SK382001A3 - Pharmaceutical uses of nab1 and nab2 - Google Patents

Abstract

The invention relates to the use, particularly in gene therapy, of an NAB1 or NAB2 polypeptide or a biologically active fragment thereof, and to nucleic acid molecules encoding such polypeptides, in the manufacture of a medicament for the treatment of cell proliferation disorders associated with wound healing in a mammal, including human.

Description

Use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide

Technical field

The present invention relates to the use of gene therapy techniques in the treatment of wounds. In particular, it relates to a new 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, to inhibit restenosis following percutaneous transluminal coroplast, calcification of the vascular wall and for inhibiting cell proliferation in cancer.

BACKGROUND OF THE INVENTION

Skin healing involves a wide range of cellular, molecular, physiological and biochemical events. During the healing process, cells migrate to the wound sites, where they proliferate and synthesize extracellular matrix components to reconstitute tissue that is very similar to the uninjured original tissue, this process being driven by mediators secreted from cells at the edges of the wound, such as platelet-derived growth factor ( plateled 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 models of diabetes (Brown). et al., J. Surg. Res. 56; 562-570, 1994).

Over the past five years, many growth factors have been shown to accelerate cell proliferation in vitro and aid wound healing in animal models. Most attention was paid to TGF beta in relation to wound repair, differentiation and matrix production. TGF beta, administered either locally or systemically, accelerates the rate of repair in animal models. · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · · ·

-2dermal injuries (Ashcroft et al., Nature Median, 3; 1209-1215, 1997; Sporn and Roberts, J. Cell Biol., 119; 1017-1021, 1997; Beck et al., J. Clin. Invest. 92; 2841 to 2849 (1993). Similarly, PDGF has been reported to promote reepithelialization and revascularization of ischemic tissue in animals with diabetes (Uhl et al., Langenbecks Archive Fur Surgery-Supplement-Kongressband 114; 705-708, 1997, summarized in Dirks and Bloemers, Mol. Biol. Reports 22). ; 1 to 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 (reviewed in Liu et al., Crit. Rev. Oncogenesis 7; 101-125, 1996; Khachigian and Collins, Cirs. 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 to activate transcription of many genes including EGF, platelet-derived growth factor A (PDGFA), basal 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-like 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, 1137-1157; 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 exerts enhanced VEGF expression control (Finkenzeller, Oncogene 15; 669-676, 1997). Transcription of mRNA 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 neointimal formation, accelerate reendothelialization and increase vasomotor activity (Asahara et al., Circulation; 94, 3291-3302).

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Expression is reported to occur in wound healing and 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; N2100-2107, 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 by Dermer et al., Trends Cardiovasc. Med. 4: 45-49, 1994). Regulators of osteoblast and / or osteoclast formation may modulate the degree of vascular wall calcification. NAB, NAB1 and NAB2 family proteins (NGFIA binding corepressors) interact with the conserved R1 domain of the Egr-1 and Egr-2 activators (Svaren et al., EMABO J., 17; 6010-5019, 1998). NAB2 has previously been shown to repress NGF-induced PC12 differentiation (Qu, Z. et al., J. Cell Biol. 142; 1075-1082, 1998) and Egr-1-induced basal FGF activation (Svaren et al., EMBO J. 17, 6010-5019, 1998).

One of the problems associated with the treatment of wounds is the formation of 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 that promotes the development of tissue fibrosis is at least partially controlled in principle by a variety of growth factors but not limited to TGF beta (Muir Eur. J. Plast. Surg. 21; 1-7, 1998), a PDGF isoform (Katou et al., J. Pathol. 186: 201-208, 1998, Heldin et al., in The Molecular and Cellular Biology of Wound Repair, Clark, ed .; 249-264, 1996 Plenum Press); ···········································

-4VEGF (Jones et al., Frontiers in Bioscience 4; D303-309, 1999). Therapy that would reduce, but not eliminate, the expression of growth factors at the wound site would have a significant effect on healing tissue with reduced scars and improving quality of life.

PCT International Patent Application 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 in vitro activation of growth factors mediated by Egr-1, b) represses basal levels of growth factor gene expression in vitro, and c) at rodent wound sites and its subsequent expression decreases TGF-βΙ but not TGF-β3 expression and has utility in reducing scar formation in healing (e.g., Shah et al., J. Cell Science 107; 1137-1157, 1994).

Therefore, debilitating regulation slows the healing process and reduces the occurrence of hypetrophic 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 restenosis following percutaneous coronary angioplasty, modulation of vascular wall calcification, and inhibition of cell proliferation in cancer.

SUMMARY OF THE INVENTION

The present invention provides the use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, for the manufacture of a medicament for the treatment of cell proliferative disorders associated with the treatment of injury in a mammal, including a human.

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

In 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 the treatment of wounds.

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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 are associated with wound healing selected from the formation of 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 hypetrophic 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 polynucleotides and nucleic acid sequences encoding NAB1 or NAB2 polypeptides of any origin or species. The human DNA sequence is listed in Genbank accession number U47007, which sequence encodes a core protein of 486 amino acids in length. Rat NAB1 is a core protein of 570 amino acids in length and is described in PNAS USA 92, 1995, p. 6873-6877. The rat DNA sequence is listed in the Genbank under accession number U17253.

The sequence of the murine and human NAB2 protein is described in Molecular and Cellular Biology, 1996, 16, 3545-3553. The human DNA sequence is listed in the Genbank under accession number U48361.

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

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

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Generally, a genetic element 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 a polypeptide in a host cell, or a nucleotide comprising both a polypeptide coding region and a region operably linked to an expression control region. The genetic elements may be contained within a vector that replicates as an episomal element, i. as a molecule physically independent of the genome of the host cell. Genetic elements may be contained within plasmids. The 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 being transformed or transfected 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 and polynucleotide sequences as determined by aligning the sequences of these sequences. Identity can be easily calculated (Computational Molecular Biology, Gloss, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM, and Griffin HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Acadamic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J. ed., M. Stockton Press, New York, 1991). There are a variety of methods for measuring identity between two polynucleotides or two polypeptides, and this term is well known to those skilled in the art (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. Devereux, J. ed., M. Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). sequences include, but are not limited to, the method;

And a and a and a and 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. Identity determination methods 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 FASTA (Atschul, SF and et al., J. Molec Biol 215: 403 (1990)).

Isolated means changed by the "hand of man" from the natural state, i. if the substance occurs in nature, it has been altered or removed from the 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, 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 may be performed in a host cell. Isolated polynucleotides, 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 still 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, inter alia, to single-stranded and double-stranded DNA, DNA which is a mixture of single and double stranded regions or single and triple stranded regions, single and double stranded RNA and RNA which is a mixture of single and double stranded regions. , hybrid molecules • ························································

Containing DNA and RNA, which may be single-stranded or more typically double-stranded or triple-stranded, or mixtures of single- and double-stranded regions. In addition, a polynucleotide, as used herein, means triple-stranded regions containing 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 contain 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, for the purposes of the terminology used herein, "polynucleotides". 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 a modified base such as tritylated bases. It will be appreciated 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, single and complex cells. The term polynucleotides also includes short polynucleotides, often referred to as oligonucleotides.

Polypetides 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, which in the art are often referred to as peptides, oligopeptides and oligomers, as well as long chains, which are commonly referred to in the art as proteins and of which many types exist. It will be appreciated that polypeptides often contain amino acids other than twenty amino acids, which are commonly referred to as occurring in nature, and that many amino acids, including • · · · · · · · · · · · · · · · · · · · · · · · ·

The 9-terminal amino acids can be modified in a given polypeptide either by natural pathways, such as Processing and other post-translational modifications, but also by chemical modification techniques well known in the art. Common modifications that occur naturally to polypeptides are too numerous to list exhaustively, but are described in basic texts and more detailed monographs, as well as in the voluminous research literature, and are well known to those skilled in the art.

Modifications that may be present in the polypeptides for use in the present invention include, for example, acetylation, acylation, ADP-ribozylation, amidation, covalent attachment of a flavin, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, phosphatidylinositol binding, crosslinking, cyclization, disulfide bond formation, demethylation, covalent crosslinking, cystine formation, pyroglutamate formation, formylation, gamacarboxylation, glycosylation, GPI anchorage formation, hydroxylation, iodination, methylation, phosphorylation, proteristolization, myristolelation, racemization, selenoylation, sulfation, addition of amino acids to protein-mediated transfer RNAs such as arginylation and ubiquitination. These modifications are well known to those skilled in the art and have been described in detail in the scientific literature. Several particularly common treatments such as glycosylation, lipid binding, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribozylation are described in the most basic texts such as "Proteins - structure and molecular Properties", 2nd edition, TE Creighton, WH Freeman et al., New York (1993). There are many detailed abstracts on this subject, such as Wold. F., Posttranslational Protein Modifications: Perspectives and Prospects, p. 1 to 12, in Posttranslational covalent modification of proteins, B.C. Johnson, ed. Academic Press, New York (1983); Seifert 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 be generally engineered as a result of post-translational events, including in nature, and, in nature, β · 9 · 9 ··· · · ···· ····· · · ·

-10 occurring processing and events that are performed by artificial manipulation that does not occur in nature. It is also possible to include circular, branched and branched circular polypeptides synthesized by both non-translational natural and fully synthetic methods. Modifications can occur anywhere in the polypeptide, including the polypeptide backbone, amino acid side chains, and amino or carboxyl termini. Blocking of the amine or carboxyl group, or both, is common in the polypeptide and such modifications may also be present in the polypeptides of the present invention. For example, the amino-terminal residue of polypeptides formed in E. coli or other cells will be almost always N-formylmethionine by proteolytic processing. During post-translational modification of the peptide may result in cleavage of the methionine residue at the NH ₂ -terminal. Thus, the present invention encompasses both the use of the amino-terminal methionine variant protein as well as the methionine-free variant. Modifications that occur with a polypeptide will often depend on how it is made. For polypeptides produced by expression of a cloned gene in, for example, a host, the nature and extent of the modifications will largely depend on the post-translational modifying ability of the host cell and the modifying signals present in the amino acid sequence of the polypeptide. 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 insect cell-based expression systems have been developed for efficient expression of mammalian proteins with, inter alia, the natural glycosylation spectrum. Similar assumptions apply to other modifications. It will be appreciated that the same type of modification may be present to the same or different extent at different sites in a given polypeptide. A given polypeptide may also contain many types of modifications. As used herein, the term polypeptide generally encompasses all of these modifications, particularly those present in polypeptides synthesized in a recombinant manner by expression of a polynucleotide in a host cell.

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For the purposes of the present invention, polynucleotide or polypeptide variant (s) refers to polynucleotides or polypeptides that differ from the reference polynucleotide or polypeptide. Variants in this sense are described in more detail below and elsewhere. 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 chain and variants are generally closely similar and in many regions identical. As discussed below, changes in the nucleotide sequence of the variant may not be externally (they may be silent). That is, they do not need to change the amino acid sequence encoded by this 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 shown below, changes in the nucleotide sequence of the variant may alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. These nucleotide changes may result in subsitutions, additions, deletions, fusions, and truncation of the polypeptide encoded by the reference sequence, as discussed below. 2) A polypeptide having a different amino acid sequence from another reference polypeptide. Generally, the differences are limited so that the sequence of the reference chain and the variants are quite 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 nucleic acid molecules 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 a NAB1 or NAB2 polypeptide, or polynucleotide sequence variants that encode functional, i.e., degenerate, genetic code. biologically active fragments of NAB1 or NAB2 and functionally equivalent allelic variants and related sequences modified by single or multiple substitution, addition and / or deletion of bases that encode polypeptides with NAB1 or NAB2 activity.

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

Polynucleotides encoding the 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 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 patient's wound site. Polynucleotides can also be used to produce an NAB1 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, the 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. including 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.

By definition, the term "polynucleotide encoding a polypeptide includes within the scope of the present invention polynucleotides comprising 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.

The present invention further relates to variants of the polynucleotides described above that encode fragments, analogs and derivatives of the polypeptide. Variant poly ····

The -13 nucleotide may be a variant that occurs in nature, such as a naturally occurring allelic variant, or it may be a variant 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. Replacements, 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 consist of polynucleotides having at least 70% identity over its entire length to a polynucleotide encoding polypeptides having the amino acid sequence described above and polynucleotides that are complementary to these polynucleotides. Alternatively, most preferred are polynucleotides that comprise a region with at least 80% full length identity to a polynucleotide encoding a polypeptide of the present invention. 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. Moreover, chains with at least 95% identity are more preferably chains with at least 97% identity, and chains with at least 98% identity are preferably chains with at least 99% identity, with at least 99% identity being most preferred.

A preferred embodiment in this regard is polynucleotides that encode polypeptides that retain substantially the same biological function or activity as the mature NAB1 or NAB2 polypeptide 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 95% and

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Preferably at least 97%. Preferably, sequences which in this way hybridize to sequences of the invention encode a polypeptide with biological activity of NAB12 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 the 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 a gene therapy, wherein the polynucleotide is administered to the site of injury or other tissues in need of healing in a form capable of controlling production of NAB1 or NAB2 or a biologically active fragment thereof. in situ.

Preferably, in gene therapy, the polynucleotide is administered such that it is expressed in the patient to be treated, e.g. Thus, such a vector will include suitable 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, a promoter is preferred, and this term includes not only the sequence necessary for directing RNA polymerase to the transcription start site, but also the need for additional 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) promoter, the HSV thymidine kinase promoter (HSV), the SV40 early and late promoters, the promoters SV40, the promoters

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-15 retroviral LTRs such as the Rous sarcoma virus (RSV) promoters and metallothionein promoters such as the mouse metallothionein-1 promoter.

The polynucleotide sequence and the transcriptional control sequence 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 3 '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 treating a human, for example SV40 virus. Other known transcriptional control sequences may 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. Examples of such topical administration include administration of a "naked" nucleic acid vector in a suitable vehicle, for example in a solution of 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 technology "Gene gun" in the art by known methods, for example as described in U.S. Pat. No. 5371015, wherein inert particles such as vector-coated gold spheres are accelerated to sufficient velocities to allow penetration into the surface at the site of the wound, such as skin cells, by firing at high pressure from a suitable device.

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

The nucleic acid sequences 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.

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

The nucleic acid sequence encoding NAB1 or NAB2 can also be delivered to the site of injury by transformed host cells. These cells include cells taken from a subject into which a nucleic acid sequence is introduced by known gene transfer techniques, followed by growth of the transformed cells in culture and grafting into the patient.

Expression constructs such as those described above can be used in a variety of ways in the therapy of the present invention. They can be administered directly to the patient's wound site, or they can be used to produce the recombinant NAB1 or NAB2 polypeptide itself, which can then be administered to the wound site, as described in more detail below. The invention also relates to host cells which are engineered by genetic engineering methods using constructs comprising the polynucleotide or polynucleotides NAB1 or NAB2 of the present invention or genetic elements as defined above, and the use of these vectors and cells for 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 (ie for NAB1 or NAB2 synthesis) 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 using known published procedures. Many plasmids and other cloning and expression vectors that can be used according to the present invention are well known and readily available to those skilled in the art.

NAB1 or NAB2 polypeptide expression vectors for use in the present invention generally contain cis-acting control regions effective

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-17 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 or only in some cell types, or both inducible and cell specific. Particularly preferred among the inducible vectors are those vectors which can be induced for expression by environmentally easy to manipulate factors such as temperature and nutritional supplements. A variety of 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 vectors include, but are not limited to, chromosomal, episomal, and virus-derived vectors, e.g. , 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 phagmids, 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 can be introduced into the vector by any of the well known routine methods, such as those set forth in Sambrook et al., Molecular Cfoning and Laboratory Manual, 2nd Edition; 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, including, for example, a promoter.

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-18 for direct transcription of mRNA. Representatives of these promoters include, but are not limited to, the lambda phage PL lac, 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 include transcription initiation and termination 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 contain an AUG translation initiation site at the beginning and a termination codon conveniently located at the end of the translational 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 and Laboratory Manual, 2nd Edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 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 Aspargillus cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 cells and Bowes melanoma cells; and plant cells.

Examples are the following commercially available vectors. Preferred vectors for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, 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).

Preferred eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG from Stratagene; and pSVK3, pBPV, pMSG and pSVL from Pharmacia.

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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 of the embodiments of the present invention. It will be appreciated that any other plasmid or vector suitable, for example, for introducing, maintaining, propagating or expressing a polynucleotide or polypeptide 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 a 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, that 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 the restriction site or sites for insertion of a possible promoter fragment, i. fragment, which may contain a promoter. As is known, the introduction of a promoter-containing fragment into a vector at a restriction site upstream of the cat gene will allow the generation of CAT activity, which can be detected by standard CAT assays. 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.

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Known prokaryotic promoters suitable for expression of the polynucleotides and polypeptides of the present invention include the E. coli lacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR promoters, the PL promoter, 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 structural sequence located downstream, and a selectable marker to allow isolation of cells containing the vector for vector exposure.

Polynucleotides of the invention encoding a heterologous structural sequence of a polypeptide 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 'to the ribosome binding site. The ribosome binding site will be 5 'to the AUG codon that initiates translation of the expressed polypeptide.

Generally, there will be no other open reading frames that start with the 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 polypeptide, and a polyadenylation signal will also be present in constructs for use in eukaryotic hosts. The transcription termination signal, which must be located at the 3 'end of the transcriptional region, can also be included in the polynucleotide construct.

For secretion of the present protein into the lumen of the endoplasmic reticulum, into the periplasmic space, or into the extracellular environment, secretory 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, in order to improve stability and persistence in a 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. Also, a region may be added to the polypeptide of interest. · · · * ·· ·· ··· ·· ·

-21 allowing purification. 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 a heterologous immunoglobin region that is useful for solubilizing or purifying polypeptides. The cells are then typically collected by centrifugation, broken up by physical or chemical means, and the crude extract obtained is stored for further purification.

Microbial cells used for protein expression can be broken by any conventional method, including freeze-thaw cycles, sonication, mechanical shredding, or the use of 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 regions, donor and acceptor cleavage sites, termination transcription sequences, and non-transcriptional sequences that are required for expression at the 5 'end. .

Genetically engineered host cells may be used to prepare NAB1 or NAB2 polypeptides for use in the present invention. Introduction of polynucleotides into the host cell may be achieved by calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, lipid-mediated cation transfection, electroporation, transduction, scratching, infusion, or ballistic delivery.

These methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Bioiogy, (1986) and Sambrook et al., Molecular Clonning, and Laboratory Manual, 2nd Edition; 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. For the production of such proteins using RNAs derived from the DNA constructs of the present invention may also be

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-22used cell-free translation systems. Suitable cloning and expression vectors for use with prokaryotic and eukaryotic host cells are described in Sambrook et al., Molecular Clonning, and Laboratory Manual, 2nd Edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

The polypeptide can be isolated and purified from recombinant cell cultures by known methods including ammonium sulfate or ethanol precipitation, acid extraction, anionic or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, and hydroxylapatite chromatography lecithin chromatography. Most preferably, high performance liquid chromatography will be used for purification. Well-known refolding methods can be used to regenerate the active conformation of a polypeptide 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 art-known methods such as column chromatography as described in Sambrook et al., Molecular Clonning, and Laboratory Manual, 2nd Edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).

As mentioned 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 and translates to NAB1 or NAB2 at the site of injury, i.e. as gene therapy, or the protein may be directly administered to repress transcription.

Thus, according to a further embodiment 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 the treatment of 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 to the mammal a therapeutically effective amount of a NAB1 or NAB2 polypeptide or a biologically active fragment thereof. In another aspect, the invention provides: • • • • • • • • • •.

Use of a NAB1 or NAB2 polypeptide or biologically active fragment thereof for the treatment of wounds and wound healing.

As used herein, the term "NAB1 or NAB2 polypeptide" includes naturally and recombinantly produced NAB1 or NAB2, natural, synthetic and biologically active analogs or variants of polypeptides or derivatives thereof, or biologically active fragments and variants thereof, including derivatives and analogues of these fragments. .

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

NAB1 or NAB2 and the above-mentioned fragments and derivatives thereof for use in the treatment of the invention can be extracted from natural sources by known methods in the art. These methods include purifying sequence-specific DNA affinity chromatography using the 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 may 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 "analogue" mean a polypeptide that retains substantially the same biological function or activity as the polypeptide. Thus, the analog will include a proprotein that can be activated by cleaving the proprotein moiety to yield an active mature polypeptide.

The fragment, derivative, or analog of the polypeptide may be i) a polypeptide in which one or more amino acid residues are substituted by 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, or ii) polypeptide wherein the one or more amino acid residues comprise a group of substituents; or iii) a polypeptide in which the mature polypeptide is fused to another

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-24 a compound, such as a compound providing an increase in the half-life of a polypeptide (e.g., polyethylene glycol); or iv) a polypeptide in which additional amino acids, such as an initial or secretory sequence, or a sequence used to purify the mature polypeptide or for protein are fused to the mature polypeptide. sequence. 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. Substitutions are those changes 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, Val, Leu, and Ile are replaced one after the other; interchange of hydroxyl residues of Ser and Thr, exchange of residues of Asp and Glu, substitution between amide residues Asn and Gin, 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 of these fragments having the amino acid sequence of a polypeptide in which a substitution, deletion or addition has been performed in several, in a few, 5 to 10, 1 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. fragments that retain the ability to heal wounds of the parent polypeptide.

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) with the above-described polypeptide sequences and also comprise portions of these polypeptides,

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Where such a portion of the polyeptide 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 polypeptide by a peptide synthesis method; therefore, 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 NAB1 or NAB2 polypeptide fragments 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 may be "free-standing", i. they do not appear as part of, or are not fused to, other amino acids or polypeptides, or may be part of another polypeptide of which they form part or region. When these fragments are contained within a longer polypeptide, the fragments described in the present invention most preferably form a single continuous region. However, several fragments may be contained within a single longer polypeptide. 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 at a self-terminus 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.

Fragments characterized by the structural or functional properties of the polypeptide of the present invention are also preferred in this embodiment. Preferred embodiments of the invention in this regard include fragments that comprise a coil and α-helix forming regions, a β-sheet and β-sheet forming regions, a tum, and a tuft forming region, a coiling and a coiling forming region, hydrophilic regions, hydrophobic regions , α-amphipathic regions, β-amphipathic regions, elastic ·· ···· · · · · ··· ·· ·

26 regions, surface forming regions, substrate binding regions on regions of high antigenic index of a polypeptide of the present invention, and combinations of these fragments.

Preferred regions are those that mediate the activity of the polypeptide of the present invention. In this regard, fragments having the chemical, biological or other activity of a 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 under stringent conditions, and polynucleotides such as PCR primers to amplify polynucleotides encoding these fragments. Preferred polynucleotides in this sense are those which correspond 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 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 that allows targeting to wound sites, including, for example,

-27 ···································· • Local, intravenous, intramuscular, intranasal or intradermal administration.

In 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 drops, ear drops, mouthwashes, impregnated bandages and threads and aerosols and may contain suitable conventional additives, including, for example, preservatives, penetration aids medicaments 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.

These carriers may comprise from about 1 wt. % to about 98 wt. % of the composition will generally comprise up to about 80 wt. means.

For administration to mammals, and in particular to humans, it is expected that the daily dose of active ingredient will be from 0.01 mg / kg to 10 mg / kg, typically about 1 mg / kg. In any event, the actual dose which will be most favorable to the individual and will vary according to the age, weight and response of the particular patient will be determined by the attending physician.

The above dosages are examples of average dosages. However, there may be individual instances in which higher or lower dosage ranges are justified, and are also within the scope of the present invention. The dose selected 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 repressor proteins in the treatment of wounds 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.

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Where the NAB1 or NAB2 polynucleotide is used in the treatment of the present invention, it may be used as part of an expression construct, 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 the treatment of wounds including ulcers in the extremities of diabetes and peripheral arterial occlusive diseases, for the treatment of postoperative scar formation, burns, psoriasis, for inhibiting restenosis after percutaneous transluminal coronary angioplasty, modulating vascular calcification and .

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 administering nucleic acid products is to use 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 wound site. 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 NAB2 immobilized on gold particles.

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

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1a to 1d show trans-repression of 1a) PDGF-AB production, 1b) TGF3, 1c) HGF, and 1d) VEGF in HVSMC mediated by NAB2 by Egr-1.

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-29Obr. 2 depicts the transpression of Ngr2-mediated Egr-1 mediated HGF production in HVSMC.

Fig. 3 shows the effect of NAB2 on Egr-1-induced angiogenesis.

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

Fig. 4b shows the effect of NAB2 transfection on growth factor levels in epidermis cut lesions in rat after 7 days.

Fig. 4c shows the effect of NAB2 transfection on growth factor levels in granulation tissue of cut lesions in rat after 7 days.

Fig. 4d depicts the effect of NAB2 transfection on angiogenesis in rat cuts after 7 days.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Use of NAB2 to repress Egr-1-mediated growth factor transactivation

techniques

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 NAB2 cDNA derived from the human cytomegalovirus promoter (hCMV; Svaren 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) into HVSMCs in the proportions shown in the figures using FUGENE (Boehringer Mannheim). A FUGENE.DNA ratio of 3: 1 (v / v) was used for all experiments and transfection was normalized using CMV driven β-galactosidase expression plasmid. Secreted growth factors were detected in tissue medium by ELISA (R&D Systems) using appropriate controls.

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-30Výsledky

Fig. Figures 1 to 1d show repression of Egr-1 mediated activation of PDGF-AB, TGFβ, HGF and VEGF, respectively. 6 µg of the Egr-1 expression plasmid was cotransfected either alone (Fig. 1a, Fig. 1b and Fig. 1d) or with 500, 100 or 25 ng of NAB2 (Fig. 1a and Fig. 1d). For PDGF-AB, TGFβ and VEGF, there was a small increase in the amount of secreted growth factor detected when transfecting Egr-1 alone. Co-transfection with NAB2 completely eliminated the PDG-AB response to Egr-1 activation and decreased basal expression, resulting in a five-fold reduction in total PDGF-AB production (Fig. 1a). Transfection of NAB2 completely stopped the TGFβ response to Egr-1 activation and caused a 30% reduction in total TGFβ production (see Fig. 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 of NAB2 as well as PDGF-AB and TGFβ blocked Egr-1 mediated VEGF activation and caused a 40% reduction in the total amount of VEGF generated (Fig. 1d).

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 to repress basal levels of growth factors

techniques

Cell culture, transfection and detection of growth factors were performed as described in the "Methods" section of the previous example. The NAB2 expression vector was transfected at final concentrations of 0, 250, 500, 1000 or 3000 ng.

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-31 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.

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 to repress induction of angiogenesis in vitro

techniques

NAB2 (wild type and dominant negative) expression constructs derived from the hCMV promoter have been described (Svaren 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 (Cambridge 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 of NAB2 - a dominant negative expression plasmid. After 11 days of co-culture, the degree of angiogenesis was determined by staining the cells to detect the presence of the endothelial cell marker PECAM-1 and for visualization using the BCIP / NBT substrate.

Representative images of tubule formation using all four doses of Egr-1 expression plasmid together with VEGF (positive control) and suramine

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-32 (negative control) were captured and processed by image analysis using the Quantimet 600 image analyzer and the included software.

The 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 decrease in the ability of Egr-1 to induce dose-dependent angiogenesis, which was removed by co-transfection with NAB2 with a dominant negative expression plasmid.

conclusion

NAB2 can be used to block angiogenesis against the background of acute injury, exemplified by the induction of growth factor by Egr-1.

Example 4

Use of NAB2 to reduce scar formation in a rodent cut injury model

techniques

1. Effect of NAB2 transfection using gene gun technology on rat growth levels of cut lesions

Transfection of NAB2 into rat cut lesions counteract scar formation by directly suppressing the expression of key scarring growth factors, 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.

2. Particle-mediated gene transfer

Eight male Sprague Dawley rats weighing approximately 250 g were anesthetized with isoflorane in a 2: 1 oxygen / nitrous oxide mixture. Two transfection sites were prepared (5 cm from the base of the skull, 1.5 cm on each side of the medial

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-33 lines) and two checkpoints (8 cm from the base of the skull, 1.5 cm on each side of the midline) on the back of the rat by first clamping and then shaving with a razor blade. One transfection was performed at each transfection site by accelerating plasmid / gold complexes with either NAB2 or Egr-1 (positive control for growth factor activation) to the skin at a pressure of 2.41 MPa (350 psi). Typically, 0.5 to 1.5 µg of DNA was delivered to the transfection site. At the control site, gold particles (without DNA) were accelerated to the skin at a pressure of 2.41 MPa (350 psi); 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.

3. Cutting wound healing model hours after transfection, animals were anesthetized and a 1 cm straight incision was made in parallel to the spine at full skin thickness 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 wound sites excised and used for routine histological and immunohistochemical tests.

4. Histological analysis

Each wound was cut horizontally into two parts over a period of time after excision. One half was placed in 4% paraformaldehyde for 24 hours and treated with wax histology. 5 mm sections from each wound were made using a microtome and sections were stained according to van Geison. Sections were observed by light microscopy and the effect of NAB2 on healing was determined.

5. Immunohistochemical examination

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 WF using the following protocol.

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J · · · · · · · · · · · · · · · · · · · · ·

-341. Wash sections with PBS.

2. Apply 30 ml of primary antibody for 1 h.

3. Wash 3x5 min in PBS.

4. Apply 30 ml secondary antibody (FITC directly bound) for 45 min.

5. Wash 3x5 min in PBS.

6. Fastening the sections with an aqueous fixture.

Immediately after immunostaining, each section was placed under a fluorescent microscope and the wound area was 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.

The results

Effect of NAB2 on Healing of Cut Wound in the Rat

A) Injury contractions and histological analysis of injuries after seven days

According to FIG. 4a, the wounds treated with NAB2 contracted at a similar width to those of the transfected Egr-1 and control wounds, and histology showed similar granular tissue maturity.

conclusion

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

B) Profiles of growth factors

Egr-1, TGFβ1, TGFp3, and PDGF-AB immunostaining was performed on frozen skin tissue cryopreservation 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 wound site. Sections were examined for growth factor expression both in the epidermis immediately after injury and at the site of injury (granulation tissue).

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-35a) 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. Compared to the unmanipulated control, NAB2 transfection reduced TGFβ1 expression in the epidermis, while both Egr-1 and gold delivery activated TGFβ1 production. Transfection of NAB2 increased TGFβ3 levels in the epidermis compared to both gold alone and non-manipulated controls. NAB2 transfection 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 TGFp1 levels in granulation tissue. However, NAB2 increased TGFp3 levels in the granulation tissue compared to both gold alone and non-manipulated controls.

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 the 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.

2. 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 quantitated using von Willebrand Factor immunostaining on tissue cryosections and image analysis to measure the area of positive staining at the wound site. As shown in FIG. 4d, seven days after injury, sites transfected with NAB2 contained fewer blood vessels compared to control (gold treated wounds). Thus, Egr1 promoted angiogenesis in vivo, which supported the in vitro finding in Example 3.

conclusion

NAB2 blocked Egr-1 stimulated activation of angiogenesis in vivo, thus 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.

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-36t

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Claims (20)

PATENT CLAIMS Use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a biologically active fragment thereof for the manufacture of a medicament 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. The use of any one of claims 1 or 2, wherein the cell proliferative disorders associated with wound healing are the formation of hypertrophic and keloid scars. The use of 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 polynucleotide sequence NAB1 or NAB2. The use of any one of claims 1 to 5, which comprises a combination of nucleic acid molecules comprising sequences encoding both NAB1 polypeptide and NAB2 polypeptide, or biologically active fragments thereof. The use according to any one of claims 1 to 5, wherein the nucleic acid molecule comprises a sequence encoding a NAB2 polypeptide or a biologically active fragment thereof. The use of any one of claims 1 to 7, wherein the nucleic acid molecule is arranged for administration to a mammal by physical methods. • ···· · · ···· · · · · • • · • • · • · • • • · · · · • · • • • · • • · · · · • · · · · · · · · · -389. The use of claim 8, wherein the nucleic acid molecule is arranged to be administered to a mammal by particle bombardment. 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 to be administered by microencapsulation. The use of 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. 14. 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. 15. A nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a biologically active fragment thereof for use in gene therapy. 16. A nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, for use in treating cell proliferative disorders associated with wound healing in a mammal, including a human. Use of a NAB1 or NAB2 polypeptide or a biologically active fragment thereof for the manufacture of a medicament for the treatment of cell proliferative disorders related to wound healing in a mammal, including a human. • ···· · · ···· · · • · • • • • · • · • · • • · · · • e • · e • • • · • · · • 3 · · · · · · · -3918. The use of claim 17, wherein the NAB1 or NAB2 polypeptide or 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 has an identity of at least 70% or 80% or 90% or 95% over its entire length to the polynucleotide sequence NAB1 or NAB2. 21. A pharmaceutical composition comprising a NAB1 and / or NAB2 polypeptide or a biologically active fragment thereof together with one or more pharmaceutically acceptable carriers. 22. A NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, for use in treating a cell proliferative disorder related to wound healing in a mammal, including a human.