Omg) was blotted and probed with ³²-P labelled myogenim, p21 cDNA probes and 18S rRNA using an oligonucleotide probe.

The Sox-18 sense cell line differentiated more efficiently with an increased percentage of ultinucleated myotubes visible after serum withdrawal. The induction of myogenin and p21 mRNA after serum withdrawal was significantly induced relative to the induction of these mRNAs in wild type cells (and relative to the equivalent levels of 18S rRNA) . To verify the positive effects on the induction/expression of myogenin and p21 mRNAs that were ascribed to Sox-18 from 'over-expression' studies, the effects of constitutive 'anti-sense' Soχ-18 cDNA expression in C2C12 cells were examined. The Sox-18 anti-sense cell line failed to morphologically differentiate after serum withdrawal. The induction of myogenin and p21 mRNA expression was dramatically abrogated after serum withdrawal, relative to the wild type and Sox-18 sense cell lines reflecting the absence of morphological differentiation in this cell line. Methods:

Myogenic mouse C2C12 cells: Proliferating myogenic C2C12 myoblasts can be induced to biochemically and morphologically differentiate into post-mitotic multinucleated myotubes (that have acquired a contractile phenotype) by serum withdrawal in culture over a 48-96 h period. This transition from a non-muscle to a contractile phenotype is associated with the repression of non-muscle protein and the activation/expression of a structurally diverse group of genes. This gene activation encodes a functional sarcomere responsible for the major activity of this specialized cell type, i.e. contraction. These events are characterized by the activation of p21 and myogenin mRNAs, that regulate cell cycle exit and trans-activation of muscle specific genes and subsequent differentiation, respectively. Construction of the Sox-18 Sense and anti-sense cell lines: C2C12 cells were stably transfected at «40% confluence using the DOTAP (Boehringer Mannheim) mediated procedure by co-transfection of pCMV-NEO. Briefly, a 1 mL DNA/DOTAP mixture (containing 20 mg of either pSG5- Sox-18 sense or pSG5-Soχ-18 anti-sense and 1.5 mg of pCMV-NEO, 150 ml of DOTAP in Hepes 20 mM, NaCl, 150 mM, pH 7.4) was added to the cells in 25 mLs of fresh culture medium (20% FCS in DMEM). The cells were then grown for a further 24h, before selection with 400 mg/mL G418, to allow cell recovery and for high level pCMV-NEO expression. Stable transformants as a polyclonal pool were selected after 7-14 days of selection in DMEM supplemented with 20% FCS and 400 mg/mL G418.

The constructs, pSG5-Sox-18 Sense and Anti-sense were expression vectors that contained the Sox-18 cDNA full length insert [(i.e. the 1588 bp fragment, encompassing a reading frame of 1134 nt, 378 amino acids, and the 5' and 3' untranslated regions; (GenBank accession number L35032)], in the sense and anti sense orientation, respectively, under the control of the SV40 promoter in the vector pSG5 (Stratagene).

RNA Preparation and Northern Analysis: Total RNA was extracted by the acid guanidinium thiocyanate-phenol- chloroform method (Chomczynski and Sacchi, 1986) (Chomczynski, P. and Sacchi, N. (1986)). Single step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction. Anal . Biochem. , 162, 156- 159. Northern blots, random priming and hybridizations were performed as described in Sambrook et al . (1989, supra ) .

These studies showed that inhibition of expression of Sox-18 dramatically blocked morphological and

SlJBSTrrUTE SHEET Rule 26 biochemical myogenesis (i.e., muscle differentiation in culture). Furthermore, stable and constitutive over¬ expression of anti-sense Sox-18 abrogated the induction of p2i^{cipl Wafl} and myogenin mRNAs after serum withdrawal (see Figure 8). Accordingly, these studies indicate that Sox-18 is absolutely required for the formation of differentiated skeletal muscle (myogenesis) in culture.

Sox-18 'gain of function' studies in myogenic C2C12 cells by constitutive over-expression of pSG5-Sox 18 S (an expression vector that contained Sox-18 in the sense orientation under the control of the SV40 promoter in the vector pSG5) resulted in the production of a cell line that differentiated more efficiently. Furthermore, Northern analysis demonstrated that constitutive over- expression of Sox-18 resulted in the hyper-induction and accumulation of the myogenin and p21 mRNAs after serum withdrawal.

Sox-18 activation of myogenin gene promoter Transfection studies suggested that Sox-18 directly activated the promoter of the myogenin gene (Figure 9) .

All cells were transfected with the reporter pmyo- 1565-CAT (containing the mouse myogenin promoter linked to the chloramphenicol acetyl transferase gene; (Edmondson et al . , Mol . Cell . Biol . , 12, 3665-3677) and pSG5 (vector/vehicle alone); pSG5-Sox-18 S (Sense); pSG5- Sox-18 AS (antisense); pSG5-Soxl8-Box (Sox-18 HMG box alone); and pSG5-Sox 9 (sense). This experiment demonstrates that full length Sox-18 specifically/and directly trans-activates the mouse myogenin promoter, whereas, the vector alone, Soχ-18 antisense, Sox-18 HMG box alone or full length Sox 9 are incapable of efficient trans-activation.

The methods used in these experiments are as follows: Transfections: 60mm dish of cells were transiently transfected with 6 mg of reporter plasmid DNA expressing CAT, mixed with additional amount of the pSG5

SUBSTTTUTE SHEET Rule26 Sox-18 expression vectors or pSG5 alone. The total amount of DNA in each transfection experiment (10 mg) was kept constant. The DNA mixtures were cotransfected by the liposome mediated procedure. We used the cationic lipid DOTAP, N-[l-(2,3-Dioleoylox ) propyl]-N, N, N-tri- methyl-ammonium-methyl-sulphate. Unilamellar vesicles were formed by mixing the appropriate DNAs with 30-40 L of DOTAP and lx Hepes Buffered Saline to a total volume of 200 mL. After a 10 min incubation at room temperature this mixture was added to 6 mLs of fresh culture medium and added to the cells which were between 50 and 70% confluence. After a period of 20-24h, fresh medium was added to the cells. The cells were harvested for the assay of CAT enzyme activity 60-72h after the transfection period.

CAT Assays: The cells were harvested and the CAT activity was measured as previously described (Gorman, CM., Moffat, L.F., Howard, B.H. (1982). Recombination genomes which express chlormphenicol cetyltransferease in mammalian cells. Mol . Cell . Biol . , 2 , 1044-1051). Aliquots of the cell extracts were incubated at 37°C, with 0.1-0.4 mCi of ¹⁴C-Chloramphenicol (Amersham) in the presence of 5mM Acetyl CoA and 0.25 M Tris-HCl pH 7.8. After a 2-4 h incubation period the reaction was stopped by the addition of l L ethyl acetate which was used to extract the chlormphenicol and its acetylated forms. The extracted materials were analyzed on Silica gel then layer chomotagraphy plates as described previously (Gorman et al . , 1982, supra ) . Quantitation of CAT assays was performed by an AMBIS β-scanner. These studies demonstrated that Sox-18 , functions as a positive regulator of muscle differentiation, by regulating the expression of p21 and myogenin that are critical regulators of the cell cycle and muscle differentiation, respectively.

(B) Anti-proliferative properties of Sox 18 in relation to skeletal cardiac and smooth muscle As discussed above, Sox-18 positively regulates the induction and expression of p21 (also known as WAF-1, CIP-1, sdll, Picl, and CAP-20). p21 is a potent inhibitor of cyclin-dependent kinases, tumour progression and cellular proliferation. (El-Dlery, WST et al . , 1993 Cell, 75, 817-825; Gu, Y. et al . , 1993, Nature , 366, 707- 710; Harper, JW et al . , 1993, Cell , 75:805-816; Harper, JW et al . , 1995, Mol . Cell . Biol . , 6, 387-400; Nakanishi, M. et al . , 1995, Proc. Natl . Acad. Sci . USA , 92, 4352- 4356; Di ri, GP. et al . , 1996, Mol . Cell . Biol . , 16, 2987-2997 and references therein) . Accordingly, when mammalian cells are transformed with a Sox-18 , proliferation of skeletal, cardiac and smooth muscle tissues is substantially inhibited. (C) Induction of differentiation of smooth muscle in pluripotential cells by Sox-18

In addition, the studies described in Exampl 5A show that (i) inhibition of expressin of Sox-18 dramatically blocks morphological and biochemical myogenesis (i.e., muscle differentiation in culture) and (ii) stable and constitutive over-expression of antisense Sox-18 abrogates the induction of p2i^{Cιpl Wafl} and myogenin mRNAs after serum withdrawal. Accordmgly, these studies indicate that Sox-18 is absolutely required for the formation of differentiated skeletel muscle in culture (myogenesis) .

In mammalian cells transformed with a sense orientation Sox- 18 gene, the differentation of smooth muscle in pluripotential cells is induced. In addition, Sox-18 induces vascularization in smooth muscle cells.

EXAMPLE 6

Therapeutic Applications of Sox-18 Treatment of Atherosclerosis, Restonosis etc. Proliferation, abnormal growth and migration of smooth muscle cells leads to inti al hyperplasia and plays a major role in a number of cardiovascular disorders, including atherosclerosis. This characteristic clinical injury is also the basis for "restonosis" or reblockage of arteries after clogged or stenotic arteries are mechanically dilated with a balloon on a catheter to restore circulation in arteries (i.e., balloon angioplasty) . This localised proliferation of smooth muscle cells impinges on the arterial 'endothelial cell-populated" lumen and circulatory blood flow.

The expression of Sox-18 in endothelial cells (during mouse embryogenesis at 8.519.5 d.p.c. between the nascent somites and in the adult) and smooth muscle; and its ability to promote differentiation (e.g., myogenin mRNA induction), cell cycle exit, and the induction of p21 mRNA expression indicates that Sox-18 can be used for "gene therapy applications" in a number of occlusive cardiovascular disorders.

Sox-18 delivered by two-balloon catheters via direct gene transfer or liposome mediated techniques and expressed by plasmid or viral vectors can prove useful in regulating smooth muscle proliferation after angioplasty or arterial injury and stimulating/accelerating endothelial cell re-population to re-surface the arterial lumen which could reduce vasoconstriction and thrombogenesis (the methodologies pertaining to these techniques, i . e . , two-balloon catheters via direct gene transfer or liposome mediated techniques and expressed by plasmid or viral vectors, are described in detail in articles by Nabel, E. et al . (1990, Science , 249, 1285- 1287; and 1993, Nature, 362, 844-846) and Ohno, T. et al . (1994, Science 265, 781-785) which articles are hereby incorporated by reference.

For example, direct intra-arterial gene transfer of Sox- 18 can be performed according to the following procedure: A double balloon intravascular catheter (CR Baid

Inc., Billerica, MA) is inserted into the iliofemoral arteries after anesthesization and sterile surgical exposure of the artery. Both balloons are inflated, and the segment is irrigated with 5 mL heparinised saline and 5 mL opti-MEM BRL to rinse blood from the vessel. The arterial segmented is partially denuded by inflation and passage of the proximal balloon. Liposomes containing 30 mg of DNA comprising a Sox-18 expression vector (for an example of a Sox-18 expression vector, see Example 2) are mixed with liposomes (DOTMA and DOPE, BRL) and instilled into the arterial segment between the two balloons at 150 mm Hg in the left and right iliofemoral arteries and incubated for 20 min.

Alternately, the Sox-18 gene could be introduced directly with a replication defective Sox-18 retroviral vector. A Sox-18 transducing Moloney murine leukemia virus vector, prepared from yCRIP cells as, for example, described by 0'Danos et al . (1988, Proc. Natl. Acad. Sci. USA, 85, 6460-6464, which is hereby incorporated by reference) is used to generate viral particles which are filtered and concentrated by centrifugation as, for example, described by Price et al (1987, Proc. Natl. Acad. Sci. USA, 84, 156-159, which is hereby incorporated by reference). The viral supernatant is instilled for 30 min in the central space of the catheter, with polybrene (u mg/mL) added after introduction of the virus. The catheter is then removed and antegrade blood flow is restored.

This technology has delivered the herpes virus thymidine kinase gene and the retinoblastoma gene product (a cell cycle regulator), separately simultaneous with balloon angioplasty to reduce smooth muscle cell proliferation and neointima formation in rat and porcine femoral artery models of restonosis (Ohno, T. et al . , 1994, Science , 265, 781-785; Chang, MW et al, 1995, Science , 267, 518-622, which articles are hereby incorporated by reference).

Direct introduction of p21 with adenoviral vectors into malignant cells, completely suppresses growth in vitro and in vivo (Yang, ZY et al . , 1995, Nature Medicine , 1, 1052-1056, which is hereby incorporated by reference) . Since Sox-18 is a regulator of this potent cell cycle inhibitor, Sox-18 can also be used for treatment of restonosis using a similar approach to that described above.

EXAMPLE 7

Treatment of Pulmonary Diseases using Sox-18 Sox-18 could be delivered and expressed in the pulmonary vasculature/endothelium and lung parenchyma for the treatment of pulmonary diseases such as, for example, pulmonary fibrosis/thrombosis by delivery into the pulmonary artery of viral/non-viral vectors, cationic liposomes and/or adenoviral vectors comprising Sox-18 using percutaneous right heart catheterization such as, for example, described by Muller, DWM et al . (1994, Circulation Research , 75, 1039-1049, which is hereby incorporated by reference).

For example, the species/animal is sedated, intubated, and anaesthetized. With the use of sterile techniques, percutaneous catheterization is performed through the right femoral vein. An 8F introducer sheath is placed in the right femoral vein by using the seldinger technique and a 7F end hole balloon tipped catheter (Meditech) is inserted through the sheath, inflated with 1 cm³ of air and advanced through the inferior vena cava, right atrium, and right ventricle into the left pulmonary artery under fluoroscopic guidance. The catheter is then floated into the left posterior basal artery and lodged into the pulmonary artery to occlude blood flow. Five mLs of contrast is subsequently delivered through the catheter to confirm the catheter position and the arrest of blood flow. Confirmation of the arrest of blood flow is performed and delivery of DNA and vectors is allowed subsequently to proceed antegradely in the artery and not diffuse retrogradely. The artery is flushed with 20 L sterile saline through the end hole of the catheter. The non¬ viral DNA expression vector, pSG5-Sox-18 and liposomes are prepared 15 minutes before transcatheter injection by dilution of 5 mg DNA in 0.5 mL Opti-MEM (Gibco BRL) and 10 mg Lipofectamine (2,3-dioleyloxy -N-[2(sperminecarbox- a ido ) ethyl ] -N, N-dimeth l- 1-propanaminium trifluroactetate and diolecylphosphatidylethanolamine) in 0.5 mL of Opti-MEM. The DNA and liposome solution are then mixed by vortexing and diluted in Opti-MEM to a final volume of 1.6 L.

If Sox-18 is introduced in the form of an adenoviral vector (eg ADV-Sσx-18), viral stocks of 1.2 x IO⁹ pfu/mL, are prepared by diluting 1 mL of virus lysate in 0.6 mL PBS to a total volume of 1.6 mL. The plasmid/liposome mix or the viral stock of the Sox- 18 expression vectors are infused through the distal end of the balloon into the left posterior basal artery, flushed with 1.0 mL of Opti-MEM to clear the dead space of the catheter and incubated for 20 min. After incubation, the balloon is deflated, the catheter and introducer sheath removed and the right femoral vein is compressed to obtain haemostasis. This technique ensures deliver to the pulmonary vasculature and the alveolar speta. (See Muller et al . , 1994, supra ) .

EXAMPLE 8

Neovascularization/Treatment of ischemic heart injury, atherosclerotic plaques, etc using Sox-18

Delivery of recombinant Sox-18 into arterial walls can also have utility in the stimulation of vascular smooth muscle cells to improve blood supply and flow in a several cardiovascular disorders including ischaemic heart injury and the neo-vascularisation of atherosclerotic plaques. This can be achieved using a similar double balloon intravascular catheter mediated gene transfer approach to that described above for Example 6. In support of this potential, double balloon intravascular catheter mediated gene transfer of FGF-1 (Nabel, E.G. et al . , 1993, Nature, 362, 844-846) and PDGF (Pompili, V.J. et al . , 1995, Arteriosclerosis , Thrombosi and Vascular Biology, 15, 2254-2264) into the femoral arteries has resulted in induced intimal hyperplasia, angiogenesis and matrix deposition.

Thus, Sox-18 can be used to induce vascularisation of skeletal muscle tissue that is used to heal/patch weakened myocardial wells in cardiac injury patients.

EXAMPLE 9

Growth and Proliferation of Vascular Endothelial

Cells using Sox-18

Delivery of recombinant Sox-18 into arterial walls during angioplasty can also have utility in the growth/proliferation of vascular endothelial cells that are denuded from the vessels by this process. This would aid in the generation of paracrine inhibitors of vascular smooth muscle migration and proliferation. Introduction of endothelial cell products after balloon injury has substantially inhibited cell proliferation, migration, matrix formation required for neointima formation (H.E. von der Leyen et al . , 1995, Proc. Natl . Acad. Sci . USA, 92 , 1137-1141).

EXAMPLE 10

Tissue Engineering using Sox-18

Sox- 18 can also have utility in the design and construction of artificial blood vessel and vascular grafts. Sox-18 could be used to selectively support/promote/induce endothelial attachment to synthetic polymers which would aid in hemocompatibility. Artificial blood vessel and vascular grafts have been, for example, in an article by Langer, R. and Vacanti, J. (1993, Science , 260, 920-926; and reference therein, which are hereby incorporated by reference). Furthermore, Sox-18 can have utility in inducing endothelialisation of vascular grafts in vivo mediated by gene therapy protocols.

********************** The present invention has been described in terms of particular embodiments found or proposed by the present inventors to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and ahanges can be made in the partifular embodiments exemplified without departing from the scope of the invention. For example, due to codon redundancy, changes can be made in the underlying DNA sequence witout affecting the protein sequence. Moreover, All such modifications are intended to be included within the scope of the appended claims.

Claims

WHAT IS CLAIMED IS:

1. An isolated nucleotide sequence comprising nucleotide 566 through nucleotide 778 of Figure 2.

2. An isolated polypeptide comprising amino acids 160 to 255 of Figure 2.

3. A homolog of the nucleotide sequence according to claim 1.

4. A homolog according to claim 3, wherein said homolog is obtained from a mammal selected from the group consisting of human, mouse, rat, chicken, guinea pig and rabbit.

5. A homolog according to claim 3, wherein said homolog is obtained from muscle tissue.

6. A homolog according to claim 3, wherein said homolog is obtained from lung tissue.

7. A homolog according to claim 3, wherein said homolog is obtained from cardiac tissue.

8. A polypeptide homolog of the polypeptide according to claim 2.

9. A polypeptide homolog according to claim 8, wherein said homolog is obtained from muscle tissue.

10. A polypeptide homolog according to claim 8, wherein said homolog is obtained from lung tissue.

11. A polypeptide homolog according to claim 8, wherein said homolog is obtained from cardiac tissue.

12. A polypeptide homolog according to claim 8, wherein said homolog is obtained from a mammal selected from the group consisting of human, mouse, rat, chicken, guinea pig and rabbit.

13. A method for inducing transcription of a gene, comprising the steps of:

(a) transforming a mammalian cell with a plasmid comprising a Sox-18 gene comprising nucleotide 89 through nucleotide 1222 of Figure 2, wherein the expression of said gene is under the control of a constitutive promoter; and

14. A method for inducing myogenesis in a mammalian cell, comprising transforming said cell with a plasmid comprising a Sox- 18 nucleotide sequence comprising nucleotide 89 through nucleotide 1222 of Figure 2, wherein said sequence is operably linked, in the sense orientation, to a constitutive promoter.

15. A method for inhibiting myogenesis in a mammalian cell, comprising transforming said cell with a plasmid comprising a Sox-18 nucleotide sequence comprising nucleotide 89 through nucleotide 1222 of Figure 2, wherein said sequence is operably linked, in the antisense orientation, to a constitutive promoter.

16. A method for inhibiting the proliferation of skeletal muscle proliferation in a mammalian cell, comprising transforming said cell with a plasmid comprising a Sox-18 nucleotide sequence comprising nucleotide 89 through nucleotide 1222 of Figure 2, wherein said sequence is operably linked, in the sense orientation, to a constitutive promoter.

17. A method for inhibiting the proli eration of cardiac muscle proliferation in a mammalian cell, comprising transforming said cell with a plasmid comprising a Sox-18 nucleotide seguence comprising nucleotide 89 through nucleotide 1222 of Figure 2, wherein said sequence is operably linked, in the sense orientation, to a constitutive promoter.

18. A method for inducing differentiation of smooth muscle in a mammalian smooth muscle cell, comprising transforming said cell with a plasmid comprising a Sox-18 nucleotide sequence comprising nucleotide 89 through nucleotide 1222 of Figure 2, wherein said sequence is operably linked, in the sense orientation, to a constitutive promoter.

19. A pharmaceutical composition comprising a nucleotide sequence encoding a Sox- 18 polypeptide.

20. A method for the treatment of atherosclerosis, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of the composition of claim 19.

21. A method for the treatment of restenosis, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of the composition of claim 19.

22. A method for the treatment of pulmonary disease, comprising administering to a patient in need of such treatment a pharmaceutically effective amount of the composition of claim 19.

23. A method according to claim 22, wherein said pulmonary disease is selected from the group consisting of pulmonary thrombosis and pulmonary fibrosis.

24. A method for stimulation of vascularization in skeletal muscle tissue in vivo , comprising adminstering to a patient in need of said stimulation a pharmaceutically effective amount of the composition of claim 19.

25. A method for stimulation of vascular endotherlial cell proliferation in vivo , comprising administering to a patient in need of said stimulation a pharmaceutically effective amount of the composition of claim 19.

26. A method for inducing endothelialization of vascular grafts in vivo, comprising administering to a patient in need of such endothelialization a pharmaceutically effective amount of the composition of claim 19.

27. An isolated DNA sequence comprising the sequence of nucleotides as shown in FIG. 2 including biological fragments and homologs thereof.

28. An isolated polypeptide sequence comprising the sequence of amino acids as shown in FIG. 2 including biological fragments and homologs thereof.