CZ2001142A3 - Use of nucleic acid molecule - Google Patents

Abstract

The present invention relates to the use of the NAB1 or NAB2 polypeptide, or its biologically active fragment, and nucleic acid molecules acids encoding these polypeptides, especially in the gene therapy, in the manufacture of a medicament for treatment cell proliferative disorders associated with wound healing including mammals.

Description

Technical field

The present invention relates to the use of gene therapy techniques in the treatment of injuries. In particular, it relates to a novel use of polynucleotides encoding NAB1 or NAB2 transcriptional repressor proteins in the down-regulation of cell proliferative disorders that slow skin healing, to prevent the formation of hypertrophic and keloid scars, psoriasis, inhibition of restenosis following percutaneous transluminal coronary angioplasty, modulation vascular walls and inhibition of cell proliferation in cancer.

BACKGROUND OF THE INVENTION

Skin healing involves a wide range of cellular, molecular, physiological and biochemical processes. During the healing process, cells migrate to wound sites where they proliferate and synthesize extracellular matrix components to reconstitute tissue that is closely similar to the uninjured original tissue. This process is driven by mediators secreted from cells at the edges of the lesions, such as platelet derived growth factor (PDGF), epidermal growth factor (EGF), transforming growth factor beta (TGF) beta) and other cytokines. The beneficial effects of these agents on cells have been shown both in vitro and in vivo (reviewed in Moulin, Eur. J. Cell Biol. 68; 1-7, 1995), including the beneficial effects of PDGF administration in rat diabetes models (Brown et al., J. Surg. Res. 56: 562 570, 1994).

Over the past five years, numerous growth factors have been shown to accelerate cell proliferation in vitro and aid healing

injuries in animal models. Most attention was paid to TGF beta in connection with injury repair, differentiation and matrix production. TGF beta administered either locally or systemically accelerates skin repair rate in animal models (Ashcroft et al., Nature Medicine, 3; 1209-1215, 1997; Sporn and Roberts, J. Cell Biol., 119; 1017-1021, 1997; Beck et al., J. Clin. Invest. 92: 2841-2849 (1993). Similarly, PDGF has been reported to promote reepithelialization and revascularization of ischemic tissue and animals with diabetes (Uhl et al., Langenbecks Archives Forum Surgeryio Supplement-Kongressband 114; 705-708, 1997, reviewed in Dirks and

Bloemers, Mol. Biol. Reports 22; 1-24, 1996).

The transient factor Egr-1 (early growth response gene) is a potential regulator of more than thirty genes and plays a role in cell proliferation, cell development and differentiation (review in Liu et al.

Crit. Roar. Oncogenesis 7; 101-125, 1996; Khachigian and Collins, Circ. Res. 81; 457-461, 1997). Egr-1 is induced after injury to the vascular endothelium (e.g., Khachigian et al., Science; 271, 1427-1431, 1996) and aims at activating the transcription of numerous genes including EGF, platelet-derived growth factor A (PDGF20 A), growth factor basic fibroblast growth factor (bFGF), induction of PDGF-A, platelet-derived growth factor B (PDGF-B), TGF beta, bFGF, uro-plasminogen activator (u-PA), tissue factor and growth factor-2 insulin-like growth factor-2 (IGF-2). Blockade of Egr-1 inducible TGF beta can find use in preventing scar formation. Anti-TGF beta antibodies reduce scar formation in rodent cut injuries (Shah et al., J. Cell Science; 107, 1137-1177; Shah et al., Lancet; 339, 213-214, 1992).

The transcription complex that mediates vascular endothelial growth factor (VEGF) induction is directly dependent on AP2 and not Egr-1 (Gille et al., EMBO J 16; 750-759, 1997). However, PDGF B performs booster control

VEGF expression (Finkenzeller, Oncogene 15; 669-676, 1997). VEGF mRNA transcription is enhanced by a number of factors including PDGF B, bFGF, keratinocyte growth factor (KGF), EGF, tumor necrosis factor,

TNF) alpha and TGF beta1. In animal models, VEGF-supported passivation of metal stents has been shown to inhibit neointima formation, accelerate reendothelialization and increase vasomotor activity (Asahara et al., Circulation; 94, 3291 - 3302).

Expression of VEGF has been reported to occur in wound healing and in apsoriatic skin, both cases in which the TGF alpha and its ligand EGFr are upregulated. EGF expression induces Egr-1 (Iwami et al., Am. J. Physiol. 270; H2100-2107, 1996; Fang et al., Calcified Tissue International 57; 450-455, 1995; J. Neuroscience Res. 36; 58-65 , 1993). There is currently undisclosed evidence that Egr-1 can activate expression of intracellular adhesion molecule-1 (ICAM-1) in phorbol ester-stimulated B cells (Maltzman et al., Mol. Cell. Biol. 16; 2283 - 2294, 1996) and can activate TNF alpha expression due to the presence of the Egr-1 binding site in the TNF alpha promoter (Kramer et al., Biochim. Biophys. Acta 1219; 413-421, 1994). Finally, mice without Egr-1 are infertile and have Luteinizing Hormone (LH) deficiency (Lee et al., 273; 1219-1221, 1996), suggesting that the LH promoter may also be a target for activation of Egr-1. Vascular calcification is an actively regulated process similar to bone formation, which includes cells and factors known to be important in the regulation of bone metabolism (reviewed in Dermer et al., Trends Cardiovasc. Med. 4; 45-49, 1994). Regulators of osteoblast and / or osteoclast formation can modulate the degree of vascular wall calcification. The NAB, NAB1, and NAB2 family proteins (NGFI-A binding corepressors) interact with the conserved R1 domain of Egr-1 and Egr-2 trans-activators (Weld et al., EMBO J., 17; 6010 6019, 1998). It has previously been shown that NAB2 will reprimate ·····················

4 NGF induced differentiation of PC12 cells (Qu, Z. et al., J. Cell Bioi. 142; 1075-1082, 1998) and Egr-1 induced basal FGF activation (Welded et al., EMBO J., 17; 6010 - 6019 , 1998).

One of the problems associated with the treatment of injuries is the formation of 5 hypertrophic and keloid scars. This is extremely undesirable, especially after surgery for cosmetic reasons. Tissue fibrosis (for example, as used in kidney, liver or skin) is manifested by accumulation of the extracellular matrix. The unregulated formation of the extracellular matrix, which promotes the development of tissue fibrosis, is at least in part controlled by a number of growth factors, but not limited to TGF beta (Muir Eur. J. Plast. Surg. 21; 1-7, 1998), PDGF isoforms ( Katou et al., J. Pathol. 186: 201-208, 1998, Heldin et al., The Molecular and Cellular Biology of Wound Repair, Clark, ed .; 249-264, 1996 Plenum Press) and VEGF (Jones et al. Frontiers in Bioscience 4;

D303-309, 1999). Therapy that would reduce, but not eliminate, the expression of growth factors at the site of injury would have a significant effect on tissue healing with reduced scars and improved quality of life.

International patent application PCT GB 99/01722 describes the use of the transcription factor Egr-1 in promoting wound healing. The present inventors have now found that administration of a polynucleotide encoding a NAB1 or NAB2 transcriptional repressor: (a) represses Egr-1-mediated growth factor activation in vitro; (b) represses basal growth factor gene expression levels in vitro; and (c) reduces the expression of TGF-βΙ but not TGF-β3 at the rodent wound site and its subsequent expression, and has utility in reducing healing scarring (e.g., Shah et al., J. Cell Science 107; 1137-1157, 1994).

Therefore, the debilitating regulation of Egr-1 slows the healing process and reduces the occurrence of hypertrophic and keloid scars and psoriasis. The debilitating regulation of Egr-1 may also be useful in the treatment of other wound healing disorders such as percutaneous restenosis

transluminal coronary angioplasty, modulation of vascular wall calcification, and inhibition of cell proliferation in cancer.

SUMMARY OF THE INVENTION

Accordingly, in one aspect of the invention there is provided the use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, in the manufacture of a pharmaceutical composition for treating cell proliferative disorders associated with treating injuries in mammals, including humans.

According to a further aspect of the invention there is provided a method of treating cell proliferative disorders associated with treating injuries in mammals, including a human, comprising administering a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a biologically active fragment thereof.

According to another embodiment, the invention provides a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, for use in the treatment of cell proliferative disorders associated with wound healing.

In another embodiment, the invention is directed to the use of a combination of a nucleic acid molecule comprising a sequence encoding a NAB1 polypeptide and another molecule comprising a sequence encoding a NAB2 polypeptide.

In a preferred embodiment of the present invention, cell proliferative disorders associated with wound healing are selected from hypertrophic and keloid scars, psoriasis, restenosis following percutaneous transluminal coronary angioplasty, vascular wall calcification, and cancer cell proliferation. In a particularly preferred embodiment of the present invention, the cell proliferative disorder is the formation of hypertrophic and keloid scars.

······· · · «• • · <<<<<<<<

Thus, the present invention relates to the therapeutic use of polynucleotides encoding NAB1 or NAB2 in wound healing processes. The invention also relates to the therapeutic use of NAB1 or NAB2 alone in the wound healing process, as described in more detail below.

The present invention relates to the use of NAB1 or NAB2 polypeptides and nucleic acid sequences encoding NAB1 or NAB2 polypeptides of any origin or species. The human DNA sequence is shown in Genbank accession number U47007, which sequence encodes a nuclear protein of 486 amino acids in length. Rat NAB1 is a 570 amino acid nuclear protein and is described in PNAS USA 92, 1995, pp. 6873-6877. The rat DNA sequence is given in Genbank Accession No. U17253.

The sequences of murine and human NAB2 protein are described in Molecular and Cellular Biology, 1996, 16, 3545-3553. The human DNA sequence is listed in Genbank Accession No. U48361.

References to the NAB1 or NAB polypeptides and polynucleotides described below are generally applicable to sequences of any origin, particularly the human sequence described above. As described below, the term NAB1 or NAB2 also includes variants, fragments, and analogs of NAB1 or NAB2.

The following illustrative explanations are provided to facilitate understanding of some of the terms used in the application.

The explanations are provided for convenience and are not limiting to the invention.

The biologically active fragments of NAB1 or NAB2 referred to are those fragments that have repressive effects on Egr-1 transcription.

Genetically, an element generally means a polynucleotide comprising a polypeptide coding region or a polynucleotide region that directs replication, transcription or translation, or other processes important for expression of polypeptides in a host cell, or a polynucleotide comprising both a polypeptide coding region and a region operably linked to an expression regulating region. Genetic elements may be contained within a vector that replicates as an episomal element; ie as a molecule physically independent of the host cell genome. Genetic elements may be contained within plasmids. Genetic elements may also be contained within the genome of the host cell; not in their natural state, but after manipulation such as isolation, cloning and introduction into the host cell, inter alia in the form of purified DNA or in a vector.

A host cell is a cell that has been transformed or transfected, or capable of transforming or transfecting with an exogenous polynucleotide sequence.

Identity is, as is known in the art, a relationship between two or more polypeptide sequences, or two or more polynucleotide sequences, as determined by sequence comparison.

In the art, identity also means the degree of sequence alignment between polypeptide or polynucleotide sequences as determined by comparison between the chains of these sequences. Identity can be easily calculated (Computational Molecular Biology, Lesk, A.M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and

Genome Projects, Smith, D.W., eds., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin H.G., ed., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., ed., M.

Stockton Press, New York, 1991). There are a number of methods for measuring identity between two polynucleotides or two polypeptides, and this term is well known to those of skill in the art (Sequence Analysis in Sequence Analysis). · · «···

Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods commonly used to determine identity between sequences include, but are not limited to, those described in Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Preferred methods for determining identity are designed to give the highest agreement among the sequences tested. Methods for determining identity are encoded in computer programs. Preferred program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN, and BLASTN.

FASTA (Atschul, S.F. et al., J. Molec. Biol. 215: 403 (1190)).

Isolated means changed by the "hand of man" from the natural state; ie, if the substance occurs in nature, it has been altered or removed from its original environment, or both. For example, a naturally occurring polynucleotide or polypeptide that is naturally present in a living organism in its natural state is not "isolated", but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated" within the term used in the present application. As part of subsequent isolation, the polynucleotides may be joined to form other polynucleotides, such as DNA, mutagenesis may be performed, fusion proteins may be made, and for example propagation or expression in the host cell may be performed. Isolated polynucleotides, whether alone or linked to other polynucleotide sequences, such as in the form of vectors, can be introduced into host cells, culture, or the whole organism. Introduced into host cells in culture or whole organisms, these DNAs can still be isolated within the meaning of the term used in the application because they are not in their naturally occurring form or environment, they are not naturally occurring mixtures and the polynucleotides or polypeptides remain isolated as used herein.

Polynucleotide (s) generally refer to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA, or cDNA. For example, polynucleotides as used herein refer to, inter alia, single-stranded and double-stranded DNA, DNA that is a mixture of single- and double-stranded regions or single-, double-, and triple-stranded regions, single- and double-stranded RNA, and RNA that is a single- and double-stranded regions, hybrid molecules comprising DNA and RNA, which may be single-stranded, or more typically double-stranded or triple-stranded, or mixtures of single-stranded and double-stranded regions. In addition, a polynucleotide as used herein is a triple strand region comprising RNA or DNA or both RNA and DNA. The chains in these regions may originate from the same molecule or from different molecules. The regions may comprise one or more molecules in their entirety, but typically contain only a portion of any of the molecules. One of the molecules of the triple helix region is often an oligonucleotide. As used herein, the term polynucleotide includes DNA or RNA molecules as described above that comprise one or more modified bases. For example, DNA or RNA molecules with backbones modified for stability, or for other reasons, are termed "polynucleotides" as used herein. In addition, for the purposes of the terminology used herein, polynucleotides are also, for example, DNA or RNA molecules containing unusual bases such as inosine or modified bases such as tritylated bases. It will be understood that DNA and RNA may be subjected to a variety of modifications that serve many different purposes known to those skilled in the art. The term polynucleotide, as used herein, includes such chemically, enzymatically or metabolically modified forms of polynucleotides as well as chemical forms of DNA and RNA characteristic of viruses and cells, including but not limited to simple

- 10 and complex cells. The term polynucleotides also includes short polynucleotides, often referred to as oligonucleotides.

Polypeptides means, as used herein, all of the polypeptides described below. The basic structure of polypeptides is well known and has been described in countless textbooks and other publications in the art. In this context, the term is used herein to refer to any peptide or protein comprising two or more amino acids linked together in a linear chain by peptide bonds. As used herein, the term refers to both short chains, often referred to in the art as peptides, oligopeptides and oligomers, as well as long chains, generally referred to in the art as proteins, of which there are many types. It will be appreciated that polypeptides often contain amino acids other than twenty amino acids, which are commonly referred to as naturally occurring, and that many amino acids, including terminal amino acids, can be modified in a given polypeptide by either natural pathways such as Processing and other post-translational modification, but also chemical modification techniques well known in the art. Even conventional modifications that occur naturally to polypeptides are too numerous to list exhaustively, but are described in the basic texts and in more detailed monographs, as well as in the voluminous research literature, and are well known to those skilled in the art.

Known modifications that may be present in polypeptides for use in the present invention include, for example, acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of a flavin, covalent attachment of a heme group, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment lipid or lipid derivative, covalent attachment of phosphatidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gamma-carboxylation, glycosylation, formation

GPI anchoring, hydroxylation, iodination, methylation, myristoleaction, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, addition of amino acids to proteins mediated by transfer RNA such as arginylation and ubiquitination. Such modifications are well known to those skilled in the art and have been similarly described in the scientific literature. Several particularly common treatments such as glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation are described in the most basic texts such as PROTEINS - STRUCTURE

AND MOLECULAR PROPERTIES, 2nd ed., Τ. E. Creighton, W.H. Freeman and Company, New York (1993). There are many detailed summaries on this subject, such as Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pp. 1-12, in the POSTTRANSLATIONAL COVALENT MODIFICATION OF

PROTEINS, B.C. Johnson, ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182: 626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. N.Y. Acad. Sci. 663: 48-62 (1992). It will be appreciated that, as is known in the art and as noted above, polypeptides are not always completely linear.

For example, polypeptides may generally be engineered as a result of post-translational events, including naturally occurring processing and events that are performed by non-naturally occurring artificial manipulation. Likewise, circular, branched and branched circular polypeptides synthesized by both non-translational natural and fully synthetic methods can be included. Modifications can occur anywhere in the polypeptide, including the polypeptide backbone, amino acid side chains, and amino or carboxyl termini. Blocking of the amino or carboxyl group, or both, in a polypeptide by covalent modification is common to both synthetic and natural polypeptides, and such modifications may also be present in the polypeptides of the present invention. For example, the amino-terminal residue of polypeptides formed by the term &quot; amino acids &quot;

In E. coli or other cells, N-formylmethionine will almost always be prior to proteolytic processing. Cleavage of the methionine residue at the Nbh-terminus may occur during post-translational modification of the peptide. Thus, the present invention encompasses both the use of the amino-terminal methionine variant protein of the invention and the methionine-free variant. Modifications that occur with polypeptides will often depend on how it is made. For polypeptides produced by expressing a cloned gene in, for example, a host, the nature and extent of the modifications will largely depend on the posttranslational modifying ability of the host cell and the modifying signals present in the amino acid sequence of the polypeptides. For example, as is well known, glycosylation often does not occur in bacterial hosts such as E. coli. Thus, if glycosylation is desired, the polypeptide should be expressed in a glycosylating host, generally in a eukaryotic cell. Insect cells often perform the same posttranslational glycosylations as mammalian cells and, therefore, an insect cell-based expression system has been developed to efficiently express mammalian proteins with, inter alia, the natural spectrum of glycosylation. Similar assumptions apply to other modifications. It will be appreciated that at the various sites of a given polypeptide, the same type of modification may be present to the same or different extent. A given polypeptide may also contain many types of modifications. As used herein, the term polypeptide generally encompasses all of these modifications, particularly those that are present in polypeptides synthesized in a recombinant manner by expressing a polynucleotide in a host cell.

For the purposes of the present invention, polynucleotide or polypeptide variant (s) refers to polynucleotides or polypeptides that differ from the reference polynucleotide (s). Variants in this sense are described in more detail below and elsewhere in the specification.

(1) A polynucleotide having a different nucleotide sequence from another reference polynucleotide. Generally, the differences are limited so that the nucleotide sequences of the reference strand and the variants are 9 9 9 9 9

- 13 • 9 ··· 99 «9 * 99

9 9 9 9 9

999 * 99 9999 9 «999 generally closely similar, and in many areas identical. As discussed below, changes in the nucleotide sequence of the variant need not be externally (may be silent). That is, they do not need to alter the amino acid sequence encoded by the polynucleotide. If the changes are limited to "silent changes" of this type, the variant will encode a polypeptide with the same amino acid sequence as the reference chain. As discussed below, changes in the nucleotide sequence of a variant may alter the amino acid sequence of the polypeptides encoded by the reference polynucleotide. These nucleotide changes may result in substitutions, additions, deletions, fusions, and truncations of the polypeptides encoded by the reference sequence, as discussed below. (2) A polypeptide having a different amino acid sequence from another, reference polypeptide. In general, the differences are limited so that the sequences of the reference chain and variants are generally closely related and identical in many regions. A variant and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions, and truncations, which changes may be present in any combination.

The present invention relates to therapeutic uses of the molecules

20 nucleic acids comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a combination thereof. The invention also relates to therapeutic uses of fragments of this polynucleotide sequence that encode biologically active fragments of NAB1 or NAB2 polypeptides or variants of a polynucleotide sequence that encode functional, i.e., biologically active fragments of NAB1 or NAB2, and functionally equivalent allelic variants and related sequences modified by single or multiple base substitution, addition, and / or deletion that encode polypeptides with NAB1 or NAB2 activity.

Variants can be obtained by standard cloning methods known to those skilled in the art.

· · · ·

9 · · ··· 9 99 ····

Polynucleotides encoding NAB1 or NAB2 transcriptional repressor proteins may be in the form of DNA, cDNA or RNA, such as mRNA obtained by cloning or produced by chemical synthesis methods. The DNA may be single stranded or double stranded.

The single-stranded DNA may be a coding or sense strand or a non-coding or anti-sense strand. For therapeutic use, the polynucleotide is in a form capable of being expressed to a functional repressor protein of NAB1 or NAB2 transcription at the site of injury in a patient. Polynucleotides can also be used to produce an NAB110 or NAB2 polypeptide in vitro for administration in another therapeutic embodiment of the invention, which will be described in more detail below.

Polynucleotides of the present invention that encode a NAB1 or NAB2 polypeptide may include, without limitation, the coding sequence of the NAB1 or NAB2 polypeptide, or biologically active fragments thereof. Thus, a polynucleotide may be provided along with additional, non-coding sequences, including, but not limited to, non-coding 5 'and 3' sequences, such as transcribed, non-translated sequences that play a role in transcription (e.g., termination signals), ribosome binding, mRNA stability elements. and other coding sequences that encode additional amino acids, such as amino acids providing additional functions. Polynucleotides of the invention also include, but are not limited to, polynucleotides comprising a structural gene for NAB1 or NAB2 and naturally associated genetic elements thereof.

According to the foregoing definition, the term "polynucleotide encoding a polypeptide" within the scope of the present invention includes polynucleotides that comprise a sequence encoding a NAB1 or NAB2 polypeptide. The term includes polynucleotides that comprise a single contiguous region or discontinuous regions encoding a polypeptide (for example, interrupted by an integrated phage or insertion sequence or editing) together with other regions, which may also include coding and / or non-coding sequences.

• 4 44 · • 44 • • 4 4 4 4 4 4 4 4 4 • 44 · 4 • 4 4 4 4 · • 4 • • • • 4 4 4 4 4 4 4 4 4 4 4 4 44 4 4

The present invention further relates to variants of the polynucleotides described above that encode fragments, analogs and derivatives of the polypeptide. The polynucleotide variant may be a variant that occurs in nature, such as an allelic variant in nature, or it may be a variant that is known not to occur in nature. These non-naturally occurring variants can be made by mutagenesis techniques including methods applied to polynucleotides, cells or organisms.

Variants in this regard include variants that differ from the above polynucleotides by nucleotide substitutions, deletions, or additions. The substitutions, deletions, or additions may include changes in one or more nucleotides. Variants may be changed in the coding or non-coding regions, or both. Changes in the coding regions can create conservative or non-conservative amino acid substitutions, deletions, or additions.

Further preferred embodiments of the invention are polynucleotides having at least 70% identity over their entire length to a polynucleotide encoding the polypeptides having the amino acid sequence described above, and polynucleotides that are complementary to these polynucleotides.

Alternatively, polynucleotides that comprise a region with at least 80% identity over the full length with a polynucleotide encoding a polypeptide of the present invention are most preferred. In this sense, polynucleotides having at least 90% identity to the reference sequences over the entire length are particularly preferred, with at least 95% identity being even more preferred. In addition, chains with at least 95% identity are more preferred chains with at least 97% identity, and mehi chains with at least 98% identity are preferred chains with at least 99% identity, with at least 99% identity being most preferred.

In addition, preferred embodiments in this regard are polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature (mature) NAB1 polypeptide.

- 16 44 4444 44 444 or NAB2 encoded by the DNA sequences described above (Genbank Accession Numbers U47007 and U48361).

The present invention further relates to polynucleotides that hybridize to the sequences described above. In this sense, the present invention relates in particular to polynucleotides which hybridize under stringent conditions to the aforementioned polynucleotides. As used herein, the term "stringent conditions" means that hybridization occurs when the identity between the sequences is at least 95%, and preferably at least 97%. Preferably, sequences which in this manner hybridize to a sequence of the invention encode a polypeptide with biological activity of NAB1 or NAB2.

Polynucleotides may encode a polypeptide that is a mature protein plus additional amino- or carboxy-terminal amino acids. These additional sequences may play a role, for example, in prolonging or shortening the half-life of the protein, or may allow manipulation of the protein for testing or production purposes or for other reasons. In general, in vivo, additional amino acids can be cleaved by cellular enzymes from the mature protein.

Polynucleotides for use in gene therapy of the invention may be provided alone or as part of a vector, such as an expression vector, which are well known in the art.

A polynucleotide encoding NAB1 or NAB2 may be therapeutically used in the method of the invention in the form of gene therapy, wherein the polynucleotide is administered to the site of injury or other tissues in need of healing in a form capable of directing production of NAB1 or NAB2 or a biologically active fragment thereof. in šitu.

Preferably, in gene therapy, the polynucleotide is administered by being expressed in a treated patient, for example, in the form of a recombinant DNA molecule comprising a polynucleotide encoding NAB1 or NAB2 operably linked to an expression control nucleic acid sequence such as an expression vector. Thus, such a vector will include · vektor · · zahrnovat vektor vektor vektor vektor vektor vektor vektor vektor vektor vektor vektor

17 appropriate transcriptional control signals, including a promoter region capable of expressing a coding sequence, wherein said promoter may function in the subject to be treated. In the case of human genetic therapy, the promoter is preferably a term which includes not only the sequence necessary for directing RNA polymerase to the transcription start site, but also, if necessary, other functional or control sequences including enhancers, human promoter sequences derived from the human gene , or from a gene that is typically expressed in humans, such as the human CMV promoter. Among the known eukaryotic promoters useful in this regard are the CMV immediate early promoter (CMV), the HSV thymidine kinase promoter (HSV), the SV40 early and late promoters, the retroviral LTR promoters, such as the Rous sarcoma virus (RSV) promoters. and metallothionein promoters such as the mouse metallothionein-1 promoter.

The polynucleotide and transcriptional control sequences may be cloned into a replicable plasmid vector based on commercially available plasmids, such as pBR322, or may be constructed from available plasmids by routine using known methods.

The vector may also include transcriptional control signals located in the 3 'direction relative to the coding sequence of NAB1 or NAB2, as well as polyadenylation signals that are recognized by the subject to be treated, such as the corresponding viral sequences, in the case of human treatment, for example SV40. Other known sequences for directing transcription can also be used.

Expression vectors may also contain selectable markers, such as for antibiotic resistance, which allow vector propagation.

Expression vectors capable of synthesizing NAB1 or NAB2 in situ can be introduced directly into the wound by physical methods. Between 9 9 9

999999 9 9 99 9

Examples of such topical administration include administration of a "naked" nucleic acid vector in a suitable vehicle, for example, in a solution in a pharmaceutically acceptable excipient such as phosphate buffered saline (PBS) or administration of the vector by physical methods such as particle bombardment, also known as a gene gun technology in a manner known in the art, for example as described in U.S. Pat. No. 5,371,015, wherein inert particles such as vector-coated gold spheres are accelerated to a sufficient velocity to allow penetration into the surface at the site of the injury, e.g., skin cells, by firing at high pressure from a suitable device.

Other physical methods of administering DNA directly to a recipient include ultrasound, electrostimulation, and electroporation.

The nucleic acid sequence encoding NAB1 or NAB2 for use in the therapy of the invention may also be delivered by delivery vectors. These include viral delivery vectors such as adenoviral or retroviral delivery vectors known in the art.

Other non-viral delivery vectors include lipid delivery vectors, including liposome delivery devices known in the art.

The nucleic acid sequence encoding NAB1 or NAB2 can also be delivered to the site of injury through transformed host cells. Such cells include cells harvested from a subject into which nucleic acid sequences are introduced by art-recognized gene transfer methods, followed by growth of the transformed cells in culture and grafting to the patient.

Expression constructs such as those described above can be used in a variety of ways in the therapy of the present invention. Thus, they can be directly administered to the patient's wound site or used to produce the recombinant NAB1 or NAB2 polypeptide itself, which can then be

• · • · • · · « • · • ·· • • · • 9 • ·· · ♦ 9 9 9 • • • 9 9 · * 9 9 99 • • · · · • ·· • • «

- 19 administered to the site of injury as discussed in more detail below. The invention also relates to host cells that are engineered by genetic engineering methods using constructs comprising the NAB1 or NAB2 polynucleotide or polynucleotides of the present invention or genetic elements as defined above, and the use of these vectors and cells in methods of treatment. These constructs may be used as such in therapeutic methods or may be used to prepare a NAB1 or NAB2 polypeptide for use in therapeutic methods, as described in more detail below.

For example, the vector may be a plasmid vector, a single-stranded or double-stranded phage vector, a single-stranded or double-stranded viral vector of RNA or DNA, depending on whether the vector is to be administered directly to the site of injury (i.e., for synthesis of NAB1 or NAB2 in situ) or to be used for the synthesis of recombinant NAB1 or NAB2. The starting plasmids described in the present invention are either commercially available, publicly available, or can be made from available plasmids by routine use of known published procedures. Many plasmids and other cloning and expression vectors,

Which can be used according to the present invention is well known and readily available to those skilled in the art.

NAB1 or NAB2 polypeptide expression vectors for use in the present invention generally comprise cis-acting control regions effective for expression in a host operably linked to the expressed polynucleotide. Suitable trans-acting factors are either delivered by the host, the complementing vector, or the vector itself upon introduction into the host.

In some embodiments, the vectors provide specific expression in this regard. For the production of recombinant NAB1 or NAB2, this specific expression can be inducible expression or expression only in some cell types or expression as either a cell type or an expression of either a cell type or an expression of either cell type or expression. · · · · · · · · · · · · · · · · · · · · · ·

Inducible as well as cell specific. Particularly preferred among the inducible vectors are those vectors that can be induced for expression by environmental factors that are easy to manipulate, such as temperature and nutritional supplements. Many vectors suitable for use in this embodiment of the invention include constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts, which vectors are well known and commonly used by those skilled in the art.

A wide variety of expression vectors can be used to express NAB1 or NAB2 of the present invention. These include, but are not limited to, chromosomal, episomal, and virus-derived vectors, such as vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculoviruses, papovaviruses such as SV40, vaccinia viruses , adenoviruses, varicella viruses, pseudorabies and retroviruses, and vectors derived from combinations thereof, such as vectors derived from plasmid and bacteriophage genetic elements such as cosmids and phagemids, all of which elements can be used for expression according to this embodiment of the present invention. . In this regard, any vector suitable for the maintenance, multiplication or expression of polynucleotides in order to express the polypeptide in a host may generally be used for expression.

A suitable DNA sequence may be introduced into the vector by any of the well known routine methods, such as those set forth in Sambrook et al., MOLECULAR CLONING, and LABORATORY MANUAL, 2nd ed .; Cold Spring Harbor Laboratory Press, New York (1989).

The nucleic acid sequence in the expression vector is operably linked to a suitable expression control sequence or sequences, including, for example, a promoter for direct transcription of mRNA. Representatives of these

• 9 • 9 99 9 · 9 9 99 9 9 9 9 9 9> 999 9 9 9 • 9 9 9 9 9 9 9 9 9 9 99 9 99 9999 99 99

The 21 promoters without limitation include the lambda phage PL promoter, lac, trp and tac E. coli promoters for recombinant expression and the early and late SV40 promoters and retroviral LTR promoters for in situ expression.

Expression constructs will generally contain 5 transcription start and stop transcription sites, and a ribosome binding site in the transcribed region to allow translation. The coding portion of the mature transcripts expressed by the constructs will comprise an AUG translation initiation site at the beginning and a termination codon suitably located at the end of the translation polypeptide.

The constructs may additionally comprise control regions that direct and prepare expression. According to many commonly used methods, such regions will generally function by controlling transcription, such as transcription factors, repressor binding sites, and terminators.

Vectors for propagation and expression will generally include selectable markers and amplification regions, such as those set forth in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd ed .; Cold Spring Harbor Laboratory Press, New York (1989).

Representative examples of suitable hosts for recombinant expression of NAB1 or NAB2 include bacterial cells such as streptococci, staphylococci, E. coli, streptomyces and Bacillus subtilis' cells, fungal cells such as yeast cells and Aspergillus cells, insect cells such as Drosophila S2 cells and Spodoptera Sf9; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK cells,

293 and Bowes melanoma cells; and plant cells.

Examples include the following commercially available vectors. Preferred vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and vectors ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia, and vector pBR322 (ATCC 37017).

9 9

999999 9 9 ·

Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG from Stratagene; and pSVK3, pBPV, pMSG and pSVL from Pharmacia. These vectors can be used for both recombinant and in situ expression, and are given solely to illustrate the many commercially available and known vectors available to those skilled in the art for use in any embodiment of the present invention. It will be appreciated that any other plasmid or vector suitable, for example, for introducing, maintaining, propagating or expressing the polynucleotide or polypeptides of the invention in a host cell may be used for these embodiments of the invention.

Examples of vectors for use in the present invention include expression vectors in which the NAB1 or NAB2 cDNA sequence is inserted into a plasmid, wherein gene expression is derived from the human early cytomegalovirus enhancer-promoter (Foecking and

Hofstetter, Cell 45: 101-105 (1986). These expression plasmids may contain SV40 RNA treatment signals, such as polyadenylation and termination signals. Expression constructs that use the CMV promoter and which are commercially available are pCDM8, pcDNA1 and derivatives, pcDNA3 and derivatives (Invitrogen). Other expression vectors, such as pSVK3 and pSVL, which contain the SV40 promoter and mRNA cleavage site and SV40 polyadenylation signals (pSVK3) and SV40 VP1 processing signals (pSVL; Pharmacia vectors) can also be used.

Promoter regions can be selected from any gene of interest using vectors that contain a reporter transcriptional unit without a promoter region present, such as a chloramphenicol acetyltransferase ("CAT") transcriptional unit located downstream of a restriction site or sites for insertion of a possible promoter fragment; i.e., a fragment that may contain a promoter. As is known, the introduction of a promoter-containing fragment into a vector at the upstream site of the cat gene will allow the generation of CAT activity, which may be

- 23 detected by standard CAT tests. Vectors suitable for this purpose are well known and readily available, such as pKK232-8 and pCM7. Promoters for the expression of the polynucleotides of the present invention include not only known and readily available promoters, but also promoters that can be readily obtained by the above methods using a reporter gene; for in situ expression, such a promoter should be recognized by the subject being treated.

Known prokaryotic promoters suitable for expression of the polynucleotides and polypeptides of the present invention include E.

the co / lac and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, PL promoters, and the trp promoter.

Recombinant expression vectors will include, for example, origins of replication, a promoter derived preferably from a high-expression gene for direct transcription of a downstream structural sequence, and an optional marker to allow isolation of cells containing the vector for vector exposure.

Polynucleotides of the invention encoding a heterologous structural sequence of polypeptides of the invention will generally be inserted into a vector using standard methods so that they will be operably linked to an expression promoter. The polynucleotide will be positioned such that the transcription start site is located 5 'relative to the ribosome binding site. The ribosome binding site will be in the 5 'direction relative to the AUG codon that initiates translation of the expressed polypeptide.

Generally, there will be no other open reading frames that start with an initiation codon, usually AUG, and lie between the ribosome binding site and the initiation codon. There will also generally be a translation stop codon at the end of the polypeptides, and a polyadenylation signal will also be present in constructs for use in eukaryotic hosts. The transcription termination signal, which

- 24 must be located at the 3 'end of the transcriptional region, can also be included in the polynucleotide construct.

For secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, appropriate secretion signals can be inserted into the expressed polypeptide during recombinant synthesis. These signals may be endogenous to the polypeptide or may be heterologous signals.

The polypeptide may be expressed in a modified form, such as a fusion protein, and may contain not only secretion signals but also other heterologous functional regions. For example, to improve stability and persistence in the host cell, during purification or during subsequent manipulation and storage, for example, a region of additional amino acids, particularly charged amino acids, may be added to the N- or C-terminus of the polypeptide. A purification region may also be added to the polypeptide. These regions may be removed prior to final preparation of the polypeptide. Addition of peptide moieties to polypeptides to facilitate secretion or excretion, to improve stability, or to facilitate purification are examples of conventional and routine modifications in the art. A preferred fusion protein comprises an immunoglobulin letherologic region that is useful for solubilizing or purifying polypeptides. The cells are then typically harvested by centrifugation, broken up by physical or chemical means, and the crude extract obtained is retained for further purification.

Microbial cells used for protein expression can be broken by any conventional method, including freeze-thaw cycles, sonication, mechanical shredding or lysing agents, all of which are well known to those skilled in the art.

Mammalian expression vectors may contain an origin of replication, a suitable promoter and enhancer sequence, as well as any necessary ribosome binding site, polyadenylation region, donor

- 25 • ··· ·· and acceptor cleavage sites, transcription termination sequences, and downstream 5 'non-transcribed sequences required for expression.

Genetically engineered host cells may be used to prepare NAB1 or NAB2 polypeptides for use in the context of the present invention. Introduction of polynucleotides into the host cell can be achieved by calcium phosphate transfection, DEAEtextran-mediated transfection, transfection, microinjection, lipid-mediated cationic transfection, electroporation, transduction, scratching delivery, ballistic delivery, infection or other methods. These methods are described in many standard laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrook et al., MOLECULAR CLONING, A LABORATORY

MANUAL, 2nd ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

The mature proteins may be expressed in host cells including mammalian cells such as CHO, yeast cells, bacterial cells or other cells under the control of the corresponding promoters. Cell-free translation systems can also be used to produce such proteins using RNAs derived from the DNA constructs of the present invention. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic host cells are described in Sambrook et al., MOLECULAR CLONING, A

LABORATORY MANUAL, 2nd ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

The polypeptide can be isolated and purified from recombinant cell cultures by methods known in the art including ammonium sulfate or ethanol precipitation, acid extraction, anionic or cation exchange chromatography, phosphocellulose chromatography, chromatography using

- 26 hydrophobic interactions, affinity chromatography, hydroxylapatite chromatography and lecithin chromatography. Most preferably, high performance liquid chromatography will be used for purification. Well-known refolding methods can be used to regenerate active conformation of polypeptides when such a polypeptide is denatured during isolation and / or purification.

For therapy, a polynucleotide encoding NAB1 or NAB2, for example, in the form of a recombinant vector, can be purified by methods known in the art, such as column chromatography as described in Samrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

As noted above, the NAB1 or NAB2 polypeptide may be administered at the site of injury either in the form of a nucleic acid encoding

NAB1 or NAB2, which transcribes at the injury site itself and translates to NAB1 or NAB2, i.e. as gene therapy, or a protein for repressing transcription can be directly administered.

Accordingly, in a further aspect of the invention there is provided the use of a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, in the manufacture of a pharmaceutical composition for treating cell proliferative disorders in a mammal, including a human.

According to another embodiment of the invention there is provided a method of treating a cell proliferative disorder in a mammal including a human comprising administering a therapeutically effective amount of NAB1 polypeptides or

NAB2 or a biologically active fragment thereof to a mammal. In another aspect, the invention provides the use of NAB1 or NAB2 polypeptides or a biologically active fragment thereof in the treatment of wound healing and wound healing.

As used herein, the term "NAB1 or NAB2 polypeptide" includes both naturally and recombinantly produced

NAB1 or NAB2, natural, synthetic and biologically active analogs or variants of polypeptides or derivatives thereof, or biologically biologically active

- 27 active fragments and variants, including derivatives and analogs thereof.

NAB1 or NAB2 protein products including biologically active fragments of NAB1 or NAB2 can be generated and / or isolated by general methods known in the art.

NAB1 or NAB2 and the above-mentioned fragments and derivatives thereof for use in the treatment of the invention may be extracted from natural sources by methods known in the art. These methods include purifying sequence-specific DNA affinity chromatography using methods described, for example, in Briggs et al., Science 234, 47-52, 1986, using a DNA binding oligonucleotide that recognizes NAB1 or NAB2. The polypeptide can also be prepared by recombinant DNA technology methods known in the art as described above, for example, by expression of the described constructs in host cells. Alternatively, the polypeptides of the invention may be produced synthetically using conventional peptide synthesizers.

The invention also relates to the use of fragments, analogs and derivatives of NAB1 or NAB2. The term "fragment", "derivative" and "analog" mean a polypeptide that retains substantially the same biological function or activity as the polypeptide. Thus, the analog will include a protein that can be activated by cleavage of the proprotein moiety to yield active mature (mature) polypeptides.

The fragment, derivative, or analog of the polypeptides may be (i) a polypeptide in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue), and such substituted amino acid residue may or may not be a genetic code encoded; (ii) a polypeptide in which one or more amino acid residues comprise a group of substituents; or (iii) a polypeptide in which:

The parent polypeptide is fused to another compound, such as a compound providing an increase in half-life of the polypeptide (e.g., polyethylene glycol), or (iv) a polypeptide in which it is with the mature polypeptide. other amino acids, such as an introductory or secretory sequence, or a sequence that is used to purify the mature polypeptide or for protein sequences, are fused. Such fragments, derivatives, and analogs are considered to be within the skill of the art.

Preferred variants include those that differ from naturally occurring NAB1 or NAB2 by conservative amino acid substitutions. Substitution is such substitutions that replace a given amino acid in a polypeptide with another amino acid of similar properties. Typically, conservative substitutions are those in which the aliphatic amino acids Ala are replaced,

Val, Leu and Ile one after another; interchange of hydroxyl residues Ser and Thr, exchange of Asp and Glu residues, substitution between amide residues Asn and Gln, exchange of basic residues Lys and Arg and exchange between aromatic residues Phe, Tyr.

Also particularly preferred in this regard are variants, analogs, derivatives and fragments, and variants, analogs and derivatives thereof, having the amino acid sequence of a polypeptide in which a substitution, deletion, or addition in a few, a few, 5 to 10, 1 has been performed. to 5, 1 to 3, 2, 1, or any amino acid residue, in any combination. Among these substitutions, "silent" substitutions, additions, or deletions that do not alter the properties and activities of the polypeptide of the present invention are particularly preferred. Conservative substitutions are particularly preferred in this context.

Particularly preferred fragments are biologically active fragments, i.e., fragments that retain the ability to heal the injury of the parent polypeptide.

- 29 • 999999 9 9 99 9

9 9 9 9 9

999 99 99 99999 99

The polypeptides and polynucleotides of the present invention are preferably provided in isolated form, and in particular in a form purified to homogeneity.

NAB1 or NAB2 polypeptides for use in the present invention include a NAB1 or NAB2 polypeptide as well as polypeptides having at least 70% identity, preferably at least 80% identity, and more preferably at least 90% identity and even more preferably 95% similarity (even more preferably at least 95% identity). identity) with the above-described polypeptide sequences, and also comprise portions of these polypeptides, wherein such portion of polypeptides generally comprises at least 30 amino acids, and more preferably at least 50 amino acids.

Fragments or portions of the polypeptides of the present invention can be used to produce the corresponding full-length polypeptides by the peptide synthesis method; therefore, the fragments can be used as intermediates to produce full-length polypeptides. Fragments or portions of the polynucleotides of the present invention can be used to synthesize the full-length polynucleotides of the present invention.

The invention also relates to the use of fragments of NAB1 or

NAB2 as defined above and fragments or variants and derivatives thereof.

In this context, the fragment is a polypeptide having exactly the same amino acid sequence as part, but not the complete amino acid sequence of the NAB1 or NAB2 polypeptides or variants or derivatives thereof.

These fragments can be free-standing, i.e.

not to be part of or fused to other amino acids or polypeptides, or may be part of a longer polypeptide of which they form part or region. If fragments are contained within longer polypeptides, these fragments are discussed in the frame

3o of the present invention most preferably forms a single continuous region. However, several longer polypeptides may contain several · ♦ ·· ·· · · · · ·

• · · · · · · · · · · · · · · · · · · · · · · · ·

- 30 fragments. For example, certain preferred embodiments pertain to a fragment or polypeptide of the present invention contained within a precursor polypeptide designed for expression in a host cell and comprising heterologous pre- and propolypeptide regions fused to the amino terminus of the fragment and another region fused to the carboxyl terminus of the fragment. Therefore, fragments, in one embodiment of the meaning contemplated herein, refer to portions or portions of a fusion peptide or fusion protein derived from the polypeptide of the present invention.

Also preferred in this embodiment of the invention are fragments characterized by the structural or functional properties of the polypeptide of the present invention. Preferred embodiments of the invention in this regard include fragments comprising alpha-helices and alpha-helix-forming regions, beta-sheet and beta15-sheet-forming regions, turn and turn-forming, folding and fold-forming regions, hydrophilic regions, hydrophobic regions regions, alpha-amphipathic regions, beta-amphipathic regions, elastic regions, surface-forming regions, substrate binding regions, and regions with a high antigenic index of a polypeptide of the present invention, and combinations of these fragments.

Preferred regions are those that mediate the activities of the polypeptide of the present invention. In this regard, fragments having chemical, biological or other activity of the polypeptide of the invention, including fragments with similar or improved activity or reduced undesired activity, are most preferred. Other preferred polypeptide fragments are fragments that include or contain antigenic or immunogenic determinants of an animal, particularly a human.

It will be understood that the invention also relates, inter alia, to polynucleotides encoding the above fragments, polynucleotides that hybridize to polynucleotides encoding the fragments, particularly those that hybridize

- 31 ······ * * · ·· · 9 9 9 9 9

Under stringent conditions, and polynucleotides such as PCR primers to amplify the polynucleotides encoding these fragments. Preferred polynucleotides in this sense are those corresponding to the preferred fragments mentioned above.

In other embodiments of the present invention, biologically, prophylactically, clinically or therapeutically useful variants, analogs or derivatives or fragments, including fragments of variants, analogs and derivatives, and mixtures containing these fragments, are included. Biologically active variants, analogs or fragments are also within the scope of the present invention.

The invention also relates to compositions comprising the polynucleotides or polypeptides discussed above. Therefore, the polynucleotides or polypeptides of the present invention may be used in combination with a pharmaceutically acceptable carrier or carriers.

Such carriers include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.

Polypeptides and polynucleotides may be used alone or together with other compounds, such as therapeutically active agents, in the present invention.

The pharmaceutical compositions may be administered in any effective conventional manner which allows targeting to sites of injury including, for example, topical, intravenous, intramuscular, intranasal or intradermal administration.

For treatment or prevention, the active ingredient may be administered to an individual as a mixture for injection, for example, in the form of a sterile aqueous dispersion, preferably isotonic.

Alternatively, the composition may be formulated for topical administration, for example, in the form of ointments, creams, lotions, eye ointments, eye

3o drops, ear drops, mouthwashes, impregnated bandages and threads ······ · · · · ·

And may contain suitable conventional additives, including, for example, preservatives, drug penetration solvents and emollients in ointments and creams. Such topical formulations may also contain compatible carriers, for example, ointment or cream bases, and ethanol or oleyl alcohol in the case of milk. Such carriers may comprise from about 1% by weight to about 98% by weight of the composition; more typically, they will comprise up to about 80% by weight of the composition.

For administration to mammals, and in particular to humans, it is expected that the daily dose of the active ingredient will be from 0.01 mg / kg to 10 mg / kg, typically about 1 mg / kg. In any case, the physician will determine the actual dosage which will be most suitable for the individual and will vary according to the age, weight and response of the particular patient. The above dosages are examples of average dosages. However, there may be individual cases in which higher or lower dosage ranges are warranted, and are also within the scope of the present invention. The dose chosen by the skilled artisan will ultimately have the function of reducing cell proliferation without preventing wound healing.

In another embodiment, there is provided a pharmaceutical composition comprising a NAB1 or NAB2 polypeptide or a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide together with one or more pharmaceutically acceptable carriers.

The therapeutic advantage of using repressor transcription proteins in the treatment of injuries is in the inactivation of multiple target genes that promote accelerated healing. NAB1 or NAB2 is naturally activated in response to injury, and an enhancement of the natural response is also an advantage. DNA-based treatment provides a reliable and reproducible delivery system.

Where a NAB1 or NAB2 polynucleotide is used in the treatment of the present invention, it may be used as part of an expression vector.

• 9 9 9 99

For example, in the form of an expression vector. In such a method, the construct is introduced into a site of injury where NAB1 or NAB2 is produced in situ. The constructs used can be standard vectors and / or gene delivery systems such as liposomes, receptor-mediated delivery systems, and viral vectors.

The present invention is suitable for all aspects of wound healing including ulcers in limbs in diabetes and peripheral arterial occlusive diseases, post-operative scar formation, burns, psoriasis, inhibition of restenosis after percutaneous transluminal and coronary angioplasty, modulation of vascular wall calcification and inhibition of cell proliferation.

As described above, the NAB1 or NAB2 polypeptides or nucleic acids of the present invention can be administered topically to the site of tissue damage by any suitable means, for example, by topical administration. A preferred method for delivering nucleic acid products is through the use of gene gun technology in which an isolated Egr-1 nucleic acid molecule, for example in the form of cDNA or in an expression vector, is immobilized on gold particles and directly injected into the site of injury. Accordingly, in a preferred embodiment of the present invention there is provided the use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide in a gene gun method for treating cell proliferative disorders associated with wound healing. Further provided is a composition suitable for gene gun treatment comprising a sequence encoding NAB1 or NAB1

NAB2 immobilized on gold particles.

The present invention will now be described by way of the following non-limiting examples with reference to the accompanying drawings.

- 34 Overview of the Drawings

Giant. 1 a) - 1 d) shows the repression of Egr-1 mediated activation of PDGF-AB, TGFβ, HGF and VEGF.

Giant. 2 depicts the transgression of HGF production in HVSMC mediated by Egr-1 by NAB2.

Giant. 3 describes the effect of NAB2 on Egr-1-induced angiogenesis.

Giant. 4a describes the effect of NAB2 transfection on the contraction of direct incision injury 7 days after injury.

Giant. 4b describes the effect of NAB2 transfection on growth factor 10 levels in epidermis cut wounds in rat after 7 days.

Giant. 4c describes the effect of NAB2 transfection on growth factor levels in granulation tissue of cut wounds in rat after 7 days.

Giant. 4d describes the effect of NAB2 transfection on angiogenesis in 7-day cut wounds in rat.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Use of NAB2 for Egr-1 mediated repression of growth factor transactivation

1.1 Methods

Human vascular smooth muscle cells (HVSMC; Clonetics) were cultured according to the manufacturer's recommendations. Cells were cultured in six-well microtiter plates (Costar) for transfection. An expression plasmid containing the NAB2 cDNA derived from the human cytomegalovirus promoter (hCMV; Welded et al., Mol. Cell. Biol., 16; 3545-3553, 1996) was transfected together with an expression plasmid containing the Egr-1 cDNA (Houston et al., Arterioscler. Thromb. Vasc. Biol., 19; 281-289, 1999) to HVSMCs at ratios indicated in &lt; RTI ID = 0.0 &gt; ft. &Lt; / RTI &gt;

- 35 in the pictures, using FUGENE (Boehringer Mannheim). A 3: 1 (v / v) FUGENE: DNA ratio was used for all experiments and transfection was normalized using CMV-driven βgalactosidase expression plasmid. Secreted growth factors were detected in tissue medium by ELISA (R&D Systems) using appropriate controls.

1.2 Results

Giant. 1a) to 1d) show the repression of Egr-1 mediated activation of PDGF-AB, TGFβ, HGF, and possibly VEGF. 6 µg of the Egr-1 expression plasmid was cotransfected either alone (Fig. 1a, Fig. 1b and Fig. 1b).

d), or with 500, 100 or 25 ng of NAB2 (Fig. 1a and Fig. 1d). For PDGFAB, TGF [beta] and VEGF, there was a small increase in the amount of secreted growth factor detected by separate Egr-1 transfection. Co-transfection with NAB2 completely abolished the PDGF-AB response to Egr-1 activation and decreased basal expression, resulting in a five-fold reduction in total PDGF-AB production (Figure 1a). Transfection with NAB2 completely stopped the ΤΰΡβ response to Egr-1 activation and caused a 30% reduction in total TGFβ production (see Figure 1b).

Using the DNA concentrations shown in the figure, Egr-1 transfection resulted in a 50% increase in HGF production, which was partially blocked by cotransfection with a low dose of NAB2 and completely blocked by a higher dose (Fig. 1c). Higher effects were observed one day after transfection, although the inhibitory effects of NAB2 were evident on days 2 and 3. Transfection

NAB2, like PDGF-AB and TGFβ, blocked Egr-1 mediated VEGF activation and caused a 40% reduction in the total amount of VEGF generated (Fig. 1d).

··· Φ · ·

- 36 1.3 Conclusion

These data show that the Egr-1 suppressor, NAB2, can block Egr-1-mediated growth factor activation that occurs at sites of acute injury.

Example 2

Use of NAB2 for repression of basal levels of growth factors

2.1 Methods

Cell culture, transfection and detection of growth factors were performed as described in section 1.1 above. NAB2 expression vector was transfected at final concentrations of 0, 250, 500, 1000 or

3000 ng.

2.2. Results

As shown in Fig. 2a) transfection of NAB2 into HVSMC caused a drastic reduction in PDGF-AB production. At the highest dose of NAB2, a 10-fold decrease in PDGF-AB concentration was observed.

2.3 Conclusion

These data show that the Egr-1 suppressor can reduce the basal level of growth factors produced by both PDGF-AB promoters.

Example 3

Use of NAB for repression of induction of angiogenesis in vitro

3.1 Methods

NAB2 (wild-type and dominant-negative) expression constructs derived from the hCMV promoter have been described. 4 < tb &gt; ______________________________________ &lt; tb &gt; ______________________________________ &lt; tb &gt; • «« »·» »» »» »

37 (Welded et al., EMBO J. 17; 6010-5019, 1998). The authors of the present application have shown that transfection of the transcription factor Egr-1 when expressed from the hCMV promoter promoted angiogenesis (patent application PG3412). In these experiments, DNA was transfected into an angiogenesis research kit according to the manufacturer's instructions (TCS Biologicals). VEGF protein (2 ng / ml) was used as a positive control and suramine (20 μΜ) as an inhibitor of angiogenesis. CMV Egr-1 DNA was transfected at 0.5 µg per well in triplicate in a 24-well microtiter plate using Mirus Transit reagent (Cambridge io Biosciences) at a 2: 1 v / v DNA ratio. To test the ability of NAB2 to repress Egr-1 mediated angiogenesis, 0.5 mg of Egr-1 DNA was co-transfected with 10, 25 or 100 ng per well of NAB2 expression plasmid in the absence or presence of 100 ng NAB2 - a dominant negative expression plasmid. After 11 days of co-culture, the degree of angiogenesis was determined by staining the cells for the presence of the endothelial cell marker PECAM-1 and visualizing using the BCIP / NBT substrate.

Representative images of tubule formation using all four batches of Egr-1 expression plasmid together with VEGF (positive control) and suramine (negative control) were captured and processed by image analysis using the Quantimet 600 image analyzer and the included software.

Results

Egr-1 DNA has been shown to have proangiogenic effects (International Patent Application No. PCT GB 99/01722, column 4, Fig. 3a). As shown in Fig. 3a), co-transfection with NAB2 provided a reduction in the ability of Egr-1 to induce dose-dependent angiogenesis, which was removed by co-transfection with NAB2 - a dominant negative

3o expression plasmid.

• · · · ·

- 38 3.3 Conclusion

NAB2 can be used to block angiogenesis in the background of acute trauma, an example of which is induction of growth factor by Egr-1.

Example 4

Using NAB2 to reduce scar formation in a rodent cut injury model ¹⁰ 4.1 Methods

4.1.1 Effect of NAB2 transfection using gene gun technology on growth factor levels of cut injuries in rat

Transfection of NAB2 into rat cut lesions countered scar formation by directly suppressing the expression of key growth factors for scar formation, namely TGFβ1. In this experiment, NAB2 cDNA was transfected into rat cut lesions using Biorad gene gun technology, and the effects on healing and growth factor levels were tested using routine histological and immunocytochemical procedures.

4.1.2 Particle-mediated gene transfer

Eight male Sprague Dawley rats weighing -250 g were anesthetized with isoflorane in a 2: 1 oxygen / nitrous oxide mixture. Two transfection sites (5 cm from the base of the skull, 1.5 cm on each side of the midline) and two control sites (8 cm from the base of the skull, 1.5 cm on each side of the midline) were prepared on the back of the rat by clamping and then shave with a razor blade. One transfection was performed at each transfection site by accelerating the plasmid / gold complexes with either NAB2 or Egr-1 (positive control for growth factor activation) into the skin at 350 psi (2.41 MPa). Typically it was «· · · · · · · · · · ·

- 39.5 to 1.5 µg of DNA were added to the transfection site. At the control site, gold particles (without DNA) were accelerated into the skin at a pressure of 350 psi (2.41 MPa); the second control site was left intact. The transfection and control sites were rotated clockwise for each additional animal to compensate for time differences in wound healing.

4.1.3. Cutting wound healing model hours after transfection, animals were anesthetized and a 1 cm straight incision in full skin thickness was made parallel to the spine using a scalpel at the transfection site. Animals were allowed to recover after anesthesia and the wounds were allowed to heal without sewing. Seven days after the injury, all eight animals were sacrificed and the lesions were excised and used for routine histological and immunohistochemical tests.

4.1.4 Histological analysis

Each wound in a certain period of time after excision was horizontally cut into two pieces. One half was inserted into

4% paraformaldehyde for 24 hours and processed for wax histology.

mm sections from each wound were made using a microtome and sections were stained by van Geison. Sections were observed by light microscopy and the effect of NAB2 on healing was examined.

4.1,5 Immunohistochemistry

After freezing in OCT, the other half of each injury was cut to 7 mm using a cryostat. Two sections from each wound were fixed in cold acetone and fluorescent immunostaining was performed with primary antibodies against Egr-1, PDGF, TGFβ1,

TGFp3 and vWF using the following protocol.

·· 4 4 · 44 44 4

444 4,444 4,444

444 44 4,444

4444444 4 44 44 4 4

4,444,444

444 44 4444 44 444

- 40 1. Wash sections with PBS.

2. Inject 30 ml of primary antibody for 1 hr.

3. Wash 3 x 5 min in PBS.

4. Application of 30 ml secondary antibody (FITC directly bound)

45 min.

5. Wash 3 x 5 min in PBS.

6. Fastening the cuts with an aqueous fixture.

Immediately after immunological staining, each section was placed under a fluorescent microscope and the area of injury was also captured at 100X magnification. The image was then integrated and the resolution level was adjusted to minimize the background. The area and color intensity were measured by image analysis and the results were plotted graphically.

4.2 Results

4.2.1 Effect of NAB2 on Cutting Injuries in the Rat

4.2.1.1. Injury contraction and histological injury analysis after seven days

Referring to Fig. 4a), the wounds treated with NAB2 20 contracted at a similar width to that of the Egr-1 transfected and control wounds, showing similar maturity of the granulation tissue histologically.

Conclusion

The delivery of NAB2 does not impair the rate of healing in the rodent wound healing model.

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Growth Factor Profiles

Egr-1, TGFβ1, TGFp3, and PDGF-AB immunostaining was performed on frozen skin tissue cryopreserves at the site of injury and the staining intensity for each growth factor was measured using image analysis. Two different immunohistochemical measurements were performed at the site of injury. Sections were examined for growth factor expression both in the epidermis immediately after injury and at the site of injury (granulation tissue).

a) Positive staining for growth factors in the epidermis

As shown in Fig. 4b), seven days after injury, NAB2 transfection significantly reduced TGFβ1 expression in the epidermis compared to Egr-1 and single gold control. NAB2 transfection decreased expression as compared to non-manipulated control

Epidermis TGFβ1, while both Egr-1 and gold delivery activated TGFβ1 production. Transfection with NAB2 increased ΤΰΡβ3 levels in the epidermis compared to both gold and non-manipulated controls. Transfection with NAB2 did not significantly alter the epidermal expression of Egr-1 and PDGF.

b) Positive staining for growth factors in granulation tissue

As shown in Fig. 4c), seven days after injury, NAB2 transfection had no effect on Egr-1, PDGF-AB and TGFβ1 levels in granulation tissue. However, NAB2 increased TGFβ3 levels in granulation tissue compared to both gold alone and non-manipulated controls.

9

- 42 Conclusion

Transfection of NAB2 cut lesions reduced TGFβ1 levels in the epidermis and increased TGFβ3 levels in the epidermis and granulation tissue seven days after injury. TGFβ1 is a known scar enhancer, while TGFβ3 counteracts scar formation (Shah et al., J. Cell Science 107; 1137-1177, 1994). Therefore, delivery of NAB2 may have scar suppressing properties.

Effect of NAB2 on angiogenesis

Seven days after injury, skin sections were stained and evaluated for vWF expression using immunohistochemistry and image analysis. Angiogenesis was quantified using von Willebrand factor immunostaining on tissue cryo-sections and image analysis to measure the area of positive staining at the injury site. As shown in Fig. 4d), seven days after the injury, the sites transfected with NAB2 contained fewer blood vessels compared to the control (gold treated wounds). Thus, Egr-1 promoted angiogenesis in vivo, which supported the in vitro finding in Example 3.

Conclusion

NAB2 blocked Egr-1-stimulated activation of angiogenesis in vivo, thereby supporting the role of NAB2 as a repressor of growth factor activation by an acute stimulus, such as activation of growth factor production by Egr-1.

Claims (5)

PATENT CLAIMS Use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB polypeptide 5 2 or a biologically active fragment thereof in the manufacture of a pharmaceutical composition for the treatment of cell proliferative disorders associated with wound healing in a mammal, particularly a human. The use of claim 1, wherein the NAB1 or NAB2 polypeptide is a human NAB1 or NAB2 polypeptide. Use according to any one of claims 1 or 2, wherein the cell proliferative disorders associated with wound healing are formation 15 hypertrophic and keloid scars. Use according to any one of claims 1 to 3, wherein the nucleic acid molecule is operably linked to an expression control nucleic acid sequence. Use according to any one of claims 1 to 4, wherein the nucleic acid molecule has an identity of at least 70% or 80% or 90% or 95% over its entire length to the NAB1 or NAB2 polynucleotide sequence. Use according to any one of claims 1 to 5, which comprises a combination of nucleic acid molecules comprising sequences. 44 encoding both NAB1 polypeptide and NAB2 polypeptide, or biologically active fragments thereof. Use according to any one of claims 1 to 5, wherein the molecule The nucleic acid 5 comprises a sequence encoding a NAB2 polypeptide or a biologically active fragment thereof. Use according to any one of claims 1 to 7, wherein the nucleic acid molecule is arranged for administration to a mammal by physical methods. The use of claim 8, wherein the nucleic acid molecule is arranged for administration to a mammal by particle bombardment. 15 Dec The use of claim 9, wherein the nucleic acid molecule is immobilized on the gold particles. The use of claim 8, wherein the nucleic acid molecule is arranged for administration by microarray. Use according to any one of claims 1 to 7, wherein the nucleic acid molecule is a vector. The use of any one of claims 1 to 7, wherein the nucleic acid molecule is in a cell. · · * 9 9 9 9 • • • 9 9 9 9 9 9 9 9 9 9 · ··· · 99 9999 99 999 - 45 A nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, for use in gene therapy. 15. A pharmaceutical composition comprising a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, together with one or more pharmaceutically acceptable carriers. A method of treating cell proliferative disorders associated with wound healing in a mammal including a human, the method comprising administering a nucleic acid molecule comprising a sequence of NAB1 or NAB2 polypeptides, or a biologically active thereof 15 fragment, mammals. Use of a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, in the manufacture of a pharmaceutical composition for the treatment of healing-related cell proliferative disorders 20 injuries in a mammal, including a human. The use of claim 17, wherein the NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, is obtained by natural, synthetic or recombinant means. The use of claim 17 or 18, wherein the NAB1 or NAB2 polypeptide is a human NAB1 or NAB2 polypeptide. Use according to any one of claims 17 to 19, wherein the polypeptide is at least 70% or 80% or 90% or 95% identical over its entire length to the NAB1 or NAB2 polynucleotide sequence. 21. A method of treating a cell proliferative disorder associated with wound healing in a mammal, including a human, comprising administering to the mammal a therapeutically effective amount of a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof. 22. A pharmaceutical composition comprising a NAB1 and / or NAB2 polypeptide, or a biologically active fragment thereof, together with one or more pharmaceutically acceptable carriers.