# Patent

## Title
Perlecan and growth factor for wound and cutaneous injury healing

## Abstract

The present disclosure describes the use of the heparan sulfate proteoglycan perlecan as treatment for the improvement of wound healing and/or cutaneous injury.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
The present disclosure is a continuation of U.S. patent application Ser. No. 11/604,633, filed Nov. 27, 2006, which is a continuation of U.S. patent application Ser. No. 10/420,270, filed Apr. 22, 2003, now U.S. Pat. No. 7,141,551, issued Nov. 28, 2006, which claims benefit of U.S. Provisional Patent Application No. 60/374,553, filed Apr. 22, 2002.


FIELD OF DISCLOSURE
The present disclosure relates to biomolecules involved in wound healing and angiogenesis.
BACKGROUND
Perlecan, originally named heparan sulfate proteoglycan, is now known to be an important component of all basement membranes (along with collagen type IV and laminin) and is thought to play a role in wound healing and angiogenesis. Perlecan consists of three heparan sulfate side chains linked to a large core protein of approximately 450 kDa (32, 36). This sequence has a single open reading frame of at least 3,707 amino acids that encodes for a protein of 396-466 kDa. Sequence analysis of the deduced sequences show the protein consists of five different domains, most of which contain internal repeats. Domain I contains a start methionine followed by a typical signal transfer sequence and a unique segment of 172 amino acids that contains the three probable sites of heparan sulfate attachment (of the amino acid sequence SGD). Domain II contains four strictly conserved cysteine-rich and acidic amino acid repeats that are very similar to those found in the LDL receptor and proteins such as megalin/GP330. Domain III consists of cysteine-rich and globular regions, both of which show similarity to those in the short arm of the laminin A chain. Domain IV contains 14 repeats of the immunoglobulin superfamily that are most highly similar to the immunoglobulin-like repeats in the neural cell adhesion molecule, and it appears domain IV has the capacity for differential splicing. Recombinant domain IV of perlecan binds to nidogens, laminin-nidogen complex, fibronectin, fibulin-2 and heparin (24). Domain V is the COOH-terminal domain and contains three repeats with similarity to the laminin A chain G domain. The repeats are separated by epidermal growth factor-like regions not found in the laminin A chain. Perlecan domain V is considered important in the supramolecular assembly of, and cell connections to, basement membranes (5).
The primary structural data agree with the appearance of the molecule in the electron microscope as a series of globules separated by rods, or “beads on a string.” Therefore, the name perlecan was adopted for this molecule (36). In summary, the variety of domains in perlecan suggests multiple interactions with other molecules, and each domain of native perlecan has the potential for separate functional activities related to wound healing and/or angiogenesis.
Expression of Perlecan
Native perlecan has been purified from the mouse Englebroth Holm Swarm (EHS) tumor (22), porcine kidneys (16), bovine kidneys (21), from the conditioned medium of bovine aortic endothelial cells (39), and from the extracellular matrix of cultured human fetal lung fibroblasts (2). Other sources of perlecan may also be available and the above list is not meant to be inclusive. The individual domains of perlecan have also been individually expressed in native configuration from bacterial hosts (33) and eukaryotic hosts (8, 10, 20). The obvious domain structure and limited susceptibility to proteolysis (11) contribute to the ability to produce each of the perlecan domains individually. For example, domain 1 has been cloned and expressed alone (10, 19), and with portions of domain II (20). It is known that sequences in the protein core affect the amount and type of glycosaminoglycan (such as heparan sulfate) which is attached when perlecan is produced in a eukaryotic host. However, the biological significance of the different glycosylation patterns remains unknown.
In situ hybridization studies and immunoenzymatic studies show a close association of perlecan with a variety of cells involved in the assembly of basement membranes, in addition to being localized within the stromal elements of various connective tissues. Perlecan has been demonstrated in periodontal ligament fibroblasts (29). Perlecan has also been detected in the basement membranes of human tissues including pituitary gland, skin, breast, thymus, prostate, colon, liver, pancreas, spleen, heart, and lung. All vascular basement membranes reported to be tested contained perlecan. In addition, sinusoidal vessels of liver, spleen, lymph nodes, and pituitary gland expressed high levels of perlecan in the subendothelial region. In situ hybridization, using as probe human cDNA-encoding Domain III, localized perlecan mRNA to specific cell types within the tissues and demonstrated that in skin, perlecan appears to be synthesized exclusively by connective tissue cells in the dermal layer (32, 33). Perlecan is also highly expressed in human bone marrow (28) and in synovium (13). An immunohistochemical study confirmed the location of perlecan on the apical cell surface of endothelial cells, and additionally as a dense fibrillar network surrounding the cells. In this context, the binding of thromobospondin 1 to the apical surface of endothelial cells, which is critical in angiogenesis (44), was found to be dependent upon the NH2-terminal heparan sulfate chains of perlecan (45). These patterns of perlecan expression clearly implicate a role in wound healing regulation and/or angiogenesis.
Cell and Growth Factor Binding
Perlecan is adhesive for fibroblasts and endothelial cells (28). Expression of perlecan in the mouse was coordinated with development of attachment competence by mouse embryos in vitro and in utero (7). Purified perlecan and laminin were found to promote attachment of immortalized rat chondrocytes in vitro (43). Perlecan is thought to modulate binding between the basement membrane structure and various cells, including smooth muscle cells and aortic endothelial cells, through a non-RGD cell binding region in one of the perlecan domains (possibly domain III in the mouse; amino acid sequence LPASFRGDKVTSY (SEQ ID NO: 1), as well as by GRGDSP (SEQ ID NO: 2), but not GRGESP (SEQ ID NO: 3)) and integrins β-I and α-5, β-3 (23). Alternatively, other investigators have found that the attachment of Rugli cells (a rat glioma derived cell line), mesenchymal, and epithelial derived tumor cell lines to mouse perlecan did not involve the protein core and was totally dependent on the presence of the heparan sulfate, although binding through the β-1 integrin of the cells was involved (4). Cell adhesion to perlecan was low compared to perlecan core alone (23). Human endothelial cell-derived perlecan was shown to bind endothelial cells in vitro with contributions from the heparan sulfate and from the protein core (46). The attachment of cells to the protein core of human perlecan further supports the implication of alternative cell binding pathways as the human homologue does not have the RGD sequence in domain III. Significant evidence for a role in cell binding further implicates perlecan in wound healing processes.
Proteoglycans such as perlecan, once thought to primarily serve as structural components of extracellular matrix, are now being focused on for their role in tissue and cell regulation, particularly angiogenesis and wound healing. Many growth factors, notably the fibroblast growth factor family (FGF) which now numbers 19 members, bind to heparin and heparan sulfate proteoglycans, and this binding has been shown to have a significant impact on the availability and activity of these growth factors. Importantly, perlecan has been shown to specifically bind to FGF-2 (also known as bFGF), which is critical in vascular development and wound healing (37). Perlecan was found to induce high affinity binding of FGF-2 both to cells deficient in heparan sulfate and to soluble FGF receptors. Further, in a rabbit ear model for in vivo angiogenesis, perlecan was a potent inducer of FGF-2-mediated neovascularization (2). It has been shown that FGF-2 binds to the heparan sulfate on domain I of perlecan (2) and that FGF-2 is released by biologically relevant enzymes such as plasmin, collagenase, and heparinases, which may have a role in the regulation of the growth factor activity (47). Mitogenic keratinocyte growth factor (FGF-7) was recently shown to bind to domains III and V of the perlecan protein core (31). Binding of FGF-2 to the heparan sulfate chain of perlecan is thought to involve three-way coordinated binding between FGF, heparan sulfate, and the FGF receptor, and to involve specific sites of sulfation (unpublished data). The FGF receptor is probably the FGF-binding protein that was recently reported to bind perlecan specifically in domain III (30).
Perlecan is also able to bind the growth factor granulocyte/macrophage-colony stimulating factor and present it to hematopoietic progenitor cells in a semi-solid colony assay (28). Bound growth factors can be released by enzymes, which are present during wound healing, such as the matrix metalloproteinases (47). Ligand binding itself can lead to internalization through a perlecan-mediated process, as has been shown for ligands such as lipoproteins (15). Binding of growth factors is clearly an important role of perlecan in wound healing and angiogenesis.
Breached basement membranes in vascular, corneal, and dermal tissues must respond quickly to the injury with repair. Such a rapid response suggests storage of needed biomolecules to effect repairs and remodeling. Cells involved in the wound healing and angiogenic process may have a ready and ample storage of FGF and other growth factors bound to perlecan in the basement membrane adjoining the wound. It has been demonstrated that enzymes which are turned on in the remodeling wound and angiogenesis, such as the matrix metalloproteinases (MMPs), stromelysin, rat collagenase, plasmin, urokinase, heparitinase I, and heparin, may modulate the bioavailability of the growth factors by degrading the protein core and removing the glycosaminoglycans (47). The MMPs are required in correct wound healing and in angiogenesis, and have been shown to bind to cells. The extracellular binding of MMPs could position the enzyme for directed proteolytic attack, for activation of other MMPs, and for regulation of other cell surface proteins. It has recently been demonstrated that heparin binds (5-10 nM) MMP-7, MMP-2, MMP-9, and MMP-13. This suggests that the MMPs may be positioned on the cell surface or retained in the ECM by perlecan heparan sulfate chains (48).
Growth and Wound Healing
The effectiveness of perlecan as an exogenously added promoter of growth and neovascularization was demonstrated with anti-sense perlecan knockouts in colon carcinoma cells (40). Growth of colon carcinoma cells was markedly attenuated upon obliteration of perlecan gene expression and these effects correlated with reduced responsiveness to, and affinity for, FGF-7. Exogenous perlecan effectively reconstituted the activity of FGF-7 in the perlecan-deficient cells. Moreover, soluble FGF-7 specifically bound immobilized perlecan in a heparan sulfate-independent manner. In both tumor xenografts induced by human colon carcinoma cells and tumor allografts induced by highly invasive mouse melanoma cells, perlecan suppression caused substantial inhibition of tumor growth and neovascularization. Thus, perlecan is a potent inducer of cell growth and angiogenesis in vivo and therapeutic interventions targeting this key modulator of tumor progression may improve wound healing.
In cells that were expressing antisense perlecan, responses to increasing concentrations of FGF-2 were dramatically reduced in comparison to wild-type or vector-transfected cells as measured by thymidine incorporation and rate of proliferation (3). Furthermore, receptor binding and affinity labeling of cells expressing antisense perlecan indicated that eliminating perlecan expression (by expressing antisense perlecan) results in reduced high-affinity FGF-2 binding. Both the binding and mitogenic response of cells expressing antisense perlecan to FGF-2 could be rescued by exogenous perlecan (3).
Poor wound healing in diabetics and in diabetes-related periodontitis may be related to perlecan expression. High levels of glucose can decrease perlecan expression in some cells, probably through transcriptional and post-transcriptional mechanism (27). Further, it has recently been demonstrated that FGF-2 bound to perlecan is protected from inactivation by non-enzymatic glycation, which occurs during the course of diabetes (35). The relationship between poor expression and protection from inactivation in diabetes has not been investigated.
Evidence indicates that in cases of poor surgical wound closure and healing by frank secondary intention, as results in many barrier membrane surgeries during oral and periodontal surgeries, for example, the presence of perlecan in the subepithelial matrix is associated with healing of the wound up to 14 days post surgery, at which time the healed wound can appear normal in terms of perlecan core protein. Subsequently, sulfation levels of the heparan sulfate chains increase over the ensuing year of healing (1). Another investigation, however, reported that perlecan is not detectable under epithelial cells migrating over connective tissue and only appears when the wound is covered and a new basement membrane is deposited (38). In wounds that are remodeling over a period of weeks, perlecan levels in the subepithelial connective tissues appear to remain relatively high (42). The functional significance of various perlecan expression patterns during healing is not yet known, but it is clear the biomolecule has an important and fundamental role in the healing process.
In summary, a complex interplay exists between 1) extracellular matrix proteins, including perlecan, 2) the cells considered key in establishing dermal integrity such as fibroblasts, endothelial cells, epithelial cells, and 3) growth factors such as the FGF s. It is clear, however, that perlecan has the potential to affect several aspects of wound healing and, to that end, is an excellent candidate for application or induction in various forms, with or without the addition of various growth factors to modulate wound healing.
SUMMARY
The present disclosure describes the use of perlecan as an adjunct for the improvement of wound healing and/or angiogenesis. The disclosure describes the application of perlecan at an effective concentration in a pharmaceutically acceptable carrier, such as a paste, for use in a variety of processes. For example, perlecan may be used during periodontal procedures, such as flap surgery, or may be applied to sites of cutaneous injury such as burns and cuts, or may be applied to sites of surgical procedures, such as incisions, implants or grafts. Perlecan may be applied directly in a pharmaceutically acceptable carrier, or may be applied to a wound bandage or dressing, or may be absorbed to surgical implants, grafting materials or surgical barrier membranes. Perlecan may also be applied by delivery of nucleic acid encoding for perlecan. Perlecan nucleic acid may be delivered via viral vectors, cells expressing perlecan molecules, via naked nucleic acid (such as mRNA) or via liposome technology. The present disclosure also encompasses the application of perlecan in conjunction with growth factors, such as FGF-2 or FGF-7, or other heparan sulfate binding growth factors. Evidence suggests that perlecan or its derivatives has the potential to effect the interplay between cells of the dermis and epidermis, the extracellular matrix, and various growth factors, each of which has a role in wound healing and or angiogenesis. Therefore, perlecan is an excellent candidate for application or delivery in various forms, possibly in conjunction with various growth factors, to modulate wound healing and/or angiogenesis.


DETAILED DESCRIPTION
In the present disclosure, the use of perlecan as an adjunct to wound healing and angiogenesis is described. As used in this specification and claims, the term “perlecan” refers to the polypeptide listed in SwissProt Protein Data Base named Heparan Sulfate Proteoglycan (accession number CACI 8534.1) (SEQ ID NO: 4) or the nucleic acid coding sequence named Human Heparan Sulfate Proteoglycan (HSPG2) mRNA (GenBank accession number M85289.1) (SEQ ID NO: 5). In addition to the perlecan nucleic acid sequences disclosed in SEQ ID NO: 5, the present disclosure also is directed to nucleic acid sequences encoding functional derivatives of the perlecan nucleic acid. As used herein, a “functional derivative” includes the “fragments,” “degenerate variation,” “mutants,” “analogs,” “variants” and “chemical derivatives”. The term “fragment” is meant to refer to any nucleic acid subset of the perlecan nucleic acid comprising at least 9 contiguous nucleic acid residues. The term “variant” is meant to refer to a molecule substantially similar in structure to either the entire perlecan gene, or to a fragment thereof. The term “chemical derivative” describes a nucleic acid or protein that contains additional chemical moieties which are not normally a part of the base molecule. Such moieties may alter the solubility, binding characteristics (to molecules such as growth factors) half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington The Science and Practice of Pharmacy, 20th edition.
It is known that there is a substantial amount of redundancy in the codons which code for specific amino acids. Therefore, this disclosure is directed to those nucleic acid sequences which contain alternative codons which code for the eventual translation of the identical amino acid in SEQ. ID NO. 4. For purposes of this specification, a nucleic acid sequence coding for one or more alternative codons will be defined as a “degenerate variation.” Also included within the scope of this disclosure are mutations either in the perlecan nucleic acid sequence which do not substantially alter the ultimate physical properties of the polypeptide encoded by the perlecan nucleic acid, or functional derivatives thereof (referred to as an “analog”). For example, an analog may comprise a conservative amino acid changes, such as, but not limited to, substitution of valine for leucine or asparagine for glutamine. Other examples of conservative amino acid changes may be found in Biochemistry, 2cd Edition Geoffrey Zubay (Macmilliam Publishing Company, New York, N.Y., Table 1-1, p 33) and include substitutions of an amino acids with other amino acid listed within the same grouping. It is known that DNA sequences coding for a polypeptide may be altered so as to code for a peptide having properties that are different than those of the naturally-occurring peptide (referred to as “mutants”). Mutant nucleic acid sequences may be isolated from nature. Methods of producing mutant nucleic acid sequences include, but are not limited to site directed mutagenesis. Examples of altered properties include, but are not limited to changes in the affinity of an enzyme for a substrate or a receptor for a ligand (such as but not limited to growth factors) and altered sulfation patterns. A molecule is “substantially similar” to the perlecan nucleic acid if both molecules have substantially similar structures, or if both molecules possess similar biological activity.
The disclosure also directed to polypeptides encoded by perlecan nucleic acid, or functional derivatives thereof. Therefore, the disclosure covers polypeptides coded for by fragments of the perlecan nucleic acid. In addition, the disclosure covers polypeptides coded for by analogs, degenerate variants and mutants of the perlecan nucleic acid.
A molecule is “substantially similar” to perlecan in terms of structure if both molecules have more than 50% similarity or 70% identity between their respective nucleotide or amino acid sequences. Forms of perlecan that varied in glycosaminoglycan profiles and sulfation patterns, including those forms of perlecan totally without glycosaminoglycan or sulfated constituents, would be considered molecules substantially similar to perlecan. As used in this disclosure, the term “percent homology” of two amino acid sequences or of two nucleic acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990), modified as in Karlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (J. Mal. Biol. 215:403-410, 1990). Blast nucleotide searches are performed with the NBLAST program, score 100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. Blast protein searches are performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to a referenced polypeptide. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (Nucleic Acids Res. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (XBLAST and NBLAST) are used.
Perlecan may be derived from purification from either natural or recombinant sources, standard cloning procedures, from proteolysis of cloned or native molecules, or by induction within host cells, or by delivery from cells expressing perlecan nucleic acid sequences. For example, a derivative of perlecan would include a recombinant perlecan protein expressed from a host cell where the fragment comprised both domains I and III of the native perlecan molecule, and where the nucleic acid coding for the recombinant perlecan molecule was mutated to alter the sulfation pattern when compared to native perlecan. In addition, perlecan may be used either alone or in the presence of a modulating compound (defined as a protein, nucleic acid or organic compound that modulates the structure and/or function and/or activity of perlecan). Examples of modulating compounds are growth factors, especially those growth factors of the fibroblast growth factor family, such as FGF-2 and FGF-7, and other growth factors that interact with the heparan sulfate chains.
As used in this disclosure, an isolated nucleic acid is a nucleic acid the structure of which is not identical to that of any naturally occurring nucleic acid, or to that of any fragment of a naturally occurring genomic nucleic acid spanning more than three separate genes. The term therefore covers, by way of example only, 1) a DNA which has the sequence of part of a naturally occurring genomic DNA molecule, but is not flanked by both of the sequences that flank that part of the molecule in the genome of the organism in which it naturally occurs; 2) a nucleic acid incorporated into a vector or into the genomic DNA of a prokaryote or eukaryote in a manner such that the resulting molecule is not identical to any naturally occurring genomic DNA; 3) a separate molecule such as a cDNA, a genomic fragment, a fragment produced by PCR, or a restriction fragment; and 4) a recombinant nucleotide sequence that is part of a hybrid gene (i.e., a gene encoding a fusion protein). Specifically excluded from this definition are nucleic acids present in mixtures of DNA molecules, transfected cells and cell clones, as these occur in a DNA library such as a cDNA or genomic library.
Perlecan may be recombinantly expressed either alone or in combination with a modulating compound by molecular cloning into an expression vector containing a suitable promoter and other appropriate transcription regulatory elements, and transferred into prokaryotic or eukaryotic host cells to produce recombinant molecules. Techniques for such manipulations are within the ordinary skill of one in the art, and representative techniques can be found described in Sambrook, J., et al., Molecular Cloning Second Edition, 1990, Cold Spring Harbor Press. Expression vectors are defined herein as the nucleic acid sequences that are required for the transcription of cloned copies of genes and the translation of their mRNAs in an appropriate host. Such vectors can be used to express eukaryotic genes in a variety of hosts such as bacteria, blue green algae, plant cells, insect cells, fungal cells and animal cells.
An appropriately constructed expression vector should contain, at the minimum: an origin of replication for autonomous replication in host cells, selectable markers, a limited number of useful restriction enzyme sites, a potential for high copy number, and active promoters. A promoter is defined as a DNA sequence that directs RNA polymerase to bind to DNA and initiate RNA synthesis. Expression vectors may include, but are not limited to, cloning vectors, modified cloning vectors, specifically designed plasmids or viruses.
A variety of expression vectors may be used for the expression of perlecan. These include, but are not limited to commercially available mammalian expression vectors such as pcDNA3 (Invitrogen), pMC1neo (Stratagene), pXT1 (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593) pBPV-1(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and IZD35 (ATCC 37565).
Commercially available bacterial expression vectors that may be suitable for recombinant expression of perlecan include, but are not limited to, pET11a (Novagen), lambda gt11 (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia). Commercially available insect cell expression vectors that may be suitable for recombinant expression of perlecan include, but are not limited to, pBlue Bac III (Invitrogen). The expression vectors may also be produced according to specific needs. The choice of the appropriate expression vector is within the ordinary skill of one in the art. The expression vector may contain nucleic acid coding only for the perlecan, or may encode for the perlecan, either alone or in combination with a modulating compound.
Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, and mammalian cells, including, but not limited to, cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including, but not limited to, Drosophila and silkworm derived cell lines. A variety of cell lines derived from mammalian species which may be suitable for use as host cells are commercially available and include, but are not limited to, L cells L-M(TK.sup.−) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C1271 (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171). The choice of host cells is within the ordinary skill of one in the art.
The expression vectors may be introduced into host cells via any one of a number of techniques including but not limited to transformation, transfection, lipofection, protoplast fusion, and electroporation. The expression vector-containing cells are clonally propagated and individually analyzed to determine whether they produce the compound of interest. Identification of cells expressing the perlecan product, can be accomplished by a variety of means, including but not limited to, immunological reactivity, or the presence of host cell-associated perlecan activity.
Expression of perlecan, either alone or in combination with a modulating compound, may also be performed using in vitro produced synthetic mRNA or isolated native mRNA. Synthetic mRNA or mRNA isolated from perlecan producing cells can be efficiently translated in various cell-free systems, including but not limited to wheat germ extracts and reticulocyte extracts, as well as efficiently translated in cell based systems, including but not limited to microinjection into frog oocytes.
Following expression of perlecan in a recombinant host cell, which host cell may also be expressing a modulating compound, purified perlecan, or purified perlecan in association with a modulating compound may be obtained. Purification methods for isolating expressed protein are well known in the art and within the ordinary skill in the art. Techniques include salt fractionation, ion exchange chromatography, size exclusion chromatography, hydroxylapatite adsorption chromatography, hydrophobic interaction chromatography, immunoaffinity chromatography and affinity chromatography.
Mono-specific antibodies to human perlecan can be purified from mammalian antisera containing antibodies reactive against perlecan, or are prepared as monoclonal antibodies reactive with perlecan. Mono-specific antibody is defined as a single antibody species, or multiple antibody species with binding characteristics for perlecan, and includes polyclonal antibodies. Homogenous binding as used herein refers to the ability of the antibody species to bind to a specific antigen or epitope. Specific antibodies are raised by immunizing animals such as mice, rats, guinea pigs, rabbits, goats, horses and the like, with rabbits being preferred, with an appropriate concentration of perlecan, either with or without an immune adjuvant.
Preimmune serum is collected prior to the first immunization to establish baseline immunoreactivity. Each animal receives between about 0.1 mg and about 1000 mg of perlecan, either with or without an acceptable immune adjuvant. Such acceptable adjuvants include, but are not limited to, Freund's complete, Freund's incomplete, alum-precipitate, water in oil emulsion containing Corynebacterium parvum and tRNA. The initial immunization consists of perlecan, and preferably Freund's complete adjuvant, at multiple sites either subcutaneously (SC), intraperitoneally (IP), or both. Each animal is bled at regular intervals, preferably weekly, to determine antibody titer. The animals may or may not receive booster injections following the initial immunization. Those animals receiving booster injections are generally given an equal amount of the initial antigen in Freund's incomplete adjuvant by the same route. Booster injections are given at about three week intervals until maximal titers are obtained. At about 7 days after each booster immunization or about weekly after a single immunization, the animals are bled, the serum collected, and aliquots are stored at about −20 degree C.
Monoclonal antibodies (mAb) reactive with perlecan are prepared by immunizing inbred mice, preferably Balb/c, with the appropriate antigen. The mice are immunized by the IP or SC route with about 0.1 mg to about 10 mg, preferably about 1 mg, of antigen in about 0.5 ml buffer or saline incorporated in an equal volume of an acceptable adjuvant, as discussed above. Freund's complete adjuvant is preferred. The mice receive an initial immunization on day 0 and are rested for about 2 to about 30 weeks. Immunized mice are given one or more booster immunizations of about 0.1 to about 10 mg of antigen in a buffer solution such as phosphate buffered saline by the subcutaneous or intravenous (IV) route. Lymphocytes, from antibody positive mice, preferably splenic lymphocytes, are obtained by removing spleens from immunized mice by standard procedures known in the art. Hybridoma cells are produced by mixing the splenic lymphocytes with an appropriate fusion partner, preferably myeloma cells, under conditions which will allow the formation of stable hybridomas. Fusion partners may include, but are not limited to: mouse myelomas P3/NS1/Ag 4-1; MPC-11; S-194 and Sp 2/0, with Sp 2/0 being preferred. The antibody producing cells and myeloma cells are fused in polyethylene glycol, about 1000 molecular weight, at concentrations from about 30% to about 50%. Fused hybridoma cells are selected by growth in hypoxanthine, thymidine and aminopterin supplemented Dulbecco's Modified Eagles Medium (DMEM) by procedures known in the art. Supernatant fluids are collected from growth positive wells on about days 14, 18, and 21 and are screened for antibody production by an immunoassay using perlecan as the antigen. The culture fluids are also tested in the Ouchterlony precipitation assay and ELISA to determine the isotype of the mAb. Hybridoma cells from antibody positive wells are cloned by techniques well know in the art.
It is readily apparent to those skilled in the art that the above described methods for producing monospecific antibodies may be utilized to produce antibodies specific for the isolated perlecan, either alone or in combination with a modulating compound.
The antibodies produced above may be used as affinity columns by adding the antibodies to Affigel-10 (Biorad, Hercules, Calif.), according to the manufacturer's directions. After coupling of the antibodies to the column, the column is washed with water followed by 0.23M glycine HCl (pH 2.6) to remove any non-conjugated antibody or extraneous protein. The column is then equilibrated in phosphate buffered saline (pH 7.3) with appropriate detergent, and the cell culture supernatants or cell extracts containing perlecan, ether alone or in combination with a modulating compound, made using appropriate membrane solubilizing detergents are slowly passed through the column. The column is then washed with phosphate buffered saline/detergent until the optical density (A280) falls to background, then the protein is eluted with 0.23M glycine-HCl (pH 2.6)/detergent. The purified protein is then dialyzed against phosphate buffered saline/detergent.
The present invention is also directed to methods for screening for compounds which modulate the expression of nucleic acid encoding perlecan, and/or the function of perlecan protein, in vivo and in vitro, either alone or in combination with a modulating compound. Compounds which modulate these activities may be DNA, RNA, peptides, proteins, or non-proteinaceous organic molecules. Compounds may modulate by increasing or attenuating the expression of DNA or RNA encoding perlecan or the function of the perlecan protein, either alone or in combination with a modulating compound. Compounds that modulate the expression of nucleic acid encoding perlecan, and/or the function of perlecan protein may be detected by a variety of assays. The assay may be a simple “yes/no” assay to determine whether there is a change in expression or function. The assay may be made quantitative by comparing the expression or function of a test sample with the levels of expression or function in a standard sample. The expression of perlecan protein, either alone or in combination with a modulating compound, is performed as described above. Such an assay is directed to direct inhibitors of perlecan nucleic acid expression or perlecan activity, as well as indirect inhibitors of this activity.
Kits containing perlecan nucleic acids or antibodies to perlecan may be prepared. Such kits can be used to detect nucleic acids which hybridize to the perlecan nucleic acids, or to detect the presence of perlecan protein or proteoglycan in a sample. In addition, such kits would contain the accessory reagents required to complete the assay contemplated by the kit.
Nucleotide sequences complementary to the nucleotide sequences coding for perlecan can be synthesized for antisense therapy. These antisense molecules may be DNA, stable derivatives of DNA such as phosphorothioates or methylphosphonates, RNA, stable derivatives of RNA such as 2′-O-alkylRNA, or other antisense oligonucleotide mimetics. These antisense molecules may be introduced into cells by microinjection, liposome encapsulation or by expression from vectors harboring the antisense sequence. Antisense therapy may be particularly useful for the treatment of diseases where it is beneficial to reduce perlecan activity.
Gene therapy may be used to introduce perlecan either alone or in combination with a modulating compound, into the cells. The nucleic acid coding for the appropriate protein can be ligated into viral vectors which mediate transfer of the DNA by infection of recipient host cells. Suitable viral vectors include retrovirus, adenovirus, adeno-associated virus, herpes virus, vaccinia virus, polio virus and the like. Alternatively, DNA can be transferred into cells for gene therapy by non-viral techniques including receptor-mediated targeted DNA transfer using ligand-DNA conjugates or adenovirus-ligand-DNA conjugates, lipofection membrane fusion or direct microinjection. These procedures and variations thereof are suitable for ex vivo, as well as in vivo gene therapy. Gene therapy may be particularly useful for the treatment of diseases where it is beneficial to elevate perlecan activity.
Pharmaceutically useful compositions comprising nucleic acids and their complements, and proteins corresponding to perlecan, either alone or in combination with modulating compounds, may be formulated according to known methods such as by the admixture of a pharmaceutically acceptable carrier. Examples of such carriers and methods of formulation may be found in Remington's Pharmaceutical Sciences. To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of the protein, nucleic acid, or modulator compound.
Therapeutic or diagnostic compositions of the invention are administered to an individual in amounts sufficient to treat and/or diagnose disorders related to perlecan (defined as the “effective amount”). The effective amount may vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode or site of administration. The pharmaceutical compositions may be provided to the individual by a variety of routes such as subcutaneous, topical, oral, intraosseous, and intramuscular. Compounds identified according to the methods disclosed herein may be used alone at appropriate dosages defined by routine testing in order to obtain optimal activity, while minimizing any potential toxicity. In addition, co-administration or sequential administration of other agents may be desirable.
The therapeutic or diagnostic derivatives of perlecan discussed herein may be used with or without chemical derivatives. Such moieties may improve the solubility, half-life, absorption, etc. of the base molecule. Alternatively the moieties may attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule. Examples of such moieties are described in a variety of texts, such as Remington's Pharmaceutical Sciences.
The compositions containing compounds identified according to this invention as the active ingredient for use in the modulation of wound healing and/or angiogenesis can be administered in a wide variety of therapeutic dosage forms in conventional vehicles for administration. For example, the compounds can be administered in such forms as tablets, capsules (each including timed release and sustained release formulations), pills, powders, granules, elixirs, tinctures, solutions, suspensions, syrups, pastes and emulsions, or by injection internally.
In the present disclosure, the compounds herein described in detail can form the active ingredient, and are typically administered in admixture with suitable pharmaceutical diluents, excipients or carriers, which are collectively referred to herein as “carrier” materials, suitably selected with respect to the intended form of administration, that is, oral tablets, capsules, elixirs, syrups and the like, and consistent with conventional pharmaceutical practices. For instance, for oral administration in the form of a tablet or capsule, the active drug component can be combined with an oral, non-toxic pharmaceutically acceptable inert carrier such as ethanol, glycerol, water and the like. Moreover, when desired or necessary, suitable binders, lubricants, disintegrating agents and coloring agents can also be incorporated into the mixture. Suitable binders include, without limitation, starch, gelatin, natural sugars such as glucose or beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose, polyethylene glycol, waxes and the like. Lubricants used in these dosage forms include, without limitation, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and the like. Disintegrators include, without limitation, starch, methyl cellulose, agar, bentonite, xanthum gum and the like.
For oral forms, the active biologic component can be combined in suitably flavored suspending or dispersing agents such as the synthetic and natural gums, for example, tragacanth, acacia, methylcellulose and the like. Other dispersing agents that may be employed include glycerin and the like. For internal injection, sterile suspensions and solutions are desired. Isotonic preparations that generally contain suitable preservatives are employed when internal injection is desired.
Topical preparations containing the active drug component can be admixed with a variety of carrier materials well known in the art, such as, e.g., alcohols, aloe vera gel, allantoin, glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate, and the like, to form, e.g., alcoholic solutions, topical cleansers, cleansing creams, skin gels, skin lotions, and shampoos in cream or gel formulations.
The compounds of the present disclosure can also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine or phosphatidylcholines.
Compounds of the present disclosure may also be delivered by the use of monoclonal antibodies as individual carriers to which the compound molecules are coupled. The compounds of the present disclosure may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include, but are not limited to, polyvinyl-pyrrolidone, pyran copolymer, polyhydroxypropylmethacryl-amidephenol, polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysine substituted with palmitoyl residues. Furthermore, the compounds of the present invention may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates and cross-linked or amphipathic block copolymers of hydrogels.
All references to articles, books, patents, websites and other publications in this disclosure are considered incorporated by reference. The following examples illustrate certain embodiments of the present disclosure without, however, limiting the same thereto.
PROPHETIC EXAMPLES
Use of Perlecan in Surgical Procedures including Periodontal Procedures
In one embodiment, perlecan would be purified from eukaryotic or bacterial cells via standard cell culture and protein purification methodology. Exemplary cell types, cell growth conditions and antibodies for affinity purification of perlecan from cultured eukaryotic cells have been described (2, 16, 21, 22, 39). Eukaryotic cells may be from primary culture or may be derived from cloned or immortalized cell lines (8, 10, 20). Antibody affinity chromatography may be used to purify perlecan from either cell source (25, 34). Various monoclonal antibodies that are specific for different epitopes of perlecan are available commercially and could be used in sequence to optimize purity. Purity would be established by molecular sieve chromatography and SDS-PAGE electrophoresis, or by other standard techniques (17). Purification of perlecan may be from cell culture media directly, or may involve fractionation of cells by standard methods and purification, such as isolation of and purification from intracellular inclusion bodies (26). Once released from inclusion bodies by standard techniques, affinity purification would follow as described above. Cloning of perlecan would involve standard PCR and ligation procedures into any number of suitable eukaryotic or bacterial expression vectors (6). Importantly, when perlecan is expressed in bacteria, it may be necessary to to alter the nucleic acid sequence of perlecan to meet the requirements of the transcriptional and translation system of the bacterial host. These forms of perlecan, and the nucleic acid sequences coding for them, are included in the definition of perlecan derivative.
As discussed above, perlecan would include those with different sulfation patterns and differing amounts of glycosaminoglycan chains. The presence or absence of particular modifications may differ between eukaryotic and bacterial cells, and within different cells types within each class. In addition, perlecan may be administered with or without various growth factors. As discussed above, perlecan has been shown to bind certain growth factors and may play a role in regulating the activity of these growth factors. Therefore, it may be advantageous to administer perlecan with growth factors in certain situations. It is preferred that the perlecan is mixed with the growth factor prior to being incorporated into a pharmaceutically acceptable formulation. The molar ratio of perlecan to growth factor can range from 0.1 to 1 to 20 to 1, but a preferred ration is 1 to 1. Growth factors that may be used with perlecan include, but are not limited to, members of the fibroblast growth factor family, such as FGF-2 and FGF-7, and GM-CSF. Other growth factors determined to be active in either wound healing, tissue remodeling or angiogenesis may also be used with perlecan.
Perlecan may be incorporated into various pharmaceutically acceptable formulations for use as discussed above. In one embodiment perlecan may be mixed with a carrier paste. Various carrier pastes for dermal or surgical application are available and many are inert formulations based on cellulose. Perlecan is included in the paste at an effective concentration as discussed above. In one embodiment, the range of perlecan concentrations within the carrier paste is 1 pM to 1 μM. The perlecan (preferably in a pharmaceutically acceptable formulation, such as the paste described above) may be used during a variety of purposes. For example, during periodontal procedures, perlecan may be applied at the time of surgical closure between and below flaps. Approximately 1 ml of paste containing perlecan would be applied for every 6 cm2 of wound surface, and would be applied liberally.
A variety of systems have been developed to accomplish expression of exogenous proteins via the delivery of nucleic acid sequences coding for the given protein into target cells. In one embodiment, perlecan would be delivered as an adjunct to wound healing or surgical healing by placing into the wound or surgical site cells which have been induced to express perlecan. These cells would have preferably been derived from the recipient, but may be of an allograft or xenograft nature, being derived from other human donors, or from animals. Cells may include stem cells, endothelial cells, epithelial cells, fibroblasts, monocytes or macrophages. The target cells will be collected from the blood or tissues of the donor by standard methods and nucleic acid coding for perlecan will be introduced into the cell. Methods of introducing the perlecan encoding nucleic acid into cells are well known in the art as described above. One such method involves infecting the target cells with a viral-based expression vector, such as adenovirus. Adenovirus containing perlecan encoding nucleic acid and target cells are mixed for 30 minutes in serum-free cell culture media. After this infection period, the cells will be collected by centrifugation and applied to the wound or surgical site at a density of between 103-106 cells per cm2.
In another embodiment, a nucleic acid fragment coding for perlecan would be delivered to the desired site in a viral-based expression vector. There are many choices for viral-based expression vectors which are well known in the art. However, a preferred viral vector is the adenovirus. There are several good reasons for utilizing recombinant adenovirus vectors for the delivery of nucleic acid encoding perlecan. First, stocks of adenovirus containing high titers of virus (greater than 1011 pfu per ml) can be prepared, which makes it possible to transduce cells in situ at high multiplicity of infection for high localized protein production. Also, the adenovirus is capable of inducing high levels of transgene expression, at least as an initial burst. Third, the vector can be engineered to incorporate a high degree of versatility. Finally, the adenovirus vector is safe based on its long-term use as a vaccine. Recombinant adenovirus vectors have been utilized as vaccine carriers by intranasal, intratracheal, intraperitoneal, intravenous, subcutaneous, or intramuscular routes (9, 12, 18). In this embodiment, nucleic acid encoding perlecan, such as cDNA will be ligated into a replication-incompetent, E1/E3defective human adenovirus serotype 5-derived vector under the transcriptional control of the human cytomegalovirus early promoter as described previously (41). For inducing perlecan expression from said cDNA expression system, 105 to 109 adenovirus particles will be delivered per cm2 of wound surface area, or per cm3 of a surgical grafting site.
The exogenous perlecan encoding nucleic acid may also be incorporated into the pharmaceutically acceptable carriers described above for the uses discussed above. For example, perlecan encoding nucleic acid may be incorporated into a paste formulation as discussed above and used in surgical and periodontal procedures, such as flap surgery, or applied to cutaneous injuries, such as burns or cuts. In addition, perlecan encoding nucleic acids may be applied to grafting materials, implant materials, surgical barrier membranes and dressings for the purposes described below. As an example, perlecan encoding nucleic acid may be adsorbed onto the surface of an implant before implant placement by painting, spraying, or soaking the implant material in a sterile solution of exogenous perlecan encoding nucleic acid for periods of time ranging from 1 second to 24 hours.
Use of Perlecan for Cutaneous Injury
In this embodiment, perlecan is expressed and purified as discussed above. As discussed in Example 1, perlecan may be used in a variety of forms and in a variety of pharmaceutically acceptable formulations. As another example of perlecan use, perlecan may be applied to the surface of a site of cutaneous injury, such as a burn, a non-healing ulcer, or a cut or similar injury. Approximately 1 ml of paste containing perlecan would be applied for every 6 cm2 of wound surface and would be applied liberally. In addition, the perlecan-containing paste may be delivered in association with a bandage or other dressing applied to the site of injury.
Use of Perlecan in Conjunction with Surgical Implants and Similar Materials
In this embodiment, perlecan is expressed and purified as discussed above. As discussed in Example 1, perlecan may be used in a variety of forms and in a variety of pharmaceutically acceptable formulations. As another example of perlecan use, perlecan may be applied to and absorbed on the surface of surgical implants, grafting material or barrier membranes.
Surgical implants include, but are not limited to, those made of titanium or titanium coated with hydroxy-apatite material. Bone grafting materials include, but are not limited to, hydroxyl apatite in various crystal sizes and forms, and autologous, autogenous, or xenograft donar bone, either cortical or cancellous, and freeze-dried with demineralization. Barrier membrane materials include, but are not limited to, poly vinyl difluoride (Gore-Tex), poly galactic acid, or collagen (BioMend). The surgical implants, grafting materials and barrier membrane would be pre-treated in a solution of perlecan, approximately 1 pM to 1 μM in concentration, preferably in a sterile buffer such as phosphate buffer, pH 7.4. The pre-treatment may be performed during surgery, or at any time prior to the surgery provided the coated surgical implant material is not subject to temperatures above 37° C. at any time. The pre-treatment procedure would involve immersion in the perlecan solution for at least 10 seconds, preferably 5 minutes. The treated membrane would then be surgically placed. As discussed above, various growth factors may also be incorporated into the perlecan solution for incorporation into and absorption onto the surgical implants, grafting materials and barrier membranes. Once prepared, the surgical implants, grafting materials and barrier membranes are used according to standard procedures.
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## Claims

1. A method for the effective healing of a wound or a cutaneous injury, the method comprising administering to a subject in need of such healing a first nucleic acid sequence encoding a perlecan polypeptide containing at least one a heparan sulfate attachment site and a second nucleic acid sequence encoding a growth factor or a fragment of a growth factor capable of binding to a heparan sulfate-decorated polypeptide, whereby the heparan sulfate-decorated perlecan polypeptide is generated by the subject, said nucleic acid being delivered in an amount sufficient to deliver an effective amount of the heparan sulfate-decorated perlecan polypeptide and said growth factor or fragment of growth factor to the wound or cutaneous injury.

2. The method of claim 1 wherein the administration results in an improvement in healing of the wound or cutaneous injury in the subject.

3. The method of claim 1 wherein the first and second nucleic acid constructs are administered to the wound or cutaneous injury in the subject.

4. The method of claim 1 where the perlecan polypeptide encoded by the first nucleic acid sequence has the sequence of SEQ ID NO. 4.

5. The method of claim 1 where the perlecan polypeptide encoded by the first nucleic acid sequence is 70% homologous to the sequence of SEQ ID NO. 4.

6. The method of claim 1 where the perlecan polypeptide encoded by the first nucleic acid sequence is a fragment of SEQ ID NO. 4.

7. The method of claim 1 wherein the perlecan polypeptide encoded by the first nucleic acid sequence contains amino acids 1-172 of SEQ ID NO. 4.

8. The method of claim 1 wherein the growth factor is a heparan sulfate binding growth factor.

9. The method of claim 1 wherein the growth factor binds to the heparan sulfate-decorated perlecan polypeptide.

10. The method of claim 1 wherein said nucleic acid construct is delivered in a pharmaceutically acceptable carrier or diluent.

11. The method of claim 1 where the first and second nucleic acid constructs are delivered together or separately.

12. The method of claim 1 where the first and second nucleic acid constructs are administered as a polycistronic nucleic acid construct.

13. A method for the effective healing of a wound or a cutaneous injury, the method comprising administering to a subject in need of such healing a first nucleic acid construct encoding a perlecan polypeptide containing at least one a heparan sulfate attachment site, whereby a heparan sulfate-decorated perlecan polypeptide is generated by the subject, said first nucleic acid construct being delivered in an amount sufficient to deliver an effective amount of the heparan sulfate-decorated perlecan polypeptide to the wound or cutaneous injury.

14. The method of claim 13 where the perlecan polypeptide encoded by the first nucleic acid sequence has the sequence of SEQ ID NO. 4.

15. The method of claim 13 where the perlecan polypeptide encoded by the first nucleic acid sequence is 70% homologous to the sequence of SEQ ID NO. 4.

16. The method of claim 13 where the perlecan polypeptide encoded by the first nucleic acid sequence is a fragment of SEQ ID NO. 4.

17. The method of claim 13 wherein the perlecan polypeptide encoded by the first nucleic acid sequence contains amino acids 1-172 of SEQ ID NO. 4.

18. The method of claim 13 further comprising administering a growth factor or a fragment of a growth factor capable of biding to the heparan sulfate-decorated polypeptide, said growth factor or a fragment of a growth factor being delivered in an amount sufficient to deliver an effective amount of at least one growth factor or a fragment of a growth factor to the wound or cutaneous injury.

19. The method of claim 18 wherein the growth factor is a heparan sulfate binding growth factor.

20. The method of claim 18 wherein the growth factor binds to the heparan sulfate-decorated perlecan polypeptide.

21. The method of claim 18 wherein said first nucleic acid construct and the growth factor or a fragment of a growth factor are delivered in a pharmaceutically acceptable carrier or diluent.

22. The method of claim 18 wherein the growth factor or a fragment of a growth factor is administered to the wound or cutaneous injury in the subject.

23. The method of claim 18 wherein the administration results in an improvement in healing of the wound or cutaneous injury in the subject.

24. A composition for healing a wound or cutaneous injury, said composition comprising a first nucleic acid construct encoding a perlecan polypeptide containing at least one heparan sulfate attachment site, and a second nucleic acid sequence encoding a growth factor or a fragment of a growth factor capable of binding to the perlecan polypeptide, whereby on administration to a subject, the first nucleic acid construct generates a heparan sulfate-decorated perlecan polypeptide, the composition comprising an amount of the first nucleic acid construct sufficient to deliver an effective amount of the heparan sulfate-decorated perlecan polypeptide to the wound or cutaneous injury.