KR20010071832A - Pharmaceutical Uses of NAB1 and NAB2 - Google Patents

Abstract

The present invention relates to the use of NAB1 or NAB2 polypeptides or biological fragments thereof, in particular as gene therapeutics, and to nucleic acid molecules encoding such polypeptides for the manufacture of a medicament for the treatment of cell proliferative diseases associated with wound healing in mammals, including humans.

Description

Pharmaceutical Uses of NAB1 and NAB2}

The present invention relates to the use of gene therapy techniques to treat wounds. More specifically, the present invention specifically down-regulates cell proliferative disorders that slow down the treatment of the skin, thereby inhibiting hypertrophic keloid scar formation, psoriasis and percutaneous trans-luminal coronary angioplasty. A novel use of a polynucleotide encoding a NAB1 or NAB2 transcription repression protein, which inhibits restenosis after angioplasty, regulates calcination of blood vessel walls and inhibits proliferation of cancer cells.

Skin healing is associated with a wide range of cellular, molecular, physiological and biochemical outcomes. During the healing process, cells migrate to the wound site where they proliferate and synthesize extracellular matrix components to reconstruct the tissue most similar to the original intact tissue. This activity is regulated by mediators secreted from cells around the wound, such as platelet induced growth factor (PDGF), epidermal growth factor (EGF), modified growth factor beta (TGF beta) and other cytokines. The effects of these agents on cells in vivo and in vivo, including the benefits of PDGF administration in a model of diabetic rats (Brown et al J. Surg. Res. 56; 562-570, 1994) Et al. (Reviewed in Moulin, Eur. J. Cell Biol. 68; 1-7, 1995).

Over the past five years, many growth factors have been identified that promote cell proliferation in vitro and promote wound healing in animal models. TGF beta has received the most attention in wound healing because it promotes cell proliferation, differentiation, and substrate production. Topically or systematically administered TGF beta accelerates the rate of treatment of skin wounds in animal models (Ashcroft et al Nature Medicine, 3; 1209-1215, 1997; Sporn and Roberts J. Cell Biol. 119; 1017-1021, 1997; Beck et al J. Clin. Invest. 92; 2841-2849, 1993). Likewise, PDGF has been reported to promote epithelial remodeling and revascularization in ischemic tissues and animals with diabetes (Uhl et al Langenbecks Archiv fur Chirurgie-Supplement-Kongressband 114; 705-708, 1997), and literature (Reviewed in Dirks and Bloemers Mol. Biol. Reports 22; 1-24, 1996)

The transcription factor Egr-1 (early growth response gene) is a potential regulator with more than 30 genes and plays a role in cell proliferation, development and differentiation (Liu et al Crit. Rev. Oncogenesis 7; 101-125, 1996; Khachigian and Collins Circ.Res. 81; 457-461, 1997). Egr-1 is induced shortly after vascular endothelial injury (e.g. khachigian et al Science; 271, 1427-1431, 1996), and targets of transcriptional activation are many genes, including EGF and platelet-induced growth factor-A. (PDGF-A), basic fibroblast growth factor (bFGF), induction of PDGF A, platelet-induced growth factor-B (PDGF B), TGF beta, bFGF, uro-plasminogen activator (u-PA) , Tissue factor and insulin-like growth factor-2 (IGF-2). Blockers of Egr-1 inducible TGF can be used to prevent scars. Antibodies augmented by TGF beta reduce scarring in Ben wounds in rodents (Shah et al J. Cell Science; 107; 1137-1157; Shah et al Lancet; 339, 213-214, 1992).

Transcription complexes that mediate vascular endothelial growth factor (VEGF) induction are dependent on AP2 and not directly on Egr-1 (Gille et al EMBO J 16; 750-759, 1997). However, PDGF B directly upregulates VEGF expression (Finkenzeller Oncogene 15; 669-676, 1997). Transcription of VEGF mRNA is increased by many factors including PDGF B, bFGF, keratinocyte growth factor (KGF), EGF, tumor necrosis factor (TNF) alpha and TGF beta1. In animal models, immobilization of metal stents by VEGF-induction has been shown to inhibit neo-intima formation, promote re-endothelialization, and increase the activity of vasomotor factors (Asahara et al. Circulation; 94, 3291-3302).

VEGF expression has been reported to treat two symptoms that are upregulated in TGF alpha and its ligand, EGFr, namely wound and psoriasis skin. Expression of EGF induces Egr-1 (lwami 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). Current anecdotal evidence suggests that Egr-1 can activate the expression of intercellular adhesion molecule-1 (ICAM-1) in phorbol ester-stimulated B lymphocytes (Maltzman et al Mol. Cell Biol; 16; 2283-2294, 1996), the presence of an Egr-1 binding site in the TNF alpha promoter allows for the activation of expression of TNF alpha (Kramer et al Biochim. Biophys. Acta 1219; 413-421, 1994). Finally, mice with Egr-1 have no fertility and exhibit luteinizing hormone (LH) deficiency (Lee et al 273; 1219-1221, 1996), suggesting that the LH promoter may also be a target of Egr-1 activation. Imply that there is. Vascular calcification is an activity regulation process similar to bone formation associated with cells and factors known to be important for the regulation of bone metabolism (Dermer et al Trends Cardiovasc. Med. 4; 45-49, 1994). Modulators of osteoblastogenesis and / or osteoclastogenesis can regulate the degree of calcification of blood vessel walls. NAB proteins NAB1 and NAB2 ( N GFI- A binding inhibitors) interact with the conserved R1 domains of Egr-1 and Egr-2 trans-activators (Svaren et al EMBO J., 17; 6010-6019, 1998). NAB2 is NGF-induced differentiation of PC12 cells (Qu, Z et al J. Cell Biol. 142; 1075-1082, 1998) and Egr-1 mediated activation of basic FGF (Svaren et al EMBO J., 17; 6010-6019 , 1998).

One problem faced in treating wounds is the formation of hypertrophic keloid scars. This problem is particularly undesirable after plastic surgery. Fibrosis of tissues (eg in the case of kidney, liver or skin tissue) is manifested by the accumulation of extracellular matrix. Unregulated production of extracellular matrix, which underlies the development of fibrosis of tissue, has been described in 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 .; 249-264, 1996 Plenum Press) and BEGF (Jones et al Frontiers in Bioscience 4; D303-309 , 1999), and is at least partly regulated by many growth factors. Although it does not eliminate the expression of growth factors at the wound site, reducing treatment has a significant impact on the treatment of reducing scars on the tissue and will improve quality of life.

International Patent Application No. PCT. GB99.01722 discloses the use of Egr-1 transcription factors to promote wound healing. At present, the present inventors have found that administration of a transcriptional repressor NAB1 or NAB2 polynucleotide inhibits (a) Egr-1 mediated activation of growth factors in vitro and (b) expression of growth factor genes in vitro. It can be used to inhibit the basal level of and (c) reduce the expression of TGF-β1 but not TGF-β3 at wound sites and subsequent expression of rodents and reduce scarring while healing the wound (eg, Shan). et al J. Cell Science 107; 1137-1157, 1994).

Thus, downregulation of Egr-1 slows down the course of treatment, reduces thickening and keloid scar formation, and the occurrence of psoriasis. Down regulation of Egr-1 can also be used for other diseases related to wound healing, such as restenosis after conjunctival trans-luminal coronary angioplasty, regulation of vascular wall calcification, inhibition of cancer cell proliferation.

Accordingly, in one aspect of the invention, the use of a nucleic acid molecule or a biologically active fragment thereof comprising a sequence encoding a NAB1 or NAB2 polypeptide in the manufacture of a medicament for treating a cell proliferative disease associated with wound treatment in a mammal, including humans It is about.

According to another aspect of the invention, the treatment of a cell proliferative disease associated with wound treatment in a mammal comprising administering to a mammal, including a human, a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a biologically active fragment thereof Provide a method.

According to another aspect of the invention, 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 treating cell proliferative diseases associated with wound treatment.

According to another aspect of the invention, the invention extends to the use of a combination of nucleic acid molecules comprising a sequence encoding a NAB1 polypeptide and other nucleic acid molecules comprising a sequence encoding a NAB2 polypeptide.

In a preferred aspect of the invention, the cell proliferative disease associated with wound treatment is selected from thickening keloid scar formation, psoriasis, restenosis after percutaneous trans-luminal coronary angioplasty, calcification of the vascular wall and cancer cell proliferation. In a particularly preferred aspect of the invention, the cell proliferative disease is thickening keloid scar formation.

Accordingly, the present invention relates to the therapeutic use of polynucleotides encoding NAB1 or NAB2 in the course of wound healing. The present invention also relates to the use of NAB1 or NAB2 itself as a therapeutic agent in the course of wound healing, as described in detail below.

The present invention relates to the use of a NAB1 or NAB2 polypeptide and nucleic acid sequences encoding NAB1 or NAB2 from a specific origin or species. Human DNA sequences are listed in the GenBank (Registration No. U47007), which encodes 486 amino acid nuclear proteins. Murine NAB1 is a 570 amino acid nuclear protein and is described in PNAS USA 92 , 1995 p6873-6877. The rat DNA sequence is listed in the Sperm Bank, and its registration number is U17253.

Mouse and human NAB2 protein sequences are described in molecular and Cellular Biology, 1996 16, 3545-3553. Human DNA sequences are listed in Gene Bank No. U48361.

Techniques for NAB1 or NAB2 polypeptides and polynucleotides described below are generally applicable to sequences of particular origin, in particular those of humans. As described below, the terms NAB1 or NAB2 also include variants, fragments and analogs thereof.

The following description is provided to assist in understanding the terminology used herein. The description is made for convenience and is not intended to limit the invention.

Biologically active fragments of NAB1 or NAB2 as referred to herein refer to fragments having Egr-1 transcriptional inhibitory activity.

GENETIC ELEMENT generally refers to a polynucleotide, or a site encoding a polypeptide, comprising a site encoding a polypeptide or polypeptide site that modulates replication, transcription or translation, or other processes important for expression of the polypeptide in a host cell; By polynucleotide is meant to include all sites operably linked thereto to regulate expression. Genetic components may be included in the vector, which is replicated as an episomal component, ie a molecule physically independent of the host cell genome. The genetic component may also be included in the host cell in the following manipulations rather than in its natural state, in particular in the form of, for example, isolated, cloned and purified DNA forms or introduction into the host cell in a vector.

Host cells (HOST CELLs) are transformed or transfected cells and can be transformed or transfected by exogenous polynucleotide sequences.

Identity, as is known in the art, is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences, determined by comparing sequences. In the art, identity also refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences, which in some cases is determined by the match between strings of sequences (Conputaional Molecular Biology). , Lesk, 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 1, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds , M Stockton Press, New York, 1991). While there are many methods for measuring identity between two polynucleotides or two polypeptides, the 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. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Commonly used methods for determining identity between sequences include those described in Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988), It is not limited to this. Preferred methods of determining identity are designed to achieve the greatest match between the sequences to be tested. Preferred computer programming methods for determining identity between two sequences include the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BLASTP, BLASTN and FASTA [Atschul, S.F. et al., J. Molec. Biol. 215: 403 (1990), but is not limited thereto.

ISOLATED is an artificial change of nature. That is, if it occurs in its natural state, either modified or removed from its original environment, or both. For example, naturally occurring polynucleotides or polypeptides that are naturally present in living organisms in their natural state are not "isolated," but as used herein, provided that the same polynucleotide or polypeptide has been separated from its natural coexistent material, During or after separation, such polynucleotides can be linked to other polynucleotides, eg, DNA, for mutation formation to form fusion proteins and for growth or expression in host cells. The polynucleotide may be introduced into the host cell alone or in combination with other polynucleotides, for example, in a vector state, or introduced into the host cell in a culture state or in an intact organism state. Is still seen As used herein, the term "isolated" means that the DNA is not in a naturally occurring form or environment that is not a naturally occurring composition, and that the polynucleotide or polypeptide has the same meaning as the term used herein. It will exist there separately.

Polynucleotide (s) generally refers to any polyribonucleotide or polydeolibonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA or cDNA. Thus, for example, as used herein, polynucleotide means, in particular, single and double stranded DNA, a mixture of single and double stranded sites or a mixture of single, double and triple stranded sites, DNA, single and double stranded. Refers to a hybrid molecule comprising DNA and RNA which may be stranded RNA and single stranded or more typically double stranded or triple stranded or a mixture of single and double stranded sites. Moreover, as used herein, polynucleotide refers to triple stranded sites comprising RNA or DNA or both RNA and DNA. Strands at these sites can come from the same or different molecules. The moiety may comprise all of one or more molecules, but more typically only the moiety of some of the molecules. One of the molecules of the triple helical region is often an oligonucleotide. The term polynucleotide as used herein includes DNA or RNA comprising one or more modified bases as described above. Thus, DNAs or RNAs having a backbone modified for stabilization or other reasons are also polynucleotides intended herein. Moreover, DNA or RNA comprising an unusual base such as inosine or a modified base such as tritylated base as two examples are also polynucleotides referred to herein. Importantly, various modifications have been made to DNA and RNA that serve many useful purposes known in the art. The term polynucleotide, as used herein, includes chemical, enzymatic or metabolic modified forms of polynucleotides, in addition to chemical forms having DNA and RNA traits of viruses and cells, particularly including simple and complex cells. do. Polynucleotides include short polynucleotides, often called oligonucleotide (s).

As used herein, a polypeptide includes all polypeptides as described below. The basic structure of a polypeptide is known and described in numerous books and other publications in the art. In this respect, the term herein refers to any peptide or protein comprising two or more amino acids linked to each other by peptide bonds in a linear chain. As used herein, the term usually refers to both short chains, also referred to in the art as eg peptides, oligopeptides and oligomers, and longer chains commonly referred to in the art as having many forms. Importantly, polypeptides contain amino acids other than twenty amino acids, often referred to as twenty naturally occurring amino acids, and many amino acids, including terminal amino acids, are known in the art, in addition to natural processes such as processing and other posttranslational modifications. It can be modified in a given polypeptide by a chemical modification technique known in the art. Although there are so many common modifications naturally occurring in polypeptides, they cannot be listed here unnecessarily, but the modifications are based on a number of other research papers. It is well described in textbooks and more detailed monographs.

Some of the known modifications that may be made to the polypeptides for use in the present invention may be exemplified by acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavins, heme moieties. Covalent attachment of nucleotides or derivatives thereof, covalent attachment of lipids or derivatives thereof, covalent attachment of phosphotidylinositol, crosslinking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinking, cystine Formation, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodide, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation Prenylation, lasimilation, selenolation, sulfated, transfer-RNA mediated addition of amino acids to proteins, e.g. arginylation ation) and ubiquitination. Such variations are well known to those skilled in the art and have been described in detail in scientific papers. Some particularly common modifications, glucose, lipid attachment, sulfate, gamma-carboxylation, hydroxylation and ADP-ribosylation of glutamic acid residues are the most basic textbooks, for example, PROTEINS. STRUCTURE AND MOLECULAR PROPERTIES, 2nd ED., TE Creighton, WH Freeman and Company, New York (1993). Many of the literature available on this subject, for example, Wold, F., Posttranslational Protein Modifications: Perspectives and Prospects, pgs. 1-12 in POSTTRANSLATIONAL COVALENT MODIFICATION OF PROTEINS, BC Johnson, Ed., Academic Press, New York (1983); Seifter et al., Meth. Enzymol. 182 626-646 (1990) and Rattan et al., Protein Synthesis: Posttranslational Modifications and Aging, Ann. NY Acad. Sci. 663: 48-62 (1992). It will be appreciated that, as known and described above, polypeptides are not always completely linear. For example, polypeptides can generally occur as a result of post-translational processes, including processes by natural processing events and by artificial manipulations that do not occur naturally. Cyclic, branched and branched cyclic polypeptides can be synthesized by fully synthetic methods in addition to untranslated natural processes. Modifications can occur anywhere on the polypeptide backbone, amino acid side chains and amino or carboxyl termini. Indeed, blocking an amino group or a carboxyl group or both by covalent modification is common in naturally occurring and synthetic polypeptides, such modifications may also be present in the polypeptides of the invention. For example, prior to proteolytic treatment, the amino terminal residues of polypeptides produced in E. coli or other cells are almost without exception N-formylmethionine. During post-translational modification of the peptide, the methionine residues at the NH ₂ -terminus can be removed. Thus, the present invention is intended for both methionine containing amino terminal variants and methionine free amino terminal variants of the proteins of the invention. Modifications occurring in a polypeptide will often be a function of the method of production. For example, for a polypeptide produced by expressing a cloned gene in a host, the nature and extent of the modification will primarily be determined by the modification signal present in the polypeptide amino acid sequence and the ability of the host cell post-translational modification. For example, as known, glucoseification does not occur in bacterial hosts such as E. coli. Thus, if glycosylation is desired, the polypeptide must be expressed in glucoseylated cells, generally eukaryotic cells. Insect cells often perform post-translational glucoseification, such as mammalian cells, and for this reason insect cell expression systems have been developed, in particular, to efficiently express mammalian proteins with natural patterns of glucoseification. Similar considerations apply to other variations. It is important to note that modifications of the same type can exist to the same or varying degrees at several sites in a given polypeptide. In addition, certain polypeptides may include many forms of variants. The term polypeptide as used herein includes all such variants, particularly those present in a recombinantly synthesized polypeptide by expressing the polynucleotide in a host cell.

As used herein, variant (s) of a polynucleotide or polypeptide are polynucleotides or polypeptides different from the reference polynucleotide or polypeptide, respectively. Variants in this sense are described in detail in some sections below herein. (1) a polynucleotide different from another reference polynucleotide in a nucleotide sequence. The nucleotide sequences of the reference and variants are very similar throughout and the differences are limited to be identical at many sites. As described below, changes in the nucleotide sequence of the variant may be silent. That is, the amino acid encoded by the polynucleotide may not be altered. If the alteration is limited to this form of quiet change, the variant will encode a polypeptide having the same amino acid sequence as the reference polynucleotide. In addition, as described below, the nucleotide sequence of a variant can be altered to alter the amino acid sequence of a polypeptide encoded by a reference polynucleotide. Such nucleotide changes can result in amino acid substitutions, additions, deletions, fusions and cleavage in the polypeptides encoded by the reference sequences, as described below. (2) A polypeptide different from another reference polypeptide in the amino acid sequence. The sequences of the reference polypeptides and variants are extremely similar throughout, and differences are generally limited to be identical at many sites. Variants and reference polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions, fusions, and truncations, which may be present in any combination.

The present invention relates to a therapeutic use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a combination thereof. The invention also relates to fragments of the polynucleotide sequence encoding biologically active fragments of NAB1 or NAB2 polypeptides, or variants of polynucleotides encoding functional, ie biologically active fragments of NAB1 or NAB2 by degeneracy of the genetic code. For therapeutic use and functionally equivalent allelic variants and related sequences modified by simple or multiple base substitutions, additions and / or deletions encoding polypeptides having NAB1 or NAB2 activity.

These can be obtained by standard cloning procedures known to those skilled in the art.

The polynucleotides encoding NAB1 or NAB2 transcription inhibitory proteins may be in the form of DNA, cDNA or RNA, eg mRNA, obtained by cloning or produced by chemical synthesis techniques. DNA can be single or double stranded. The single stranded DNA may be coding or sense strands, or may be non-coding or antisense strands. As a therapeutic use, the polynucleotide is present in a form that can be expressed as a functional NAB1 or NAB2 transcription repression protein at the wound site of the subject to be treated. Polynucleotides can also be used for ex vivo production of NAB1 or NAB2 polypeptides, which are administered in a further therapeutic aspect of the invention as described below.

Polynucleotides of the invention encoding a polypeptide of NAB1 or NAB2 include, but are not limited to, coding sequences for NAB1 or NAB2 polypeptide or biological fragments thereof. Thus, polynucleotides are transcribed and untranslated, which play a role in, for example, additional noncoding 5 'and 3' sequences such as transcription (e.g. including termination signals), ribosomal binding, mRNA stable components. Sequences, and additional coding sequences that encode additional amino acids, such as additional noncoding sequences, including but not limited to providing additional functionality. In addition, polynucleotides of the present invention include, but are not limited to, polynucleotides comprising structural genes for NAB1 or NAB2 and naturally related genetic components thereof.

As mentioned above, "polynucleotide encoding a polypeptide" as used herein includes a polynucleotide comprising a sequence encoding a polypeptide of NAB1 or NAB2. The term refers to a single contiguous site or discontinuous site (e.g., integrated phase or insertion sequence or editing), with additional sites that may also contain coding and / or non-coding sequences. Polynucleotides);

Furthermore, the present invention relates to variants of said polynucleotides encoding fragments, analogs and derivatives of the polypeptide. Variants of the polynucleotides may be naturally occurring variants, for example naturally occurring allelic variants, or may be variants known to not occur naturally. Such non-naturally occurring variants of polynucleotides can be prepared by mutagenesis techniques, including those applied to polynucleotides, cells, or organisms.

One of the variants consistent with this aspect is a variant that is distinguished from the polynucleotide by nucleotide substitution, deletion or addition. Substitutions, deletions or additions may include one or more nucleotides. Variants may be modified at the coding or non-coding site or both. Modifications at the coding site can produce conservative or non-conservative amino acid substituents, deletions or adducts.

A further preferred embodiment of the invention relates to polynucleotides encoding polypeptides having said amino acid sequences and to polynucleotides at least 70% of the total length of which is complementary to these polynucleotides. Optionally, most preferred is at least 80% of the total length of the polynucleotide encoding a polypeptide of the invention. In this respect, polynucleotides of at least 90% of the total length are particularly preferred, and particularly preferably at least 95% of these particularly preferred polynucleotides. Furthermore, it is very preferable that more than 97% are the same among the same 95% or more, It is very preferable that 98% or more are the same among these, It is especially very preferable that 99% or more are the same.

Moreover, preferred embodiments in this respect are polynucleotides that retain almost the same biological function or activity as mature NAB1 or NAB2 polypeptides encoded by the DNA sequences (GenBank accession numbers U47007 and U48361).

Furthermore, the present invention relates to polynucleotides that hybridize to such sequences. In this respect, the present invention relates to polynucleotides that hybridize to such polynucleotides under stringent conditions. As used herein, the term "strict conditions" means that hybridization can occur if there is at least 95%, preferably at least 97%, identity between sequences. In this way, the sequences that hybridize to the sequences of the present invention preferably encode polypeptides having a biological activity of NAB1 or NAB2.

The polynucleotide may encode a mature protein and a polypeptide that is an additional amino or carboxyl-terminal amino acid. Such additional sequences can extend or reduce certain roles, for example, protein half-life, or can facilitate manipulation of proteins for testing or production. Generally in vivo, additional amino acids can be separated from mature proteins by cellular enzymes.

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

The polynucleotide encoding NAB1 or NAB2 is administered to a method of the present invention by gene therapy in which the polypeptide is administered to a wound site or other tissue in a form that can induce the production of NAB1 or NAB2 or a biologically active fragment thereof in situ. It can be used therapeutically.

For example, administering the polynucleotide to be expressed in a subject to be treated in the form of a recombinant DNA molecule comprising a polynucleotide encoding NAB1 or NAB2 operably linked to a nucleic acid sequence that controls expression in an expression vector. Preferred in gene therapy. Thus, such vectors will include transcriptional control signals that can express coding sequences and include promoters that act in the subject to be treated. Thus, for human gene therapy, a promoter (the term includes sequences necessary for inducing RNA polymerase to a transcription initiation site, as well as operational or regulatory sequences, including an enhancer, if appropriate) is a human gene or It is preferred that it is a human promoter sequence from a gene that is typically expressed in humans, for example a promoter from human CMV. Known as suitable eukaryotic promoters in this respect are the CMV immediate early expression promoter, the HSV tamidin kinase promoter, early and late SV40 promoters, promoters of retroviral LTRs such as Loose sarcoma virus ( RSV) promoter and metallothionein, such as the mouse metallothionein-1 promoter.

Polynucleotide sequences and transcriptional control sequences can be provided in cloned form into a commercially available plasmid, eg, a replicable plasmid vector based on pBR322, or can be prepared from plasmids available by routine application of known and published procedures. .

In addition, the vector contains a transcriptional regulatory signal located 3 ′ to the NAB1 or NAB2 coding sequence and a corresponding polyadenylation signal recognizable in the subject to be treated, such as a corresponding sequence from a virus such as the SV40 virus for treating humans. Include. Other transcriptional regulatory sequences are known in the art and can be used.

In addition, the expression vector includes a selectable marker capable of growing the vector, such as antibiotic resistance.

Expression vectors capable of synthesizing NAB1 or NAB2 in situ can be introduced directly to the wound site by physical methods. Such examples include topical administration of “naked” nucleic acid vectors in a solution in a suitable excipient, for example, a pharmaceutically acceptable excipient such as phosphate buffered saline (PBS), or methods known in the art. US Pat. No. 5371015, which accelerates at a rate sufficient to pass through the surface of a wound by releasing inert particles such as, for example, gold beads coated with a vector from a projecting device under high pressure. Administration of the vector by particle bombardment (or also known as a "gene gun" technique).

Other physical methods of administering DNA directly to the receptor include ultrasound, electrical stimulation, and electroporation.

Nucleic acid sequences encoding NAB1 or NAB2 for use in the treatment of the present invention may also be administered by a delivery vector. Such viruses include viral delivery vectors, such as adenovirus or retrovirus delivery vectors known in the art.

Other nonviral delivery vectors include lipid delivery vectors, including known liposome delivery vehicles.

Nucleic acid sequences encoding NAB1 or NAB2 can also be administered to the wound site by modified host cells. Such cells include cells harvested from a subject, wherein the nucleic acid sequence is introduced into the subject by gene delivery methods known in the art, and then the modified cells are grown in the medium and transplanted into the subject.

Expression constructs such as those described above can be used in various therapies of the invention. Thus, the constructs of the present invention can be used to administer directly to a wound site of a subject, or after removal of the recombinant NAB1 or NAB2 polypeptide itself, and then to the wound site as described in detail below. In addition, the invention provides host cells genetically engineered with NAB1 or NAB2 polynucleotides or polynucleotides of the invention or constructs comprising the genetic components defined above, and the use of these vectors and cells for use in the methods of treatment of the invention. It is about. This construct may be used by itself to prepare a NAB1 or NAB2 polypeptide for use in the therapeutic methods of the invention or for use in the therapeutic methods of the invention as described in detail below.

The vector is for example a plasmid vector, single or double stranded vector or double, depending on whether the vector is administered directly to the wound site (ie in case of synthesis of NAB1 or NAB2 in situ) or used to synthesize recombinant NAB1 or NAB2. Strand RNA or DNA viral vectors. The starting plasmids disclosed herein can be prepared from plasmids that are commercially or publicly available, or are available by routine application of known and published procedures. Many plasmids and other cloning and expression vectors usable in accordance with the present invention are known and readily available to those skilled in the art.

Vectors for expressing NAB1 or NAB2 polypeptides for use in the present invention comprise cis-acting sites that are effective for expression in a host operably linked to the polynucleotide to be expressed. Suitable trans agonists are supplied by the host cell or complementary vector, or by the vector itself immediately after introduction into the host cell.

In this aspect, in some embodiments, the vector is provided for specific expression. To produce recombinant NAB1 or NAB2, such specific expression may be inducible expression, or expression only in specific types of cells, or expression in inducible and specific cells. Particularly preferred among inducible expressions are vectors which can be expressed by easy to manipulate environmental factors such as temperature and nutrient additives. Constitutive and inducible expression vectors for use in prokaryotic and eukaryotic hosts are 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. Such vectors include, in particular, chromosomes, episomes and viral induction vectors such as bacteriophages, transposons, yeast episomes, insertion components, yeast chromosomal components, baculovirus, papova virus, SV40, Vectors derived from viruses such as baccinia virus, adenovirus, chicken pox virus, rabies virus and retrovirus, and vectors derived from combinations thereof, such as plasmids and bacteriophages, cosmids and phagemids Vectors may be cited, and all such vectors may be used for expression according to this aspect of the invention. Any vector suitable for maintaining, growing or expressing a polynucleotide to express a polypeptide in a host cell can be used for expression in this respect.

Suitable DNA sequences are one of a variety of known and conventional techniques, including, for example, Sambrook et al., MOLECULAR CLONING, A LABORATORY MANUAL, 2nd Ed .; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989).

Nucleic acid sequences in an expression vector can be operably linked with appropriate expression control sequence (s), including, for example, a promoter for inducing mRNA transcription. Representative examples of such promoters include the phage lambbda PL promoter for recombinant expression, the E. coli lac, trp and tac promoters, and the promoters of early and late SV40 and retroviral LTRs for in situ expression. But it is not limited thereto.

Expression constructs may include transcription initiation and termination sites, and ribosomal binding sites for translation at the transcribed site. The coding portion of the mature transcript expressed by the construct will include a translation that initiates AUG at the initiation and termination codons that are suitably positioned at the ends of the polypeptide to be translated.

Moreover, the construct may contain regulatory sites that not only induce expression but also regulate expression. In general, according to the many procedures practiced, such sites will work, in particular, by controlling transcription (eg, transcription factors), inhibitory binding sites, and terminations.

Vectors for growth and expression include selectable markers and amplification sites, such as 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 or NAB2 include bacterial cells (eg, streptococci, staphylococci, E. coli, Streptomyce and Bacillus subtilis). Cells], fungal cells (eg yeast cells and Aspergillus cells), insect cells (eg Drosophila S2 and Spodoptera Sf9 cells), animal cells [For example, CHO, COS, HeLa, C127, 3T3, BHK, 293 and Bowes melanoma cells] and plant cells.

The following commercially available vectors are provided by the methods of the examples. Preferred vectors for use in bacterium are pQE70, pQE60 and pQE-9 (available from Kiagen), Phagescript vector, Bluescript vector, pNH8A, pNH16a, pNH18A, pNH46A (stratagen Available from) and pptrc99a, pKK223-3, pDR540, pRIT5 (available from Pharmacia) and pBR322 (available from ATCC 37017). Preferred among eukaryotic cells include pWLNEO, pSV2CAT, pOG44, pXT1 and pSG (available from stratagen) and pSVK3, pBPV, pMSG and pSVL (available from Pharmacia). These vectors that can be used in both recombinant expression and expression in situ are only listed by way of example of many commercially known and known vectors available to those skilled in the art for use consistent with this aspect of the invention. Importantly, any other plasmid or vector suitable for, for example, introduction, maintenance, growth or expression of a polynucleotide or polypeptide of the invention into a host cell is usable in this aspect of the invention.

Vectors for use in this aspect of the invention include expression vectors in which NAB1 or NAB2 cDNA sequences are inserted into the plasmid, whereby gene expression is derived from human early early expression cytomegalovirus enhancer-promoter (Foecking and Hofstetter, Cell, 45, 101-105, 1986). Such expression plasmids can include SV40 RNA processing signals such as polyadenylation and termination signals. Using the CMV promoter, commercially available expression constructs are pCDM8, pcDNA1 and derivatives (invitrogen). Other expression vectors available and available are pSVK3 and pSVL (vectors from Pharmacia) comprising the SV40 promoter and mRNA splice sites and polyadenylation signals from the SV40 and SV40VP1 processing signals.

The downstreem of a reporter transcription unit without a promoter site, for example, a chloramphenicol acetyl transferase (CAT) transcription unit, a restriction point or a point for introducing a candidate promoter fragment (ie, a fragment that may include a promoter) Including vectors can be used to select promoter sites from any desired gene. As is known, introduction of a promoter containing fragment into the upper restriction point of the cat gene can generate CAT activity, which activity can be eliminated by standard CAT tests. Suitable vectors for this purpose are known and are readily available, such as pKK232-8 and pCM7. The vector for expressing the polynucleotide of the present invention includes a promoter which can be easily obtained by the above technique using a reporter gene, in addition to a known and readily available promoter, and for in situ expression, such a promoter is preferred in the subject to be treated. Should be recognized.

Among the prokaryotic promoters suitable for expression of the polynucleotides and polypeptides of the invention are the E. coli lacl and lacZ and promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR, the PL promoter and the trp promoter.

Recombinant expression vectors include promoters, preferably derived from highly expressed genes to induce transcription of underlying structural sequences, and selectable markers that allow separation of cell containing vectors after exposure to the vector.

Heterologous structural sequences of the polypeptides of the invention will generally be inserted into the vector using standard techniques so that they can be operably linked to and expressed in the promoter. The polynucleotide will be positioned such that the transcription site is located about 5 'to the ribosomal binding site. The ribosomal binding site will be 5 'to the AUG that initiates the transcription of the polypeptide to be expressed.

It will be common for the initiation codon, usually starting with AUG, to be free of other open reading frames located between the ribosome binding site and the initiation codon. In addition, translation termination codons will generally be present at the terminal end of the polypeptide, and polyadenylation will be present in the construct for use in a eukaryotic host. Transcription termination signals suitably located at the 3 ′ end of the transcribed site may also be included in the polynucleotide construct.

In order to secrete the translated protein into lumen, protoplasty or extracellular environment of the endoplasmic reticulum, a secretion signal suitable for recombinant synthesis can be incorporated into the expressed polypeptide. These signals may be endogenous to the polypeptide or may be heterologous signals.

The polypeptide may be expressed in a modified form, eg, a fusion protein, and may further comprise heterologous functional sites in addition to the secretion signal. Thus, for example, sites of additional amino acids, especially charged amino acids, can be added to the N- or C-terminus of the polypeptide to enhance stability and durability in the purification or subsequent handling and storage steps. In addition, the site can be added to the polypeptide to facilitate purification. In particular, the addition of peptide moieties to polypeptides to trigger secretion, enhance stability, or facilitate purification is well known and conventional in the art. The fusion protein preferably comprises a heterologous site from immunoglobin useful for dissolving and purifying the polypeptide. Cells are then typically harvested by centrifugation, destroyed by physical or chemical means, and the resulting extracts are maintained for further purification.

Microbial cells used in protein expression can be disrupted using any convenient method, including freeze thaw circulation, sonication, mechanical disruption, or cell lysates, which methods are known to those skilled in the art.

Mammalian expression vectors include a replicator, a suitable promoter and enhancer, and any necessary ribosome binding points, polyadenylation sites, splice donor and receptor sites, transcription termination sequences, and non-transcribed sequences located on the 5 'side necessary for expression. can do.

Genetically engineered host cells can be used to prepare NAB1 or NAB2 polypeptides for use in the present invention. Upon introduction of polynucleotides into host cells, calcium phosphate transfection, DEAE-dextran mediated transfection, transfection, microinjection, cationic lipid mediated transfection, electroporation, transduction, scrap loading It may be affected by scrape loading, ballistic introduction, infection and other methods. Such methods are described in many standard laboratory manuals, such as, for example, Davids et al., BASIC METHODS IN MOLECULAR BIOLOGY, (1986) and Sambrooke et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989).

Mature proteins can be expressed in host cells, including mammalian cells, such as CHO cells, yeasts, bacteria or other cells by regulating suitable promoters. In addition, translational systems in the absence of cells can be used to produce mature proteins using RNA derived from the DNA constructs of the invention. Suitable cloning and expression vectors for use in 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, N.Y. (1989).

Polypeptides are known by methods such as ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography. Can be recovered and purified from recombinant cell medium. Most preferred is the use of high performance liquid chromatography for purification purposes. Upon denaturation of the polypeptide during separation and / or purification, known techniques may be used to refold the protein to regenerate the active conformation.

For therapeutic purposes, for example, polynucleotides encoding NAB1 or NAB2 in the form of recombinant vectors are known in the art, such as, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUNNAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)] can be used to purify using tube chromatography.

As noted above, the NAB1 or NAB2 polypeptide may be administered to the wound site as a nucleic acid encoding NAB1 or NAB2 that is transcribed and translated into NAB1 or NAB2 in a gene therapy form at the wound site, or the transcription repression protein itself may be administered directly. have.

Thus, in a further aspect of the invention, the invention relates to the use of a NAB1 or NAB2 polypeptide or a biological fragment thereof for use in the manufacture of a medicament for the treatment of cell proliferative diseases in mammals, including humans.

In a further aspect of the invention, a method is provided for treating a cell proliferative disease in a mammal comprising administering to a mammal, including a human, a therapeutically effective amount of NAB1 or NAB2 polypeptide or a biological fragment thereof.

Accordingly, in a further aspect, the present invention provides the use of a NAB1 or NAB2 polypeptide or active fragment thereof for use in the treatment and healing of a wound.

As used herein, "NAB1 or NAB2 polypeptide" means a naturally and recombinantly produced NAB1 or NAB2, a natural, synthetic and biologically active polypeptide analog or variant or derivative thereof or a biologically active fragment and variant thereof, a derivative of the fragment. And variants.

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

NAB1 or NAB2 and the fragments and derivatives thereof for use in the treatment of the present invention can be extracted from natural sources by methods known in the art. Such methods may be performed by sequence specific DNA affinity chromatography using methods such as those described in Briggs et al, Science 234, 47-52, 1986 and using DNA binding oligonucleotides that recognize NAB1 or NAB2. Tablets are included. In addition, polypeptides can be prepared using methods of recombinant DNA techniques known to those skilled in the art, such as those described above, ie, expression in host cells of the construct. Alternatively, the polypeptides of the present invention can be produced synthetically by conventional pentide synthesizers.

The present invention also relates to the use of fragments, analogs and derivatives of NAB1 or NAB2. The terms "fragment", "derivative" and "analogue" refer to a polypeptide having the biological function or activity necessary as the polypeptide. Thus, analogs include proteins that can be cleaved and activated at portions of the protein to produce active mature polypeptides.

Fragments, derivatives, or analogs of the polypeptide include (i) one or more amino acid residues are replaced with conserved or non-conserved amino acid residues (preferably with conserved amino acid residues), and such substituted amino acid residues are replaced by a genetic code. May or may not be encoded or (ii) one or more amino acid residues may contain substituents, or (iii) other compounds for which the mature polypeptide increases the half-life of the polypeptide (e.g., polyethylene Glycol), or (iv) an additional amino acid is fused to a mature polypeptide, such as a leader or secretory sequence, or a sequence used to purify a mature polypeptide or protein sequence. Such fragments, derivatives and analogs are within the scope of those skilled in the art from the teachings herein.

Preferred of the variants include mutations from naturally occurring NAB1 or NAB2 by conservative amino acid substitutions. Such substituents include substituting predetermined amino acids in a polypeptide by other amino acids having similar characteristics. Typical conservative substitutions include replacement of aliphatic amino acids Ala, Val, Leu and lle one by one, interchange of the hydroxyl residues Ser and Thr, and exchange of the acidic residues Asp and Glu. ), Substitution of Asn and Gln as amide residues with each other, exchange of Lys and Arg as basic residues, and substitution at Phe and Tyr as aromatic residues.

Particularly preferred in this respect, further examples include amino acids of polypeptides in which several, minor, 5 to 10, 1 to 5, 1 to 3, 2, 1 or 0 amino acid residues are substituted, deleted or added in any combination. Variants, analogs, derivatives and fragments having a sequence, and variants, analogs and derivatives of the fragments. Particularly preferred among these are silent substitutions, additions and deletions that do not alter the properties and activities of the polypeptides of the invention. Particularly preferred in this respect are also conservative substitutions.

Particularly preferred fragments are biologically active fragments, ie fragments which retain the wound healing properties of the parent polypeptide.

The polypeptides and polynucleotides of the present invention are preferably provided in isolated form and are preferably purified to be homogeneous.

NAB1 or NAB2 polypeptides used in the present invention have at least 70% identity, preferably at least 80% identity, more preferably at least 90% identity, even more preferably at least 95% identity (more More preferably in addition to polypeptides having at least 95% identity) and also include portions of a polypeptide comprising a NAB1 or NAB2 polypeptide, and generally comprising at least 30 amino acids and more preferably at least 50 amino acids.

Fragments or portions of polypeptides of the invention can be used to produce polypeptides of corresponding intact lengths by peptide synthesis. Thus, fragments can be used as intermediates to produce polypeptides of intact length. Fragments or portions of the polynucleotides of the invention can be used to synthesize polynucleotides of the entire length of the invention.

The invention also relates to the use of fragments and variants of NAB1 or NAB2 polypeptides as defined above and the derivatives thereof.

In this respect, fragments are polypeptides in which some but not all of the amino acid sequences of NAB1 or NAB2 polypeptides and variants or derivatives thereof have amino acid sequences that are completely identical.

Such fragments may not be “free-standing”, ie, part of, or fused to, other amino acids or polypeptides, and may be included in larger polypeptides that form certain portions or sites. When included in larger polypeptides, the fragments currently discussed most preferably form a single contiguous site. For example, certain preferred embodiments of the invention include a pre- and pro-polypeptide fused to an amino terminus of a fragment and an additional site fused to the carboxyl terminus of a fragment, which is included in a precursor polypeptide designed for expression in a host. A fragment of a polypeptide of Thus, in one sense intended herein, a fragment relates to a portion (s) of a fusion polypeptide or fusion protein derived from a polypeptide of the invention.

In addition, fragments characterized by the structural or functional properties of the polypeptides of the invention are preferred in this aspect of the invention. In this respect, preferred embodiments of the present invention include alpha-helix and alpha-helix forming sites, beta-sheets and beta-sheet forming sites, turn and turn-forming sites, coils and coils- of the present invention. Fragments comprising a formation site, a hydrophilic site, a hydrophobic site, an alpha amphoteric site, a beta amphoteric site, a flexible site, a surface forming site, a substrate binding site, and a high antigenicity indicator site and combinations thereof.

The site preferably mediates the activity of the polypeptides of the invention. Most preferred in this respect are fragments having chemical, biological or other activity of the polynucleotides of the present invention, including those having similar or increased activity, or unwanted reduced activity. Further examples of preferred polypeptide fragments are those comprising antigenic or immunogenic determinants in animals, especially humans.

The invention also relates to polynucleotides, such as polynucleotides encoding said fragments, polynucleotides hybridizing to polynucleotides encoding fragments, in particular polynucleotides hybridizing under stringent conditions and PCR primers for amplifying polynucleotides encoding fragments. You will recognize that it is about. In this respect, the polynucleotide preferably corresponds to the preferred fragment as described above.

In this aspect of the invention, further embodiments include variants, analogs or derivatives thereof that are biologically, prophylactically, clinically or therapeutically useful, including fragments of variants, analogs and derivatives and compositions comprising the same. Biologically active variants, analogs or fragments are within the scope of the present invention.

The present invention also relates to a composition comprising said polynucleotide or polypeptide. Accordingly, the polynucleotides or polypeptides of the present invention can be used with pharmaceutically acceptable carrier (s).

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

Polypeptides and polynucleotides may be used in the present invention alone or in combination with other compounds, such as therapeutic compounds.

The pharmaceutical compositions can be administered using effective and conventional methods to target the wound site, including, for example, by topical, intravenous, intramuscular, intranasal or subcutaneous routes.

As a therapeutic or prophylactic agent, the active agent can be administered to the subject as an injectable composition, for example, a sterile aqueous acid solution, preferably isotonic.

Optionally, the composition may be formulated for topical administration, eg in the form of ointments, creams, lotions, eye ointments, eye drops, ear drops, water solubles, impregnated dressings and sutures and aerosols, for example preservatives, Conventional and suitable additives may be included, including solvents to aid drug penetration and emollients in ointments and creams. Such topical formulations may also contain conventional compatible carriers, such as creams or ointment bases, and ethanol or oleyl alcohols for lotions. Such carriers comprise about 1 to 98% by weight of the formulation, and typically up to about 80% by weight of the formulation.

For administration to mammals, especially humans, it is expected that the daily dosage of the active agent will be 0.01 to 10 mg / kg, typically about 1 mg / kg. In the end, however, the physician will determine the actual dosage that will be best suited for a particular subject, which will vary depending on the age, weight and response of the particular subject. The dosages illustrate the average case. Of course, there may be cases where a higher or lower dose is desired for each individual than the upper or lower limit, and such cases are also within the scope of the present invention. Eventually, the dosage chosen by those skilled in the art will act to reduce cell proliferation without interfering with wound healing.

In a further aspect, a pharmaceutical composition comprising a NAB1 or NAB2 polypeptide, or a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, is provided with one or more pharmaceutically acceptable carriers thereof.

The therapeutic benefit of healing wounds using transcription inhibitory proteins lies in the inactivation of multiple target genes that promote accelerated wound healing. NAB1 or NAB2 is naturally activated in response to a wound, and an increased natural response is also an advantage of the present invention. The treatment is based on DNA and provides a reliable and reproducible delivery system.

When a NAB1 or NAB2 polynucleotide is used in the therapeutic methods of the invention, the polynucleotide is available as part of an expression construct, eg in the form of an expression vector. In such a method, the construct is introduced at the wound site where NAB1 or NAB2 is produced in situ. Constructs used may be standard vectors and / or gene delivery systems such as liposomes, receptor mediated delivery systems and viral vectors.

Wound healing, including limb ulceration, postoperative scars, burns, psoriasis, restenosis after trans-luminal coronary angioplasty, regulation of vascular wall calcification and inhibition of cancer cell proliferation in diabetic and peripheral arterial obstructive diseases Suitable for all aspects of

As noted above, the NAB1 or NAB2 polypeptides or nucleic acids of the invention can be administered locally to the site of injury by any conventional method, such as topical administration. Preferred among methods of delivering nucleic acid products is the use of gene gun techniques, i.e., in which Egr-1 isolated nucleic acid proteins, e.g., cDNA form or expression vector, are immobilized on gold particles and administered directly to the wound site. Thus, in a preferred aspect of the present invention, there is provided the use of a nucleic acid protein comprising a sequence encoding a NAB1 or NAB2 polypeptide, for treating a cell proliferative disease associated with wound healing. Further provided are compositions suitable for the treatment of gene guns comprising sequences encoding NAB1 or NAB2, immobilized on gold particles.

In the following, the invention will be described by the following non-limiting examples with reference to the accompanying drawings.

1a) -1d) show inhibition of Egr-1 mediated activity of PDGF-AB, TGF-β, HGF and VEGF, respectively.

2 shows NAB2 trans-inhibition of Egr-1 mediated HGF production in HVSMC.

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

4A shows the effect of NAB2 transfection on the contraction of the linear venous wound, on day 7 after wound development.

4B shows the effect of NAB2 transfection on the extent of growth factor in the epidermis of Ben wounds of rats on day 7 post wound development.

4C shows the effect of NAB2 transfection on the extent of growth factors in the slaughtered tissues of rat wounds on day 7 after wound development.

4D shows the effect of NAB2 transfection on angiogenesis in Ben wounds of rats on day 7 post wound development.

Example 1

Use of NAB2 to Inhibit Egr-1 Mediated Trans-Activation of Growth Factors

1.1 method

Human smooth muscle cells (HVSMC; clonetics) were cultured according to the manufacturer's recommendations. Cells were cultured for transfection in 6-well microtitre plates (Costa). Expression plasmids comprising NAB2 cDNA derived from the human cytomegalovirus promoter (hCMV; Svaren et al Mol. Cell. Biol., 16; 3545-3553, 1996) were designated as Egr-1 cDNA (Houston et al Arterioscler. Thromb). FUGENE (Boehringer Mannheim) was used to transfect HVSMC at the rates described in the figure with an expression plasmid comprising Vasc. Biol., 281-289, 1999). FUGENE: DNA was used in a 3: 1 ratio (v / w) for all experiments and transfection was normalized using CMV induced β-galactosidase expression plasmids. Secreted growth factors were detected in tissue medium by ELISA (R & D system) using appropriate controls.

1.2 Results

1A) to 1D) show inhibition of Egr-1 mediated activation of PDGF-AB, TGFβ, HGF and VEGF, respectively. 6 μg of Egr-1 expression plasmids were transfected alone (FIGS. 1A, 1B and 1C) or with 500, 100 or 25 ng NAB2 (FIGS. 1A and 1D). For PDGF-AB, TGFβ and VEGF, the detected amount of secreted growth factor slightly increased when transfected with Egr-1 alone. Co-transfection with NAB2 completely eliminated PDGF-AB's response to Egr-1 activation and reduced basal expression, resulting in a 5-fold reduction in total production of PDGF-AB (FIG. 1A). NAB2 transfection completely eliminated TGFβ's response to Egr-1 activation, resulting in a 30% decrease in total production of TGFβ (FIG. 1B). Using DNA concentrations such as those shown in the figure, the production of HGF was increased by 50% by transfecting Egr-1, but this was partially blocked by cotransfection with lower doses of NAB2, and covalently with higher doses of NAB2. It was completely blocked by transfection (FIG. 1C). Although the inhibitory effect of NAB2 is clear on days 2 and 3, maximal effect is seen on day 1 after transfection. As in PDGF-AB and TGFβ, NAB2 transfection blocked Egr-1 mediated activation of VEGF, resulting in a 40% decrease in the total production of VEGF (FIG. 1D).

1.3 Conclusion

From the data it can be seen that Egr-1 suppressor NAB2 can block Egr-1 mediated growth factor activation such as can be found at the site of acute trauma.

Example 2

Use of NAB2 to Inhibit Basal Levels of Growth Factors

2.1 method

Cell culture, transfection and detection of growth factors were performed as described in 1.1 above. NAB2 expression vectors were transfected to final concentrations of 0, 250, 500, 1000 or 3000 ng.

2.2 Results

As shown in FIG. 2A, transfection of NAB2 with HVSMC resulted in a sharp decrease in PDGF-AB production. At the maximum dose of NAB2, PDGF-AB decreased 10-fold.

2.3 Conclusion

These data show that Egr-1 suppressors can reduce basal levels of growth factors produced from PDGF-AB promoters.

Example 3

Use of NAB to Induce Induction of In Vitro Angiogenesis

3.1 method

NAB2 expression constructs (wild-type and dominant negative) derived from the hCMC promoter have been described in Svaren et al, EMBO J. 17; 6010-6019, 1998. We have shown that transfection of the Egr-1 transcription factor upon expression from the hCMV promoter promotes angiogenesis (patent application PG3412). In this experiment, DNA was transfected into the angiogenesis kit supplied and maintained according to the manufacturer's instructions (TCS biologic). VEGF protein (2ng / ml) was used as a positive control, and suramin (20 μm) was used as an angiogenesis inhibitor. CMV Egr-1 DNA was tripled in a microtitre plate with 24 wells using DNA and Myrus Transit reagent (Cambridge Bioscience) in a 2: 1 ratio (v / w), 0.5 per well The cells were transfected at μg. To assess the potential of NAB2 for inhibition of Egr-1 mediated angiogenesis, in the presence or absence of 100 ng NAB2 dominant negative expression plasmid, 0.5 mg of Egr-1 DNA was added to NAB 2 expression plasmid (12, 25 or 100 ng per well). Co-transfection). After 11 days of co-culture, angiogenesis was determined by staining cells for endothelial cell marker PECAM-1 and visualizing using BCIP / NBT substrate.

Representative images of tubule formation were included by administering four Egr-1 expression vectors with VEGF (positive control) and suramin (negative control) and analyzed using Quantimet 600 image analyzer and related software.

3.2 Results

Egr-1 DNA has been found to be pro-angiogenic as shown in Figures 3a, 4 of International Patent Application No. PCT.GB99.01722. As shown in FIG. 3A, cotransfection with NAB2 resulted in a dose-dependent decrease in angiogenic capacity of Egr-1, but this phenomenon was eliminated by co-transfection with NAB2 dominant negative.

3.3 Conclusion

NAB2 can be used to block angiogenesis in acute trauma, such as induction of growth factors by Egr-1.

Example 4

Use of NAB2 to Reduce Scars in Ben Wound Models in Rodents

4.1 Method

4.1.1 Effect of NAB2 Transfection with Gene Gun on Rat Growth Factor Levels in Ben Wounds

Transfection of NAB2 into Ben wounds in rodents results in a direct inhibitory action on the expression of the major scar growth factor, TGFβ1, whereby scars can be prevented. In this experiment, NAB2 cDNA was transfected into ben wounds of rats using a Biorad gene gun, and the effect on healing and growth factor levels was assessed using conventional histology and immunocytochemistry.

4.1.2 Particle-Mediated Gene Transfer

Eight Sprague Dawley rats weighing approximately 250 g were anesthetized with isofloran in a 2: 1 mixture of oxygen / nitrous oxide. First, the hides are cut and then shaved with a razor and two transfection sites (8 cm from the base of the skull and 1.5 cm from the centerline) and two control sites (8 cm from the base of the skull and from the center) on the rat's back. On one side 1.5 cm). Transfection was performed once at each transfection site by accelerating the plasmid / gold complex of NAB2 or Egr-1 (positive control for growth factor activation) to the skin at 350 psi. Typically, 0.5-1.5 μg of DNA per transfection is delivered. At each control site, gold particles (without DNA) were accelerated to the skin at 350 psi and the other control site was left unmanaged. The transfection site and control site were rotated clockwise in each additional animal to control differences in the anterior and posterior portions in the treatment of rodent wounds.

4.1.3 Venture Healing Model

24 hours after transfection, the animals were anesthetized and a wound scalpel of sufficient length equal to 1 cm in length was equilibrated with the spine using a surgical scalpel at the exact site to be transfected. The animals were woken up from anesthesia and allowed to heal without closing the wound. On the seventh day after wounding, all eight animals died, the wounds were dissected and harvested for conventional histology and immunocytochemistry.

4.1.4 Operational Analysis

At each time point after the incision, each wound site was bisected vertically. One nutrient portion was soaked in paraformaldehyde (4%) for 24 hours and treated for wax histology. 5 mm sections from each wound were cut using a microtome and the sections stained with van Geison. Sections were observed using an optical microscope and the effect of NAB2 on the healing response was evaluated.

4.1.5 Immunocytochemistry

Once frozen in OCT, the other half of each wound site was cut to 7 mm using a cooler. Two sections from each wound site were fixed in ice-cold acetone and fluorescent immunostaining was performed with primary antibodies against Egr-1, PDGF, TGFβ, TGFβ and vWF using the following protocol.

1. Wash slides with PBS

2. Apply 30 ml of primary antibody for 1 hour

3. Wash 3 times for 5 minutes in PBS

4. Apply 30 ml of secondary antibody (directly bound to FITC) for 45 minutes

5. Wash 3 times for 5 minutes in PBS

6. Create a slide using an aqueous mount

Immediately after immunostaining, each slide was placed under a fluorescence microscope and wound sites were examined using a × 100 magnification. The images were intergrated and the threshold set to minimize background. Staining area and intensity were measured using phase analysis and plotted graphically.

4.2 Results

4.2.1 Effect of NAB2 on Venture Healing in Rats

4.2.1.1 Wound contraction and histology 7 days after wound development

According to FIG. 4A, wounds treated with NAB2 contracted to a similar width as the Egr-1 transformed control and showed histologically similar slaughter tissue maturity.

conclusion

Delivery of NAB2 does not reduce the healing rate in rodent wound healing models.

4.2.1.2 Growth Factor Profile

Immunostaining was performed on Egr-1, PDGF, TGFβ, TGFβ, and PDGF-AB on frozen sections of skin tissue at the wound site, and staining intensity for each growth factor was measured by phase analysis. Immunohistochemical analysis was performed twice each at the wound site. Sections were tested for growth factor expression in the epidermis just above the wound and in the wound (sac tissue).

a) positive staining for growth factors in the epidermis

As shown in FIG. 4B, 7 days after wounding, NAB2 transfection significantly reduced the expression of TGFβ1 on the epidermis compared to the control of Egr-1 and gold alone. Compared to the unmanaged control, NAB2 transfection reduced the expression of TGFβ1 on the epidermis, whereas both Egr-1 and gold shear activated the production of TGF β1. NAB2 transfection increased the concentration of TGFβ3 on the epidermis compared to gold alone and untreated controls. NAB2 transfection did not significantly alter epidermal expression of Egr-1 and PDGF.

b) positive staining for growth factors in slaughter tissue

As shown in FIG. 4C, at day 7 after wound development, NAB2 transfection did not affect the concentrations of Egr-1, PDGF-AB and TGFβ1 in slaughter tissue. However, NAB2 increased the concentration of TGFβ3 in slaughter tissue compared to gold and the untreated control.

conclusion

On day 7 post wound development, NAB2 transfection of Ben wounds reduced TGFβ1 concentrations in the epidermis and increased TGFβ1 concentrations in epidermal and anal tissues. TGFβ1 is a known scarring agent and TGFβ3 has antiscarring properties (Shan et al J. Cell Science 107; 1137-1157, 1994). Thus, delivery of NAB2 has antiscarring properties.

4.2.2 Effect of NAB2 on Angiogenesis

Seven days after wound development, skin sections were stained and scored for vWF expression using immunocytochemistry and phase analysis. Von Willebrand factor immunostaining and phase analysis of the wound site were used to quantify angiogenesis and determine the area of positive staining within the wound site. As shown in FIG. 4D, seven days after wound development, NAB2 transformed wounds had less new blood vessels than the control (gold treated wound). Egr-1 promoted angiogenesis in vivo and supported the ex vivo results in Example 3.

conclusion

NAB2 prevented Egr-1 stimulatory activation of angiogenesis in vivo and supported the role of NAB2 as an inhibitor of growth factor activation when induced by stimulation, for example Egr-1 activation of growth factor production. .

Claims (22)

Use of a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a fragment thereof having a biological activity for use in the manufacture of a therapeutic for a cell proliferative disease associated with wound healing in a mammal, including humans. The use of claim 1, wherein the NAB1 or NAB2 polypeptide is a human NAB1 or NAB2 polypeptide. Use according to claim 1 or 2, wherein the cell proliferative disease associated with wound healing is hypertrophic keloid scarring. The use according to any one of claims 1 to 3, wherein said nucleic acid molecule is operably linked to a nucleic acid sequence as long as it regulates expression. 5. The use according to claim 1, wherein at least 70%, 80%, 90% or 95% of the nucleic acid molecule is identical to the NAB1 or NAB2 polypeptide sequence. 6. 6. The use according to claim 1, comprising a combination of nucleic acid molecules comprising sequences encoding NAB1 polypeptides and NAB2 polypeptides, or fragments thereof having biological activity. 7. 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 fragment thereof having biological activity. 8. Use according to any of the preceding claims, wherein the nucleic acid molecule is arranged to be administered to a mammal by a physical method. The use of claim 8, wherein the nucleic acid molecule is arranged to be administered to the mammal by particle bombardment. 10. The use according to claim 9, wherein said nucleic acid molecule is immobilized on gold particles. The use of claim 8, wherein the nucleic acid molecule is arranged to be administered by microseeding. 8. Use according to any one of the preceding claims, wherein the nucleic acid molecule is in a vector. The use according to any one of claims 1 to 7, wherein said nucleic acid molecule is in a cell. A nucleic acid molecule for use in gene therapy, comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a fragment thereof having biological activity. A pharmaceutical composition comprising a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide, or a fragment thereof having biological activity, and at least one pharmaceutically acceptable carrier. A method of treating a cell proliferative disease associated with wound healing in a mammal comprising administering to a mammal, including a human, a nucleic acid molecule comprising a sequence encoding a NAB1 or NAB2 polypeptide or a fragment thereof having a biological activity. Use of a NAB1 or NAB2 polypeptide, or fragment thereof having biological activity, for use in the manufacture of a therapeutic for a cell proliferative disease associated with wound healing in a mammal, including humans. 18. The use according to claim 17, wherein the NAB1 or NAB2 polypeptide, or fragment thereof having biological activity, is produced naturally, produced synthetically, or produced recombinantly. The use according to claim 17 or 18, wherein said NAB1 or NAB2 polypeptide is a human NAB1 or NAB2 polypeptide. The use of claim 17, wherein the polypeptide is at least 70%, 80%, 90% or 95% of its full length identical to the NAB1 or NAB2 polypeptide sequence. A method of treating a cell proliferative disease associated with wound healing in a mammal comprising administering to a mammal, including a human, a therapeutically effective amount of a NAB1 or NAB2 polypeptide, or a fragment thereof having biological activity. A pharmaceutical composition comprising NAB1 and / or NAB2 polypeptide, or a fragment thereof having biological activity, and one or more pharmaceutically acceptable carriers.