HRP20010025A2 - Pharmaceutical uses of nab1 and nab2 - Google Patents

Description

This invention relates to the use of gene therapy techniques in wound healing. More specifically, it refers to the new use of polynucleotides encoding NAB1 or NAB2 protein transcription repressors in the reduction of cell proliferation disorders that particularly slow down skin healing, thus preventing hypertrophy and formation of keloid scars, psoriasis, inhibition of restenosis followed by percutaneous transluminal coronary angioplasty, modulation of vessel wall calcification and inhibition of cancer cell proliferation.

Skin healing involves a wide range of cellular, molecular, physiological and biochemical processes. During the healing process, cells migrate to the site of the wound, where they proliferate and synthesize components of the extracellular matrix, in order to reconstruct a tissue very similar to the one that was injured. This action is regulated by mediators secreted by border cells near the wound, such as platelet-derived growth factors (PDGF), epidermal growth factor (EGF), transforming growth factor beta (TGF beta), and other cytokines. Beneficial effects of these substances on cells have been demonstrated both in vitro and in vivo (published in Moulin, Eur. J. Cell Biol. 68; 1-7, 1995), including benefit from the administration of PDGF in diabetic rats (Brown et al. colleagues, J. Surg. Res. 56; 562-570, 1994).

In the last five years, numerous growth factors have been shown to accelerate proliferation in vitro and promote wound healing in animals. TGF beta has received the most attention in the context of wound healing as it promotes cell proliferation, differentiation and matrix production. TGF applied either locally or systemically accelerates the rate of wound healing in animal skin. (Ashcroft et al., Nature Medicine, 3; 1209-1215, 1997; Sborn and Roberts, J. Cell Biol. 119; 1017-1021, 1997; Beck et al., J. Clin. Invest. 92; 2841-2849, 1993 ). PDGF has also been reported to promote reepithelialization and revascularization of ischemic tissue and diabetic animals (Uhl et al., Langenbecks Archiv für Chirurgie-Supplement-Kongressband 114; 705-708, 1997 and Drinks and Bloemers, Mol. Biol. Reports 22; 1-24, 1996).

The transcription factor Egr-1 (early growth response gene) is a potential regulator of over 30 genes, and has a role in cell proliferation, development and differentiation (published in Liu et al., Crit. Rev. Oncogenesis 7; 101-125, 1996; Khachigian and Collins, Circ. Res. 81; 457-461, 1997). Egr-1 is induced by vascular endothelial injury (Khachigan et al., Science; 271, 1427-1431, 1996), and numerous genes are targets of transcriptional activation, including EGF, platelet-derived growth factor A (PDGF-A), basic fibroblast factor growth factor (bFGF), induction of PDGF A, platelet-derived growth factor B (PDGF B), TGF beta, bFGF, uroplasminogen activator (u-PA), tissue factor and insulin-like growth factor 2 (IGF-2). Blockade of inducible TGF beta by the Egr-1 gene may be used to prevent scarring. Antibodies raised to TGF beta reduce incisional scarring in rodents (Shah et al., J. Cell Science; 107; 1137-1157; Shah et al.; Lancet; 339, 213-214, 1992).

The transcriptional complex that mediates the induction of vascular endothelial growth factor (VEGF) depends on AP2, and indirectly on Egr-1 (Gille et al., EMBO J 16; 750-759, 1997). PDGF B directly regulates the increase in VEGF expression (Finkenzeller, Oncogene 15; 669-676, 1997). Transcription of VEGF 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. It has been shown in animals that passivation of metal "stents" due to VEGF inhibits the formation of neo-intima, accelerates reendothelialization and increases vasomotor activity (Asahara et al., Circulation; 94; 3291-3302).

VEGF expression has been observed in wound healing and psoriatic skin, both conditions in which TGF alpha and its ligand EGFr are up-regulated. 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). It is quite evident that Egr-1 can activate the expression of intracellular adhesion molecule-1 (ICAM-1) in the phorbol ester of stynulated B lymphocytes (Maltzman et al., Mol. Cell. Biol; 16; 2283-2294, 1996), and can activate the expression TFN alpha by the Egr-1 binding site on the TNF alpha protomer (Kramer et al., Biochim. Biophys. Acta 1219; 413-421, 1994). Finally, Egr-1 knockdown mice are infertile, and the lack of luteinizing hormone (LH) (Lee et al. 273; 1219-1221, 1996) implies that the LH promoter may also be a target for Egr-1 activation. Vascular calcification is an actively regulated process similar to bone formation involving cells and factors known to be important in the regulation of bone metabolism (published in Dermer et al., Trends Cardiovasc. Med. 4; 45-49, 1994). Regulators of osteoblastogenesis and/or osteoclastogenesis can modulate the degree of calcification of vessel walls. The NAB proteins NAB1 and NAB2 (NGFI-A binding corepressors) interact with the conserved R1 domain of Egr-1 and Egr-2 transactivators (Svarwen et al., EMBO J. 17; 6010-6019, 1998). NAB2 has previously been observed to repress NGF-induced PC12 cell differentiation (Qu, Z. et al., J. Cell Biol. 142, 1075-1082, 1998) and Egr-1-driven activation of basic FGF (Svaren et al., EMBO J 17; 6010-6019, 1998).

The problem encountered in the treatment of wounds is the formation of hypertrophy and the formation of keloid scars. This is particularly undesirable, especially after cosmetic surgery. Tissue fibrosis (which occurs in the kidneys, liver or skin) is manifested by the accumulation of extracellular matrix. Deregulated extracellular matrix production, which is subject to the development of tissue fibrosis, is at least partially regulated by a number of growth factors, primarily, but not exclusively, by TGFβ (Muir, Eur. J. Plast. Surg. 21; 1-7, 1998), PDGF isoforms (Katou et al. J. Pathol. 186; 201-208, 1998, Heldin et al. in The molecular and cellular biology of wound repair, Clark, ed.; 249-364, 1996 Plenum Press) and VEGF (Jones et al., Frontiers in Bioscience 4; D303-309, 1999). A therapy that would reduce, but not eliminate, growth factor expression at wound sites would have a significant impact on the healing of scar-reduced tissue, and would improve quality of life.

International patent application PCT.GB99.01722 describes the use of Egr-1 transcription factors to promote wound healing. These inventors have found that administration of polynucleotides encoding the transcriptional repressor NAB1 or NAB2: (a) repress Egr-1-driven growth factor activation in vitro; (b) repress basal levels of growth factor gene expression in vitro; and (c) at rodent wound sites, as well as their subsequent expression, reduces the expression of TGF-β but not TGF-β3 and is administered to reduce scarring during healing (Shah et al. J. Cell Science 107; 1137 -1157, 1994).

Therefore, downregulation of Egr-1 slows down the healing process and reduces the appearance of hypertrophy and the formation of keloid scars and psoriasis. Downregulation of the Egr-1 gene may also be useful in the treatment of other disorders associated with wound healing, such as restenosis followed by percutaneous transluminal coronary angioplasty, modulation of vessel wall calcification, and inhibition of cancer cell proliferation.

Therefore, according to an aspect of the invention, it is possible to use a nucleic acid molecule that includes a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, in the production of drugs for cell proliferation disorders associated with wound healing in mammals, including humans.

According to the next aspect of the invention, a procedure for the treatment of cell proliferation disorders associated with wound healing in mammals, including humans, is enabled, which includes the application to mammals of a nucleic acid molecule, which includes a sequence encoding the NAB1 or NAB2 polypeptide, or its biologically active fragment.

According to a further aspect, the invention provides a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a biologically active fragment thereof, which is used in the treatment of cell proliferation disorders associated with wound healing.

Another aspect of the invention relates to the use of a combination of a nucleic acid molecule comprising a sequence encoding the NAB1 polypeptide and another comprising a sequence encoding the NAB2 polypeptide.

The most convenient aspect of the present invention is that cell proliferation disorders associated with wound healing form a group that includes hypertrophic and keloid scars, psoriasis, restenosis followed by percutaneous transluminal coronary angioplasty, vessel wall calcification, and cancer cell proliferation. A particularly suitable aspect of this invention is represented by cell proliferation disorders, that is, the formation of hypertrophic and keloid scars.

Therefore, the present invention relates to the therapeutic use of polynucleotides encoding NAB1 or NAB2 in wound healing procedures. The invention also relates to the therapeutic use of NAB1 or NAB2 alone in wound healing procedures, as detailed below.

The invention relates to the use of NAB1 or NAB2 polypeptides and nucleic acid sequences encoding NAB1 or NAB2 polypeptides regardless of species origin. The human DNA sequence is listed in the gene bank under accession number U47007, and encodes a nuclear protein of 486 amino acids. Rat NAB1 is a nuclear protein with 570 amino acids and is described in PNAS USA 92, 1995 p. 6873-6877. The rat DNA sequence is listed in the gene bank under accession number U17253.

Mouse and human NAB2 protein sequences are described in Molecular and Cellular Biology, 1996 16, 3545-3553. The human DNA sequence is listed in the gene bank under accession number U48361.

The literature data related to NAB1 or NAB polypeptides and polynucleotides, described below, can generally be applied to sequences regardless of origin, and in particular to the human sequences described above. As described below, the terms NAB1 or NAB2 also include variants, fragments, and analogs of NAB1 or NAB2.

The following illustrative explanations are provided to facilitate understanding of certain terms used herein. The explanations are given by way of preference and do not limit the invention.

Biologically active fragments of NAB1 or NAB2, as referred to herein, are those fragments that have a repressive effect on Egr-1 transcription.

GENETIC ELEMENT generally means a polynucleotide that includes a region that codes for a polypeptide, or a polynucleotide region that governs replication, transcription or translation, or other processes that are important for the expression of a polypeptide in a host cell, or a polynucleotide that includes both a region that codes for a polypeptide and a region adjacent to it operably bound, and regulates expression. Genetic elements may be included within a vector that replicates as an episomal element; which actually represents a molecule physically independent of the genome of the host cell. They can be included within a plasmid. Genetic elements may also be included within the host cell genome; not in its natural form but more likely, inter alia, in the form of purified DNA or in a vector after manipulation, such as isolation, cloning and introduction into a host cell.

A HOST CELL is a cell that has been transformed or transfected, or that can be transformed or transfected using an exogenous polynucleotide sequence.

As is known in the art, identity represents the relationship between two or more polypeptide sequences, or two or more polynucleotide sequences, and is determined by comparing the sequences. Technically, identity also means the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, and is determined by matching the strands of those sequences. Identity can be easily calculated (Computational Molecular Biology, Lesk, A.M. editor, Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D.W., editor, Academic Press, New York, 1993; Computer Analysis of Sequence Data Part I, Griffin, A.M. and Griffin, H.G. editors, Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology ”, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer “Introduction to Sequence Analysis”, Gribskov, M. and Devereux, J. editors, M Stockton Press, New York, 1991). Given that there are a number of procedures for measuring the identity between two polynucleotide or two polypeptide sequences, the term is well known to those skilled in the art (Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer "Introduction to Sequential Analysis", Gribskov, M. and Devereux, J. editors, M Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 ( 1988).Procedures commonly used to determine identity between sequences include, but are not limited to, those described in and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). for determining identity are designed to provide the broadest match between the sequences examined. Suitable computer program procedures for determining identity between sequences include the GCG program package (Devereux, J. et al., Nucleic Acids Research 12(1): 3 87 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S.F. and associates, J. Molec. Biol. 215: 403 (1990)), but are not limited to it.

ISOLATED means changed “by the hand of man” from its natural state; eg if it occurs in nature, then it has been changed or removed from its original environment, or both. For example, a naturally occurring polynucleotide or a polypeptide that is naturally present in a living organism in its natural state is not “isolated,” as used herein. As part of, or after isolation, such polynucleotides can be joined to other polynucleotides, such as DNA molecules, for mutagenesis, to create fusion proteins, and for example for propagation or expression in the host. An isolated polynucleotide, single or joined to other polynucleotide sequences in the form of a vector, can be introduced into a host cell, into a culture, or into the whole organism. Introduced into host cells, in culture, or in the whole organism, such DNA molecules would still be isolated, in the sense used here, because they would not be in their natural form in which they appear, or in the natural environment, because they are not mixtures that appear in nature, and therefore remain isolated polynucleotides or polypeptides within the meaning of the term used herein.

POLYNUCLEOTIDE(S) generally refers to any polynucleotide or polydeoxyribonucleotide, which can be unmodified RNA or DNA, or modified RNA or DNA or cDNA. Therefore, for example, polynucleotides in the sense used here refer, among others, to single-stranded and double-stranded DNA, DNA representing a mixture of single-stranded and double-stranded regions, i.e. single-stranded, double-stranded and triple-stranded regions, single-stranded and double-stranded RNA, and RNA representing a mixture of single-stranded and double-stranded regions, hybrid molecules that include DNA and RNA that can be single-stranded or more often double-stranded or triple-stranded, i.e. a mixture of single-stranded and double-stranded regions. Additionally, polynucleotide as used herein refers to triple regions that include RNA or DNA or both, i.e., RNA and DNA. The chains in such areas can be of the same or different molecules. Regions can include one entire molecule or more, but more often they only include a region of individual molecules. Often, one of the molecules with a triple helical region is an oligonucleotide. As used herein, the term polynucleotide includes DNA or RNA molecules, as described above, that contain one or more modified bases. Therefore, DNA and RNA molecules with a modified base, to achieve stability or for other reasons, are “polynucleotides” as used herein. Furthermore, DNA or RNA molecules containing unusual bases, such as inosine, or modified bases, such as tritium bases, the above being only two examples, are polynucleotides as used herein. It is estimated that there is a wide variety of DNA and RNA modifications that are used for many useful 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 among others simple and complex cells. Polynucleotides include short polynucleotides, which often refer to oligonucleotide(s).

POLYPEPTIDES, as used herein, includes all polypeptides described below. The basic structure of polypeptides is well known and has been described in countless books and other professional publications. In this context, the term used herein refers to any peptide or protein that includes two or more amino acids linked together by a peptide bond in a straight chain. The term used here refers to short chains, whereby in the art it is usually understood, for example, peptides, oligopeptides and oligomers, as well as to long chains, whereby in the art it is generally understood proteins of various types. Conveniently, polypeptides contain amino acids that differ from the 20 amino acids that normally occur in nature, and then that many amino acids, including terminal amino acids, may be modified in a given polypeptide either by natural processes, such as processing and other post-translational modifications, but also by chemical modification techniques that are well known in the art. Even simple modifications that occur naturally, in polypeptides, are so numerous that listing them here would take a long time, but they are well described in basic texts and in significantly more exhaustive monographs, as well as extensive research literature, and are well known to experts.

An illustrative number of known modifications that may be encountered in the polypeptides used in this invention are listed, namely acetylation, acylation, ADP-ribosylation, amidation, covalent binding of flavin, covalent binding of heme structure, covalent binding of nucleotides or nucleotide derivatives, covalent binding of lipids or lipid derivatives, covalent binding of phosphotidylinositol, cross-linking, formation of cystine, formation of pyroglutamate, formylation, gammacarboxylation, glycosylation, formation of GPI anchor structure, hydroxylation, iodination, methylation, myristoylation, oxidation, protolytic processing, phosphorylation, prenylation, racemization, selenoylation, introduction of sulfate groups, transfer-RNA-mediated addition of amino acids to proteins, such as arginylation, and ubiquitination. Such modifications are well known to experts and are very exhaustively described in the scientific literature. For example, several particularly common modifications such as glycosylation, lipid attachment, introduction of a sulfate group, carboxylation of the gamma terminus of glutamic acid, hydroxylation and ADP-ribosylation are described in most basic texts, such as for example PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES "PROTEINS - STRUCTURE AND MOLECULAR PROPERTIES”, 2nd edition, T. E. Creighton, W. H- Freeman and Company, New York (1993). Many comprehensive reviews are available on this subject, such as Wold, F., Posttranslational Protein Modification: Perspectives and Prospects, p. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, B. C. Johnson, editor, Academis 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). Polypeptides are preferably not always completely planar, as mentioned above. For example, polypeptides can be the general result of post-translational events, including natural processes as well as events caused by human manipulation, which do not normally occur in nature. Circular, branched and branched-circular polypeptides can be synthesized by a non-translational natural process, and also entirely by synthetic processes. Modifications can occur anywhere within a polypeptide, including in the polypeptide backbone, amino acid side chains, and terminal amino or carboxyl groups. In fact, blocking of the amino or carboxyl group of a polypeptide, or both groups by covalent modification is common for natural and synthetic polypeptides, and such modifications may also be present in the polypeptides of this invention. For example, the amino terminus of a polypeptide synthesized in E. coli or in other cells, prior to proteolytic processing, will almost always be N-formylmethionine. During the post-translational modification of the polypeptide, deletion of the methionine end at the NH2-terminal can occur. Accordingly, the invention contemplates the use of methionine-containing, as well as methionine-free, amino-terminal variants of the protein of this invention. The modification that occurs to a protein will often depend on how it is performed. For example, with polypeptides made by expression of a cloned gene in the host, the nature and extent of the modification will be largely determined by the post-translational modification capacity of the host cell, and the modification signals present in the polypeptide amino acid sequence. For example, it is well known that glycosylation often does not occur in a bacterial host, such as, for example, E. coli. Accordingly, when glycosylation is to be achieved, the polypeptide must be expressed in a host cell, generally a eukaryotic cell. Insect cells often perform the same post-translational glycosylation as mammalian cells, and therefore the insect cell expression system was developed to efficiently express mammalian proteins that, among other things, have natural ways of glycosylation. Similar considerations were applied to other modifications. Preferably, the same type of modification is present to the same or variable degree at several sites in a given polypeptide. Also, a given polypeptide can contain many types of modifications. In general, the term polypeptide as used herein encompasses all such modifications, particularly those present in recombinantly synthesized polypeptides by expression of the polynucleotide in a host cell.

VARIANT(S) of a polynucleotide or polypeptide, as used herein, refers to polynucleotides or peptides that differ from a standard polynucleotide or polypeptide. Variants in this sense are described in detail in the rest of this text. (1) A polynucleotide that differs in nucleotide sequence from another standard polynucleotide. In general, the differences are limited so that the nucleotide sequences of the standards and variants are very similar throughout, and in many areas identical. As noted below, changes in the nucleotide sequences of the variant may be inactivating. This means that they do not have to change the amino acids encoded by the polynucleotide. Where the differences are limited to inactivating changes of this type, the variant will encode a polypeptide with the same amino acid sequence as the standard. Also as noted below, changes in the nucleotide sequence of a variant can alter the amino acid sequence of the polypeptide encoded by the standard polynucleotide. Such nucleotide changes can result in amino acid substitution, addition, deletion, fusion, and truncation of the polypeptide encoded by the standard sequence, as noted below. (2) A polypeptide that differs in amino acid sequence from another, standard polypeptide. In general, the differences are limited so that the sequences of standards and variants are very similar throughout, and in many areas identical. The variant and standard polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions and truncations, and may be present in any combination.

The invention relates to the therapeutic use of a nucleic acid molecule that includes the sequence encoding the NAB1 or NAB2 polypeptide, or their combination. The invention also relates to the therapeutic use of fragments of the aforementioned polynucleotide sequence that encodes biologically active fragments of the NAB1 or NAB2 polypeptide, i.e. variants of the polynucleotide sequence that, with the help of degeneration of the genetic code, encode functional or biologically active fragments of NAB1 or NAB2, and refers to functionally equivalent allelic variants and related sequences modified by single or multiple base substitution, addition and/or deletion encoding polypeptides having NAB1 or NAB2 activity.

This can be accomplished by standard cloning procedures known to those skilled in the art.

Polynucleotides encoding NAB1 or NAB2 protein transcription repressors can be in the form of DNA, cDNA or RNA, such as mRNA obtained by cloning or produced by chemical synthetic techniques. DNA can be single-stranded or double-stranded. Single-stranded DNA can be coding or with a sense strand, that is, it can be non-coding or with an anti-sense strand. For therapeutic use, the polynucleotide is in such a form that it can be expressed to functional NAB1 or NAB2 protein repressors at the wound site of the subject being treated. The polynucleotides can also be used for in vitro production of NAB1 or NAB2 polypeptides for use in a further therapeutic aspect of the invention, as described in detail below.

Polynucleotides of the present invention that encode NAB1 or NAB2 polypeptides may include the coding sequence of NAB1 or NAB2 polypeptides, or a biologically active fragment thereof, but are not limited thereto. Thus, a polynucleotide may be obtained together with (but not limited to) additional, non-coding sequence, including, for example, non-coding 5' and 3' sequences, such as a transcribed, untranslated sequence that plays a role in transcription (including, for example, termination signals), ribosomal binding, mRNA stabilizing elements, and an additional coding sequence that encodes additional amino acids, such as those that enable additional functionality. Polynucleotides of the invention also include, but are not limited to, polynucleotides that include the structural gene for NAB1 or NAB2, and their naturally associated genetic elements.

In accordance with the foregoing, the term “polynucleotide encoding a polypeptide” as used herein includes a sequence encoding a NAB1 or NAB2 polypeptide. The term encompasses polynucleotides that include a single continuous region of a polypeptide (eg interrupted by an associated phage or inserted sequence or editing) together with additional regions, which may also contain coding and/or non-coding sequences.

The present invention further relates to variants of the above-described polynucleotides that encode fragments, analogs, and derivatives of polypeptides. A polynucleotide variant can be a naturally occurring variant, such as a naturally occurring allelic variant, or it can be a variant that is not known to occur in nature. Such polynucleotide variants that do not occur in nature can be prepared by mutagenic techniques, including those applied to polynucleotides, cells or organisms.

Variants from this domain include variants that differ from the aforementioned polynucleotides by nucleotide substitutions, deletions or additions. Substitutions, deletions and additions may involve one or more nucleotides. Variants may differ in coded or non-coded regions or both. Differences in coding regions can produce a conservative or non-conservative substitution, deletion or addition of an amino acid.

Further suitable compounds of this invention are polynucleotides that are at least 70% identical over their entire length to the polynucleotide encoding the polypeptides, and which have the previously described amino acid sequence, and polynucleotides that are complementary to such polynucleotides. Alternatively, polynucleotides that include a region that is at least 80% identical over its entire length to a polynucleotide encoding a polypeptide of the present invention are most suitable. From this point of view, polynucleotides that are at least 90% identical over their entire length to said polynucleotide are particularly suitable, and among such, those polynucleotides that are at least 95% identical are particularly suitable. Furthermore, those which are at least 97% identical are highly preferred among polynucleotides which are at least 95% identical, and among such polynucleotides which are at least 98% identical and at least 99% identical are even more suitable, with the most suitable polynucleotides which are at least 99% identical.

Suitable compounds from this point of view are polynucleotides encoding polypeptides that retain the essential biological function or activity as the mature NAB1 or NAB2 polypeptides encoded by the previously described DNA sequences (GenBank Accession Numbers U47007 and U48361).

The present invention further relates to polynucleotides that hybridize to the sequences described above. From this point of view, this invention particularly relates to polynucleotides that hybridize under very well-established conditions to the polynucleotides described above. As used herein, the term "well-established conditions" means hybridization that occurs if there is at least 95%, but more preferably 97%, identity between the sequences. It is convenient that the sequences that hybridize in this way to the sequences of the invention encode a polypeptide that has a biological activity like NAB1 or NAB2 compounds.

Polynucleotides can encode a polypeptide that is a mature protein as well as an additional amino or carboxyl-terminal amino acid. Such additional sequences can, for example, play a role in prolonging or shortening the half-life of the protein, or they can, among other things, facilitate the manipulation of the protein for the purpose of determination or production. As is generally the case in vivo, additional amino acids may be processed away from the mature protein by cellular enzymes.

Polynucleotides of the present invention used in gene therapy can be administered alone or as part of a vector, such as an expression vector, examples of which are well known in the art.

NAB1 or NAB2 encoding polynucleotide can be used therapeutically in the method of the invention in gene therapy, in which the polynucleotide is applied to the site of a wound, or to other tissues that should heal, in a form that is able to control the production of NAB1 or NAB2, or their biologically active fragments in situ.

Conveniently, in gene therapy, the polynucleotide is administered so that it is expressed in the treatment subject, e.g. in the form of a recombinant DNA molecule that includes a polynucleotide encoding NAB1 or NAB2, operably linked to a nucleic acid sequence that directs expression as in an expression vector. Such a vector will therefore include appropriate transcriptional control signals, including promoter regions capable of expressing the encoded sequence, said promoters having to be operable in the treatment subject. Therefore, for human gene therapy, the term "promoter" includes not only the sequence necessary to direct the RNA polymerase to the transcription start site, but also includes, if appropriate, other operative or control sequences, including enhancers, and primarily means the human promoter sequence of the human gene, or a gene that is typically expressed in humans, such as a promoter from human CMV. Known eukaryotic promoters suitable from this point of view include the CMV proximal start promoter, the HSV thymidine kinase promoter, SV40 early and late promoters, promoters of retroviral LTRs, such as those from Rous sarcoma virus (“RSV”), and metallothionein promoters, such as mouse promoter of metallothionein-I.

The polynucleotide sequence and the transcription control sequence can be used cloned in a replicable plasmid vector, based on commercially available plasmids, such as pBR322, or can be made from available plasmids by routine use of well-known published procedures.

The vector may also include transcriptional control signals located 3' to the NAB1 or NAB2 coding sequence, as well as polyadenylation signals, which can be recognized in the subject to be treated, such as for example the corresponding virus sequences, such as the SV40 virus when treating people. Other transcriptional control sequences are well known in the art and may be used.

Expression vectors may also include selectable markers for antibiotic resistance that enable the vectors to be propagated.

Expression vectors capable of synthesizing NAB1 or NAB2 in situ can be directly introduced into the wound site by physical procedures. Such examples include topical application of a "naked" nucleic acid vector in an appropriate vehicle, eg, in a solution of a pharmaceutically acceptable excipient, such as phosphate buffer solution (PBS), or delivery of the vector by physical procedures, such as particle bombardment, also known as "gene gun" technology, according to procedures known in the art, eg as described in US patent no. 5371015, in which inert particles, such as vector-coated gold beads, are accelerated to velocities sufficient to allow them to penetrate the surface of a wound site, eg, skin cells, by means of a high-pressure charge from a projecting device.

Other physical procedures for applying DNA directly to the recipient include ultrasound, electrical stimulation, and electroporation.

The NAB1 or NAB2 encoding nucleic acid sequence used in the therapy of the present invention can also be administered using delivery vectors, such as adenovirus or retrovirus delivery vectors known in the art.

Other non-viral delivery vectors include lipid delivery vectors, including liposomal delivery vehicles known in the art.

The NAB1 or NAB2 encoding nucleic acid sequence can also be administered to the wound site using transformed host cells. Such cells include cells obtained from a subject and introduced into them with a nucleic acid sequence using methods known in the art, which include gene transfer, followed by growth of the transformed cells in culture, and grafting to the subject.

Expression constructs, such as those described above, can be used in a variety of ways in the therapy of this invention. Therefore, they can be applied directly to the wound site of the subject, or they can be used to prepare a recombinant NAB1 or NAB2 polypeptide that can then be applied to the wound site, as will be discussed in more detail below. The invention also relates to host cells generally genetically engineered with constructs that include the NAB1 or NAB2 polynucleotide or polynucleotides of the invention, or the genetic elements defined above, as well as to the use of these vectors and cells in the therapeutic methods of the invention. These constructs can be used per se in the therapeutic methods of the invention, that is, they can be used to prepare NAB1 or NAB2 polypeptides for use in the therapeutic methods of the invention which are described in more detail below.

The vector can for example be a plasmid vector, a single or double phage vector, a single or double RNA or DNA viral vector, depending on whether the vector is to be applied directly to the wound site (eg for in situ synthesis of NAB1 or NAB2), or should be used to synthesize recombinant NAB1 or NAB2. The starting plasmids described herein may either be commercially available, or publicly available, or may be prepared from available plasmids by routine use of well-known, published procedures. Many plasmids and other cloning and expression vectors that can be used in accordance with the present invention are well known and readily available to those skilled in the art.

In general, vectors for expression of NAB1 or NAB2 polypeptides for use in the invention include cis-control regions effective for expression in the host and operably linked to the polynucleotide to be expressed. Appropriate trans-acting factors are obtained either from the host, or from the complementary vector, or from the vector itself after introduction into the host.

For certain compounds from this domain, vectors enable specific expression. For the production of recombinant NAB1 or NAB2, such specific expression may include inducible expression or expression only in certain cell types, or both: both inducible and cell-specific. Particularly suitable among inducible vectors are vectors that are easy to manipulate, such as temperature and food additives. A variety of vectors suitable for this aspect of the invention include constitutive and inducible expression vectors used in prokaryotic and eukaryotic hosts, and are well known and routinely employed by those skilled in the art.

A wide variety of expression vectors can also be used to express NAB1 or NAB2 of the present invention. Such vectors include, but are not limited to, chromosomal, episomal, and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophages, transposons, yeast episomes, insert elements, yeast chromosomal elements, viruses such as baculoviruses, papoviruses, such as SV40, vaccinia virus, adenovirus, pox virus, pseudorabies virus, and retroviruses, as well as vectors derived from combinations thereof, such as those derived from plasmids and genetic elements of bacteriophages, such as cosmids and phagmids, all of which can be used for expression according to this aspect of the present invention. In general, any vector, which is suitable for maintaining, propagating or expressing a polynucleotide to effect expression of a polypeptide in a host, can be used for expression from this point of view.

The appropriate DNA sequence can be inserted into the vector using any of a variety of well-known routine techniques, such as those described in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

The nucleic acid sequence in the expression vector is operably linked to appropriate expression control sequence(s), including for example promoters for direct mRNA transcription. Representatives of such promoters include, but are not limited to, the phage lambda PL promoter, E. coli lac, trp and tac promoters for recombinant expression, and SV40, early and late promoters, and retroviral LTRs promoters for in situ expression.

In general, expression constructs will contain sites for initiation and termination of transcription, and in the transcribed region, a ribosome binding site for translation. Encoding the fraction of mature transcripts that have been expressed using the constructs, they will include a translational initiation AUG at the start and end codons that are placed right at the end of the polypeptide being translated.

Additionally, constructs may contain control regions that direct and cause expression. In general, according to many commonly used procedures, such regions will act, among other things, to control transcription, as transcription factors, repressor binding sites, and termination sites.

Propagation and expression vectors generally include selectable markers and extension amplification regions, such as those described in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Representative examples of suitable hosts for recombinant expression of NAB1 and NAB2 include bacterial cells, such as streptococci and staphylococci, E. coli, streptomyces, and Bacillus subtilis cells; fungal cells, such as yeast cells and Aspergillus cells; insect cells, such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells; and plant cells.

The following vectors, which are commercially available, are obtained by way of example. Suitable vectors for use in bacteria include pQE70, pQE60, and pQE-9, available from Qiagen; pBS vectors, fagscript vectors, bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagana; ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia, and pBR322 (ATCC 37017). Suitable eukaryotic vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL which can be obtained from Pharmacia. Those vectors that can be used for both recombinant expression and in situ expression are listed only to illustrate the many commercially available, well-known vectors available to those skilled in the art for use in accordance with aspects of the present invention. Preferably, any other plasmid or vector suitable for introducing, maintaining, propagating, or expressing a polynucleotide or polypeptide of the present invention in a host may be used according to an aspect of the present invention.

Examples of vectors useful in this aspect of the invention include expression vectors in which the NAB1 or NAB2 cDNA sequence is inserted into a plasmid, wherein expression of the gene is derived from the human proximal cytomegalovirus enhanced promoter (Foecking and Hofstetter, Cell, 45, 101-105, 1986). Such expression plasmids may contain SV40 RNA processing signals such as polyadenylation and termination signals. Commercially available expression constructs using the CMV promoter are pCDM8, pcDNA1 and derivatives, pcDNA3 and derivatives (invitrogen). Other available expression vectors that can be used are pSVK3 and pSVL, which contain the SV40 promoter and mRNA binding sites and polyadenylation signals from SV40 (pVSK3) and SV40 VP1 as well as processing signals (pSVL; vectors from Pharmacia).

Promoter regions can be selected from any desired gene by using vectors containing a reporter transcription unit lacking a promoter region, such as a chloramphenicol acetyltransferase (CAT) transcription unit, downstream of a restriction site or a site for introducing a fragment with a potential promoter; i.e. a fragment that may contain a promoter. As is well known, the introduction into the vector of a fragment containing the promoter at the restriction site upstream of the cat gene causes the production of CAT activity, which can be determined by standard CAT assays. The vectors corresponding to this end are well known and readily available, namely pKK232-8 and pCM7. The polynucleotide expression promoters of the present invention include not only well-known and readily available promoters, but also promoters readily obtained by prior techniques using reporter genes; for in situ expression, it would be desirable to recognize such a promoter in the treated subject.

Known prokaryotic promoters suitable for expression of polynucleotides and polypeptides of the present invention include E. coli lacI and lacZ and promoters, T3 and T7 promoters, gpt promoter, lambda Pr, PL promoters and trp promoter.

For example, recombinant expression vectors include replication origins, then a promoter, which is preferably derived from a gene with a significant degree of expression, and serves for direct expression of the downstream structural sequence, and selected markers that allow isolation of the vector containing cells after exposure to the vector.

Polynucleotides of the invention encoding a heterogeneous structural sequence of a polypeptide of the invention are generally inserted into a vector using standard techniques, such that the vector is operably linked to an expression promoter. The polynucleotide is positioned so that the transcription start site is located appropriately 5' to the binding site on the ribosome. The binding site on the ribosome is at the 5' position in relation to the AUG that initiates the translation of the expressed polypeptide.

In general, there are no long open reading frames that begin with the start codon, usually AUG, and are located between the ribosome binding site and the start codon. Also, there is a translational stop codon at the end of the polypeptide, and there is also a polyadenylation signal in the constructs used in eukaryotic hosts. A transcription terminal signal that is located at the appropriate 3' end of the transcribed region can also be included in the polynucleotide construct.

For the secretion of the translated protein into the lumen of the endoplasmic reticulum, into the periplasmic space, that is, into the extracellular environment, suitable signals for secretion can be incorporated into the polypeptide on which expression was performed during recombinant synthesis. These signals can be endogenous to the polypeptide, or they can be heterogeneous signals.

The polypeptide may be expressed in a modified form, such as a fusion protein, and may contain not only a secretion signal but also additional heterogeneous signals. Therefore, regions of additional amino acids, especially charged amino acids, can be added to the N- or C-terminus of the polypeptide to improve stability and resistance in the host cell during purification, or during subsequent handling and storage. The region can also be added to the polypeptide to facilitate purification. Such regions can be removed before the final preparation of the polypeptide. Additions of peptide structures to a polypeptide to induce secretion or shedding, and to increase stability or facilitate purification, are common routine professional techniques. A suitable coupling protein includes a heterogeneous region of the immunoglobulin that is useful for melting or purifying the polypeptide. It is typical that after that the cells are collected by centrifugation, dissolved by physical or chemical means, and the obtained crude extract is left for further cleaning.

Microbial cells used in protein expression can be disrupted by conventional methods, including cyclic freezing and thawing, sonication, mechanical disruption, or the use of lytic agents, procedures well known to those skilled in the art.

Mammalian expression vectors may include an origin of replication, an appropriate promoter, an enhancer, and also any necessary ribosome binding sites, polyadenylation regions, donor bonds, and acceptor sites, transcription termination sequences, and 5' flanking non-transcribed sequences necessary for expression.

Genetically engineered host cells can be used to prepare the NAB1 or NAB2 polypeptides used in the invention. Introduction of polynucleotides into a host cell can be induced by calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid mediated transfection, electroporation, transduction, thin layer application, ballistic introduction, infection, or other procedures. Such procedures 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. laboratory manual”, 2nd edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

The mature proteins can be synthesized by expression in a host cell including mammalian cells, such as CHO cells, yeast cells, bacteria or other cells under the control of an appropriate promoter. Cell-free translation systems can also be used to produce such proteins using RNA molecules derived from the DNA constructs of the present invention. Suitable cloning and expression vectors used for prokaryotic and eukaryotic hosts are described in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

The polypeptide can be recovered and purified from recombinant cell cultures using well known procedures, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, lecithin chromatography. The most suitable for use in cleanup is high performance chromatography. Well-known techniques for protein recovery can be used to regenerate the active conformation when a polypeptide is denatured during isolation and purification.

For therapy, the NAB1 or NAB2 encoding polynucleotide in recombinant vector form can be purified by techniques known in the art, such as column chromatography as described in Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL. ”, 2nd edition; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

As noted above, the NAB1 or NAB2 polypeptide can be administered to the wound site either as a NAB1 or NAB2 encoding nucleic acid that is transcribed and translated into NAB1 or NAB2 at the wound site itself in the form of gene therapy, or the transcriptional repressor protein itself can be administered directly.

Therefore, according to the following aspect of the invention, the use of the NAB1 or NAB2 polypeptide, or its biologically active fragment, is enabled in the production of drugs for the treatment of cell proliferation disorders in mammals, including humans.

According to the next aspect of the invention, a method of treating cell proliferation disorders in mammals, including humans, is enabled, which includes the administration to mammals of a therapeutically effective amount of NAB1 or NAB2 polypeptide, or its biologically active fragment.

Thus viewed from the following aspect, the invention enables the use of NAB1 or NAB2 polypeptides, or their biologically active fragments, in wound treatment and wound healing.

The term "NAB1 or NAB2 polypeptide" used here includes naturally and recombinantly produced NAB1 or NAB2, natural, synthetic and biologically active polypeptide analogs, or variants, or their derivatives, or their biologically active fragments and variants, and derivatives and analogs of said fragments.

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

NAB1 or NAB2, and the aforementioned fragments and derivatives thereof used in the treatment of this invention, can be extracted from natural sources using methods known in the art. Such methods include purification by sequence-specific DNA affinity chromatography using the methods described in Briggs et al., Science 234, 47-52, 1986, in which a DNA-binding oligonucleotide that recognizes NAB1 or NAB2 is used. The polypeptide can also be prepared by methods of recombinant DNA technology known in the art, as described above. Alternatively, the polypeptides of the present invention can be produced synthetically by conventional peptide synthesizers.

The invention also relates to the use of fragments, analogs and derivatives of NAB1 or NAB2. The terms "fragment", "derivative" and "analog" mean a polypeptide that retains essentially the same biological function or activity as such polypeptide. Therefore, the analog includes a proprotein that can be activated by cleavage of a portion of the proprotein to produce an active mature polypeptide.

A fragment, derivative or analogue of a polypeptide can be one in which one or more amino acid residues are substituted with a conserved or non-conserved amino acid residue (it is more convenient for the amino acid residue to be protected), and such a substituted amino acid residue may or may not be the one encoded by genetic code, or (ii) one in which one or more amino acid residues include a substituent, or (iii) one in which the mature polypeptide is linked to another compound, such as, for example, a compound that increases the half-life of the polypeptide (eg, polyethylene glycol), or ( iv) one in which additional amino acids are joined to the mature protein, such as the leader sequence or secretory sequences, i.e. the sequence used to purify the mature polypeptide or proprotein sequence. Such fragments, derivatives and analogs are considered by experts to be within the target area of the teachings listed here.

Suitable variants include those that differ from natural NAB1 or NAB2 by conservative amino acid substitutions. Such substitutions replace a given amino acid in the polypeptide with another amino acid with similar properties. Typical conservative substitutions include replacing aliphatic acids Ala, Val, Leu and Ile; mutual substitutions of hydroxyl residues Ser and Thr, substitutions of acidic residues Asp and Glu, substitutions between amino residues Asn and Gln, substitutions of basic residues Lys and Arg, and substitutions between aromatic residues Phe, Tyr.

From this point of view, the following particularly suitable variants, analogues, derivatives and fragments, and variants, analogues and derivative fragments have an amino acid sequence of the polypeptide in which several, 5 to 10, 1 to 5, 1 to 3, 2, 1 have been substituted, deleted or added or else no amino acid residue, in any combination. Particularly suitable among them are inactive substitutions, additions or deletions, which do not change the properties and actions of the polypeptide of this invention. Conservative substitutions are also particularly suitable from this point of view.

Particularly suitable fragments are biologically active fragments, i.e. fragments that retain the properties of the original polypeptide related to wound healing.

It is convenient that the polypeptides and polynucleotides of this invention are obtained in isolated form, and that they are preferably purified to homogeneity.

NAB1 or NAB2 polypeptides used in the present invention include NAB1 or NAB2 polypeptides, as well as polypeptides having at least 70% identity, and more preferably at least 80% identity, and more preferably at least 90% identity, and even more preferably at least 95% similarity ( more preferably at least 95% identity) to the polypeptide sequence described above, and also include portions of such polypeptides with such polypeptide portions generally containing at least 30 amino acids, or more preferably 50 amino acids.

Fragments or parts of the polypeptides of the present invention can be used in the production of the corresponding full-length polypeptides by peptide synthesis; therefore, the fragments can be used as intermediates in the production of full-length polypeptides. Fragments or parts of polynucleotides of the present invention can be used in the synthesis of corresponding full-length polynucleotides.

The invention also relates to the use of the above-defined fragments of NAB1 or NAB2 polypeptides, and fragments or variants and their derivatives.

From this point of view, a fragment is a polypeptide having an amino acid sequence that is completely identical to part, but not all, of the amino acid sequence of NAB1 or NAB2 polypeptides and variants and derivatives thereof.

Such fragments can be "independent", i.e. they are not part of something, or they can be joined with other amino acids or polypeptides, or they can be included within a larger polypeptide in which they form a part or region. If they are included in a larger polypeptide, it is most convenient when the previously discussed fragments form single continuous regions. For example, certain suitable structures relate to a fragment or polypeptide of the present invention that is included within a polypeptide precursor engineered for expression in the host, and which has heterogeneous pre- and pro-polypeptide regions fused to the amino terminus of the fragment, as well as an additional region fused to the carboxyl fragment terminus. Thus, fragments in one aspect refer to a portion or portions of a fusion polypeptide or fusion protein that is derived from a polypeptide of the present invention.

From this aspect, fragments that characterize the structural or functional features of the polypeptide of this invention are also suitable. Suitable compounds of this invention, from this point of view, include fragments comprising alpha-coil regions and alpha-coil-forming regions, beta-pleated regions and beta-pleated regions, substitution regions and substitution-forming regions, helical regions and helix forming regions, hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface forming regions, substrate binding regions, distinct antigenic index regions of the polypeptides of this invention, and combinations of such fragments.

Those regions that affect the activity of the polypeptides of this invention are suitable. The most suitable fragments from this point of view are fragments that have a chemical, biological or other activity of the polypeptide of this invention, including those with similar activity or improved activity, or with reduced side effects. Further suitable polypeptide fragments are those which include or contain antigenic or immunogenic determinants in animals, particularly in humans.

It is also preferable that the invention relates, among others, to polynucleotides that encode the above-mentioned fragments, polynucleotides that hybridize polynucleotides that encode fragments, especially those that hybridize under well-established conditions, and polynucleotides, such as PCR primers, for amplifying polynucleotides that encode fragments. From this point of view, those polynucleotides which correspond to suitable fragments, as described above, are suitable.

In the following compounds, with respect to this aspect of the invention, are included biological, prophylactic, clinically or therapeutically useful variants, analogs or derivatives thereof, or fragments thereof, including fragments of variants, analogs and derivatives, and mixtures including the same. Biologically active variants, analogs or fragments are included within the scope of the present invention.

The invention also relates to mixtures including the polynucleotides or polypeptides discussed above. Therefore, the polynucleotides and polypeptides of the present invention may be used together with a pharmaceutically acceptable vehicle or vehicles.

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

Polypeptides and polynucleotides may be used in this invention alone or in conjunction with other compounds, such as, for example, therapeutic compounds.

The pharmaceutical compositions may be administered by any effective means capable of penetrating the wound site, including, but not limited to, for example, topical, intravenous, intramuscular, intranasal, or intradermal routes of administration.

In treatment, i.e. as prophylaxis, the active substance can be given to the individual in the form of an injection containing a mixture, for example a sterile aqueous dispersion, preferably isotonic.

Alternatively, the mixtures may be prepared for local application, e.g. in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, mouthwashes, impregnated bandages and sutures, and aerosols, and may contain appropriate common additives, including, for example, preservatives, solvents that enable drug penetration, and emollients in ointments and creams. Such preparations for topical application may also contain compatible, conventional vehicles, eg bases for creams and ointments, and ethanol or oleyl alcohol for lotions. Such vehicles can make up from about 1% to about 98% of the mass of the preparation; it is more common for them to make up to about 80% of the mass of the preparation.

When applied to mammals, and especially to humans, the daily dose level of the active substance is expected to be from 0.01 mg/kg to 10 mg/kg, or typically 1 mg/kg. In each individual case, doctors will determine the most suitable dose for an individual, which will vary depending on the age, weight and reaction of a particular person. The above doses represent examples of average cases. Of course, there may be individual examples in which higher or lower doses are required, and these too fall within the scope of the present invention. Finally, the dose selected by the expert must reduce cell proliferation without preventing wound healing.

As a further aspect, there is provided a pharmaceutical mixture 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 vehicles thereof.

The therapeutic advantage of using a protein transcription repressor in wound healing is the deactivation of multiple target genes that promote accelerated healing. NAB1 or NAB2 is naturally activated in the wound response, and augmenting the natural response is also an advantage. The treatment is based on DNA and enables a reliable and repeatable delivery system.

Where a NAB1 or NAB2 polynucleotide is used in a therapeutic method of the present invention, the polynucleotide may be used as part of an expression construct in the form of an expression vector. In such a procedure, the construct is introduced into the wound site 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.

This invention is suitable for all aspects of wound treatment including limb suppuration in diabetes and peripheral arterial occlusion disease, postoperative scars, burns, psoriasis, inhibition of restenosis followed by percutaneous transluminal coronary angioplasty, modulation of vessel wall calcification, and inhibition of cancer cell proliferation.

As described above, the NAB1 or NAB2 polypeptides or nucleic acids of the invention can be administered locally at the site of tissue damage by any conventional method, eg, topical application. A suitable way of applying products containing nucleic acid is the use of gene gun ("gun") technology in which the Egr-1 nucleic acid molecule is isolated, e.g. in the form of cDNA or expression vector, immobilized on gold particles, and "fired" directly at the site injuries. Therefore, a convenient aspect of the present invention is to enable the use of nucleic acid molecules that include the sequence encoding the NAB1 or NAB2 polypeptide in a “gene gun” for the treatment of cell proliferation disorders associated with wound healing. Furthermore, a mixture suitable for gene gun ("gun") therapy was obtained which includes the NAB1 or NAB2 coding sequence immobilized on gold particles.

The present invention will now be described by means of the following non-limiting examples with reference to the accompanying figures, where:

Figures 1a)-1d). respectively, they show the repression of the activation of PDGF-AB, TGFβ, HGF and VEGF compounds, which occurs through the mediation of Egr-1;

Figure 2 describes NAB2 trans-repression of HGF production mediated by Egr-1;

Figure 3 describes the effect of NAB2 on Egr-1-driven angiogenesis;

Figure 4a depicts the effect of NAB2 transfection on linear wound shortening seven days after wounding;

Figure 4b describes the effect of NAB2 transfection on growth factor levels in rat epidermis with 7-day-old cuts;

Figure 4c describes the effect of NAB2 transfection on growth factor levels in the granulation tissue of rats with 7-day-old cuts;

Figure 4d describes the effect of NAB2 transfection on angiogenesis in rats with 7-day-old cuts;

Examples

Example 1

Use of NAB2 to repress growth factor trans-activation mediated by Egr-1;

1.1 Procedures

Human vascular smooth muscle cells (HVSM; Clonetics) were cultured according to the manufacturer's recommendation. Cells were grown for transfection in 6-well microtiter plates (Costar). An expression plasmid including NAB2 cDNA derived from the promoter of human cytomegalovirus (hCMV; Svaren et al., Mol. Cell. Biol., 16; 3545-3553, 1996) was co-transfected with an expression plasmid including Egr-1 cDNA (Houston et al., Arterioscler. Thromb. Vasc. Biol., 19; 281-289, 1999) in HVSMC at ratios described in the figures using FUGENE (Boehringer Mannheim). A 3:1 ratio (v/m) of FUGENE:DNA was used for all experiments, and transfection was normalized using a CMV-derived β-galactoside expression plasmid. Secreted growth factors were detected in tissue culture by ELISA (R&D Systems) using appropriate controls.

1.2 Results

Figures 1a). to 1d). show, in order, the repression of the activation of PDGF-AB, TGFβ, HGF and VEGF compounds, which is mediated by Egr-1. 6 μg of Egr-1 expression plasmid was countertransfected either alone (Figure 1a, Figure 1b, Figure 1d), or with 500, 100 or 25 ng of NAB2 (Figure 1a and Figure 1d). For PDGF-AB, TGFβ and VEGF, a small increase in the amount of detected secreted growth factor was observed when Egr-1 was transfected alone. In case of co-transfection, there was a complete disappearance of the PDGF-AB reaction to Egr-1 activation, and a decrease in the basic expression, which leads to a fivefold decrease in the total production of TGFβ (Figure 1a). NAB2 transfection completely abrogated the TGFβ response to Egr-1 activation, and caused a 30% reduction in total TGFβ production (Figure 1b). Using the DNA concentrations shown in the figure, Egr-1 transfection caused a 50% increase in HGF production, which was partially blocked by co-transfection of a low dose of NAB2, and completely blocked by a higher dose (Figure 1c). The greatest effect was observed on the 1st day after transfection, although inhibitory effects were evident on the 2nd and 3rd day. NAB2 transfection, as well as PDGF-AB and TGFβ transfections, blocked the activation of VEGF mediated by Egr-1, and caused a 40% decrease in the amount of total VEGF produced (Figure 1d).

1.3 Conclusion

These data indicate that the Egr-1 suppressor NAB2 blocks Egr-1-mediated activation of growth factors, as found at sites of acute injury.

Example 2

Use of NAB2 to repress basal levels of growth factors

2.1 Procedures

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 Figure 2a, transfection of NAB2 in HVSCM caused a drastic reduction in PDGF-AB production. At the highest dose of NAB2, a tenfold reduction in PDGF-AB was achieved.

2.3 Conclusion

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

Example 3

Use of NAB to repress angiogenesis induction in vitro

3.1 Procedures

NAB2 expression constructs (wild type and dominant negative) derived from the hCMV promoter have been described (Svaren et al., EMBO J. 17;6010-6019, 1998). We have shown that transfection of the Egr-1 transcription factor, when its expression was performed from the hCMV promoter, enhances angiogenesis (Patent registration PG3412). In these experiments, DNA was transfected into an angiogenetic kit as prescribed by the manufacturer's supply and maintenance instructions (TCS Biologicals). VEGF protein (2 ng/ml) is used as a control, and suramin (20 μM) as an angiogenesis inhibitor. CMV Egr-1 DNA was transfected in triplicate at 0.5 μg per site in 24-well microtiter plates using MirusTransit reagent (Cambridge Biosciences) at a ratio of 2:1 v/m DNA. To determine NAB2's potential for Egr-1-mediated repression of angiogenesis, 0.5 mg of Egr-1 DNA was cotransfected together with 10, 25, or 100 ng of NAB2 expression plasmid per site in the absence or presence of 100 ng of NAB2 dominant negative expression plasmid. After 11 days, coculture angiogenesis was determined by staining cells with the endothelial cell marker PECAM-1 and visualization using BCIP/NBT substrate.

Representative images of tube formation using all four Egr-1 expression plasmids together with VEGF (positive control) and suramin (negative control) were captured and processed with a Quantiment 600 image analyzer and associated software.

3.2 Results

Egr-1 DNA has been shown to be pro-angiogenic in International Patent Application No. PCT.GB99.01722 (as shown in column 4 of Figure 3a). As shown in Figure 3a, co-transfection with NAB2 caused a dose-dependent reduction in the ability of Egr-1 to induce angiogenesis, which was particularly reduced by co-transfection with dominant negative NAB2.

3.3 Conclusion

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

Example 4

Use of NAB2 to reduce scarring in cut rodents

4.1 Procedures

4.1.1 Effect of NAB2 transfection using the "gene gun" on growth factor levels in rat cuts

NAB2 transfection into rodent incisions may have scar removal potential through its direct suppressive effect on the expression of key scar growth factors, such as TGFβ1. In this experiment, NAB2 cDNA was transfected into rat wounds using a Biorad “gene gun”, and the effects on healing and growth factor levels were determined by routine histological and immunochemical procedures.

4.1.2 Gene transfer by means of particles

Eight male Sprague Dawley rats weighing about 250 g were anesthetized with isofluorane in a 2:1 mixture of oxygen/nitrous(I)-oxide. Two transfection sites (5 cm from the base of the skull, 1.5 cm on either side of the center) and 2 control sites (8 cm from the base of the skull, 1.5 cm on either side of the center) on the back of rats were made by cutting the skin, and then shaving with shaving preparation. Transfection is performed in the skin at each transfection site using an accelerating plasmid/gold complex and NAB2 or Egr-1 (positive control growth factor activation) at a pressure of 350 psi. It is usual to apply 0.5-1.5 μg of DNA per transfection. In one control site, gold particles are accelerated into the skin at a pressure of 350 psi, in another control site there was no manipulation. Transfection sites and control sites are rotated clockwise in each subsequent animal to control for anterior-posterior differences in wound healing in rodents.

4.1.3 Model of wound healing

Twenty-four hours after transfection, the animals are anesthetized, and a 1 cm wide linear incision parallel to the spine is made using a scalpel exactly at the transfection sites. Allow the animals to wake up from anesthesia and allow the wounds to heal without suturing. On the 7th day after wounding, all 8 animals were killed, and the wounds were dissected and used for routine histological and immunochemical procedures.

4.1.4 Histological analysis

Each wound is divided horizontally into two parts at a certain time after sectioning. One half is placed in 4% paraformaldehyde for 24 hours and used in wax histology. 5 millimeter sections are cut from each wound using a microtome, and these sections are stained using a van Geison. The sections are observed under a light microscope, and the effect of NAB2 on the healing reaction is determined.

4.1.5 Immunohistochemistry

The other half of the total wounds are frozen in OCT, and dissected at 7 mm using a cryostat. Two sections from each wound are fixed in ice-cold acetone, and fluorescent immunostaining with primary antibodies to Egr-1, PDGF, TGFβ1, TGFβ3 and vWF is performed according to the following procedure

1. Wash microscope slides with PBS

2. Apply 30 ml of primary antibody over 1 hour

3. Wash 3 times for 5 minutes with PBS

4. Apply 30 ml of secondary antibodies for 45 minutes (directly linked to FICT)

5. Wash 3 times for 5 minutes with PBS

6. Glue the object microscope slide using water glue

Immediately after immunostaining, each slide is placed under a fluorescent microscope, and the surface of the wounds is recorded using 100% magnification. The image is integrated, and the noise is corrected to minimize the background. The area and intensity of staining are measured using an image analyzer, and plotted graphically.

4.2 Results

4.2.1 Effect of NAB2 on wound healing in rats

4.2.1.1 Wound contraction and histological analysis of the seven-day wound

According to Figure 4a, wounds treated with NAB2 contract to a similar size as those transfected with Egr-1 and as control wounds, and histologically they have a similar maturity as granulation tissue.

Conclusion

Administration of NAB2 does not impair the rate of healing observed in a rodent wound healing model.

4.2.1.2 Diagrams of growth factors

Immunostaining of compounds Egr-1, TGFβ1, TGFβ3, and PDGF-AB is performed on frozen cryosections of skin tissue at wound sites, and the staining intensity of each individual growth factor is measured using an image analyzer. Two separate immunohistochemical measurements are performed at the wound site. Growth factor expression is examined in sections and in the epidermis immediately above the wound and in the epidermis within the wound site (granulation tissue).

a) positive staining for growth factors in the epidermis

As shown in Figure 4b, at the seventh day after wounding, the expression of TGFβ1 in the epidermis was significantly decreased compared to Egr-1 control and gold. Compared to the unmanipulated control, NAB2 transfection reduces TGFβ1 expression in the epidermis, since Egr-1 application as well as gold application activates TGFβ1 production. NAB2 transfection increases TGFβ3 levels in the epidermis compared with gold and unmanipulated controls. NAB2 transfection did not significantly change the epidermal expression of Egr-1 and PDGF.

b) positive staining for growth factors in granulation tissue

As shown in Figure 4c, on the seventh day after wounding NAB2 transfection had no effect on the levels of Egr-1, PDGF-AB and TGFβ1 in the granulation tissue. NAB2 increases TGFβ3 levels in granulation tissue compared to gold and non-manipulated controls.

Conclusion

NAB2 transfection of wounds decreases TGFb1 levels in the epidermis and increases TGFβ3 levels in the epidermis and granulation tissue seven days after wounding. TGFβ1 is known to be responsible for scar formation, while TGFβ3 has anti-scarring properties (Shah et al., J. Cell Science 107; 1137-1157, 1994). Therefore, NAB2 delivery may have anti-scarring properties.

4.2.2 Effect of NAB2 on angiogenesis

Seven days after wounding, skin sections were stained and marked with scored lines for vWF expression using immunohistochemical procedures and an image analyzer. Angiogenesis is quantified using von Willebrand factor immunostaining on wound cryosections and image analysis to measure the area of positive staining within the wound site. As shown in Figure 4d, 7 days after wounding, NAB2-transfected wounds had fewer new blood vessels compared to controls (gold-treated wounds). Egr-1 promotes angiogenesis in vivo, supporting the in vitro findings of Example 3.

Conclusion

NAB2 blocks Erb-1-stimulated activation of angiogenesis in vivo, thereby supporting NAB2's role as a repressor of growth factor activation when driven by acute stimuli, exemplified by Egr-1 activation of growth factor production.

Claims (22)

1. Use of a nucleic acid molecule, characterized in that it includes a sequence encoding a NAB1 or NAB2 polypeptide, or a biologically active fragment thereof, in the manufacture of a medicament for the treatment of cell proliferation disorders associated with wound healing in mammals, including humans. 2. Use as claimed in claim 1, wherein the NAB1 or NAB2 polypeptide is a human NAB1 or NAB2 polypeptide. 3. Use as claimed in any one of claims 1-2, characterized in that the cell proliferation disorders associated with wound healing represent hypertrophic and keloid scarring. 4. Use as claimed in any of claims 1-3, wherein the nucleic acid molecule is operably linked to a nucleic acid sequence that controls expression. 5. Use as claimed in any one of claims 1-4, characterized in that the nucleic acid molecule is at least 70%, respectively 80%, respectively 90%, respectively 95% identical over its entire length to NAB1 or NAB2 polynucleotide sequence. 6. Use according to any one of claims 1-5, characterized in that it includes a combination of nucleic acid molecules comprising a sequence encoding both NAB1 and NAB2 polypeptides, or biologically active fragments thereof. 7. Use as claimed in any one of claims 1-5, characterized in that the nucleic acid molecule includes a sequence encoding a NAB2 polypeptide, or a biologically active fragment thereof. 8. Use as claimed in any one of claims 1-7, characterized in that the nucleic acid molecule is physically adapted for use in mammals. 9. Use as claimed in claim 8, characterized in that the nucleic acid molecule is adapted for use in mammals by particle bombardment. 10. Use as claimed in claim 9, characterized in that the nucleic acid molecule is immobilized on gold particles. 11. Use as claimed in claim 8, characterized in that the nucleic acid molecule is adapted for use in mammals by microinoculation. 12. Use as claimed in any of claims 1-7, wherein the nucleic acid molecule is in a vector. Use as claimed in any of claims 1-7, wherein the nucleic acid molecule is expressed in a cell. 14. A nucleic acid molecule that includes a sequence encoding a NAB1 or NAB2 polypeptide or a biologically active fragment thereof, indicated to be used in gene therapy. 15. A pharmaceutical preparation, characterized in that it includes a nucleic acid molecule that includes a sequence encoding a NAB1 or NAB2 polypeptide or a biologically active fragment thereof, together with one or more pharmaceutically acceptable carriers. 16. A method of treating a cell proliferation disorder associated with wound healing in a mammal, including a human, comprising administering to the mammal a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a biologically active fragment thereof. 17. Use of NAB1 or NAB2 polypeptides or their biologically active fragment, characterized in that said are used in the production of a drug for the treatment of cell proliferation disorders associated with wound healing in mammals, including humans. 18. Use according to claim 17, characterized in that the NAB1 or NAB2 polypeptide or its biologically active fragment is produced naturally, synthetically or recombinantly. 19. Use according to claim 17 or claim 18, characterized in that the NAB1 or NAB2 polypeptides are human NAB1 or NAB2 polypeptides. 20. Use as claimed in any of claims 17-19, characterized in that the polypeptide is at least 70%, respectively 80%, respectively 90%, respectively 95% identical throughout its length to the NAB1 or NAB2 polynucleotide sequence. 21. A method of treating a cell proliferation 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. Pharmaceutical preparation, characterized in that it includes NAB1 and/or NAB2 polypeptide or their biologically active fragment together with one or more pharmaceutically acceptable carriers.