# Patent

## Title
Hepatocyte growth factor nucleic acid sequence to enhance musculocutaneous flap survival

## Abstract

The present invention relates to the use of growth factors in improving tissue survival. In particular, the invention describes methods for enhancing organ transplant, musculocutaneous flap or skin graft survival by administering a nucleic acid sequence encoding hepatocyte growth factor.

## Background
BACKGROUND
1. Field of the Invention
This invention generally relates to the use of nucleic acid sequences encoding growth factors to promote angiogenesis and wound healing. In particular, described herein are methods and compositions for enhancing flap and skin graft survival by administering a nucleic acid sequence encoding hepatocyte growth factor.
2. Background of the Invention
Hepatocyte growth factor (HGF) functions as a growth factor for particular tissues and cell types. HGF was initially identified as a mitogen for hepatocytes. Michalopoulos et al., Cancer Res., 44:4414-4419 (1984); Russel et al., J. Cell. Physiol., 119:183-192 (1984); Nakamura et al., Biochem. Biophys. Res. Comm., 122:1450-1459 (1984). Nakamura et al. (supra), reported the purification of HGF from the serum of partially hepatectomized rats. Subsequently, the subunit structure of HGF was determined when HGF was purified from rat platelets. Nakamura et al., Proc. Natl. Acad. Sci. USA, 83:6489-6493 (1986); Nakamura et al., FEBS Letters, 224:311-316 (1987). Human HGF (“huHGF”) has also been purified from human plasma. Gohda et al., J. Clin. Invest., 81:414-419 (1988).
Comparisons of the amino acid sequence of rat HGF and huHGF revealed that the two sequences are highly conserved and have the same characteristic structural features. For example, the length of the four kringle domains in rat HGF is exactly the same as in huHGF, and the location of cysteine residues are in exactly the same positions. This is an indication that the three-dimensional structure of the two proteins is similar. Okajima et al. Eur. J. Bioch., 193:375-81 (1990); Tashiro et al., Proc. Natl. Acad. Sci., USA, 87:3200-4 (1990).
Furthermore, several reports revealed close sequence homology between HGF and scatter factor (SF). Gherardi and Stoker, Nature, 346:228 (1990); Weidner et al., J. Cell Biol., 111:2097-2108 (1990); Coffer et al., Biochem J., 278:35-41 (1991). SF is a polypeptide that stimulates dissociation of epithelial cell colonies in monolayer culture. Gherardi et al., Proc. Natl. Acad. Sci. USA, 86:5844-5848 (1989). In fact, there now is evidence indicating that the two factors are identical; they are identical in structure and biological activity. Weidner et al., Proc. Natl. Acad. Sci. USA, 88:7001-5 (1991); Bhargava et al., Cell Growth Differ. 3:11-20 (1992); Naldini et al., EMBO J., 10:2867-78 (1991); Furlong et al., J. Cell Sci., 100:173-7 (1991). HGF and HGF variants are described further in U.S. Pat. Nos. 5,227,158, 5,316,921, and 5,328,837.
Binding of HGF to its receptor is believed to be conveyed by a functional domain located in the N-terminal portion of the HGF molecule. Matsumoto et al., Biochem. Biophys. Res. Commun., 181:691-699 (1991); Hartmann et al., Proc. Natl. Acad. Sci. USA., 89:11574-11578 (1992); Lokker et al., EMBO J., 11:2503-2510 (1992); Lokker and Godowski, J. Biol. Chem., 268:17145-17150 (1991). The HGF receptor is usually referred to as “c-Met” or “p190MET” and typically comprises, in its native form, a 190-kDa heterodimeric (a disulfide-linked 50-kDa α-chain and a 145-kDa β-chain) membrane-spanning tyrosine kinase protein. Park et al., Proc. Natl. Acad. Sci. USA, 84:6379-6383 (1987). The c-Met protein becomes phosphorylated on tyrosine residues of the 145-kDa β-subunit upon HGF binding.
Various biological activities have been described for HGF and its receptor. See, generally, Chan et al., HEPATOCYTE GROWTH FACTOR—SCATTER FACTOR (HGF-SF) AND THE C-MET RECEPTOR, Goldberg and Rosen, eds., Birkhauser Verlag-Basel (1993), pp. 67-79). For example, HGF has been shown to be a mitogen for certain cell types, including melanocytes, renal tubular cells, keratinocytes, certain endothelial cells and cells of epithelial origin. Matsumoto et al., Biochem. Biophys. Res. Commun., 176:45-51 (1991); Igawa et al., Biochem. Biophys. Res. Commun., 174:831-838 (1991); Han et al., Biochem., 30:9768-9780 (1991); Rubin et al., Proc. Natl. Acad. Sci. USA, 88:415-419 (1991). HGF has also been described as an epithelial morphogen, Montesano et al., Cell, 67:901-908 (1991), and therefore, HGF has been postulated to be important in tumor invasion, Comoglio, HEPATOCYTE GROWTH FACTOR—SCATTER FACTOR (HGF-SF) AND THE C-MET RECEPTOR, Goldberg and Rosen, eds., Birkhauser Verlag-Basel (1993), pp. 131-165. Until now, the intramuscular delivery of an HGF gene to promote flap and skin graft survival has not been described.
The use of skin flaps has gained increased acceptance and use in the course of reconstructive surgery, as well as in other forms of surgery. However, these techniques continue to be plagued by problems having to do with survival of the skin flaps which is, at least in part, due to the inefficient revascularization at the surgical site. Indeed, a number of approaches have been considered or evaluated for improving skin flap survival. See, for example, Waters et al., which provides a comparative analysis of the ability of five classes of pharmacological agents to augment skin flap survival in various models and species. Annals of Plastic Surgery, 23(2):117-22 (1989). Nevertheless, there still remains a need in the art for compositions and methods for enhancing survival of flap and skin grafts.
SUMMARY OF THE INVENTION
Therefore, the present invention describes the use of a nucleic acid sequence encoding HGF to enhance the survival of a flap or a skin graft.
In particular, described herein is a method for enhancing tissue survival, including survival of a flap or a skin graft following flap or skin graft surgery, comprising administering to a subject in need thereof a vector that comprises a nucleic acid sequence encoding hepatocyte growth factor. In specific embodiments, the vector is administered intramuscularly and is administered at least about 3-14 days prior to flap or skin graft surgery, at least about 5-10 days prior to surgery, or at least about 7 days prior to surgery. The instant invention is suitable for enhancing the survival of a flap, such as a skin flap, a muscle flap, a myocutaneous flap, or a cartilocutaneous flap, or a skin graft. Survival of the flap or skin graft may be enhanced by at least about 10-30% as compared to an untreated subject.
In one embodiment, the vector comprises the HGF nucleic acid sequence represented by SEQ ID NO. 1. The vector also may comprise the pcDNA3.1(−) plasmid, as set forth in SEQ ID NO. 2, or the pVAX1 plasmid, as set forth in SEQ ID NO. 3.
In another embodiment, the present invention discloses a method for enhancing organ transplant survival in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a composition that comprises a nucleic acid sequence encoding HGF.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 compares musculocutaneous flap survival in an animal model after pretreatment with normal saline solution, an empty vector, or a vector comprising a nucleic acid sequence encoding HGF.
FIG. 2 depicts the survival (1 week after surgery) of a transverse rectus abdominis musculocutaneous (TRAM) flap in rats pre-treated 7 days prior to the surgery with (A) normal saline, (B) empty vector or (C) vector comprising a nucleic acid sequence encoding HGF. (N=8 for each group).
FIG. 3 is a histogram representing mean percentage blood flow in animals given normal saline solution (NSS), empty vector, or a vector comprising a nucleic acid sequence encoding HGF one week post-injection. Groups H1, H2 and H3 represent the experimental groups treated with 125 μg, 250 μg, and 500 μg HGF, respectively.



DETAILED DESCRIPTION OF THE INVENTION
1. Introduction
The inventors have surprisingly discovered that pre-treating subjects with a nucleic acid sequence encoding HGF prior to flap, skin graft or organ transplant surgery significantly enhances survival of the tissue as compared to transplanted tissue in control subjects.
2. Definitions
Unless otherwise specified, “a” or “an” means one or more.
The terms “hepatocyte growth factor” and “HGF” as used herein include hepatocyte growth factor from humans (“huHGF”) and any non-human mammalian species of HGF, including rat HGF. The term “HGF” as used herein includes mature, pre, pre-pro, and pro forms, including forms purified from a natural source, and chemically synthesized or recombinantly produced HGF.
“Sequence identity” is defined herein with reference the Blast 2 algorithm, which is available at the NCBI (http://www.ncbi.nlm.nih.gov/BLAST), using default parameters. References pertaining to this algorithm include those found at http://www.ncbi.nlm.nih.gov/BLAST/blast_references.html; Altschul, et al., J. Mol. Biol. 215: 403-410 (1990); Gish & States, Nature Genet. 3: 266-272 (1993); Madden et al., Meth. Enzymol. 266: 131-141 (1996); Altschul et al., Nucleic Acids Res. 25: 3389-3402 (1997), and Zhang & Madden, Genome Res. 7: 649-656 (1997).
The terms “alteration,” “amino acid alteration,” “variant,” and “amino acid sequence variant” refer to HGF molecules with some differences in their amino acid sequences as compared to a native human HGF. Ordinarily, the variants will possess at least about 80%, 85%, or 90% homology with the domains of native human HGF, including sequences at least about 95% homologous or at least about 99% homologous to native human HGF.
3. Hepatocyte Growth Factor Nucleic Acid Sequence and Variants Thereof
The nucleic acid sequences encoding HGF for use in the present invention may encode a hepatic parenchymal cell growth factor. The encoded HGF may have a structure with six domains (finger, Kringle 1, Kringle 2, Kringle 3, Kringle 4 and serine protease domains).
One human HGF nucleic acid sequence suitable for use in the present invention is represented as SEQ ID NO:1 herein. Other human HGF nucleic acids also are suitable for use in the present invention, such as the hepatic parenchymal cell growth factor sequence disclosed in Kitamura et al., U.S. Pat. No. 5,500,354.
Likewise, non-mammalian HGF nucleic acids are suitable for use in the present invention. Rat HGF, for example, shares the same structural features as human HGF and is described in Tashiro et al., Proc. Nat'l. Acad. Sci. USA, 87(8):3200-4 (1990) and GenBank Accession No. NM—017017.
In other embodiments, the nucleic acid encoding HGF useful in the present invention has at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least 99% sequence identity with a native mammalian HGF gene. For example, genes having at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% sequence identity, at least about 95% sequence identity, or at least about 99% sequence identity to SEQ ID NO:1 can be used in the present invention. As used herein, two nucleic acid molecules or proteins are said to “share significant sequence identity” if the two contain regions which possess greater than 90% sequence (amino acid or nucleic acid) identity over the entire length of the gene.
The invention also includes nucleic acid sequences that encode HGF proteins that are variants of a native HGF protein. For example, such HGF variants may have at least about 80% sequence identity, at least about 85% sequence identity, at least about 90% identity, at least 95% identity, or at least about 99% sequence identity to the protein encoded by the nucleic acid sequence represented in SEQ ID NO. 1. Given the known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist readily can construct DNAs encoding the conservative amino acid variants described herein. Examples of suitable variants are described below.
Fragments of HGF constitute HGF with fewer than all six domains (finger, Kringle 1, Kringle 2, Kringle 3, Kringle 4 and serine protease domains). Variants of HGF may have some of the domains of HGF repeated. Both fragments and variants are included within the scope of the invention if they still retain their respective ability to bind a HGF receptor, as determined by means known in the art.
Substituted HGF variants are those that have at least one amino acid residue in the corresponding wild-type HGF sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. Conservative substitutions are contemplated in the present invention. For example, (a) nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; (b) polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positively charged (basic) amino acids include arginine, lysine, and histidine; and (d) negatively charged (acidic) amino acids include aspartic acid and glutamic acid, and substitutions typically may be made within groups (a)-(d). In addition, glycine and proline may be substituted for one another based on their ability to disrupt α-helices. Similarly, certain amino acids, such as alanine, cysteine, leucine, methionine, glutamic acid, glutamine, histidine and lysine are more commonly found in α-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophan and threonine are more commonly found in β-pleated sheets. Glycine, serine, aspartic acid, asparagine, and proline are commonly found in turns. Some preferred substitutions may be made among the following groups: (i) S and T; (ii) P and G; and (iii) A, V, L and I.
Insertional HGF variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in the wild-type HGF molecule. Immediately adjacent to an amino acid means connected to either the α-carboxy or α-amino functional group of the amino acid. The insertion may be of one or more amino acids. Ordinarily, the insertion will consist of one or two conservative amino acids. As stated above, amino acids similar in charge and/or structure to the amino acids adjacent to the site of insertion are defined as conservative. Alternatively, this invention includes insertion of an amino acid with a charge and/or structure that is substantially different from the amino acids adjacent to the site of insertion.
Deletional variants are those with one or more amino acids in the wild-type HGF molecule removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the HGF molecule. Such deletional variants are also contemplated in the present invention.
All variants suitable for use in the present invention retain HGF activity. Such activity can be assayed by one of skill in the art according to known methods.
4. Recombinant Vector Production
Recombinant vector production is well known in the art and is outlined in a brief exemplary fashion below.
Generally speaking, the constructs of the present invention comprise a vector, such as a plasmid or viral vector, into which a genomic DNA or DNA fragment or cDNA bearing an open reading frame is inserted, in either orientation. The invention further contemplates cells containing these vectors.
Bacterial Expression
Useful vectors for bacterial expression may be constructed by inserting a structural DNA sequence encoding a desired protein, together with suitable translation initiation and termination signals in operable reading phase with a functional promoter. The vector may comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and, if desirable, to provide amplification within the host. Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice. In one embodiment, the prokaryotic host is E. coli. 
Bacterial vectors may be, for example, bacteriophage-, plasmid- or cosmid-based. These vectors can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids typically containing elements of the well known cloning vector pBR322 (ATCC 37017). Such commercial vectors include, for example, pGEM 1 (Promega Biotec, Madison, Wis., USA), pBs, phagescript, PsiX174, pBluescript SK, pBs KS, pNH8a, pNH16a, pNH18a, pNH46a (Stratagene); pTrc99A, pKK223-3, pKK233-3, pKK232-8, pDR540, and pRIT5 (Pharmacia).
These “backbone” sections may be combined with an appropriate promoter and the structural sequence to be expressed. Bacterial promoters include lac, T3, T7, lambda PR or PL, trp, and ara.
These vectors optionally may be used for recombinant protein production. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter may be derepressed/induced by appropriate means (e.g., temperature shift or chemical induction) and cells may be cultured for an additional period. Cells are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification and isolation of the recombinant protein.
Eukaryotic Expression Vectors
In one embodiment, HGF cDNA is subcloned into a mammalian expression vector. Various mammalian cell culture systems can be employed to express recombinant protein. Examples of mammalian expression systems include selected mouse L cells, such as thymidine kinase-negative (TK) and adenine phosphoribosyl transferase-negative (APRT) cells. Other examples include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell 23: 175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines. Mammalian expression vectors may comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5′ flanking non-transcribed sequences. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early promoter, enhancer, splice, and polyadenylation sites may be used to provide the required non-transcribed genetic elements.
Mammalian promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. In one embodiment, the HGF cDNA is subcloned into any expression vector in which the expression is driven by a CMV promoter. Exemplary mammalian vectors include pWLneo, pSV2cat, pOG44, pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). In one specific embodiment, the mammalian expression vector is pcDNA3.1 (Invitrogen) or pVAX1 (Invitrogen). pVAX1 is a 3.0 kb plasmid vector designed for use in the development of DNA vaccines. HGF cDNA can be inserted, for example, into a multiple cloning site of the pVAX1 vector. pcDNA3.1(−) has the same multiple cloning site as pcDNA3.1(+) but in a reverse orientation.
In mammalian host cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, the coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing a target protein in infected hosts. (See, e.g., Logan et al., 1984, Proc. Natl. Acad. Sci. USA 81: 3655-3659). If a viral vector is chosen as the delivery vehicle it may be one which is capable of integrating into the host genome so that the gene can be expressed permanently. In cases where the vector does not integrate into the host genome, the expression of the gene may be transient rather than permanent.
Adenoviral vectors (“Ad”) are currently among the most efficient gene transfer vehicles for both in vitro and in vivo delivery, but the utilization of a first generation Ad for many gene therapy applications is limited due to the transient nature of transgene expression obtained by these vectors. Several factors have been shown to contribute to and modulate the duration of Ad-mediated gene expression as well as the immunogenicity of these vectors, including “leaky” viral protein expression and the transgene delivered. The development of Ad vectors, deleted in all viral protein coding sequences offers the prospects of a potentially safer, less immunogenic vector with an insert capacity of up to approximately 37 kb. This vector requires supplementation of viral regulatory and structural proteins in trans for packaging and rescue and is therefore helper dependent (HD). This is further described in Parks et al., Proc. Natl. Acad. Sci. USA, 93:13565-13570 (1996).
Use of retroviral vectors for protein expression are also known in the art. See, for example, Veres, et al., J. Virol., 72:1894-1901 (1998); Agarwal et al., J. Virol., 72:3720-3728 (1998); Forestell et al., Gene Therapy, 4:600-610 (1997); Plavec et al., Gene Therapy, 4:128-139, 1997; Forestell et al., Gene Therapy, 2:723-730 (1995); and Rigg et al., J. Virol., 218:290-295, 1996. The genome of a recombinant retroviral vector is comprised of long terminal repeat (LTR) sequences at both ends which serve as a viral promoter/enhancer and a transcription initiation site, and a Psi site which serves as a virion packaging signal and a selectable marker gene. In one embodiment, the HGF polynucleotide sequences disclosed herein can be cloned into a suitable cloning site in the retroviral genome. Expression is under the transcriptional control of the retroviral LTR. Tissue selectivity is determined by both the origin of the viral genome (e.g., sarcoma virus, leukemia virus, or mammary tumor virus) and the cell line used to package the virus.
The recombinant vector useful in the present invention may include the exogenous DNA and regulatory sequences necessary and sufficient for expression of the encoded product (e.g., HGF) upon entry into the target cell. In one embodiment of the present invention, the vector includes exogenous DNA encoding the desired product (i.e., HGF), and, optionally, DNA encoding a selectable marker, along with additional sequences necessary for expression of the exogenous DNA in a target cell. In one specific embodiment, the vector comprises the nucleic acid sequence of SEQ ID NO. 1 and a pcDNA3.1(−) plasmid or a pVAX1 plasmid is used. See, e.g., SEQ ID NOs. 2 and 3. In yet another embodiment, the vector does not comprise an enhancer element. Additionally, infectious vectors can be used in the present invention, such as adenoviral, retroviral, and adenovirus-associated viral vectors, to express the exogenous DNA sequence in a target cell.
In accordance with the invention, a vector encoding HGF may be administered by intramuscular injection, or by intravenous, intraperitoneal, oral or subcutaneous means, or by other means of delivery. Suitable titers will depend on a number of factors, such as the particular vector chosen, the host, and the strength of promoter used.
In accordance with the invention, the vector encoding HGF is administered (for example, by intramuscular injection) prior to organ transplant, skin flap or skin graft surgery, up to a week or more prior to surgery. Administration at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, or at least about 10 days prior to organ transplant or flap or skin graft surgery is expressly contemplated and included within the invention. Administration up to about 14 days prior to organ transplant or flap or skin graft surgery is also contemplated and included within the invention. The term “about” in this context connotes up to one day before the specified number of days. For example, the phrase “at least about 3 days” means that the construct encoding HGF is administered between 2-3 days prior to organ transplant or flap or skin graft surgery. Thus, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22 or 24 hours prior to precisely 3 days before the surgery is within the meaning of the term “about.”
Administration of the vector encoding HGF may be by direct injection, i.e., at or near the site of the flap, graft or organ transplant. In one embodiment, the construct encoding HGF is administered intramuscularly, such into the rectus abdominus muscle.
The inventive methods described herein enhance flap or skin graft survival by at least about 10%, at least about 15%, at least about 20%, at least about 25%, or at least about 30% or more compared to an untreated control subject. The term “about” in this context connotes a range of up to 5% before or after the specified percentage. Thus, the phrase “about 10%” refers to a range of 5%-15%, but also specifically includes 10%.
5. Pharmaceutically Acceptable Formulations
The HGF vector or HGF nucleic acid sequence compositions as described herein can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the inventive compositions, or their functional derivatives, are combined in admixture with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulation, inclusive of other human proteins, e.g., human serum albumin, are described, for example, in Remington's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton Pa. (1980)). To form a pharmaceutically acceptable composition suitable for effective administration, such compositions will contain an effective amount of one or more of the vectors of the present invention, together with a suitable amount of carrier vehicle.
Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. Thus, the HGF vector or HGF nucleic acid sequence compositions described herein, and their physiologically acceptable salts and solvate, may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, rectal, subcutaneous or intramuscular administration. In one embodiment, the HGF nucleic acid sequence or HGF vector compositions are formulated for intramuscular administration.
For oral administration, the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they maybe presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated to give controlled release of the active compound. For buccal administration the composition may take the form of tablets or lozenges formulated in conventional manner.
For administration by inhalation, the HGF vector or HGF nucleic acid sequence compositions for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
The HGF vector or HGF nucleic acid sequence compositions may be formulated for intravenous, subcutaneous or intramuscular administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampules or in multi-dose containers, with an added preservative. The HGF compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
In addition to the formulations described previously, the HGF vector or HGF nucleic acid sequence compositions may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
The compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration.
The present invention describes therapy with HGF nucleic acid sequences to enhance organ transplant, flap and skin graft survival. In particular, a nucleic acid sequence encoding HGF can be administered prior to organ transplant, flap or skin graft surgery. The invention is useful in any type of organ transplant, flap or skin graft surgery. For example, the invention is useful in conjunction with transplantation of a wide variety of organs, including skin, kidney, heart, liver, spleen, bone marrow, pancreas, lung, and islet of langerhans. There are many different kinds of flaps that can be used to address cutaneous defects, defects of muscle, defects of subcutaneous tissues, and defects in bone. For example, the HGF methods and compositions of the present invention can be used in conjunction with (including prior to) a cutaneous flap, muscle flap, myocutaneous flap, or cartilocutaneous flap surgery. Likewise, the HGF methods and compositions of the present invention can be used in conjunction with (including prior to) skin graft surgery.
The therapeutic methods of the present invention involve administering to a subject in need of treatment a therapeutically effective amount of the HGF vector or HGF nucleic acid sequence compositions described herein. “Therapeutically effective” is employed here to denote the amount of the HGF composition that is of sufficient quantity to promote angiogenesis and accelerate wound healing. In particular, it is desirable to administer a therapeutically effective amount of an HGF composition that will enhance organ transplant, musculocutaneous flap or skin graft survival. Some methods contemplate combination therapy with known medicaments or therapies that also promote angiogenesis or organ or flap survival.
The therapeutically effective amount of the HGF composition for use in this invention largely will depend on particular patient characteristics, the route of administration, and the nature of the disorder being treated (such as the type of organ transplant or the size, location, and thickness of the flap or skin graft at issue). General guidance can be found, for example, in the publications of the International Conference on Harmonisation and in REMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528 (Mack Publishing Company 1990). In addition, the therapeutically effective amount may depend on such factors as toxicity and efficacy of the medicament. Toxicity may be determined using methods well known in the art and found in the foregoing references. Efficacy may be determined utilizing the same guidance in conjunction with the methods described below in the Examples. A pharmaceutically effective amount, therefore, is an amount that is deemed by the clinician to be toxicologically tolerable, yet efficacious. Efficacy, for example, can be measured by the decrease in necrosis, or the increase in angiogenesis or organ transplant or flap or graft survival. As illustrated by the foregoing references, the determination of a therapeutically effective amount can be determined by those skilled in the art, as guided by this disclosure.
In one embodiment, the therapeutically effective amount is from about 0.1 to about 50 mg per treatment, such as from about 0.5 to about 25 mg per treatment, and from about 1 to about 10 mg per treatment. In a specific embodiment, the plasmid is administered in several injections to the same area of the body as part of a single treatment.
The patient may be a human or non-human mammal, or another animal. A patient typically will be in need of treatment when scheduled to receive flap or skin graft surgery.
The invention is further described by reference to the following examples, which are provided for illustration only. The invention is not limited to the examples but rather includes all variations that are evident from the teachings provided herein.
EXAMPLE 1
Plasmid Constructs
Human HGF is subcloned into the pcDNA3.1(−) (Invitrogen) into the NotI site, which produces the human HGF protein under the control of the CMV promoter. See, e.g., SEQ ID NO. 2. The HGF gene plasmid concentrate is reconstituted with normal saline solution (NSS) to obtain a 1 μg/μl concentration.
EXAMPLE 2
HGF Nucleic Acid Sequence Enhances Flap Survival
The experiment is conducted following the guidelines set by the animal laboratory of the Institute of Animal and Experimental Sciences, Osaka University. Twenty four male Sprague-Dawley rats weighing 300-350 grams are used and equally divided into three groups (N=8). Animals are anesthetized with an intraperitoneal injection of pentobarbital (2 mg/100 g). In group 1, 250 μl of NSS is injected into the left rectus abdominus muscle, while 250 μg (1 μg/μl) of empty plasmid (pcDNA3.1(−)) and 250 μg (1 μg/μl) of HGF gene containing plasmid is injected into the same site in Group 2 and Group 3, respectively. After 7 days, a simulated transverse rectus abdominus musculocutaneous (TRAM) flap measuring 4.5 cm×9 cm is elevated. TRAM flap based on the left rectus abdominus muscle as the carrier and the superior epigastric vessels as the vascular pedicle are designed on the lower half of the abdomen. The flap is sutured to its original location and monitored for 1 week for infection, necrosis and flap survival. All the animals are sacrificed using an overdose of pentobarbital given intraperitonally. Direct measurement is made on the area of necrosis and flap survival using a transparent metric template.
The data indicate the following with regard to flap survival one week post-surgery:











TABLE 1







NSS
Empty Vector
HGF IM



















% Flap Survival
54.125
53.875
87.75


Standard Deviation
22.96853438
25.41336825
17.88654722


Sample Size
8
8
8









The superiority of pre-treating a TRAM flap with a gene encoding HGF is also exemplified in FIG. 2. FIG. 2 demonstrates that flap survival is markedly enhanced one week post-surgery in animals receiving a gene encoding HGF (designated as “H” animals; n=8) at least 7 days prior to flap surgery, compared to control animals (designated as “C” animals; n=8) and animals treated with the empty vector (designated as “V” animals; n=8). (The number following the letters “H,” “C” or “V” indicate the animal number.)
In a separate study, 20 Sprague Dawley rats were randomized into 5 groups, 4 rats per group, and were directly injected with 125 μg, 250 μg, or 500 μg of a plasmid encoding HGF, NSS, or an empty vector into the left rectus abdominus muscle, (the likely TRAM flap cite). One week post-injection, skin blood flow measurements were taken by a laser color Doppler (Laser Doppler Imager, Moor Instruments), which determines blood flow velocity and correlates with capillary density. The results indicated that blood flow was higher in the treated area for the HGF treated groups compared to the groups given NSS or the empty vector (FIG. 3 and Table 2).













TABLE 2








Empty
HGF IM
HGF IM
HGF IM



NSS
Vector
(125 μg)
(250 μg)
(500 μg)





















Mean %
282.675
278.175
343.333
392.900
396.725


Blood


Flow


Standard
5.923593
16.7246
15.59202
37.70972
33.55604


Error


Sample
4
4
4
4
4


Size









FIG. 3 was generated by quantitatively converting the laser images from the laser Doppler, creating a histogram with the amount of blood flow provided on the y-axis and each experimental group, i.e., animals given NSS, the empty vector, H1(125 μg HGF), H2 (250 μg HGF) or H3 (500 μg HGF), on the x-axis. Use of the laser Doppler to visualize blood vessels and measure blood flow is described in Wong et al., Gastrointest. Endosc., 55(1): 88-95 (2002).
Additional embodiments are within the scope of the invention. For example, the invention is further illustrated by the following numbered embodiments:
1. A method for enhancing tissue survival, including survival of a flap or a skin graft following flap or skin graft surgery, comprising administering to a subject in need thereof a vector that comprises a nucleic acid sequence encoding hepatocyte growth factor.
2. The method of embodiment 1, wherein said flap is selected from the group consisting of a skin flap, a muscle flap, a myocutaneous flap and a cartilocutaneous flap.
3. The method of embodiment 1, wherein said vector is administered intramuscularly.
4. The method of embodiment 1, wherein said nucleic acid encoding HGF is represented by SEQ ID NO. 1.
5. The method of embodiment 4, wherein said vector is administered at least 3-14 days prior to flap or skin graft surgery.
6. The method of embodiment 4, wherein said vector is administered at least 5-10 days prior to flap or skin graft surgery.
7. The method of embodiment 4, wherein said vector is administered at least 7 days prior to flap or skin graft surgery.
8. The method of embodiment 5, wherein said vector is a plasmid selected from the group consisting of pcDNA3.1(−) and pVAX1.
9. The method of embodiment 5, wherein said vector comprises the sequence of SEQ ID NO. 2.
10. The method of embodiment 5, wherein said vector comprises sequence of SEQ ID NO. 3.
11. The method of embodiment 1, wherein the survival of the flap or skin graft is enhanced by at least 10-30% as compared to an untreated subject.
12. A method for enhancing organ transplant survival in a subject comprising administering to a subject in need thereof a therapeutically effective amount of a composition that comprises a nucleic acid encoding HGF.
13. The method of embodiment 3, wherein said vector is administered in the rectus abdominus muscle.
14. A method for enhancing tissue survival, including survival of a flap or a skin graft following flap or skin graft surgery, comprising administering to a subject in need thereof a vector that comprises a nucleic acid sequence encoding hepatocyte growth factor, wherein administration of the vector results in enhanced blood flow.
Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention. All of the publications, patent applications and patents cited in this specification are incorporated herein by reference in their entirety.








Hepatocyte Growth Factor Nucleic Acid Sequence









SEQ ID NO. 1









ATGTGGGTGACCAAACTCCTGCCAGCCCTGCTGCTGCAGCATGTCCTCCT



 


GCATCTCCTCCTGCTCCCCATCGCCATCCCCTATGCAGAGGGACAAAGGA


 


AAAGAAGAAATACAATTCATGAATTCAAAAAATCAGCAAAGACTACCCTA


 


ATCAAAATAGATCCAGCACTGAAGATAAAAACCAAAAAAGTGAATACTGC


 


AGACCAATGTGCTAATAGATGTACTAGGAATAAAGGACTTCCATTCACTT


 


GCAAGGCTTTTGTTTTTGATAAAGCAAGAAAACAATGCCTCTGGTTCCCC


 


TTCAATAGCATGTCAAGTGGAGTGAAAAAAGAATTTGGCCATGAATTTGA


 


CCTCTATGAAAACAAAGACTACATTAGAAACTGCATCATTGGTAAAGGAC


 


GCAGCTACAAGGGAACAGTATCTATCACTAAGAGTGGCATCAAATGTCAG


 


CCCTGGAGTTCCATGATACCACACGAACACAGCTTTTTGCCTTCGAGCTA


 


TCGGGGTAAAGACCTACAGGAAAACTACTGTCGAAATCCTCGAGGGGAAG


 


AAGGGGGACCCTGGTGTTTCACAAGCAATCCAGAGGTACGCTACGAAGTC


 


TGTGACATTCCTCAGTGTTCAGAAGTTGAATGCATGACCTGCAATGGGGA


 


GAGTTATCGAGGTCTCATGGATCATACAGAATCAGGCAAGATTTGTCAGC


 


GCTGGGATCATCAGACACCACACCGGCACAAATTCTTGCCTGAAAGATAT


 


CCCGACAAGGGCTTTGATGATAATTATTGCCGCAATCCCGATGGCCAGCC


 


GAGGCCATGGTGCTATACTCTTGACCCTCACACCCGCTGGGAGTACTGTG


 


CAATTAAAACATGCGCTGACAATACTATGAATGACACTGATGTTCCTTTG


 


GAAACAACTGAATGCATCCAAGGTCAAGGAGAAGGCTACAGGGGCACTGT


 


CAATACCATTTGGAATGGAATTCCATGTCAGCGTTGGGATTCTCAGTATC


 


CTCACGAGCATGACATGACTCCTGAAAATTTCAAGTGCAAGGACCTACGA


 


GAAAATTACTGCCGAAATCCAGATGGGTCTGAATCACCCTGGTGTTTTAC


 


CACTGATCCAAACATCCGAGTTGGCTACTGCTCCCAAATTCCAAACTGTG


 


ATATGTCACATGGACAAGATTGTTATCGTGGGAATGGCAAAAATTATATG


 


GGCAACTTATCCCAAACAAGATCTGGACTAACATGTTCAATGTGGGACAA


 


GAACATGGAAGACTTACATCGTCATATCTTCTGGGAACCAGATGCAAGTA


 


AGCTGAATGAGAATTACTGCCGAAATCCAGATGATGATGCTCATGGACCC


 


TGGTGCTACACGGGAAATCCACTCATTCCTTGGGATTATTGCCCTATTTC


 


TCGTTGTGAAGGTGATACCACACCTACAATAGTCAATTTAGACCATCCCG


 


TAATATCTTGTGCCAAAACGAAACAATTGCGAGTTGTAAATGGGATTCCA


 


ACACGAACAAACATAGGATGGATGGTTAGTTTGAGATACAGAAATAAACA


 


TATCTGCGGAGGATCATTGATAAAGGAGAGTTGGGTTCTTACTGCACGAC


 


AGTGTTTCCCTTCTCGAGACTTGAAAGATTATGAAGCTTGGCTTGGAATT


 


CATGATGTCCACGGAAGAGGAGATGAGAAATGCAAACAGGTTCTCAATGT


 


TTCCCAGCTGGTATATGGCCCTGAAGGATCAGATCTGGTTTTAATGAAGC


 


TTGCCAGGCCTGCTGTCCTGGATGATTTTGTTAGTACGATTGATTTACCT


 


AATTATGGATGCACAATTCCTGAAAAGACCAGTTGCAGTGTTTATGGCTG


 


GGGCTACACTGGATTGATCAACTATGATGGCCTATTACGAGTGGCACATC


 


TCTATATAATGGGAAATGAGAAATGCAGCCAGCATCATCGAGGGAAGGTG


 


ACTCTGAATGAGTCTGAAATATGTGCTGGGGCTGAAAAGATTGGATCAGG


 


ACCATGTGAGGGGGATTATGGTGGCCCACTTGTTTGTGAGCAACATAAAA


 


TGAGAATGGTTCTTGGTGTCATTGTTCCTGGTCGTGGATGTGCCATTCCA


 


AATCGTCCTGGTATTTTTGTCCGAGTAGCATATTATGCAAAATGGATACA


 


CAAAATTATTTTAACATATAAGGTACCACAGTCATAG
















pcDNA3.1(-)HGF Sequence









SEQ ID NO. 2




















GACGGATCGG
GAGATCTCCC
GATCCCCTAT
GGTGCACTCT
CAGTACAATC
TGCTCTGATG
CCGCATAGTT
AAGCCAGTAT
CTGCTCCCTG
CTTGTGTGTT




GGAGGTCGCT
GAGTAGTGCG
CGAGCAAAAT
TTAAGCTACA
ACAAGGCAAG
GCTTGACCGA
CAATTGCATG
AAGAATCTGC
TTAGGGTTAG
GCGTTTTGCG



CTGCTTCGCG
ATGTACGGGC
CAGATATACG
CGTTGACATT
GATTATTGAC
TACTTATTAA
TAGTAATCAA
TTACGGGGTC
ATTAGTTCAT
AGCCCATATA



TGGAGTTCCG
CGTTACATAA
CTTACGGTAA
ATGGCCCGCC
TGGCTGACCG
CCCAACGACC
CCCGCCCATT
GACGTCAATA
ATGACGTATG
TTCCCATAGT



AACGCCAATA
GGGACTTTCC
ATTGACGTCA
ATGGGTGGAG
TATTTACGGT
AAACTGCCCA
CTTGGCAGTA
CATCAAGTGT
ATCATATGCC
AAGTACGCCC



CCTATTGACG
TCAATGACGG
TAAATGGCCC
GCCTGGCATT
ATGCCCAGTA
CATGACCTTA
TGGGACTTTC
CTACTTGGCA
GTACATCTAC
GTATTAGTCA



TCGCTATTAC
CATGGTGATG
CGGTTTTGGC
AGTACATCAA
TGGGCGTGGA
TAGCGGTTTG
ACTCACGGGG
ATTTCCAAGT
CTCCACCCCA
TTGACGTCAA



TGGGAGTTTG
TTTTGGCACC
AAAATCAACG
GGACTTTCCA
AAATGTCGTA
ACAACTCCGC
CCCATTGACG
CAAATGGGCG
GTAGGCGTGT
ACGGTGGGAG



GTCTATATAA
GCAGAGCTCT
CTGGCTAACT
AGAGAACCCA
CTGCTTACTG
GCTTATCGAA
ATTAATACGA
CTCACTATAG
GGAGACCCAA
GCTGGCTAGC



GTTTAAACGG
GCCCTCTAGA
CTCGAGCGGC
CGCTCTAGAA
CTAGCTGGAT
CCTGCCAGCC
CGTCCAGCAG
CACCATGTGG
GTGACCAAAC
TCCTGCCAGC



CCTGCTGCTG
CAGCATGTCC
TCCTGCATCT
CCTCCTGCTC
CCCATCGCCA
TCCCCTATGC
AGAGGGACAA
AGGAAAAGAA
GAAATACAAT
TCATGAATTC



AAAAAATCAG
CAAAGACTAC
CCTAATCAAA
ATAGATCCAG
CACTGAAGAT
AAAAACCAAA
AAAGTGAATA
CTGCAGACCA
ATGTGCTAAT
AGATGTACTA



GGAATAAAGG
ACTTCCATTC
ACTTGCAAGG
CTTTTGTTTT
TGATAAAGCA
AGAAAACAAT
GCCTCTGGTT
CCCCTTCAAT
AGCATGTCAA
GTGGAGTGAA



AAAAGAATTT
GGCCATGAAT
TTGACCTCTA
TGAAAACAAA
GACTACATTA
GAAACTGCAT
CATTGGTAAA
GGACGCAGCT
ACAAGGGAAC
AGTATCTATC



ACTAAGAGTG
GCATCAAATG
TCAGCCCTGG
AGTTCCATGA
TACCACACGA
ACACAGCTTT
TTGCCTTCGA
GCTATCGGGG
TAAAGACCTA
CAGGAAAACT



ACTGTCGAAA
TCCTCGAGGG
GAAGAAGGGG
GACCCTGGTG
TTTCACAAGC
AATCCAGAGG
TACGCTACGA
AGTCTGTGAC
ATTCCTCAGT
GTTCAGAAGT



TGAATGCATG
ACCTGCAATG
GGGAGAGTTA
TCGAGGTCTC
ATGGATCATA
CAGAATCAGG
CAAGATTTGT
CAGCGCTGGG
ATCATCAGAC
ACCACACCGG



CACAAATTCT
TGCCTGAAAG
ATATCCCGAC
AAGGGCTTTG
ATGATAATTA
TTGCCGCAAT
CCCGATGGCC
AGCCGAGGCC
ATGGTGCTAT
ACTCTTGACC



CTCACACCCG
CTGGGAGTAC
TGTGCAATTA
AAACATGCGC
TGACAATACT
ATGAATGACA
CTGATGTTCC
TTTGGAAACA
ACTGAATGCA
TCCAAGGTCA



AGGAGAAGGC
TACAGGGGCA
CTGTCAATAC
CATTTGGAAT
GGAATTCCAT
GTCAGCGTTG
GGATTCTCAG
TATCCTCACG
AGCATGACAT
GACTCCTGAA



AATTTCAAGT
GCAAGGACCT
ACGAGAAAAT
TACTGCCGAA
ATCCAGATGG
GTCTGAATCA
CCCTGGTGTT
TTACCACTGA
TCCAAACATC
CGAGTTGGCT



ACTGCTCCCA
AATTCCAAAC
TGTGATATGT
CACATGGACA
AGATTGTTAT
CGTGGGAATG
GCAAAAATTA
TATGGGCAAC
TTATCCCAAA
CAAGATCTGG



ACTAACATGT
TCAATGTGGG
ACAAGAACAT
GGAAGACTTA
CATCGTCATA
TCTTCTGGGA
ACCAGATGCA
AGTAAGCTGA
ATGAGAATTA
CTGCCGAAAT



CCAGATGATG
ATGCTCATGG
ACCCTGGTGC
TACACGGGAA
ATCCACTCAT
TCCTTGGGAT
TATTGCCCTA
TTTCTCGTTG
TGAAGGTGAT
ACCACACCTA



CAATAGTCAA
TTTAGACCAT
CCCGTAATAT
CTTGTGCCAA
AACGAAACAA
TTGCGAGTTG
TAAATGGGAT
TCCAACACGA
ACAAACATAG
GATGGATGGT



 



TAGTTTGAGA
TACAGAAATA
AACATATCTG
CGGAGGATCA
TTGATAAAGG
AGAGTTGGGT
TCTTACTGCA
CGACAGTGTT
TCCCTTCTCG
AGACTTGAAA



GATTATGAAG
CTTGGCTTGG
AATTCATGAT
GTCCACGGAA
GAGGAGATGA
GAAATGCAAA
CAGGTTCTCA
ATGTTTTCCA
GCTGGTATAT
GGCCCTGAAG



GATCAGATCT
GGTTTTAATG
AAGCTTGCCA
GGCCTGCTGT
CDTGGATGAT
TTTGTTAGTA
CGATTGATTT
ACCTAATTAT
GGATGCACAA
TTCCTGAAAA



GACCAGTTGC
AGTGTTTATG
GCTGGGGCTA
CACTGGATTG
ATCAACTATG
ATGGCCTATT
ACGAGTGGCA
CATCTCTATA
TAATGGGAAA
TGAGAAATGC



AGCCAGCATC
ATCGAGGGAA
GGTGACTCTG
AATGAGTCTG
AAATATGTGC
TGGGGCTGAA
AAGATTGGAT
CAGGACCATG
TGAGGGGGAT
TATGGTGGCC



CACTTGTTTG
TGAGCAACAT
AAAATGAGAA
TGGTTCTTGG
TGTCATTGTT
CCTGGTCGTG
GATGTGCCAT
TCCAAATCGT
CCTGGTATTT
TTGTCCGAGT



AGCATATTAT
GCAAAATGGA
TACACAAAAT
TATTTTAACA
TATAAGGTAC
CACAGTCATA
GCTGTTAACC
CGGGTCGAAG
CGGCCGCCAC
TGTGCTGGAT



ATCTGCAGAA
TTCCACCACA
CTGGACTAGT
GGATCCGAGC
TCGGTACCAA
GCTTAAGTTT
AAACCGCTGA
TCAGCCTCGA
CTGTGCCTTC
TAGTTGCCAG



CCATCTGTTG
TTTGCCCCTC
CCCCGTGCCT
TCCTTGACCC
TGGAAGGTGC
CACTCCCACT
GTCCTTTCCT
AATAAAATGA
GGAAATTGCA
TCGCATTGTC



TGAGTAGGTG
TCATTCTATT
CTGGGGGGTG
GGGTGGGGCA
GGACAGCAAG
GGGGAGGATT
GGGAAGACAA
TAGCAGGCAT
GCTGGGGATG
CGGTGGGCTC



TATGGCTTCT
GAGGCGGAAA
GAACCAGCTG
GGGCTCTAGG
GGGTATCCCC
ACGCGCCCTG
TAGCGGCGCA
TTAAGCGCGG
CGGGTGTGGT
GGTTACGCGC



AGCGTGACCG
CTACACTTGC
CAGCGCCCTA
GCGCCCGCTC
CTTTCGCTTT
CTTCCCTTCC
TTTCTCGCCA
CGTTCGCCGG
CTTTCCCCGT
CAAGCTCTAA



ATCGGGGGCT
CCCTTTAGGG
TTCCGATTTA
GTGCTTTACG
GCACCTCGAC
CCCAAAAAAC
TTGATTAGGG
TGATGGTTCA
CGTAGTGGGC
CATCGCCCTG



ATAGACGGTT
TTTCGCCCTT
TGACGTTGGA
GTCCACGTTC
TTTAATAGTG
GACTCTTGTT
CCAAACTGGA
ACAACACTCA
ACCCTATCTC
GGTCTATTCT



TTTGATTTAT
AAGGGATTTT
GCCGATTTCG
GCCTATTGGT
TAAAAAATGA
GCTGATTTAA
CAAAAATTTA
ACGCGAATTA
ATTCTGTGGA
ATGTGTGTCA



GTTAGGGTGT
GGAAAGTCCC
CAGGCTCCCC
AGCAGGCAGA
AGTATGCAAA
GCATGCATCT
CAATTAGTCA
GCAACCAGGT
GTGGAAAGTC
CCCAGGCTCC



CCAGCAGGCA
GAAGTATGCA
AAGCATGCAT
CTCAATTAGT
CAGCAACCAT
AGTCCCGCCC
CTAACTCCGC
CCATCCCGCC
CCTAACTCCG
CCCAGTTCCG



CCCATTCTCC
GCCCCATGGC
TGACTAATTT
TTTTTATTTA
TGCAGAGGCC
GAGGCCGCCT
CTGCCTCTGA
GCTATTCCAG
AAGTAGTGAG
GAGGCTTTTT



TGGAGGCCTA
GGCTTTTGCA
AAAAGCTCCC
GGGAGCTTGT
ATATCCATTT
TCGGATCTGA
TCAAGAGACA
GGATGAGGAT
CGTTTCGCAT
GATTGAACAA



GATGGATTGC
ACGCAGGTTC
TCCGGCCGCT
TGGGTGGAGA
GGCTATTCGG
CTATGACTGG
GCACAACAGA
CAATCGGCTG
CTCTGATGCC
GCCGTGTTCC



GGCTGTCAGC
GCAGGGGCGC
CCGGTTCTTT
TTGTCAAGAC
CGACCTGTCC
GGTGCCCTGA
ATGAACTGCA
GGACGAGGCA
GCGCGGCTAT
CGTGGCTGGC



CACGACGGGC
GTTCCTTGCG
CAGCTGTGCT
CGACGTTGTC
ACTGAAGCGG
GAAGGGACTG
GCTGCTATTG
GGCGAAGTGC
CGGGGCAGGA
TCTCCTGTCA



TCTCACCTTG
CTCCTGCCGA
GAAAGTATCC
ATCATGGCTG
ATGCAATGCG
GCGGCTGCAT
ACGCTTGATC
CGGCTACCTG
CCCATTCGAC
CACCAAGCGA



AACATCGCAT
CGAGCGAGCA
CGTACTCGGA
TGGAAGCCGG
TCTTGTCGAT
CAGGATGATC
TGGACGAAGA
GCATCAGGGG
CTCGCGCCAG
CCGAACTGTT



CGCCAGGCTC
AAGGCGCGCA
TGCCCGACGG
CGAGGATCTC
GTCGTGACCC
ATGGCGATGC
CTGCTTGCCG
AATATCATGG
TGGAAAATGG
CCGCTTTTCT



GGATTCATCG
ACTGTGGCCG
GCTGGGTGTG
GCGGACCGCT
ATOAGGACAT
AGCGTTGGCT
ACCCGTGATA
TTGCTGAAGA
GCTTGGCGGC
GAATGGGCTG



ACCGCTTCCT
CGTGCTTTAC
GGTATCGCCG
CTCCCGATTC
GCAGCGCATC
GCCTTCTATC
GCCTTCTTGA
CGAGTTCTTC
TGAGCGGGAC
TCTGGGGTTC



GAAATGACCG
ACCAAGCGAC
GCCCAACCTG
CCATCACGAG
ATTTCGATTC
CACCGCCGCC
TTCTATGAAA
GGTTGGGCTT
CGGAATCGTT
TTCCGGGACG



CCGGCTGGAT
GATCCTCCAG
CGCGGGGATC
TCATGCTGGA
GTTCTTCGCC
CACCCCAACT
TGTTTATTGC
AGCTTATAAT
GGTTACAAAT
AAAGCAATAG



 



CATCACAAAT
TTCACAAATA
AAGCATTTTT
TTCACTGCAT
TCTAGTTGTG
GTTTGTCCAA
ACTCATCAAT
GTATCTTATC
ATGTCTGTAT
ACCGTCGACC



TCTAGCTAGA
GCTTGGCGTA
ATCATGGTCA
TAGCTGTTTC
CTGTGTGAAA
TTGTTATCCG
CTCACAATTC
CACACAACAT
ACGAGCCGGA
AGCATAAAGT



GTAAAGCCTG
GGGTGCCTAA
TGAGTGAGCT
AACTCACATT
AATTGCGTTG
CGCTCACTGC
CCGCTTTCCA
GTCGGGAAAC
CTGTCGTGCC
AGCTGCATTA



ATGAATCGGC
CAACGCGCGG
GGAGAGGCGG
TTTGCGTATT
GGGCGCTCTT
CCGCTTCCTC
GCTCACTGAC
TCGCTGCGCT
CGGTCGTTCG
GCTGCGGCGA



GCGGTATCAG
CTCACTCAAA
GGCGGTAATA
CGGTTATCCA
CAGAATCAGG
GGATAACGCA
GGAAAGAACA
TGTGAGCAAA
AGGCCAGCAA
AAGGCCAGGA



ACCGTAAAAA
GGCCGCGTTG
CTGGCGTTTT
TCCATAGGCT
CCGCCCCCCT
GACGAGCATC
ACAAAAATCG
ACGCTCAAGT
CAGAGGTGGC
GAAACCCGAC



AGGACTATAA
AGATACCAGG
CGTTTCCCCC
TGGAAGCTCC
CTCGTGCGCT
CTCCTGTTCC
GACCCTGCCG
CTTACCGGAT
ACCTGTCCGC
CTTTCTCCCT



TCGGGAAGCG
TGGCGCTTTC
TCATAGCTCA
CGCTGTAGGT
ATCTCAGTTC
GGTGTAGGTC
GTTCGCTCCA
AGCTGGGCTG
TGTGCACGAA
CCCCCCGTTC



AGCCCGACCG
CTGCGCCTTA
TCCGGTAACT
ATCGTCTTGA
GTCCAACCCG
GTAAGACACG
ACTTATCGCC
ACTGGCAGCA
GCCACTGGTA
ACAGGATTAG



CAGAGCGAGG
TATGTAGGCG
GTGCTACAGA
GTTCTTGAAG
TGGTGGCCTA
ACTACGGCTA
CACTAGAAGA
ACAGTATTTG
GTATCTGCGC
TCTGCTGAAG



CCAGTTACCT
TCGGAAAAAG
AGTTGGTAGC
TCTTGATCCG
GCAAACAAAC
CACCGCTGGT
AGCGGTTTTT
TTGTTTGCAA
GCAGCAGATT
ACGCGCAGAA



AAAAACGATC
TCAAGAAGAT
CCTTTGATCT
TTTCTACGGG
GTCTGACGCT
CAGTGGAACG
AAAACTCACG
TTAAGGGATT
TTGGTCATGA
GATTATCAAA



AAGGATCTTC
ACCTAGATCC
TTTTAAATTA
AAAATGAAGT
TTTAAATCAA
TCTAAAGTAT
ATATGAGTAA
ACTTGGTCTG
ACAGTTACCA
ATGCTTAATC



AGTGAGGCAC
CTATCTCAGC
GATCTGTCTA
TTTCGTTCAT
CCATAGTTGC
CTGACTCCCC
GTCGTGTAGA
TAACTACGAT
ACGGGAGGGC
TTACCATCTG



GCCCCAGTGC
TGCAATGATA
CCGCGAGACC
CACGCTCACC
GGCTCCAGAT
TTATCAGCAA
TAAACCAGCC
AGCCGGAAGG
GCCGAGCGCA
GAAGTGGTCC



TGCAACTTTA
TCCGCCTCCA
TCCAGTCTAT
TAATTGTTGC
CGGGAAGCTA
GAGTAAGTAG
TTCGCCAGTT
AATAGTTTGC
GCAACGTTGT
TGCCATTGCT



ACAGGCATCG
TGGTGTCACG
CTCGTCGTTT
GGTATGGCTT
CATTCAGCTC
CGGTTCCCAA
CGATCAAGGC
GAGTTACATG
ATCCCCCATG
TTGTGCAAAA



AAGCGGTTAG
CTCCTTCGGT
CCTCCGATCG
TTGTCAGAAG
TAAGTTGGCC
GCAGTGTTAT
CACTCATGGT
TATGGCAGCA
CTGCATAATT
CTCTTACTGT



CATGCCATCC
GTAAGATGCT
TTTCTGTGAC
TGGTGAGTAC
TCAACCAAGT
CATTCTGAGA
ATAGTGTATG
CGGCGACCGA
GTTGCTCTTG
CCCGGCGTCA



ATACGGGATA
ATACCGCGCC
ACATAGCAGA
ACTTTAAAAG
TGCTCATCAT
TGGAAAACGT
TCTTCGGGGC
GAAAACTCTC
AAGGATCTTA
CCGCTGTTGA



GATCCAGTTC
GATGTAACCC
ACTCGTGCAC
CCAACTGATC
TTCAGCATCT
TTTACTTTCA
CCAGCGTTTC
TGGGTGAGCA
AAAACAGGAA
GGCAAAATGC



CGCAAAAAAG
GGAATAAGGG
CGACACGGAA
ATGTTGAATA
CTCATACTCT
TCCTTTTTCA
ATATTATTGA
AGCATTTATC
AGGGTTATTG
TCTCATGAGC



GGATACATAT
TTGAATGTAT
TTAGAAAAAT
AAACAAATAG
GGGTTCCGCG
CACATTTCCC
CGAAAAGTGC
CACCTGACGT
C














pVAX1HGF/MGB1









SEQ ID NO. 3









GCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGA



 


CTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT


 


ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACC


 


GCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAG


 


TAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGG


 


TAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCC


 


CCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT


 


ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTC


 


ATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGG


 


ATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCA


 


ATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGT


 


AACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGA


 


GGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACT


 


GGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAG


 


CGTTTAAACTTAAGCTTGGTACCGAGCTCGGATCCGCCAGCCCGTCCAGC


 


AGCACCATGTGGGTGACCAAACTCCTGCCAGCCCTGCTGCTGCAGCATGT


 


CCTCCTGCATCTCCTCCTGCTCCCCATCGCCATCCCCTATGCAGAGGGAC


 


AAAGGAAAAGAAGAAATACAATTCATGAATTCAAAAAATCAGCAAAGACT


 


ACCCTAATCAAAATAGATCCAGCACTGAAGATAAAAACCAAAAAAGTGAA


 


TACTGCAGACCAATGTGCTAATAGATGTACTAGGAATAAAGGACTTCCAT


 


TCACTTGCAAGGCTTTTGTTTTTGATAAAGCAAGAAAACAATGCCTCTGG


 


TTCCCCTTCAATAGCATGTCAAGTGGAGTGAAAAAAGAATTTGGCCATGA


 


ATTTGACCTCTATGAAAACAAAGACTACATTAGAAACTGCATCATTGGTA


 


AAGGACGCAGCTACAAGGGAACAGTATCTATCACTAAGAGTGGCATCAAA


 


TGTCAGCCCTGGAGTTCCATGATACCACACGAACACAGCTTTTTGCCTTC


 


GAGCTATCGGGGTAAAGACCTACAGGAAAACTACTGTCGAAATCCTCGAG


 


GGGAAGAAGGGGGACCCTGGTGTTTCACAAGCAATCCAGAGGTACGCTAC


 


GAAGTCTGTGACATTCCTCAGTGTTCAGAAGTTGAATGCATGACCTGCAA


 


TGGGGAGAGTTATCGAGGTCTCATGGATCATACAGAATCAGGCAAGATTT


 


GTCAGCGCTGGGATCATCAGACACCACACCGGCACAAATTCTTGCCTGAA


 


AGATATCCCGACAAGGGCTTTGATGATAATTATTGCCGCAATCCCGATGG


 


CCAGCCGAGGCCATGGTGCTATACTCTTGACCCTCACACCCGCTGGGAGT


 


ACTGTGCAATTAAAACATGCGCTGACAATACTATGAATGACACTGATGTT


 


CCTTTGGAAACAACTGAATGCATCCAAGGTCAAGGAGAAGGCTACAGGGG


 


CACTGTCAATACCATTTGGAATGGAATTCCATGTCAGCGTTGGGATTCTC


 


AGTATCCTCACGAGCATGACATGACTCCTGAAAATTTCAAGTGCAAGGAC


 


CTACGAGAAAATTACTGCCGAAATCCAGATGGGTCTGAATCACCCTGGTG


 


TTTTACCACTGATCCAAACATCCGAGTTGGCTACTGCTCCCAAATTCCAA


 


ACTGTGATATGTCACATGGACAAGATTGTTATCGTGGGAATGGCAAAAAT


 


TATATGGGCAACTTATCCCAAACAAGATCTGGACTAACATGTTCAATGTG


 


GGACAAGAACATGGAAGACTTACATCGTCATATCTTCTGGGAACCAGATG


 


CAAGTAAGCTGAATGAGAATTACTGCCGAAATCCAGATGATGATGCTCAT


 


GGACCCTGGTGCTACACGGGAAATCCACTCATTCCTTGGGATTATTGCCC


 


TATTTCTCGTTGTGAAGGTGATACCACACCTACAATAGTCAATTTAGACC


 


ATCCCGTAATATCTTGTGCCAAAACGAAACAATTGCGAGTTGTAAATGGG


 


ATTCCAACACGAACAAACATAGGATGGATGGTTAGTTTGAGATACAGAAA


 


TAAACATATCTGCGGAGGATCATTGATAAAGGAGAGTTGGGTTCTTACTG


 


CACGACAGTGTTTCCCTTCTCGAGACTTGAAAGATTATGAAGCTTGGCTT


 


GGAATTCATGATGTCCACGGAAGAGGAGATGAGAAATGCAAACAGGTTCT


 


CAATGTTTCCCAGCTGGTATATGGCCCTGAAGGATCAGATCTGGTTTTAA


 


TGAAGCTTGCCAGGCCTGCTGTCCTGGATGATTTTGTTAGTACGATTGAT


 


TTACCTAATTATGGATGCACAATTCCTGAAAAGACCAGTTGCAGTGTTTA


 


TGGCTGGGGCTACACTGGATTGATCAACTATGATGGCCTATTACGAGTGG


 


CACATCTCTATATAATGGGAAATGAGAAATGCAGCCAGCATCATCGAGGG


 


AAGGTGACTCTGAATGAGTCTGAAATATGTGCTGGGGCTGAAAAGATTGG


 


ATCAGGACCATGTGAGGGGGATTATGGTGGCCCACTTGTTTGTGAGCAAC


 


ATAAAATGAGAATGGTTCTTGGTGTCATTGTTCCTGGTCGTGGATGTGCC


 


ATTCCAAATCGTCCTGGTATTTTTGTCCGAGTAGCATATTATGCAAAATG


 


GATACACAAAATTATTTTAACATATAAGGTACCACAGTCATAGCTGTTAA


 


CCCGGGTCGAAGCGGCCGCTCGAGTCTAGAGGGCCCGTTTAAACCCGCTG


 


ATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCT


 


CCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCC


 


TAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTAT


 


TCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACA


 


ATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTACTGGGCGG


 


TTTTATGGACAGCAAGCGAACCGGAATTGCCAGCTGGGGCGCCCTCTGGT


 


AAGGTTGGGAAGCCCTGCAAAGTAAACTGGATGGCTTTCTTGCCGCCAAG


 


GATCTGATGGCGCAGGGGATCAAGCTCTGATCAAGAGACAGGATGAGGAT


 


CGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCT


 


TGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTG


 


CTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTT


 


TTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAAGACGAGGCA


 


GCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCT


 


CGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGC


 


CGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCC


 


ATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTG


 


CCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGA


 


TGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGG


 


CTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGAGCATGCCCGACGG


 


CGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGG


 


TGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTG


 


GCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGA


 


GCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCG


 


CTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTC


 


TGAATTATTAACGCTTACAATTTCCTGATGCGGTATTTTCTCCTTACGCA


 


TCTGTGCGGTATTTCACACCGCATCAGGTGGCACTTTTCGGGGAAATGTG


 


CGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCC


 


GCTCATGAGACAATAACCCTGATAAATGCTTCAATAATAGCACGTGCTAA


 


AACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAAT


 


CTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGA


 


CCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCG


 


TAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGT


 


TTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAG


 


CAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCC


 


ACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATC


 


CTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTT


 


GGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGG


 


GGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTG


 


AGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAG


 


AAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCA


 


CGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGG


 


TTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGG


 


GCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGG


 


CCTTTTGCTGGCCTTTTGCTCACATGTTCTT

## Claims

1. A method for decreasing necrosis of a flap or skin graft in a subject undergoing flap or skin graft surgery, comprising administering to the subject prior to the surgery, by injection at or near the planned site of flap or skin graft, a vector that comprises a nucleic acid sequence encoding human hepatocyte growth factor, wherein said vector is a plasmid selected from the group consisting of pcDNA3.1(−) and pVAX1 and comprises the nucleic acid sequence of SEQ ID NO:1 and the CMV promoter, wherein the area of necrosis in the flap or skin graft is decreased as compared to a subject who has not been injected with said vector.

2. The method of claim 1, wherein said flap is selected from the group consisting of a skin flap, a muscle flap, a myocutaneous flap and a cartilocutaneous flap.

3. The method of claim 1, wherein said vector is administered at least 3-14 days prior to flap or skin graft surgery.

4. The method of claim 1, wherein said vector is administered at least 5-10 days prior to flap or skin graft surgery.

5. The method of claim 1, wherein said vector is administered at least 7 days prior to flap or skin graft surgery.

6. The method of claim 1, wherein said vector is administered intramuscularly.

7. The method of claim 6, wherein said vector is administered in the rectus abdominus muscle.

8. The method of claim 1, wherein said vector is pVAX1.

9. The method of claim 8, wherein said vector comprises the sequence of SEQ ID NO. 2.

10. The method of claim 8, wherein said vector comprises sequence of SEQ ID NO. 3.

11. The method of claim 1, wherein the subject is a human.

12. The method of claim 1, wherein said vector is pcDNA3.1(−).