HK1150779A

HK1150779A - Treatment and prevention of cardiac conditions using two or more isoforms of hepatocyte growth factor

Info

Publication number: HK1150779A
Application number: HK11104935.7A
Authority: HK
Inventors: 金锺默; 金水晶; 韩熊
Original assignee: 百疗医株式会社
Priority date: 2008-01-25
Filing date: 2009-01-28
Publication date: 2012-01-13

Description

Treatment or prevention of cardiac disorders using two or more isoforms of hepatocyte growth factor

Technical Field

The present invention relates to methods of treating or preventing a cardiac disorder in a subject comprising administering to the subject two or more isoforms of Hepatocyte Growth Factor (HGF) (isofrorm). The invention also relates to methods of promoting endothelial cell growth in a blood vessel comprising administering to the blood vessel two or more isoforms of Hepatocyte Growth Factor (HGF). In one embodiment, the two or more isoforms of HGF are administered as one or more polynucleotides encoding the isoforms.

Background

HGF is a glycoprotein that binds heparin, also known as a spreading factor or hepatocyte growth factor a (hepatopoetin a). HGF was originally identified as a potent hepatotrophic growth factor (Nakamura et al, Nature 342: 440(1989)), a heparin-binding protein from mesenchymal cells with a variety of biological effects on a variety of cell types, such as mitogenesis, morphogenesis (motogenesis) and morphogenesis (morphogenesis). The gene encoding HGF is located on chromosome 7q21.1, contains 18 exons and 17 introns, has SEQ ID NO: 1 (Seki T, et al, Gene 102: 213-219 (1991)). An approximately 6kb transcript is transcribed from the HGF gene, from which a full-length polypeptide HGF precursor consisting of 728 amino acids (flHGF) is then synthesized, which comprises the following domains: an N-terminal hairpin loop-kringle 1-kringle2-kringle 3-kringle 4-inactivated serine protease domain. Meanwhile, several other isoforms of HGF polypeptides are synthesized by selective splicing of the HGF gene. Known isoforms include deletion variants of HGF (full length HGF, but with 5 residues deleted in kringle 1), NK1 (N-terminal hairpin loop-kringle 1), NK2 (N-terminal hairpin loop-kringle 1-kringle 2), and NK4 (N-terminal hairpin loop-kringle 1-kringle2-kringle 3-kringle 4). In addition, allelic variants exist for each isoform. Biologically inactive precursors can be converted by proteases in serum into active forms of disulfide-linked heterodimers. In the heterodimer, the α -chain with high molecular weight forms 4 kringle domains as well as an N-terminal hairpin loop, like the pre-activated peptide region of plasminogen. Kringle domains of three disulfide-bonded loop structures consisting of about 80 amino acids can play an important role in protein-protein interactions. The low molecular weight beta chain forms an inactive serine protease-like domain. dHGF, consisting of 723 amino acids, is a polypeptide with 5 amino acids (i.e., F, L, P, S and S) deleted in the first kringle domain of the alpha chain due to alternative splicing between exon 4 and exon 5.

In vivo, two isoforms of HGF (flHGF having 728 amino acids and dHGF having 723 amino acids) were produced by alternative splicing between exon 4 and exon 5. Although flHGF and dHGF share several biological functions, they differ in immunology and several biological properties.

HGF has been shown to stimulate angiogenesis by modulating endothelial cell growth and vascular smooth muscle cell migration. HGF is considered as one of the promising candidates for therapeutic angiogenesis because of its angiogenic activity. "therapeutic angiogenesis" refers to the intervention of an angiogenic factor (as a recombinant protein or gene) in the treatment of ischemic diseases such as Coronary Artery Disease (CAD) or Peripheral Artery Disease (PAD). HGF is also known to stimulate not only the growth but also migration of endothelial cells (Bussolino et al, J Cell biol. 119: 629 (1992); Nakamura et al, JHP ertens 14: 1067(1996)), and has been tested for its role as a re-endothelialization stimulator (Yasuda et al, Circulation 101: 2546 (2000); Hayashi et al, Gene Ther 7: 1664 (2000)).

HGF has been used as a therapeutic angiogenic agent. The HGF gene has been used by Morishita and colleagues for the treatment of PAD and CAD. They observed some therapeutic response to PAD after administration of HGF gene, but it was unclear whether HGF gene transfer could effectively treat CAD. HGF Gene transfer has been tested to date in various animal models for CAD (Miyagawa et al, Circulation 105: 2556 (2002); Azuma et al, Gene ther.13: 1206 (2006); Aoki et al, Gene ther.7: 417 (2000); Funatsu et al, J.Thorac Cardiovasc.Surg.124: 1099 (2002)). However, whether HGF gene transfer has a beneficial effect on CAD remains controversial. For example, Miyagawa and coworkers showed that human HGF transfer did not increase the Left Ventricular Ejection Fraction (LVEF) of infarcted hearts 8 weeks after treatment in the rat myocardial infarction model (Miyagawa et al, Circulation 105: 2556(2002), FIG. 2). Moreover, HGF gene transfer had little effect on percent fractional shortening and left ventricular anterior wall thickness 8 weeks after treatment in the same model (Miyagawa et al, Circulation 105: 2556(2002), FIGS. 3 and 5).

HGF has also been used as an agent for inhibiting restenosis. Coronary angioplasty (e.g., balloons and stents) is a widely used method of treating occluded blood vessels. However, intimal thickening (e.g., coronary restenosis) is a serious problem when angioplasty is employed. One of the causes of restenosis is the hyperproliferation and migration of vascular smooth muscle cells and the concomitant synthesis of extracellular matrix, which is caused in response to vascular injury. There are indications that rapid endothelial resurfacing inhibits smooth muscle cell proliferation and thus restenosis (e.g., Bauters et al, ProgCardiovasc Dis.40: 107 (1997)). As one approach to preventing restenosis, local delivery of endothelial growth factors (e.g., Vascular Endothelial Growth Factor (VEGF) or Hepatocyte Growth Factor (HGF)) to damaged vessels has been attempted and shown to attenuate restenosis (Asahara et al, Circulation 94: 3291 (1996); Yasuda et al, Circulation 101: 2546 (2000); Hayashi et al, Gene Ther 7: 1664 (2000); Walter et al, Circulation 110: 36 (2004)).

All of the above studies on HGF gene therapy were conducted using a flHGF cDNA encoding 728 amino acids, rather than a dHGF cDNA encoding 723 amino acids (Miyagawa et al; Azuma et al; Aoki et al; Funatsu et al; Yasuda et al and Hayashi et al). In contrast to flHGF cDNA tested in most previous reports, the present invention demonstrates for the first time that transfer of a nucleotide sequence expressing multiple HGF isoforms (e.g., flHGF and dHGF) is effective in treating CAD in animals and humans. The present invention also demonstrates for the first time that the transfer of a nucleotide sequence expressing multiple isoforms of HGF accelerates the process of re-endothelialization of blood vessels.

Summary of The Invention

It is therefore an object of the present invention to provide the use of a composition comprising two or more isoforms of HGF.

One aspect of the invention relates to the use of a composition comprising two or more isoforms of HGF or one or more polynucleotides encoding the isoforms for the manufacture of a medicament for treating or preventing a cardiac disorder in a subject.

Another aspect of the invention relates to the use of a composition comprising two or more isoforms of HGF or one or more polynucleotides encoding the isoforms for the preparation of a medicament for promoting endothelial cell growth in a blood vessel.

It is another object of the invention to provide methods of treating or preventing cardiac conditions by administering two or more isoforms of HGF.

Another aspect of the invention relates to a method of increasing perfusion of ischemic heart tissue or blood vessel density in the myocardium in a subject comprising administering to the subject a composition comprising two or more isoforms of HGF.

Another aspect of the invention relates to a method of treating a cardiac disorder in a subject comprising administering to the subject a composition comprising two or more isoforms of HGF.

Yet another aspect of the invention relates to a method of enhancing endothelial repair or providing treatment for a vascular injury or diseased vascular site in a subject comprising administering to the subject a composition comprising two or more isoforms of HGF.

Yet another aspect of the invention relates to a method of promoting endothelial cell growth in a blood vessel comprising administering to the blood vessel a composition comprising two or more isoforms of HGF. In one embodiment, the blood vessel is damaged. In yet another embodiment, re-endothelialization (re-endothelialization) of the blood vessel is promoted.

In one embodiment, the two or more isoforms of HGF comprise full length HGF (hereinafter flHGF) and a deletion variant HGF (hereinafter dHGF). In another embodiment, the two or more isoforms of HGF further comprise NK 1.

In yet another embodiment, the two or more isoforms of HGF are administered in the form of a polynucleotide encoding the isoforms.

In one aspect of the invention, the composition is administered by injection.

In another aspect of the invention, the composition is administered by a delivery device. In one embodiment, the delivery device is a stent. In yet another embodiment, the stent is selected from the group consisting of a non-polymer based stainless steel stent, a non-polymer based cobalt chromium stent and a polymer based cobalt chromium stent.

In one aspect of the invention, the two or more isoforms of HGF are administered directly to ischemic heart tissue of the subject.

Yet another aspect of the invention relates to compositions comprising two or more isoforms of HGF.

In one embodiment, the composition comprises a polynucleotide encoding the two or more isoforms of HGF.

Yet another aspect of the invention relates to a composition for increasing perfusion of ischemic heart tissue in a subject comprising two or more isoforms of HGF.

Yet another aspect of the invention relates to a composition for promoting endothelial cell growth in a blood vessel of a subject comprising two or more isoforms of HGF.

In one embodiment, the composition is administered to a blood vessel of a subject to promote endothelialization of the blood vessel. In another embodiment, the composition is administered to a blood vessel of a subject to promote and/or accelerate the re-endothelialization of the blood vessel.

In yet another embodiment, the subject is in need of prevention or treatment of restenosis.

Drawings

The above and other objects and features of the present invention will become apparent from the following description of the present invention when taken in conjunction with the accompanying drawings.

FIG. 1 shows the effect of the HGF isoforms on HUVEC migration.

Fig. 2 shows the effect of HGF isoforms on C2C12 cell migration.

Fig. 3 shows the effect of HGF isoforms on H9C2 cell migration.

Fig. 4 shows the effect of HGF isoforms on HUVEC proliferation.

FIG. 5 shows a schematic diagram of experimental procedures for evaluating the efficacy of HGF in a rat ischemic heart disease model.

Fig. 6 shows the effect of HGF on the function of left ventricular ejection fraction.

Fig. 7 shows the effect of HGF on the function of the chamber spacing during systole.

Fig. 8 shows the effect of HGF injection into ischemic myocardium on capillary density.

Fig. 9 shows the effect of HGF injection into ischemic myocardium on myocardial fibrosis.

FIG. 10 shows a coronary map on a 20-segment model of MIBI-SPECT.

FIG. 11 shows the selection of myocardial regions for pCK-HGF-X7 injection. pCK-HGF-X7 was administered by intramuscular injection to both sides of the coronary arteries in the region of the myocardium assessed as perfusion reduction by MIBI-SPECT.

FIG. 12 shows the effect of pCK-HGF-X7 on myocardial perfusion under MIBI-SPECT.

Figure 13 shows myocardial perfusion (κ) assessed by myocardial contrast stress echocardiography.

Figure 14 shows acceleration of re-endothelialization by HGF plasmid eluting stents on OCT.

Fig. 15 shows acceleration of re-endothelialization by HGF plasmid eluting scaffold on SEM.

Detailed Description

The present invention is based on the discovery that administration of two or more isoforms of HGF to a subject having a cardiac disorder (e.g., CAD) is effective to increase perfusion of ischemic cardiac tissue and thereby treat or prevent the cardiac disorder. Further aspects of the invention relate to the discovery that administration of two isoforms of HGF promotes vascular endothelialization (e.g., inhibition of restenosis by rapid re-endothelialization of the blood vessel). Accordingly, it is an object of the present invention to provide methods for treating or preventing a cardiac disorder (e.g., CAD or coronary restenosis) by administering two or more isoforms of HGF. Another aspect of the invention is to provide a method for promoting endothelial cell growth in a blood vessel (e.g., wherein the blood vessel is damaged).

Another aspect of the invention relates to a method of increasing perfusion of ischemic heart tissue or increasing blood vessel density in the myocardium in a subject comprising administering to the subject a composition comprising two or more isoforms of HGF.

Another aspect of the invention relates to a method of treating or preventing a cardiac disorder in a subject comprising administering to the subject a composition comprising two or more isoforms of HGF.

Another object of the invention relates to a method of promoting endothelial cell growth in a blood vessel comprising administering to the blood vessel a composition comprising two or more isoforms of HGF, wherein endothelial cell growth of the blood vessel is promoted.

In one embodiment, the method comprises administering a composition comprising three or more isoforms of HGF (e.g., four or more isoforms of HGF). In one embodiment, the composition comprises two or more isoforms of HGF selected from flHGF, dHGF, NK1, NK2, and NK 4. In another embodiment, the composition comprises two or more isoforms of HGF selected from flHGF, dHGF, NK1, and NK 2. In yet another embodiment, the composition comprises two or more isoforms of HGF selected from flHGF, dHGF and NK 1. In yet another embodiment, the two or more isoforms of HGF comprise flHGF and dHGF. In yet another embodiment, the two or more isoforms of HGF consist of flHGF and dHGF. In another embodiment, the two or more isoforms of HGF comprise flHGF, dHGF and NK 1. In another embodiment, the two or more isoforms of HGF are administered in the form of a polynucleotide encoding the isoforms.

In the present invention, a cardiac disorder to be treated or prevented is any disorder associated with a reduction of blood flow in the heart, aorta or coronary arteries or ischemic tissue of the heart. Examples of cardiac disorders include, but are not limited to, coronary artery occlusion (e.g., caused by or associated with lipid/cholesterol deposition, macrophage/inflammatory cell recruitment, plaque rupture, thrombosis, platelet deposition, or neointimal proliferation), ischemic syndrome (e.g., caused by or associated with myocardial infarction, stable angina, unstable angina, coronary restenosis, or reperfusion injury), cardiomyopathy (e.g., caused by or associated with ischemic syndrome, cardiotoxin, infection, hypertension, metabolic disease (e.g., uremia, beriberi, or glycogen storage disease), radiation, neuromuscular disease, invasive disease (e.g., sarcoidosis, hemochromatosis, amyloidosis, fabry's disease, or huler's syndrome), trauma or congenital causes), arrhythmia or dysrhythmia (e.g., caused by or associated with ischemic syndrome, plaque rupture, thrombosis, platelet deposition, or neointimal proliferation), radiation, neuromuscular disease, or dysrhythmia, Cardiotoxin, doxorubicin, infection, hypertension, metabolic disease, radiation, neuromuscular disease, invasive disease, trauma or congenital cause or associated therewith), infection (e.g., by a pathogen such as a bacterium, virus, fungus or parasite), and inflammatory disorder (e.g., an inflammatory disorder associated with myocarditis, pericarditis, endocarditis, cardiac immune rejection, or caused by one of an idiopathic disease, an autoimmune disease, or a connective tissue disease).

In one embodiment, the methods of the invention can be used to treat or prevent atherosclerosis (e.g., in the aorta or coronary arteries), prevent complications associated with atherosclerosis (e.g., angina pectoris, myocardial infarction, arrhythmia, heart failure, renal failure, cirrhosis, childhood avascular necrosis of the femoral head (Legg-calcium-Perthes Disease), ischemic stroke, peripheral arterial occlusion, aneurysm, embolism), or reduce early symptoms and signs of atherosclerosis (e.g., inability of blood to flow to affected tissue with increased demand (e.g., in angina, excretion, or intermittent claudication).

In another embodiment, the methods of the invention can be used to treat or prevent a cardiac disorder resulting from a vascular surgical intervention, including, but not limited to, angioplasty (e.g., percutaneous transluminal coronary angioplasty, carotid percutaneous transluminal angioplasty, or stented coronary angioplasty), stent implantation, atherectomy, or transplantation (e.g., coronary artery bypass graft). In this embodiment, the method of the invention may be performed before, during and/or after the surgical intervention. In certain embodiments, the cardiac disorder is coronary restenosis.

In one aspect of the invention, two or more isoforms of HGF are administered to a subject having coronary artery disease. In one embodiment, the subject has one or more coronary arteries partially or fully occluded. In another embodiment, the subject has suffered from, is suffering from, or is at risk of having a myocardial infarction. In yet another embodiment, the subject is determined to have or is now suspected of having an ischemic heart disease, e.g., based on an angiogram, electrocardiogram, echocardiogram, or other method. In one embodiment, the subject is a candidate for Coronary Artery Bypass Graft (CABG). In another embodiment, the subject has one or more coronary arteries partially or fully occluded but not suitable for CABG. In yet another embodiment, the subject has undergone CABG but has incomplete revascularization of the myocardium.

In one aspect of the invention, two or more isoforms of HGF are administered to a blood vessel to promote endothelial cell growth. In one embodiment, the blood vessel is obstructed or damaged. In certain embodiments, the obstructed blood vessel may include an artery or vein in which the lumen of the blood vessel has been narrowed and blood flow to the blood vessel is reduced. In one embodiment, two or more isoforms of HGF are administered to promote vascular re-endothelialization, e.g., after injury to the blood vessel wall, e.g., during angioplasty. In certain embodiments, re-endothelialization can be promoted and/or accelerated, e.g., an increase in endothelial cell growth rate as compared to endothelial cell growth in the absence of two or more isoforms of HGF. In another embodiment, two or more isoforms of HGF are administered to a subject in need of prevention or treatment of restenosis to inhibit proliferation of smooth muscle cells in blood vessels.

As used herein, the term "treating" a disorder (e.g., a cardiac disorder or obstructed or damaged blood vessels) refers to administering a factor in an amount sufficient to result in an improvement in one or more symptoms of the disorder, or to prevent progression of the disorder, or to cause a decline in the disorder (e.g., due to promotion of angiogenesis or endothelial cell growth). For example, with respect to ameliorating a symptom of a cardiac disorder, treatment results in a measurable reduction in the symptom by at least 5%, preferably by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. The physiological effects that can be detected and measured to determine treatment associated with treating a cardiac disorder include, but are not limited to, cardiac efficiency (as measured by at least one clinical indicator of cardiac function, such as cardiac output, pulmonary artery pressure, and central venous pressure, or ventricular ejection fraction), transmural blood flow in the myocardium under resting or stress conditions, regeneration of myocardial tissue, formation, maturation and/or growth of collateral vessels (e.g., local neovascularization, increased capillary density, arteriogenesis, lymphangiogenesis, angiogenesis), cardiomyogenesis (e.g., striated muscle cells, smooth muscle cells, or myoepithelial cells), vascularization of myocardial tissue, cardiac contractile function, increase in left ventricular ejection fraction, or ventricular interval; or a reduction in myocardial fibrosis, intimal thickening (neointimal proliferation/hyperplasia), endothelial or smooth muscle cell proliferation, chest pain or shortness of breath.

The term "prevention" as used herein refers to a reduced incidence of one or more symptoms of a disorder in an animal, such as altered cardiac function or reduced blood flow due to obstructed or damaged blood vessels. The prevention may be complete, e.g., no symptoms at all in the subject. The prevention may also be partial, such that the incidence of symptoms in a subject is lower than without the use of the present invention.

In one embodiment, the isoform of HGF or polynucleotide encoding the isoform of HGF is administered to a blood vessel or heart of a subject, e.g., a damaged blood vessel, a partially or fully occluded coronary artery, ischemic myocardial tissue, pericardial space, or coronary sinus.

The HGF isoform or a polynucleotide encoding the HGF isoform may be delivered to the desired site using any method known in the art. Examples of delivery devices that may be used include, but are not limited to, catheters (e.g., balloon catheters, infusion catheters, stylets), needles, needleless syringes, stents, infusion cannulas, meshes, cardiac anchors (cardiac harnesss), shunts, cardiac pacemakers, implantable defibrillators, sutures, staples, perivascular sheaths (perivasular wrap), flexible sheets or membranes that may substantially conform to the contour of the wound site, tubing devices, grafts, and pumps specific examples of methods of delivering HGF isoforms include, but are not limited to, via a balloon catheter placed in a vein that flows into the coronary sinus (e.g., great cardiac vein, central vein, posterior left ventricular vein, anterior interventricular vein, or any other branch), via a catheter that leads to a lumen of one or more coronary arteries (e.g., right or left coronary artery) where the HGF isoform is coated on a balloon that is inflated at the site or injected from the tip of the catheter, and during open heart surgery or heart transplantation Needle-over delivery (e.g., into the left or right atrium or left or right ventricle); delivering to the pericardial space through the left atrium, right ventricle, or left ventricle through an internal portal or using an external portal by open heart or micro-interventional surgery, or to the pericardial space by injection, catheterization, laser induced creation of an infusion channel, cannulation, percutaneous portal created using a particle gun or using a pump; delivering by antegrade perfusion from a catheter placed within a conduit that delivers blood to the tissue, or by retrograde perfusion from a catheter placed in a conduit that receives blood from the tissue; or by intraluminal devices or endovascular prostheses (e.g., stents, grafts, stent-grafts, vena cava filters) for maintaining vessel patency. In one embodiment, the device is biodegradable, thus, it does not need to be removed after it is no longer needed. In certain embodiments, the two isoforms of HGF are delivered using a scaffold. In yet another embodiment, the stent is selected from the group consisting of a non-polymer based stainless steel stent, a non-polymer based cobalt chromium stent and a polymer based cobalt chromium stent.

In one embodiment, the polynucleotide encoding an HGF isoform is delivered in the form of a cell comprising the polynucleotide and expressing the HGF polypeptide. The cells may be autologous or non-autologous (e.g., allogeneic or xenogeneic) cells. Any cell that is viable after transplantation can be used, including, for example, fibroblasts, cardiomyocytes, endothelial cells, or stem cells (e.g., embryonic stem cells, hematopoietic stem cells, mesenchymal stem cells). The polynucleotide-containing cells may be introduced as an injectable liquid suspension, for example, by injection into the damaged myocardium or by intravenous injection. Cells may be introduced into the infarct zone to reduce the extent of scar formation and enhance ventricular function. When it is desired to introduce a polynucleotide into a cell ex vivo, the cell may be obtained from the subject by any technique known in the art, including but not limited to biopsy, scraping, and surgical tissue removal. The isolated cells can be cultured for a time sufficient to allow introduction of the polynucleotide into the cell, e.g., 2, 4, 6, 8, 10, 12, 18, 24, 36, 48 hours or more. Methods for short-term culture of primary cells are well known in the art. For example, cells can be cultured adherent to or in suspension in a plate (e.g., a microplate). In one embodiment, the presence of the polynucleotide in the cell is determined prior to introducing the cell back into the subject. In another embodiment, cells containing the polynucleotide are selected (e.g., based on the presence of a selectable marker in the polynucleotide) and only those cells containing the polynucleotide are reintroduced into the subject.

When the isoform of HGF or polynucleotide encoding the isoform of HGF is delivered by injection, the injection may be an intracardiac injection, e.g., intra-atrial (left and/or right) or intraventricular (left and/or right). The injection may also be intramyocardial. The injection site may be at or near the ischemic/hypoxic region, at the intersection of normal tissue and the ischemic/hypoxic region, or in normal tissue. The injection site may be at one or more coronary arteries, such as a blocked artery. The injection may be epicardial or endocardial. Delivery may consist of one injection or more injections at one or more sites. Delivery may be by epicardial injection. Delivery to the vascular site may be carried out by intravascular injection, e.g., intravenous or intraarterial (intracoronary, intraaortic). Can be delivered into at least two coronary arteries (e.g., at least one left coronary artery and one right coronary artery), for example at a site at least about 1cm within the lumen of a coronary artery. Vascular injections may be performed, for example, at a site near ischemic or diseased tissue, at a site of vascular injury, or at a site of restenosis.

Administration of the HGF isoform may be repeated more than once, e.g., after 0.5, 1, 2,3, 4, 5,6, 7 or more days apart, e.g., after 1, 2,3, 4, 5,6, 7, 8, 9 or 10 or more weeks. In one embodiment, the cardiac perfusion status or vascular health status of the subject is monitored after each administration of an isoform of HGF, e.g., by angiogram, electrocardiogram, echocardiogram, or other method, as necessary to provide additional administrations.

In one embodiment of the invention, two or more isoforms of HGF are administered to a subject currently experiencing an ischemic event. In another embodiment, the two or more isoforms of HGF are administered as soon as possible after the occurrence of an ischemic event, e.g., within 0.5, 1, 2,3, 4, 5,6, 12, 18, 24, 36, 48, or 72 hours after the ischemic event.

The two or more isoforms of HGF are administered at therapeutically effective doses, e.g., doses that result in a measurable improvement in a cardiac and/or vascular condition in the subject, e.g., increased perfusion of ischemic cardiac tissue, increased capillary density in ischemic cardiac tissue, decreased fibrosis in ischemic cardiac tissue, decreased extent of vascular injury, increased endothelialization, etc. The effective dose will vary from subject to subject, depending on the degree of disorder and/or need for endothelialization, the chosen route of administration, the age, sex, and weight of the individual subject, the health of the subject, and the severity of the subject's symptoms, and can be administered in a single dose or in divided doses. Therefore, the daily dose should not impose any limitation on the scope of the invention. For example, when two or more isoforms of HGF are administered in protein form, the therapeutically effective dose for each protein may range from about 1 μ g to about 100mg, such as from about 10 μ g to about 10 mg. When two or more isoforms of HGF are administered in the form of a polynucleotide, a therapeutically effective dose can range from about 1 μ g to about 10mg, e.g., from about 5 μ g to about 5mg, e.g., from about 10 μ g to about 2mg, e.g., from about 100 μ g to about 1 mg. When HGF isoforms are administered more than once repeatedly, the dose for each administration may be the same or different.

In one embodiment, the method further comprises administering to the subject an additional therapeutic agent or procedure known to be effective in treating a cardiac and/or vascular disorder (e.g., angioplasty). Examples of therapeutic agents include, but are not limited to, angiogenesis promoters (e.g., vascular endothelial growth factor, nitric oxide releasing or generating agents, fibroblast growth factor, platelet-derived growth factor, interleukin-6, monocyte chemotactic protein-1, granulocyte-macrophage colony stimulating factor, transforming growth factor-beta), anti-thrombotic agents (e.g., aspirin, heparin, PPACK, enoxaparin (enoxaprin), hirudin), anticoagulants, antibiotics, antiplatelet agents, thrombolytic agents (e.g., tissue plasminogen activator), antiproliferative agents, anti-inflammatory agents, agents that inhibit hyperplasia, agents that inhibit restenosis, smooth muscle cell inhibitors, growth factors, growth factor inhibitors, cell adhesion inhibitors, chemotherapeutic agents, and combinations thereof.

The following definitions are provided to aid in understanding the scope and practice of the invention.

The term "isolated" for the purposes of the present invention refers to biological material (cells, nucleic acids or proteins) that has been separated from its original environment (the environment in which it naturally occurs). For example, a nucleotide that is naturally occurring in a plant or animal is not isolated, however, the same polynucleotide isolated from its naturally occurring adjacent nucleic acid is considered "isolated".

"nucleic acid", "nucleic acid molecule", "oligonucleotide" and "polynucleotide" are used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules") or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxyuridine or deoxycytidine; "DNA molecules") or any phosphate ester analogs thereof (e.g., phosphorothioates and thioesters), either in single stranded form or as double stranded helices. Double-stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible. The term "nucleic acid molecule" (especially DNA or RNA molecule) relates only to the primary and secondary structure of the molecule and is not limited to any particular tertiary form. Thus, the term includes double-stranded DNA found in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA, and chromosomes. In discussing the structure of a particular double-stranded DNA molecule, sequences may be described herein according to the conventional convention of giving sequences only in the 5 'to 3' direction of the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has been subjected to molecular biological manipulations. DNA includes, but is not limited to, cDNA, genomic DNA, plasmid DNA, synthetic DNA, and semisynthetic DNA.

The term "fragment" when used in reference to a polynucleotide sequence refers to a nucleotide sequence that is reduced in length compared to a reference nucleic acid and comprises, in common, the same nucleotide sequence as the reference nucleic acid. Where appropriate, the nucleic acid fragments of the invention may be included as a component in a larger polynucleotide. These fragments comprise or consist of an oligonucleotide of at least 6, 8, 9, 10, 12, 15, 18, 20, 21, 22, 23, 24, 25, 30, 39, 40, 42, 45, 48, 50, 51, 54, 57, 60, 63, 66, 70, 75, 78, 80, 90, 100, 105, 120, 135, 150, 200, 300, 500, 720, 900, 1000, 1500, 2000, 3000, 4000, 5000 or more consecutive nucleotides in length of a nucleic acid of the invention.

"Gene" refers to a polynucleotide containing nucleotides that encode functional molecules, including functional molecules produced by transcription alone (e.g., biologically active RNA species) or by transcription and translation (e.g., polypeptides). The term "gene" includes cDNA and genomic DNA nucleic acids. "Gene" also refers to a nucleic acid fragment that expresses a particular RNA, protein or polypeptide, including regulatory sequences upstream (5 'non-coding sequences) and downstream (3' non-coding sequences) of the coding sequence. "native gene" refers to a naturally occurring gene having its own regulatory sequences. "chimeric gene" refers to any gene that is not a native gene, comprising regulatory and/or coding sequences that are not found together in nature. Thus, a chimeric gene may comprise regulatory sequences and coding sequences that are derived from different sources, or regulatory sequences and coding sequences derived from the same source, but arranged in a manner different than that found in nature. A chimeric gene may comprise coding sequences derived from different sources and/or regulatory sequences derived from different sources. "endogenous gene" refers to a native gene that occurs naturally in the genome of an organism. "foreign" gene or "heterologous" gene refers to a gene that is not normally present in a host organism, but is introduced into the host organism by gene transfer. The foreign gene may comprise a native gene inserted into a non-native organism, or a chimeric gene. A "transgene" is a gene that is introduced into a cell by gene transfer manipulation.

"heterologous DNA" refers to DNA that does not naturally occur in a cell or chromosomal site of a cell. The heterologous DNA may comprise an extracellular gene.

The term "genome" includes chromosomal as well as mitochondrial, chloroplast and viral DNA or RNA.

A nucleic acid molecule is "hybridizable" to another nucleic acid molecule (e.g., cDNA, genomic DNA, or RNA) when the nucleic acid molecule in single-stranded form can anneal to the other nucleic acid molecule under conditions of suitable temperature and solution ionic strength. Hybridization and washing conditions are well known and exemplified in Molecular Cloning by Sambrook et al, a Laboratory Manual, second edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly in chapter 11, Table 11.1 thereof (incorporated herein by reference). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.

Stringency conditions can be adjusted to screen for moderately similar fragments (e.g., homologous sequences from distantly related organisms) to highly similar fragments (e.g., genes that produce the same functional enzymes as from closely related organisms). For preliminary screening of homologous nucleic acids, low stringency hybridization conditions can be used, corresponding to a melting temperature (Tm) of 55 ℃, e.g., 5X SSC, 0.1% SDS, 0.25% milk and no formamide; or 30% formamide, 5 XSSC, 0.5% SDS. Hybridization conditions of moderate stringency correspond to higher Tm, e.g., 40% formamide, 5X or 6X SSC. High stringency hybridization conditions correspond to the highest Tm, e.g., 50% formamide, 5X or 6X SSC.

Hybridization requires that the two nucleic acids comprise complementary sequences, although mismatches between bases are also possible depending on the stringency of the hybridization. The term "complementary" is used to describe the relationship between nucleotide bases that are capable of hybridizing to each other. For example, in the case of DNA, adenosine is complementary to thymidine and cytidine is complementary to guanosine. Thus, the invention also includes isolated nucleic acid fragments that are complementary to the complete sequences disclosed or used herein, as well as those substantially similar nucleic acid sequences.

In one embodiment of the invention, polynucleotides are detected using hybridization conditions comprising a hybridization step with a Tm of 55 ℃ and the conditions described above. In other embodiments, the Tm is 60 ℃, 63 ℃, or 65 ℃.

Washing after hybridization also determines stringent conditions. One set of conditions used the following series of washes: starting from 6 XSSC, 0.5% SDS at room temperature for 15 minutes (min), then repeating with 2 XSSC, 0.5% SDS at 45 ℃ for 30min, then repeating twice with 0.2 XSSC, 0.5% SDS at 50 ℃ for 30 min. A preferred set of stringent conditions uses higher temperatures, wherein the washes are the same as described above except that the temperature of the last two 30min washes in 0.2 XSSC, 0.5% SDS is increased to 60 ℃. Another preferred set of stringent conditions utilizes two final washes at 65 ℃ in 0.1 XSSC, 0.1% SDS.

The appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The higher the degree of similarity or homology between two nucleotide sequences, the higher the Tm value of hybrids of nucleic acids containing these sequences. The relative stability of nucleic acid hybridization (corresponding to higher Tm) decreases in the following order: RNA, DNA, RNA, DNA. For hybrids greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook et al, supra, 9.50-0.51). For hybridization of shorter nucleic acids, i.e., oligonucleotides, the position of the mismatch becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al, supra, 11.7-11.8).

In one embodiment of the invention, the polynucleotide is detected using hybridization conditions comprising a hybridization step at less than 500mM salt and at least 37 ℃ and a wash step in 2XSSPE at a temperature of at least 63 ℃. In another embodiment, the hybridization conditions include a hybridization step at less than 200mM salt and at least 37 ℃. In yet another embodiment, the hybridization conditions comprise 2X SSPE and 63 ℃ in both the hybridization and wash steps.

In another embodiment, the length of the hybridizable nucleic acid is at least about 10 nucleotides. Preferably, the minimum length of the hybridizable nucleic acid is at least about 15 nucleotides, for example at least about 20 nucleotides; for example at least 30 nucleotides. In addition, one skilled in the art will recognize that the temperature and wash solution salt concentration may be adjusted as necessary depending on factors such as probe length.

The term "probe" refers to a single-stranded nucleic acid molecule that can base pair with a complementary single-stranded target nucleic acid to form a double-stranded molecule.

As used hereinThe term "oligonucleotide" refers to a short nucleic acid that can hybridize to a genomic DNA molecule, a cDNA molecule, a plasmid DNA, or an mRNA molecule. The oligonucleotides can be used, for example³²The P-nucleotide or the nucleotide covalently conjugated to a label (e.g., biotin) is labeled. The labeled oligonucleotides can be used as probes to detect the presence of nucleic acids. Oligonucleotides (one or both of which may be labeled) may be used as PCR primers for cloning full-length nucleic acids or nucleic acid fragments, for DNA sequencing, or for detecting the presence of nucleic acids. Oligonucleotides may also be used to form triple helices with DNA molecules. Generally, oligonucleotides are prepared synthetically, preferably on a nucleic acid synthesizer. Thus, oligonucleotides can be prepared using non-naturally occurring phosphate ester-like linkages (e.g., thioester linkages, etc.).

"primer" refers to an oligonucleotide that hybridizes to a target nucleic acid sequence to form a double-stranded nucleic acid region that can serve as a point of initiation of DNA synthesis under suitable conditions. These primers can be used in polymerase chain reactions or for DNA sequencing.

"polymerase chain reaction", abbreviated PCR, refers to an in vitro method for the enzymatic amplification of a specific nucleic acid sequence. PCR comprises a series of repeated temperature cycles, each cycle comprising three steps: denaturing the template nucleic acid to separate strands of the target molecule, annealing single-stranded PCR oligonucleotide primers to the template nucleic acid, and amplifying the annealed primers by DNA polymerase. PCR provides a means of detecting the presence of a target molecule and determines the relative amount of the target molecule within the starting nucleic acid mixture under quantitative or semi-quantitative conditions.

"reverse transcription polymerase chain reaction", abbreviated RT-PCR, refers to an in vitro method of enzymatically producing a target cDNA molecule or a molecule derived from an RNA molecule, followed by enzymatic amplification of a specific nucleic acid sequence within the target cDNA molecule as described above. RT-PCR also provides a means of detecting the presence of a target molecule and determining the relative amount of the target molecule within the starting nucleic acid mixture under quantitative or semi-quantitative conditions.

A DNA "coding sequence" refers to a double-stranded DNA sequence that encodes a polypeptide and which, when placed under the control of appropriate regulatory sequences, is transcribed and translated into the polypeptide either extracellularly or intracellularly. "suitable regulatory sequences" refer to nucleotide sequences located upstream (5 'non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, which affect the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and stem-loop structures. The boundaries of the coding sequence are determined by a start codon at the 5 '(amino) terminus and a translation stop codon at the 3' (carboxyl) terminus. A coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from mRNA, genomic DNA sequences, and even synthetic DNA sequences. If expression of the coding sequence in eukaryotic cells is desired, polyadenylation signals and transcription termination sequences are typically located 3' to the coding sequence.

An "open reading frame," abbreviated ORF, refers to a length of a nucleic acid sequence (DNA, cDNA, or RNA) that includes a translation initiation signal or start codon (e.g., ATG or AUG) and a stop codon, and that can be translated into a polypeptide sequence.

The term "downstream" refers to a nucleotide sequence that is 3' to a reference nucleotide sequence. In particular, the downstream nucleotide sequence generally refers to a sequence located downstream of the transcription start point. For example, the translation initiation codon of a gene is located downstream of the transcription initiation site.

The term "upstream" refers to a nucleotide sequence that is 5' to a reference nucleotide sequence. In particular, an upstream nucleotide sequence generally refers to a sequence that is located 5' to a coding sequence or transcription initiation point. For example, most promoters are located upstream of the transcription start site.

"homologous recombination" refers to the insertion of a foreign DNA sequence into another DNA molecule, for example, the insertion of a vector into a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific chromosomal recombination, the vector will contain a region of homology to the chromosomal sequence long enough for the vector to bind complementarily to the chromosome and be introduced into the chromosome. Longer regions of homology and higher degrees of sequence similarity may increase the efficiency of homologous recombination.

"vector" refers to any vehicle for cloning and/or transferring a nucleic acid into a host cell. The vector may be a replicon, which may be linked to another DNA segment for replication of the linked segment. A "replicon" refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions in vivo as an autonomously replicating unit of DNA, i.e., capable of replicating under the control of itself. The term "vector" includes both viral and non-viral vectors used to introduce nucleic acids into cells in vitro, ex vivo (ex vivo) or in vivo. Many vectors known in the art can be used to manipulate nucleic acids, introduce response elements and promoters into genes, and the like. Possible vectors include, for example, plasmids or modified viruses (including, for example, adenoviruses, retroviruses, adeno-associated viruses, herpes viruses) or plasmids (for example pBR322 or pUC plasmid derivatives) or Bluescript vectors. For example, the DNA fragments corresponding to the response elements and promoters are inserted into suitable vectors, which can be accomplished by ligating the appropriate DNA fragments into selected vectors having complementary cohesive ends. Alternatively, the ends of the DNA molecule may be enzymatically modified, or a nucleotide sequence (linker) may be ligated into the ends of the DNA to create any site. These vectors can be engineered to contain a selectable marker gene for selection of cells into which a marker has been introduced into the genome of the cell. These markers may allow the identification and/or selection of host cells into which the protein encoded by the marker is introduced and expressed.

Viral vectors have been used in a variety of gene delivery applications in cells as well as in live animal subjects. Viral vectors that may be used include, but are not limited to, adenovirus, retrovirus, vaccinia virus, poxvirus, adeno-associated virus, herpes simplex virus, lentivirus, baculovirus, sendai virus, measles virus, monkey virus 40, and EB virus vectors. Non-viral vectors include plasmids, lipid complexes (cationic liposome-DNA complexes), polymeric complexes (anionic polymer-DNA complexes), and protein-DNA complexes. In addition to the nucleic acid, the vector may also contain one or more regulatory regions and/or selectable markers for selection, measurement and monitoring of nucleic acid transfer results (tissue transferred, duration of expression, etc.).

The term "plasmid" refers to an extrachromosomal element that typically carries genes that are not part of the central metabolism of the cell, and is typically in the form of a circular double-stranded DNA molecule. These elements may be autonomously replicating sequences, genome integrating sequences, bacteriophage or nucleotide sequences, linear, circular or supercoiled single-or double-stranded DNA or RNA derived from any source in which multiple nucleotide sequences have been joined or recombined into a unique structure which enables the introduction of a promoter fragment of a selected gene product and DNA sequence into a cell together with appropriate 3' untranslated sequence.

"cloning vector" refers to a "replicon" which is a unit length of nucleic acid, preferably DNA, which is continuously replicating and contains an origin of replication (e.g., a plasmid, phage, or cosmid) which can be ligated to another nucleic acid segment to replicate the ligated segment. The cloning vector may be capable of replication in one cell type and expression in another cell type ("shuttle vector"). Cloning vectors may comprise one or more sequences useful for selecting cells containing the vector and/or one or more multiple cloning sites for insertion of a sequence of interest.

The term "expression vector" refers to a vector, plasmid, or vehicle designed to express an inserted nucleic acid sequence after transformation into a host. Cloned genes (i.e., inserted nucleic acid sequences) are typically under the control of control elements (e.g., promoters, minimal promoters, enhancers, etc.). There are many initiation control regions or promoters useful for driving expression of a nucleic acid in a desired host cell, and are familiar to those skilled in the art. Essentially, any promoter capable of driving expression of these genes can be used in the expression vector, including but not limited to viral, bacterial, animal, mammalian, synthetic, constitutive, tissue-specific, pathogenic or disease-related, development-specific, inducible, light-regulated promoters, including but not limited to the SV40 early (SV40) promoter region, the promoter contained in the 3' Long Terminal Repeat (LTR) of Rous Sarcoma Virus (RSV), E1A or the Major Late Promoter (MLP) of adenovirus (Ad), the Human Cytomegalovirus (HCMV) immediate early promoter, Herpes Simplex Virus (HSV) Thymidine Kinase (TK) promoter, baculovirus IE1 promoter, elongation factor 1 alpha (EF1) promoter, glyceraldehyde 3-phosphate dehydrogenase (GAPDH) promoter, phosphoglycerate kinase (PGK) promoter, ubiquitin c (ubc) promoter, albumin promoter, regulatory sequences and transcriptional control regions of mouse metallothionein-L promoter, ubiquitous promoters (HPRT, vimentin, β -actin, tubulin, etc.), promoters of intermediate filaments (desmin, neurofilament, keratin, GFAP, etc.), (of MDR, CFTR or factor VIII, etc.) therapeutic genes, pathogenic or disease-related promoters, and promoters that exhibit tissue specificity and have been used in transgenic animals (e.g., elastase I gene control region that is active in pancreatic acinar cells); insulin gene control region active in pancreatic beta-cells, immunoglobulin gene control region active in lymphocytes, mouse mammary tumor virus control region active in testis, breast, lymph and mast cells; albumin gene, Apo AI and Apo AII control regions active in the liver, alpha-fetoprotein gene control regions active in the liver, alpha 1-antitrypsin gene control regions active in the liver, beta-globin gene control regions active in myeloid cells, myelin basic protein gene control regions active in oligodendrocytes of the brain, myosin light chain-2 gene control regions active in skeletal muscle, and gonadotropin-releasing hormone gene control regions active in the hypothalamus, pyruvate kinase promoter, villin promoter, intestinal fatty acid-binding protein promoter, smooth muscle cell beta-actin promoter, and the like. In addition, these expression sequences may be modified by the addition of enhancers or regulatory sequences, etc.

The expression vector constructed above is then administered to a subject in the form of a pharmaceutical composition. Two or more isoforms of HGF may be administered separately (sequentially or simultaneously, i.e., co-administered); separate plasmids directed against two or more isoforms of HGF may be administered or co-administered, or a single expression plasmid comprising a gene directed against two or more isoforms of HGF and capable of expressing the two or more isoforms of HGF may be administered. For example, two isoforms flHGF and dHGF may be administered using two separate plasmids. Alternatively, two separate plasmids containing flHGF and dHGF genes may be co-administered. Finally, a single expression plasmid containing the genes for both flHGF and dHGF may be administered. In certain aspects of the invention, flHGF and dHGF on a single expression plasmid are encoded by the same polynucleotide or by separate polynucleotides. There are many ways to include more than one polynucleotide capable of expressing an isoform of HGF on a single plasmid. These methods include, for example, the use of Internal Ribosome Entry Site (IRES) sequences, dual promoters/expression cassettes, and fusion proteins. The two or more isoforms expressed from the same plasmid or two separate plasmids (as described above) are selected from flHGF, dHGF, NK1, and NK2, or from SEQ id no: 2-5 and 11-12. The two or more isoforms may also include other isoforms of HGF known to those of ordinary skill in the art.

Vectors can be introduced into a desired host cell by methods well known in the art, such as injection, transfection, electroporation, microinjection, transduction, cell fusion, lipofection, use of gene guns or DNA vector transporters (see, e.g., Wu et al, J.biol.chem.267: 963 (1992); Wu et al, J.biol.chem.263: 14621 (1988); and Hartmut et al, Canadian patent application No.2,012,311).

The polynucleotides of the invention can also be introduced in vivo by lipofection. In the past decade, liposomes have been increasingly used for encapsulation and transfection of nucleic acids in vitro. Liposomes for in vivo gene transfection can be prepared using synthetic cationic lipids designed to limit the difficulties and risks encountered with liposome-mediated transfection (Felgner et al, Proc. Natl. Acad. Sci. USA.84: 7413 (1987); Mackey et al, Proc. Natl. Acad. Sci. USA 85: 8027 (1988); and Ulmer et al, Science 259: 1745 (1993)). The use of cationic lipids facilitates encapsulation of negatively charged nucleic acids and also fusion with negatively charged cell membranes (Felgner et al, Science 337: 387 (1989)). Particularly useful lipid compounds and compositions for nucleic acid transfer are described in WO95/18863, WO96/17823 and U.S.5,459,127. The use of lipofection to introduce foreign genes into specific organs in vivo has certain practical advantages. Targeting liposomes to specific cells by molecules represents another benefit. It is clear that direct transfection into specific cell types would be particularly preferred in tissues with cellular heterogeneity (e.g., pancreas, liver, kidney, and brain). For targeting purposes, lipids can be chemically coupled to another molecule (Mackey et al, 1988, supra). Targeted peptides (e.g., hormones or neurotransmitters) and proteins (e.g., antibodies) or non-peptide molecules can be chemically coupled to the liposomes.

Other molecules may also be used to facilitate transfection of nucleic acids in vivo, such as cationic oligopeptides (e.g., WO95/21931), peptides derived from DNA binding proteins (e.g., WO96/25508), or cationic polymers (e.g., WO 95/21931).

Vectors, such as naked DNA plasmids, can also be introduced in vivo (see U.S. Pat. nos. 5,693,622, 5,589,466, and 5,580,859). Receptor-mediated DNA delivery methods (Curiel et al, hum. Gene Ther.3: 147(1992) and Wu et al, J.biol.chem.262: 4429(1987)) may also be used.

The term "transfection" refers to the uptake of exogenous or heterologous RNA or DNA by a cell. When exogenous or heterologous RNA or DNA has been introduced into a cell, then the cell has been "transfected" with such RNA or DNA. When the transfected RNA or DNA affects a phenotypic change, then the cell has been "transformed" by the exogenous or heterologous RNA or DNA. The transforming RNA or DNA may be integrated (covalently linked) into the chromosomal DNA that makes up the genome of the cell.

"transformation" refers to the transfer of a nucleic acid fragment into a host organism to form a genetically stable genetic trait. Host organisms comprising transformed nucleic acid fragments are referred to as "transgenic" or "recombinant" or "transformed" organisms.

In addition, a recombinant vector comprising a polynucleotide may comprise one or more origins of replication, markers, or selectable markers for use in a cellular host in which amplification or expression is desired.

The term "selectable marker" refers to a recognition element (typically an antibiotic or chemical resistance gene) that can be selected based on the action of the marker gene, i.e., resistance to antibiotics, resistance to herbicides, colorimetric markers, enzymes, fluorescent markers, etc., wherein the action is used to track the inheritance of a nucleic acid of interest, and/or to identify a cell or organism having a inherited nucleic acid of interest. Examples of selectable marker genes known and used in the art include genes resistant to ampicillin, streptomycin, gentamicin, kanamycin, hygromycin, bialaphos, sulfonamide, and the like; and genes used as phenotypic markers, i.e., an anthocyanin regulatory gene, an isopentyltransferase gene, and the like.

The term "reporter gene" refers to a nucleic acid encoding an identifier that can be identified based on the reporter gene's action, wherein the action is used to track the inheritance of a nucleic acid of interest, to identify a cell or organism that has inherited the nucleic acid of interest, and/or to measure gene expression induction or transcription. Examples of reporter genes known and used in the art include luciferase (Luc), Green Fluorescent Protein (GFP), Chloramphenicol Acetyltransferase (CAT), beta-galactosidase (LacZ), beta-Glucuronidase (GUS), and the like. The selectable marker gene may also be considered a reporter gene.

"promoter" and "promoter sequence" are used interchangeably and refer to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. Generally, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements from different promoters found in nature, or even comprise synthetic DNA segments. It will be appreciated by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types under most conditions are commonly referred to as "constitutive promoters". Promoters that cause the expression of genes in a particular cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters". Promoters that cause the expression of genes at a particular developmental stage or cell differentiation are commonly referred to as "developmental-specific promoters" or "cell differentiation-specific promoters". Promoters that are induced and cause gene expression after a cell is exposed to or treated with an agent, biomolecule, chemical, ligand, light, etc., are commonly referred to as "inducible promoters" or "regulated promoters". It will also be appreciated that DNA fragments of different lengths may have the same promoter activity, since in most cases the exact boundaries of the regulatory sequences have not yet been fully defined.

The promoter sequence is generally bounded by its transcription initiation site at the 3 'end and extends upstream (5' direction) to include the minimum number of bases or elements required to initiate transcription at detectable levels above background. Within the promoter sequence will be found a transcription initiation site (conveniently defined, for example, by mapping with nuclease S1) and a protein binding domain (consensus sequence) responsible for binding RNA polymerase.

A coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, followed by trans-splicing of the RNA (if the coding sequence comprises introns) and translation into the protein encoded by the coding sequence.

"transcriptional and translational control sequences" refer to DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell. In eukaryotic cells, polyadenylation signals are control sequences.

The term "response element" refers to one or more cis-acting DNA elements that confer promoter reactivity by mediating interactions with the DNA-binding domain of a transcription factor. The sequence of this DNA element may be palindromic (complete or incomplete) or consist of sequence motifs or half-sites separated by a variable number of nucleotides. Half sites may be similar or identical and arranged in either a forward or reverse repeat, or a tandem multimer of a single half site or adjacent half sites. The response element may comprise a minimal promoter isolated from a different organism, depending on the nature of the cell or organism into which the response element is to be introduced. The DNA binding domain of a transcription factor binds to a DNA sequence of a response element in the presence or absence of a ligand to initiate or inhibit transcription of a downstream gene under the control of the response element.

The term "operably linked" refers to the linkage of nucleic acid sequences to a single nucleic acid fragment such that the function of one of the sequences is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). The coding sequence may be operably linked to the regulatory sequences in sense or antisense orientation.

The term "expression" as used herein refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from a nucleic acid or polynucleotide. Expression may also refer to translation of mRNA into protein or polypeptide.

The terms "cassette", "expression cassette" and "gene expression cassette" refer to a segment of DNA that can be inserted into a nucleic acid or polynucleotide at a specific restriction site or by homologous recombination. The DNA segment comprises a polynucleotide encoding a polypeptide of interest, the cassette and restriction sites being designed to ensure that the cassette is inserted into an appropriate reading frame for transcription and translation. "transformation cassette" refers to a particular vector that comprises a polynucleotide encoding a polypeptide of interest and, in addition to the polynucleotide, elements that facilitate transformation of a particular host cell. The cassettes, expression cassettes, gene expression cassettes and transformation cassettes may further comprise elements that allow for enhanced expression of the polynucleotide encoding the polypeptide of interest in the host cell. These elements may include, but are not limited to, promoters, minimal promoters, enhancers, response elements, terminator sequences, polyadenylation sequences, and the like.

The term "gene switch" refers to the combination of a response element with a promoter and a ligand-dependent transcription factor-based system that regulates the expression of a gene into which the response element and promoter are introduced in the presence of one or more ligands.

The term "modulate" refers to the induction, reduction or inhibition of expression of a nucleic acid or gene, resulting in the induction, reduction or inhibition of production of a protein or polypeptide, respectively.

Enhancers that may be used in embodiments of the invention include, but are not limited to, the SV40 enhancer, the Cytomegalovirus (CMV) enhancer, the elongation factor 1(EF1) enhancer, yeast enhancers, viral gene enhancers, and the like.

Termination control regions (i.e., terminators or polyadenylation sequences) may also be derived from a variety of genes that are native to the preferred host. Optionally, a termination site may not be necessary, but is preferably included. In one embodiment of the invention, the termination control region may comprise or be derived from a synthetic sequence, a synthetic polyadenylation signal, the SV40 late polyadenylation signal, the SV40 polyadenylation signal, a Bovine Growth Hormone (BGH) polyadenylation signal, a viral terminator sequence, and the like.

The term "3 ' non-coding sequence" or "3 ' untranslated region (UTR)" refers to a DNA sequence that is located downstream (3 ') of a coding sequence and may include polyadenylation [ poly (a) ] recognition sequences and other sequences that encode regulatory signals capable of affecting mRNA processing or gene expression. Polyadenylation signals are generally characterized by the addition of polyadenylic acid to the 3' end of the mRNA precursor.

"regulatory region" refers to a nucleic acid sequence that regulates the expression of another nucleic acid sequence. The regulatory regions may comprise sequences naturally responsible for the expression of a particular nucleic acid (homologous regions), or may comprise sequences from different sources responsible for the expression of different proteins or even synthetic proteins (heterologous regions). In particular, the sequence may be a sequence of a prokaryotic, eukaryotic or viral gene or a derivative sequence which stimulates or inhibits the transcription of the gene in a specific or non-specific manner and in an inducible or non-inducible manner. Regulatory regions include origins of replication, RNA splice sites, promoters, enhancers, transcription termination sequences, and signal sequences that direct the polypeptide into the secretory pathway of a target cell.

Regulatory regions from "heterologous sources" refer to regulatory regions that are not naturally associated with the expressed nucleic acid. Heterologous regulatory regions include regulatory regions from different species, regulatory regions from different genes, hybrid regulatory sequences, and regulatory sequences not found in nature but designed by one of ordinary skill in the art.

"RNA transcript" refers to the product resulting from the transcription of a DNA sequence catalyzed by RNA polymerase. When the RNA transcript is a perfectly complementary copy of a DNA sequence, it is referred to as the original transcript, or it may be an RNA sequence derived from post-translational processing of the original transcript, referred to as the mature RNA. "messenger RNA (mRNA)" refers to RNA that does not contain introns and can be translated into protein by a cell. "cDNA" refers to double-stranded DNA that is complementary to and derived from mRNA. "sense" RNA refers to RNA transcript that comprises mRNA and is therefore translatable into protein by the cell. "antisense RNA" refers to RNA transcripts that are complementary to all or part of the target original transcript or mRNA and block expression of the target gene. The antisense RNA can be complementary to any portion of a particular gene transcript, i.e., the 5 'non-coding sequence, the 3' non-coding sequence, or the coding sequence. "functional RNA" refers to antisense RNA, ribozyme RNA, or other RNA that is not translated, but that affects cellular processes.

"polypeptide," "peptide," and "protein" are used interchangeably to refer to a multimeric compound composed of covalently linked amino acid residues.

An "isolated polypeptide," "isolated peptide," or "isolated protein" refers to a polypeptide or protein that is substantially free of those compounds with which it is normally associated in nature (e.g., other proteins or polypeptides, nucleic acids, carbohydrates, lipids). "isolated" is not meant to exclude the presence of artificial or synthetic mixtures with other compounds or impurities that do not interfere with biological activity, which may be present, for example, as a result of incomplete purification, addition of stabilizers, or mixing into pharmaceutically acceptable formulations.

The mutation may be performed using any of the mutagenesis techniques known in the art, including, but not limited to, site-directed mutagenesis in vitro (Hutchinson et al, J.biol.chem.253: 6551 (1978); Zoller et al, DNA 3: 479 (1984); Oliphant et al, Gene 44: 177 (1986); Hutchinson et al, Proc.Natl.Acad.Sci.USA 83: 710(1986)), using TABLinkers (Pharmacia), restriction endonuclease digestions/fragment deletions and substitutions, PCR-mediated/oligonucleotide-directed mutations, and the like. PCR-based techniques are preferred for site-directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", PCR Technology: Principles and applications for DNA Amplification, editions H. Erlich, Stockton Press, Chapter 6, pages 61-70).

A "variant" of a polypeptide or protein refers to any analog, fragment, derivative, or mutant derived from the polypeptide or protein and which retains at least one biological property of the polypeptide or protein. Different variants of a polypeptide or protein may occur naturally. These variants may be allelic variations, characterized by differences in the nucleotide sequence of the structural genes encoding the proteins, or may include differential splicing or post-translational modifications. The skilled person can prepare variants with single or multiple amino acid substitutions, deletions, additions or substitutions. These variants may include: (a) variants in which one or more amino acid residues are replaced with a conserved or non-conserved amino acid, (b) variants in which one or more amino acids are added to a polypeptide or protein, (c) variants in which one or more amino acids comprise a substituent, and (d) variants in which a polypeptide or protein is fused to another polypeptide (e.g., serum albumin), and the like. Techniques for obtaining such variants, including genetic (inhibition, deletion, mutation, etc.), chemical and enzymatic techniques, are well known to those of ordinary skill in the art.

The term "homology" refers to the percentage of identity between two polynucleotide or two polypeptide portions. The correspondence between the sequence of one portion to another can be determined by techniques well known in the art. For example, homology can be determined by aligning sequence information and directly comparing the sequence information between two polypeptide molecules using readily available computer programs. Alternatively, homology can be determined by hybridizing polynucleotides under conditions that form a stable duplex between the homologous regions, followed by digestion with a single strand specific nuclease and determination of the size of the digested fragments.

All grammatical and spelling changes of the term "homologous" as used herein refer to the relationship between proteins of "common evolutionary origin", including proteins from a superfamily (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck et al, Cell 50: 667 (1987)). These proteins (and their encoding genes) have sequence homology as reflected by their high degree of sequence similarity. However, in the usual sense and in the present application, the term "homologous" when modified with an adverb such as "high" may refer to sequence similarity, rather than a common evolutionary origin.

Thus, all grammatical forms of the term "sequence similarity" refer to the degree of identity or correspondence between amino acid sequences of nucleic acids or proteins that may or may not have a common evolutionary origin (see Reeck et al, Cell 50: 667 (1987)). In one embodiment, two DNA sequences are "substantially homologous" or "substantially similar" when at least about 50% (e.g., at least about 75%, 90%, or 95%) of the nucleotides match over the determined length of the DNA sequences. Substantially homologous sequences can be identified by aligning the sequences using standard software available in sequence databases or performing Southern hybridization experiments under stringent conditions as defined, for example, by the particular system. Defining suitable hybridization conditions is within the skill in the art (see, e.g., Sambrook et al, 1989, supra).

As used herein, "substantially similar" refers to nucleic acid fragments wherein a change in one or more nucleotide bases results in the substitution of one or more amino acids, but does not affect the functional properties of the protein encoded by the DNA sequence. "substantially similar" also refers to nucleic acid fragments wherein a change in one or more nucleotide bases does not affect the ability of the nucleic acid fragment to mediate a change in gene expression by antisense or cosuppression techniques. "substantially similar" also relates to modifications (e.g., deletion or insertion of one or more nucleotide bases) to the nucleic acid fragments of the invention that do not substantially affect the functional properties of the resulting transcripts. Thus, it is to be understood that the invention encompasses more sequences than the specific exemplary sequences. Each of the proposed modifications is within the routine skill in the art, as is the determination of the retention of biological activity of the encoded product.

However, the skilled artisan will recognize that substantially similar sequences encompassed by the invention can also be defined by their ability to hybridize to the exemplified sequences shown herein under stringent conditions (0.1 XSSC, 0.1% SDS, 65 ℃ and washed with 2 XSSC, 0.1% SDS, then 0.1 XSSC, 0.1% SDS). Substantially similar nucleic acid fragments of the invention are those whose DNA sequence is at least about 70%, 80%, 90% or 95% identical to the nucleic acid fragments reported herein.

Two amino acid sequences are "substantially homologous" or "substantially similar" when more than about 40% of the amino acids are identical or more than 60% of the amino acids are similar (functionally identical). Preferably, similar or homologous sequences are identified by alignment using, for example, the program GCG (Genetics Computer Group, the program manual of GCG package 7 th edition, Madison, Wisconsin) pileup.

The term "corresponding to" as used herein refers to similar or homologous sequences, whether identical or different in exact position to the detected molecule with similarity or homology. Nucleic acid or amino acid sequence alignments can include gaps. Thus, the term "corresponding to" refers to sequence similarity, not the numbering of amino acid residues or nucleotide bases.

A "significant portion" of an amino acid or nucleotide sequence comprises a polypeptide amino acid sequence or a gene nucleotide sequence sufficient for the putative identification of the polypeptide or gene by manual evaluation of the sequence by one of skill in the art, or by computer automated sequence comparison and identification using algorithms such as BLAST (Basic Local Alignment Search Tool; Altschul et al, J.Mol.biol.215: 403 (1993); ncbi.n.m.nih.gov/BLAST/procurement)). In general, in order to putatively identify a polypeptide or nucleic acid sequence homologous to a known protein or gene, a sequence of 10 or more contiguous amino acids or 30 or more nucleotides is necessary. Furthermore, in terms of nucleotide sequence, gene-specific oligonucleotide probes comprising 20-30 contiguous nucleotides can be used in sequence-dependent gene identification (e.g., Southern hybridization) and isolation (e.g., in situ hybridization of bacterial clones or phage plaques). In addition, short oligonucleotides of 12-15 bases can be used as amplification primers in PCR to obtain specific nucleic acid fragments comprising the primers. Thus, a "significant portion" of a nucleotide sequence comprises sufficient sequence to specifically identify and/or isolate a nucleic acid fragment comprising the sequence.

As is well known in the art, the term "percent identity" is a relationship between two or more polypeptide sequences or two or more polynucleotide sequences as determined by comparing the sequences. In the art, "identity" also refers to the degree of sequence relatedness between polypeptide or polynucleotide sequences, as the case may be, as determined by the match between strings of such sequences. "identity" and "similarity" can be readily calculated by known methods, including but not limited to those described below: computational Molecular Biology (Lesk, A.M. ed.) Oxford University Press, New York (1988); biocomputing: information and Genome Projects (Smith, D.W. eds.) Academic Press, New York (1993); computer Analysis of Sequence Data, Part I (Griffin, A.M. and Griffin, edited by H.G.) Humana Press, New Jersey (1994); sequence analysis in Molecular Biology (von Heinje, g. ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. eds.) Stockton Press, New York (1991). Preferred methods of determining identity are designed to give the best match between the sequences sequenced. Methods for determining identity and similarity are written in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using sequence analysis software, such as the Megalign program of LASERGENE bioinformatics computing suite (DNASTAR inc., Madison, WI). Multiple sequence alignments can utilize the Clustal alignment method (Higgins et al, cabaos.5: 151(1989)) and default parameters (gap penalty of 10, gap length penalty of 10). Default parameters for pairwise alignment using the Clustal method may be selected: KTUPLE 1, gap penalty 3, WINDOW 5 and DIAGONALS SAVED 5.

The term "sequence analysis software" refers to any computer algorithm or software program for analyzing a nucleotide or amino acid sequence. "sequence analysis software" can be purchased or developed on its own. Typical sequence analysis software includes, but is not limited to, the GCG program (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis.), BLASTP, BLASTN, BLASTX (Altschul et al, J.mol.biol.215: 403(1990)), and DNASTAR (DNASTAR, Inc.1228 S.park St.Madison, Wis 53715 USA). In the context of the present application, it should be understood that when sequence analysis software is used for analysis, the results of the analysis will be based on the "default values" of the referenced program, unless otherwise specified. As used herein, "default values" will refer to any value or set of parameters that are first loaded at software initialization.

"chemically synthesized," when related to a DNA sequence, refers to the assembly of component nucleotides in vitro. Manual chemical synthesis of DNA can be accomplished using well-established methods, or automated chemical synthesis can be performed using one of a variety of commercially available instruments. Thus, the gene can be adjusted to optimize gene expression based on nucleotide sequence optimization reflecting the codon bias of the host cell. The skilled artisan understands that gene expression is likely to be successful if codon usage is adjusted according to host-preferred codons. Preferred codons can be determined based on studies of genes derived from host cells for which sequence information is available.

The term "exogenous gene" refers to a gene outside the subject, that is, a gene introduced into the subject by a transformation process, which is a non-mutated form of an endogenous mutated gene or a mutated form of an endogenous non-mutated gene. The method of transformation is not critical to the present invention and may be any method known to those of skill in the art to be suitable for use with a subject. The foreign gene may be a natural or synthetic gene introduced into the subject in the form of DNA or RNA, which may act through a DNA intermediate (e.g., by reverse transcriptase). These genes can be introduced into the target cell, directly into the subject, or indirectly into the subject by transfer of transformed cells.

The term "subject" refers to an intact animal, preferably a vertebrate, most preferably a mammal.

The term "HGF isoform" refers to any HGF polypeptide having an amino acid sequence that is at least 80% identical (e.g., at least 90% or 95% identical) to an HGF amino acid sequence naturally occurring in an animal, including all allelic variants. In one embodiment, the term refers to isoforms known to have cell proliferative activity. HGF isoforms include, but are not limited to, flHGF, dHGF, NK1, NK2, and NK 4.

The term "flHGF" refers to the full-length HGF protein of an animal (e.g., a mammal), such as amino acids 1-728 of human HGF.

The term "dHGF" refers to a deletion variant of HGF protein produced by alternative splicing of the HGF gene in an animal (e.g., a mammal), such as a human HGF consisting of 723 amino acids, wherein the first kringle domain of the alpha chain of the full length HGF sequence is deleted by 5 amino acids (F, L, P, S and S).

The term "NK 1" refers to an isoform of HGF from an animal (e.g., a mammal, such as a human) consisting of an N-terminal hairpin loop and a kringle 1 domain.

The term "NK 2" refers to an isoform of HGF from an animal (e.g., a mammal, such as a human) consisting of an N-terminal hairpin loop, kringle 1 and kringle2 domains.

The term "NK 4" refers to an isoform of HGF from an animal (e.g., a mammal, such as a human) consisting of an N-terminal hairpin loop, kringle 1, kringle2, kringle 3, and kringle 4 domain.

The methods of the invention comprise administering to a subject having a cardiac and/or vascular disorder a composition comprising two or more isoforms of HGF. In one embodiment, the two or more isoforms of HGF are isoforms of mammalian HGF (e.g., human HGF). The amino acid sequences of HGF from various species are well known in the art and can be found in sequence databases, e.g., GenBank (the amino acid sequence of human HGF of accession number BAA14348, incorporated herein by reference). In one embodiment, one of the isoforms of HGF is human flHGF (SEQ ID NO: 2). In another embodiment, one of the isoforms of HGF is human dHGF (SEQ ID NO: 3). In another embodiment, one of the isoforms of HGF is human NK1(SEQ ID NO: 4). In another embodiment, one of the isoforms of HGF is human NK2(SEQ ID NO: 5). In another embodiment, one of the isoforms of HGF is human NK4(SEQ ID NO: 6). In yet another embodiment, the two or more isoforms of HGF comprise flHGF and dHGF. In yet another embodiment, the two or more isoforms of HGF consist of flHGF and dHGF.

In one embodiment, the isoform of HGF is a variant of a human wild-type isoform of HGF. For example, the isoform may be a variant of a human flHGF, dHGF, NK1, NK2, or NK4 sequence that is at least 80% sequence identical (e.g., at least 85, 90, 95, 96, 97, 98, or 99% sequence identical) to a wild-type human flHGF (SEQ ID NO: 2), dHGF (SEQ ID NO: 3), NK1(SEQ ID NO: 4), NK2(SEQ ID NO: 5), or NK4(SEQ ID NO: 6) sequence. The variants may include additions, deletions, substitutions, or combinations thereof, of the amino acid sequence of the wild-type human HGF isoform. Additions or substitutions may include the use of unnatural amino acids, and may occur anywhere within, at the N-terminus, and/or at the C-terminus.

Preferably, any substitution is a conservative amino acid substitution. "conservative amino acid substitution" refers to a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine tryptophan, histidine).

Sequence identity was calculated by the following steps: the percentage of sequence identity is determined by comparing the two optimally aligned sequences in the comparison region, determining the number of positions at which the identical amino acid residue is present in the two sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison region (i.e., the window size), and multiplying the result by 100. In one aspect, percent identity is calculated as the percentage of amino acid residues that are the smaller of the two sequences that match at the same amino acid residue in the compared Sequence (Dayhoff, in Atlas of Protein Sequence and Structure, vol.5, page 124, National Biochemical Research Foundation, Washington, d.c. (1972), incorporated herein by reference), where 4 gaps in 100 amino acid lengths can be introduced for optimal alignment. Determination of identity is generally performed by computer homology programs well known in the art. An exemplary program is a Gap program (WisconsinSequence Analysis Package, Version 8 for UNIX, Genetics computer group, University Research Park, Madison, WI) that utilizes the algorithm of Smith and Waterman (adv. appl. math.2: 482(1981)), which is incorporated herein by reference in its entirety, using default settings.

In one embodiment, the isoform variant of HGF retains substantially all of any one or more of the biological activities of the wild-type isoform protein of HGF. The term "substantially all activity of wild-type HGF" as used herein refers to an HGF isoform variant that retains at least 70% of any one or more of the biological activities of the HGF isoform variant (e.g., the ability to stimulate angiogenesis or promote cell proliferation). In some embodiments, at least 75, 80,85, 90, or 95% of the one or more biological activities of the HGF isoform is retained. HGF activity can be detected by conventional in vitro and in vivo assays well known in the art, such as Matrigel plug and corneal neovascularization in vivo assays, chick embryo allantoic membrane (CAM) assays in vivo/in vitro, cells in vitro (proliferation, migration, tube formation) and organotypic (arterial loop) assays.

The structure and function of HGF has been extensively studied and it is known to those skilled in the art that amino acids in the HGF sequence are important to retain substantially all of the biological activity of the protein, and preferably are not changed or are only conservatively changed in any HGF variant. See, e.g., Hartmann et al, proc.natl.acad.sci.usa 89: 11574 (1992); lokker et al, EMBO J.11: 2503(1992), Zhou et al, Structure 6: 109(1998), Ultsch et al, Structure 6: 1383(1998), Shimizu et al, biochem. Biophys. Res. Commun.189: 1329(1992), Yoshiyama et al, biochem. biophysis. res. commun.175: 660(1991), the entire contents of which are incorporated herein by reference. For example, the N-terminal hairpin loop and kringle 1 domain may be required for cell proliferative activity. Other amino acids not critical to biological activity may be more freely deleted and/or substituted. One skilled in the art can prepare variants of the HGF isoforms using conventional mutagenesis techniques (e.g., those described in the references cited above) and identify variants that retain substantially all of the biological activity of the HGF isoform.

In one aspect of the invention, two or more isoforms of HGF are administered to a subject in the form of a polynucleotide encoding the isoforms. In one embodiment, the polynucleotide encodes a mammalian isoform, such as human HGF. Polynucleotide sequences of HGF genes from various species are well known in the art and can be found in sequence databases, e.g., GenBank (polynucleotide sequence of human HGF gene under accession No. NM000601, incorporated herein by reference). In one embodiment, the polynucleotide encodes flHGF. In another embodiment, the polynucleotide encodes dHGF. In another embodiment, the polynucleotide encodes NK 1. In another embodiment, the polynucleotide encodes NK 2. In another embodiment, the polynucleotide encodes NK 4. In yet another embodiment, the polynucleotide encodes both flHGF and dHGF. In yet another embodiment, the polynucleotide encodes flHGF, dHGF and NK 1.

In one embodiment, the one or more polynucleotides encoding two or more isoforms of HGF comprise a wild-type human HGF gene sequence. In another embodiment, the polynucleotide comprises a sequence variant of a wild-type HGF gene, but still encodes the wild-type amino acid sequence of the HGF isoform due to codon degeneracy. In yet another embodiment, the polynucleotide encodes an HGF isoform protein sequence variant as described above. In one embodiment, the polynucleotide sequence variant of the human HGF isoform gene sequence has at least 80% sequence identity, e.g., at least 85, 90, 95, 96, 97, 98, or 99% identity, to the wild-type human HGF isoform gene sequence.

In one aspect of the invention, the two or more isoforms of HGF are encoded by a hybrid HGF gene that simultaneously expresses the two or more isoforms (e.g., flHGF and dHGF). The hybrid HGF gene described in US 2005/0079581 a1 (the entire contents of which are incorporated herein by reference) comprises a cDNA corresponding to HGF introns 1-18, and an intrinsic or exogenous intron inserted between exons 4 and 5 of said cDNA, wherein there are no other introns between the exons of the hybrid gene other than the intron between exons 4 and 5. The intron includes a fragment or recombination sequence of the inherent intron.

One embodiment of a hybrid HGF gene comprising an inherent intron is 7113bp in length and has the amino acid sequence of SEQ ID NO: 7. The hybrid HGF gene expresses both flHGF and dHGF simultaneously and has higher expression efficiency than flHGF cDNA.

Codon degeneracy allows hybrid HGF genes to be modified or altered in coding and/or non-coding regions without altering the amino acid sequence of the protein and expression of the gene. Thus, in comparison to SEQ ID NO: 7 and fragments thereof are within the scope of the invention. "substantially identical" means sequence identity of no less than 80%, such as no less than 90%, such as no less than 95%.

Hybrid HGF genes may comprise a fragment of the inherent intron, optionally containing a small recombination sequence inserted between exons 4 and 5 of the HGF cdna. Herein, these hybrid HGF genes comprising a fragment of the intrinsic intron are designated "HGF-X". Examples include those having SEQ ID NOs: HGF-X6, HGF-X7 and HGF-X8 of the nucleotide sequence of 8 to 10.

The two or more isoforms of HGF to be administered may be encoded by separate polynucleotides or a single polynucleotide. The polynucleotide may be operably linked to one or more regulatory regions (e.g., a promoter or enhancer) that controls expression of the HGF isoform. The promoter may be a constitutive promoter (e.g., the human cytomegalovirus promoter) or an inducible promoter. The regulatory sequence may be part of a gene switch that regulates expression of the HGF isoform by addition or removal of a specific ligand. Examples of ligand-dependent transcription factors that can be used in the gene switch of the present invention include, but are not limited to, nuclear receptor superfamily members activated by respective ligands (e.g., glucocorticoids, estrogens, progesterone, retinoids, ecdysones, and analogs and mimetics thereof) and rTTA activated by tetracycline. In yet another embodiment, the promoter is a cardiomyocyte-specific promoter (e.g., cardiac ankyrin repeat protein, MYBPC 3). When two or more isoforms of HGF are encoded by separate polynucleotides, each polynucleotide may be operably linked to its own regulatory sequences. In another embodiment, two or more polynucleotides may be controlled by a single regulatory sequence as part of a tandem cassette, optionally with the insertion of sequences (e.g., internal ribosome binding sites) between the two or more polynucleotides to facilitate expression of all isoforms.

In one embodiment of the invention, the one or more polynucleotides encoding two or more isoforms of HGF are part of a vector (e.g., an expression vector). The vector may be, for example, a plasmid vector (e.g., Lee et al, biochem. Biophys. Res. Commun.272: 230(2000), pCK vectors disclosed in WO 2000/040737) or a single-or double-stranded RNA or DNA viral vector. These vectors can be introduced into cells by well-known techniques for introducing DNA and RNA into cells. Viral vectors may be replication competent or replication deficient. In the latter case, viral propagation will generally occur only in the complementing host cell. The term "host cell" or "host" as used herein is intended to refer to a cell of the invention which carries one or more polynucleotides of the invention.

Thus, the vector must comprise at least the polynucleotide of the invention. Other components of the vector may include, but are not limited to, selectable markers, chromatin modification domains, other promoters that drive expression of other polypeptides (e.g., lethal polypeptides) that may also be present on the vector, genomic integration sites, recombination sites, and molecular insertion sites. The vector may contain any number of these other elements, whether within the polynucleotide or not, so that the vector may be adapted for the specific purpose of the desired therapeutic method.

In one embodiment of the invention, the vector introduced into the cell further comprises a "selectable marker gene" which, when expressed, indicates that the vector has been introduced into the host cell. Thus, the selection gene may be a positive marker for the presence of the vector. Although not critical to the methods of the invention, the presence of a selectable marker gene allows the skilled artisan to select a population of living cells into which a vector construct has been introduced. Thus, certain embodiments of the invention include selecting cells into which a vector has been successfully introduced. The term "selection" or variations thereof, as used herein, when used in conjunction with a cell, is intended to denote standard well-known methods for selecting cells containing a particular genetic make-up or phenotype. Typical methods include, but are not limited to, culturing cells in the presence of antibiotics such as G418, puromycin, and ampicillin. Other examples of selectable marker genes include, but are not limited to, genes that confer resistance to methotrexate, hygromycin or mycophenolic acid. Then, cells comprising the vector construct containing the antibiotic resistance gene will be able to tolerate the antibiotic in culture. Likewise, cells that do not contain a vector construct containing an antibiotic resistance gene will not be able to tolerate the antibiotic in culture.

As used herein, "chromatin modification domain" (CMD) refers to a nucleotide sequence that interacts with a variety of proteins associated with maintaining and/or altering chromatin structure, such as, but not limited to, DNA insulators. See, Ciavatta et al, proc.natl.acad.sci.u.s.a.103: 9958(2006), which is hereby incorporated by reference. Examples of CMDs include, but are not limited to, chicken beta-globin insulator and chicken hypersensitive site 4(cHS 4). The use of different CMD sequences between one or more gene sequences (i.e., promoter, coding sequence and 3' regulatory region) may, for example, facilitate the use of different CMD DNA sequences as "mini homology arms" in combination with various microbial or in vitro recombination techniques to "swap" gene sequences between existing multigene and monogene shuttle vectors. Other examples of chromatin modification domains are known in the art or can be readily identified.

Specific vectors that may be used in the present invention are expression vectors encoding proteins or polynucleotides. Generally, these vectors comprise a cis-acting control region operably linked to the polynucleotide to be expressed for efficient expression in a host. Suitable trans-acting factors are provided by the host, by a complementing vector or by the vector itself after introduction into the host.

Many expression vectors are available for expression of proteins or polynucleotides. These vectors include chromosomal, episomal or virally derived vectors, such as vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, viruses (e.g., adeno-associated virus, lentivirus, baculovirus, papova virus, SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retroviruses), as well as vectors derived from combinations thereof, such as vectors derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. All can be used for expression in this aspect of the invention. In general, any vector suitable for maintaining, propagating or expressing a polynucleotide or protein in a host may be used for expression in this regard.

The polynucleotide sequence in the expression vector may be operably linked to suitable expression control sequences, including for example a promoter, to direct transcription of the mRNA. Representative additional promoters include, but are not limited to, constitutive promoters and tissue-specific or inducible promoters. Examples of constitutive eukaryotic promoters include, but are not limited to, the mouse metallothionein I gene promoter (Hamer et al, J.mol.appl.Gen.1: 273(1982)), the TK promoter of herpes virus (McKnight, Cell 31: 355(1982)), the SV40 early promoter (Benoist et al, Nature 290: 304(1981)), and the vaccinia virus promoter. All references listed above are incorporated herein by reference. Other examples of promoters that can be used to drive expression of a protein or polynucleotide include, but are not limited to, tissue-specific promoters and other endogenous promoters for a particular protein, such as albumin promoters (stem cells), proinsulin promoters (pancreatic beta cells), and the like. In general, an expression construct will contain sites for transcription, initiation, and termination, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct includes a translation initiating AUG located at the start of the polypeptide to be translated and a stop codon (UAA, UGA or UAG) correctly placed at the end.

In addition, the construct may comprise control regions that regulate and initiate expression. Generally, these regions will be manipulated by controlling transcription (e.g., repressor binding sites and enhancers, etc.).

Examples of eukaryotic vectors include, but are not limited to, pW-LNEO, pSV2CAT, pOG44, pXT1 and pSG, available from Stratagene; pSVK3, pBPV, pMSG, and pSVL obtained from Amersham Pharmacia Biotech; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2-Mito, and pCMV-EGFP, obtained from Clontech. Many other vectors are well known and commercially available.

Selection of suitable vectors and promoters for expression in a host cell is a well known procedure, and the necessary techniques for vector construction and introduction into, and expression in, a host are conventional in the art.

Introduction of the polynucleotide into the cell may be transient transfection or stable transfection of the vector. Transient transfection of the vector into the host cell can be achieved by direct injection, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, or other methods. These methods are described in many standard Laboratory manuals (e.g., Davis et al, basic methods in Molecular Biology (1986); Keown et al, method.enzymol.185: 527 (1990); Sambrook et al, 2001, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, N.Y., which are incorporated herein by reference).

The two or more isoforms of HGF or one or more polynucleotides encoding the isoforms of HGF may be administered to a subject in the form of a pharmaceutical composition. In one embodiment, the composition is formulated for injection.

The pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. Any method commonly used in the pharmaceutical field can be used to prepare oral formulations such as tablets, capsules, pills, granules, suspensions and solutions; injectable formulations, such as solutions, suspensions or dry powders which may be mixed with distilled water prior to injection; formulations for topical application, such as ointments, creams and lotions; and other agents.

Carriers commonly used in the pharmaceutical field may be used in the compositions of the present invention. For example, formulations for oral administration may contain binders, emulsifiers, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants or flavorants. Injectable formulations may contain preservatives, pain-relieving agents (unagonizing agents), solubilizers or stabilizers. Formulations for topical administration may contain a base, excipient, lubricant or preservative. Any suitable agent known in the art (Remington's Pharmaceutical Science (18th edition), Mack publishing company, Eaton PA) may be used in the present invention.

The pharmaceutical composition can be clinically administered in various oral and parenteral formulations. Suitable formulations may be prepared using excipients such as additives, promoters, binders, wetting agents, disintegrants and surfactants or diluents. Solid preparations for oral administration include pills, tablets, powders, granules and capsules. These solid preparations can be prepared by mixing one or more excipients such as starch, calcium carbonate, sucrose, lactose, gelatin and dibenzylbutyrolactone (lignan) derivative. Also, lubricating agents such as magnesium stearate and talc may be included in the formulation. Liquid preparations for oral administration include suspensions, solutions, emulsions and syrups. These preparations may contain wetting agents, sweeteners, aromatics and preservatives in addition to the usual simple diluents such as water and liquid paraffin. Formulations for parenteral administration include sterile aqueous solutions, suspensions, emulsions, lyophilized substitution therapies, and suppositories. Water-insoluble excipients and suspending agents include vegetable fats such as propylene glycol, ethylene glycol and olive oil, and injectable esters such as ethyl oleate. Witepsol、Macrogol、Tween61. Cocoa butter, laurate (laurin fat) and glycerogelatin may be used as suppository bases.

The following examples are given for illustrative purposes only and should not be construed as limiting the scope of the present invention.

Example 1: construction of plasmids

pCK vectors can utilize the Human Cytomegalovirus (HCMV) promoter to drive gene expression and have been described previously (Lee et al, biochem. Biophys. Res. Commun.272: 230 (2000); WO 2000/040737).

pCK-VEGF165 was constructed by inserting VEGF165 cDNA into a pCK vector and has been described previously (Lee et al, biochem. Biophys. Res. Commun.272: 230 (2000); WO 2000/040737).

pCK-cHGF comprising an encoded HGF under the control of the HCMV promoter₇₂₈And has been previously described (US 2005/0079581).

pCK-dHGF comprising an encoded HGF under the control of the HCMV promoter₇₂₃And has been described previously (PCT/KR 03/00548).

pCK-HGF-X7 comprises a hybrid HGF cDNA (SEQ ID NO: 9) designed to express two isoforms of HGF inserted simultaneously into a pCK vector and has been previously described (US 2005/0079581).

Example 2: effect of HGF on cell migration and proliferation

The objective of this study was to evaluate the effect of HGF on cell migration and proliferation in vitro.

1. Materials and methods

(1) Preparation of HGF protein

Using FuGENE6^TM(Roche Diagnostics, Germany) pCK-HGF-X7 was transfected into 293T cells. pCK, pCK-cHGF, and pCK-dHGF were used as controls. 2 days after transfection, culture supernatants containing HGF protein were obtained and tested by human HGF ELISA (R) according to the manufacturer's recommendations&D Systems, MN, USA) to measure the amount of HGF.

(2) Cell migration assay

The effect of HGF on migration of human umbilical vein endothelial cells (HUVEC, Agiolab co., ltd., AL01-0122S), mouse skeletal muscle myoblasts (C2C12, ATCC No. crl-1772), and rat cardiac myoblasts (H9C2, atccno.crl-1446) was evaluated in a modified Boyden chamber assay. 1% Min in PBSThe inserts of a 24-well transwell cell culture chamber (Corning, NY, US) with a porous polycarbonate filter (pore size 8 μm) were gel coated. HUVEC (n-15) suspended in M199 medium supplemented with 1% FBS, C2C12 (n-10) or H9C2 (n-10) cells suspended in DMEM medium supplemented with 3% FBS, respectively, were cultured at 1 × 10⁴Individual cells/wells are added to the insert. The test substance (supernatant from 293T cells transfected with pCK, pCK-cHGF, pCK-dHGF or pCK-HGF-X7) was diluted to a final HGF concentration of 50ng/ml in M199 or DMEM supplemented with 1% or 3% FBS, respectively, and 600. mu.l of the diluted test substance was placed in the lower chamber. Make the cells in CO₂The incubators were migrated at 37 ℃ for 3 hours, then the inserts were lifted, washed with PBS, fixed with 4% formaldehyde for 10 minutes, and stained with 0.2% crystal violet. Cell migration was quantified by counting cells located on the contralateral side of the insert. Cells from 5 high power fields (. times.200) were counted in each insert. Using Image-Proplus (MediaCybernetics, US) analyzes images.

(3) Cell proliferation assay

Use of³H]Thymidine incorporation assay to evaluate the effect of HGF on HUVEC cell proliferation. HUVEC cells (n-10) suspended in M199 medium supplemented with 1% FBS were cultured at 5 × 10³Individual cells/well were plated in 96-well plates. 10ng of HGF protein (supernatant from 293T cells transfected with pCK, pCK-cHGF, pCK-dHGF or pCK-HGF-X7) was added to the cells. Make the cells in CO₂The cells were grown in an incubator at 37 ℃ for 48 hours. Then, 1. mu. Ci [ alpha ]³H]Thymidine was added to each well and the cells were incubated for a further 16 hours at 37 ℃. Cells were harvested and measured with a liquid scintillation counter (Wallac, Turku, Finland) ("Wallac, Turku")³H]Thymidine incorporation.

2. Results and discussion

To investigate the biological results of the two isoforms of HGF protein, their effect on cell migration and proliferation was investigated. These assays were performed using supernatants from 293T cells transfected with the respective expression vectors, as described in materials and methods. In all experiments, equal amounts of HGF protein were used.

As shown in figures 1, 2 and 3, the presence of two isoforms of HGF produced by pCK-HGF-X7 induced migration of HUVEC, C2C12 and H9C2 cells, respectively, more efficiently than there was only one isoform. The presence of both isoforms of HGF also promoted efficient proliferation of HUVEC cells (fig. 4). The supernatant produced by pCK-HGF-X7 transfected cells induced HUVEC cell proliferation more efficiently than the supernatant produced by pCK-dHGF. These results indicate that the combined action of the two HGF isoforms results in stronger migratory and proliferative activity in endothelial cells and stronger migratory activity in skeletal and cardiac myoblasts.

Vascular endothelial cell migration has a strong correlation with angiogenesis, and the migration of cardiac and skeletal muscle precursor cells is a key step in muscle development and muscle regeneration following injury. Thus, these results indicate that administration of two isoforms of HGF can provide a more effective method for inducing neovascularization and ischemic tissue regeneration. Vascular endothelial cell migration and proliferation are also natural processes associated with the formation of vascular walls. Thus, these results also indicate that administration of both isoforms of HGF promotes vascular wall re-endothelialization.

Example 3: evaluation of HGF efficacy in rat ischemic heart disease model

The objective of this study was to evaluate the cardioprotective effects of intramyocardial injection of HGF in a rat ischemic heart disease model. The experimental procedure is shown in figure 5.

1. Materials and methods

(1) Animal(s) production

Upon arrival, 38 Sprague-Dawley rats (male, 12 weeks old, 350-.

(2) Myocardial infarction model

To analyze the potency of HGF in this study, a rat ischemic heart disease model, one of the widely used CAD pathology models, was used. Rats were anesthetized by intramuscular injection of xylazine (5mg/kg) followed by ketamine (50 mg/kg). After sterilization with 95% alcohol and iodine, the chest was covered with sterile surgical gauze, exposing only the incision area, thus providing completely sterile conditions for the procedure. Endotracheal intubation is performed via the endotracheal intubation approach. During surgery, positive pressure ventilation is maintained. The electrocardiogram and oxygen saturation were continuously monitored. A median thoracotomy (mid thoracotomy) is performed. After opening the pericardium and examining the anterior left ventricular wall, the proximal third of the left anterior descending coronary artery (LAD) was ligated with 6-0 polypropylene suture supported by a small piece of Nelaton (5 Fr). The increase of ST segment on the monitored electrocardiogram was confirmed. 60 minutes after LAD ligation, the perfused rats had ischemic myocardium. Pericardium and thoracotomy wounds were closed. After sufficient spontaneous breathing has been resumed, the single chest tube to which the midwall aspirator (mid wall suction) is attached is removed, and the endotracheal tube is removed. The incision is then examined for the presence of bleeding. After bleeding control, the incised muscles, fascia and skin were sutured. After surgery, gentamicin (3 mg/kg/day) was administered intramuscularly for 3 days to prevent infection. 28 days after the operation, transthoracic echocardiography was performed to confirm that myocardial infarction was induced in the rats.

(3) Efficacy evaluation study design

The efficacy of HGF was tested by injecting the plasmid containing the HGF gene directly into ischemic myocardium and observing cardioprotection both physiologically and anatomically. The plasmid containing the HGF gene was administered immediately after the induction of ischemia (day 0). Each animal was injected intramyocardially with a total dose of 250 μ g of either pCK-cHGF (n-12) or pCK-HGF-X7 (n-10). For negative and positive controls, equal amounts of pCK vector (n-7) without HGF coding sequence and pCK-VEGF165 (n-9) were injected. The improvement in physiological function of the heart was assessed by transthoracic echocardiography on days 1, 14, 28 and 56 after DNA injection. Angiogenesis and anti-fibrosis levels were measured after necropsy.

(4) Cardiac physiology analysis

On day 1, left ventricular ejection fraction and ventricular interval during systole were measured using transthoracic echocardiography. The value obtained on day 1 was set as the baseline value. On days 14, 28 and 56, echocardiograms were measured again. The values obtained on days 14, 28 and 56 were compared between the pCK, pCK-HGF-X7, pCK-cHGF and pCK-VEGF165 groups. In addition, tissue sections were taken from the ischemic heart to analyze changes in capillary density and fibrosis in the left ventricle.

(5) Capillary densitometry

On day 56, myocardial tissue was obtained from the ischemic heart and fixed in 10% formalin solution for 2 days, and then embedded in paraffin. Several serial sections were prepared from each sample. On tissue sections, endothelial cells of capillaries were identified by staining with CD31 antibody. Capillary density was quantified under a 400 Xmagnification microscope and expressed as per 0.15mm²Number of capillary vessels (Image-Pro)plus, Version 4.1, Media Cybernetics, Bethesda, Maryland, USA). The values between the treatment groups are then compared.

(6) Anti-fibrosis assay

On day 56, myocardial tissue was obtained from the ischemic heart and fixed in 10% formalin solution for 2 days, and then embedded in paraffin. Several serial sections were prepared from each sample and subjected to Trichrom staining to assess collagen content. The area of fibrosis in the left ventricle was quantitatively analyzed under an 8 × magnification microscope. The values between the treatment groups are then compared.

(7) Statistics of

Results are expressed as mean ± SEM and analyzed with SPSS (version 10.0, SPSS. inc, Chicago, IL, USA). Statistical analysis of all data was performed using one-way ANOVA followed by LSD test or Tukey test to determine the significance of the difference in multiple comparisons. P values less than 0.05 were considered significant.

2. Results

(1) Induction of left ventricular myocardial infarction in rats

The transthoracic echocardiography was measured 28 days after induction of myocardial infarction to confirm the animal disease model. A significant reduction in the physiological function of the left ventricle was observed following surgically-induced myocardial infarction. Moreover, myocardial fibrosis was observed in the anterior lateral wall of the left ventricle.

(2) Effect of HGF on left ventricular function

Changes in Left Ventricular Ejection Fraction (LVEF) after DNA injection were compared between treatment groups. There were no statistically significant differences in LVEF from group to group on days 1 and 14. However, at 28 days post-myocardial DNA treatment, LVEF values (40.77 ± 2.92%) for the pCK-HGF-X7 treated group were statistically significantly higher compared to either the pCK group (31.24 ± 3.58%, p ═ 0.028) or the pCK-chf group (33.99 ± 2.26%, p ═ 0.069). The LVEF values (39.63 ± 2.44%) for the pCK-VEGF165 group appeared to be higher than either the pCK group (p ═ 0.056) or the pCK-cHGF group (p ═ 0.138), but there was no statistically significant difference. A similar situation was also observed at day 56 after treatment (fig. 6).

Changes in the intersystolic compartment (IVS) after DNA treatment were also compared. Systolic IVS was significantly increased only in the pCK-HGF-X7 treated group; on day 56, IVS was significantly higher for pCK-HGF-X7 treated group than for pCK treated group (p 0.061), pCK-VEGF165 treated group (p 0.012) or pCK-cHGF (p 0.011) (fig. 7).

(3) Effect of HGF on capillary Density

On day 56, the capillary density in myocardial tissue in the ischemic boundary region of the pCK-HGF-X7-treated group was 300.00. + -. 14.71/0.15mm². The density of the capillary vessels is obviously higher than 227.54 +/-6.16/0.15 mm of the pCK group²247.38 + -7.52/0.15 mm of (p < 0.001), pCK-VEGF165 group²231.35 + -5.55/0.15 mm for (p ═ 0.001) or pCK-cHGF groups²(p < 0.001) (FIG. 8).

(4) Effect of HGF on myocardial fibrosis

On day 56, the degree of fibrosis in the left ventricle was 18.88. + -. 1.81% in the pCK-HGF-X7-treated group. This percentage fibrosis was significantly lower than 30.20 ± 2.35% (p ═ 0.009) for the pCK group. The degree of fibrosis (20.96 ± 2.25%) was also significantly lower for the pCK-VEGF165 group than for the pCK group (p ═ 0.049), but the statistical significance of this difference was lower than for the pCK-HGF-X7 group. The degree of fibrosis of the pCK-cHGF group (25.02 ± 2.49%) was not significantly lower than that of the pCK-treated group (p ═ 0.411) (fig. 9).

3. Discussion of the related Art

The therapeutic potential of HGF was evaluated in a rat ischemic heart disease model, which is a well-known CAD animal model. On day 0, cardiac ischemia was surgically created and a total of 250 μ g of plasmid containing HGF gene or control DNA was injected into the ischemic myocardium. HGF effect was assessed by echocardiography and/or histological analysis. The function of the ischemic heart of the pCK-HGF-X7 group was significantly improved. Although pCK-cHGF expresses one isoform of HGF protein, the cardiac function of the pCK-cHGF group did not show any significant improvement.

Example 4: evaluation of the efficacy of pCK-HGF-X7 in human clinical trials

The efficacy of pCK-HGF-X7 was evaluated in human clinical trials. Two subjects who underwent Coronary Artery Bypass Graft (CABG) were injected with 0.5mg of pCK-HGF-X7.

1. Method of producing a composite material

(1) Test subject

Subjects were included in the trial when they met the following criteria: 1) age between 19 and 75 years; 2) reversible perfusion impairment (difference between resting and stress perfusion greater than 7%) was assessed by MIBI-SPECT; 3) it is estimated that areas of incomplete revascularization remain after CABG or areas of myocardial perfusion that are not suitable for CABG.

Subjects were excluded if they had the following medical history or current signs: 1) a malignant tumor; 2) uncontrolled ventricular arrhythmias; 3) late stage heart failure or Killip grading greater than grade II evidence of left ventricular dysfunction and left ventricular ejection fraction < 25% as measured by transthoracic 2D echocardiography; 4) serious infectious diseases; 5) uncontrolled blood disease; 6) valvular heart disease and the need for left ventricular massive resection; 6) proliferative retinopathy; 7) stroke; 8) uncontrolled essential hypertension assessed as JNC grade II; 9) severe liver and kidney disease; or 10) a previous CABG.

The protocol was approved by the institutional review board of seoul national university hospital and the korean food and drug administration.

(2) MIBI-SPECT myocardial perfusion study

3 months and 6 months before and after pCK-HGF-X7 and pCK-HGF-X7 treatment, all patients were rested and after pharmacological stress with adenosine^99mTc-MIBI gated SPECT (Vertex EPIC, ADAC Labs, Calif., USA). SPECT images were constructed by electrocardiographic gating and analyzed using a semi-quantitative 20-segment model and an automated quantification program (AutoQUANT, ADAC Labs, ca., USA).

The 7% difference between resting and stress perfusion fractions at SPECT is the baseline for reversible perfusion defects in myocardial ischemia. Therefore, a difference between resting and stress perfusion of > 7% indicates reversible perfusion defect in the myocardium, and a difference of < 7% indicates normal perfusion in the myocardium.

Resting and stress perfusion scores were obtained from segment 10 and 16 numbers of SPECT concentric circle plots and score differences were assessed 3 and 6 months after injection of pCK-HGF-X7 during the screening period (figure 10).

(3) Intra-myocardial pCK-HGF-X7 injection under CABG

Coronary artery bypass grafting of the left anterior descending coronary artery and circumflex coronary artery was accomplished by standard median sternotomy. 0.5mg of pCK-HGF-X7(0.125mg/0.25 mL/injection; 4 sites/patient) was administered by intramyocardial injection to both sides of the posterior descending artery which, although having reduced perfusion, was not suitable for CABG as assessed by MIBI-SPECT. The number of segments to which pCK-HGF-X7 was administered were segments 10 and 16 of the concentric circle plot obtained by MIBI-SPECT (fig. 11).

2. Results and discussion

(1) Effect of pCK-HGF-X7 on myocardial perfusion under MIBI-SPECT

The difference between resting and stress perfusion fractions at SPECT before and after injection of pCK-HGF-X7 was compared. In the first subject, the average difference between resting and stress perfusion fraction prior to injection of pCK-HGF-X7 was 16%. The mean difference between resting and stress perfusion fractions in the injected area (segment numbers 10 and 16) was 3.5% and 0.5% at 3 and 6 months after injection of pCK-HGF-X7, with significant difference from baseline values. In the second subject, the average difference between resting and stress perfusion fraction prior to injection of pCK-HGF-X7 was 9%. The mean difference between resting and stress perfusion fractions in the injected area (segment numbers 10 and 16) was 4% and 3.5% at 3 and 6 months after injection of pCK-HGF-X7, with significant differences from the baseline value (figure 12).

In summary, intramyocardial injection of pCK-HGF-X7 into disease affected subjects can change the state of myocardial perfusion from reversible defects to normal. These results indicate that administration of pCK-HGF-X7 significantly increased myocardial perfusion.

Example 5: evaluation of the efficacy of pCK-HGF-X7 in the porcine ameroid ischemia model

The objective of this study was to evaluate cardioprotection in a porcine ameroid ischemia model by percutaneous intracardiac injection of pCK-HGF-X7 using a catheter under the guidance of a cardiac imaging system.

1. Materials and methods

(1) Animal(s) production

Upon arrival, yorkshire castrated domestic pigs (n-9, male, 20-40kg) were provided ad libitum with feed and water and allowed to rest for 7 days prior to surgery.

(2) Porcine ameroid ischemia model

The porcine chronic myocardial ischemia model established by ameroid constriction is a clinically relevant and widely accepted preclinical chronic myocardial ischemia model tested for maturation in neoangiogenesis therapy. The model simulates the human coronary anatomy and the degree of vascularization in response to human ischemia. Furthermore, the pig model is a mature model for testing cardiovascular medical devices because its size and anatomy are similar to the human cardiovascular system.

An ameroid constriction device was surgically implanted proximal to the left circumflex of the pig to cause chronic ischemia. By intramuscular injection of Telazol4-6mg/kg and atropine sulfate 0.02-0.05mg/kg to calm the pig. Isoflurane is then administered through a mask to induce general anesthesia for surgical intervention. Pigs were intubated and prepared for surgery as appropriate. The pig is kept in the right side lying position. The hydration is maintained and any arrhythmia is controlled throughout the procedure by intravenous drip of plasma supplement (plasmalyte) and lidocaine. Wash solution (scrub solution) was applied to the sterile surgical site, the breast was covered with sterile drape, and the entire animal was covered with drape. After deep anesthesia, pancuronium bromide was administered intravenously for muscle relaxation. A 20cm incision was made in the left thoracic fifth intercostal space. The ribs and then the lungs were retracted and wrapped with gauze sponge soaked with saline. The pericardium was opened by horizontal incision in close proximity to the phrenic nerve ending and the heart was suspended in the pericardial bed. Circumflex coronary artery (LCX) was dissected a distance of about 0.5cm immediately adjacent to the first limbal branch and placed around it a ameroid constriction of a size appropriate for the artery. All the accessory substances affecting the LCX bed were permanently ligated. If arrhythmia control is desired, a bolus injection of lidocaine is administered. The pericardium is not closed by conventional sutures, but is used as an aid in the positioning of the ameroid over the artery. The muscle layers were closed in a continuous manner using a Chromic gut (PDS II) suture. Subcutaneous use of Dexon^TMAnd (4) weaving the suture. The skin was closed with Surgical staples (Surgical staples). Then, all pigs received enrofloxacin (5.0 mg/kg/day) in the muscle for 3 days to prevent infection.

(3) Efficacy evaluation study design

4 weeks after Ameroid transplantation, pigs were randomized to receive 1mg of pCK-HGF-X7[ lower amount group: 1mg/2ml (n ═ 3)]4mg of pCK-HGF-X7[ high-order group: 4mg/8ml (n ═ 3)]Or a control consisting of the same excipient buffer used with pCK-HGF-X7[ vehicle control 8ml (n ═ 3)]. Using NOGAThe substance was administered into the intracardiac route by a MyoStar delivery catheter (Biosense Webster, USA). Each animal received 8 times (lower amount group: 0.125mg/0.25 ml/injection site) or 16 times (higher amount group: 0.25mg/0.5 ml/injection site) pCK-HGF-X7 or control (vehicle control group: 0.5 ml/injection site) injected into the lateral wall and injection site

In the boundary region between viable myocardium and ischemic myocardium. To evaluate the functional outcome of pCK-HGF-X7 treatment, myocardial perfusion (κ) under peak stress before and after DNA treatment was determined as measured by echocardiography under myocardial contrast stress.

2. Results

Changes in myocardial perfusion under peak stress before and after plasmid DNA injection were compared between treatment groups. In the vehicle control group, day 30 showed a tendency of decreased perfusion compared to day 0. However, both pCK-HGF-X7 groups showed a tendency to maintain perfusion at day 30 compared to day 0 (fig. 13). These results indicate that transcardial (transcardial) transfer of pCK-HGF-X7 prevents a decrease in myocardial perfusion induced by myocardial ischemia.

These results show that HGF can be delivered using a catheter. Percutaneous intracardiac injection catheters have been used in clinical trials to inject various drugs, such as stem cells, adenovirus and naked DNA, intracardiac under the guidance of the electrical cardiac navigation system. These results indicate that pCK-HGF-X7 plasmid DNA can be safely administered into myocardium with perfusion disease by percutaneous intracardiac injection using a catheter under motor guidance. Therefore, a catheter injection system for the transfer of pCK-HGF-X7 into the heart can be used in patients with ischemic heart tissue (e.g., stable, unstable angina or acute, chronic myocardial infarction).

Example 6: delivery of HGF expressing cells

HGF can be delivered in the form of cells comprising pCK-HGF-X7. Mesenchymal stem cells are collected from a subject. The source of mesenchymal stem cells may be bone marrow aspirate or mobilized peripheral blood. The harvested mesenchymal stem cells were cultured and transfected with pCK-HGF-X7 by liposome. The cells were then harvested, washed with saline, resuspended in infusion solution, and infused into the subject. Mesenchymal stem cells transfected with pCK-HGF-X7 can be administered into ischemic and infarcted heart tissue: i) intramuscular injection with a syringe, or ii) percutaneous intracardiac injection with a catheter under motor guidance.

Example 7: scaffold eluted with pCK-HGF-X7

This example demonstrates the preparation of a plasmid eluting stent.

A. Preparation of stainless steel scaffold from which plasmids are eluted

1. Materials and methods

(1) Stainless steel support

Stainless steel holder (SS holder, Libert é)3.0 mm. times.20 mm) from Boston scientific (USA).

(2) Generation of an SS scaffold eluting pCK-HGF-X7

a. Generation of non-Polymer based pCK-HGF-X7 eluting SS scaffolds

To wash the surface of the SS scaffold struts, the scaffolds were sonicated in ethanol 3 times for 3 minutes each (Vibra-cell tm, sonic & Materials inc., Switzland) and dried at 37 ℃ for 30 minutes. The scaffold was then immersed 1 time for 5 minutes in 5mg/ml pCK-HGF-X7 and dried for 30 minutes at 37 ℃.

b. Generation of Polymer-based pCK-HGF-X7 eluting SS scaffolds

To prepare a polymer-based SS scaffold, the scaffold was immersed 1 time in 5mg/ml Phosphatidylcholine (PC) polymer (CM5208, Vertellus specificities inc., UK) in ethanol. The PC polymer-based scaffold was then immersed 1 time 5 minutes in 5mg/ml pCK-HGF-X7 and dried for 20 minutes at 37 ℃.

(3) Quantification of pCK-HGF-X7 eluted from SS scaffolds

To analyze the amount of pCK-HGF-X7 loaded onto the SS scaffold, a large volume of solution (0.8 ml removed from 1 ml) was selected for removal and refilling to quantify pCK-HGF-X7 elution in a rapid and efficient manner.

SS scaffolds coated with pCK-HGF-X7 were immersed in a cryopreservation tube containing 1ml of physiological saline, respectively. The tube with the stent was placed on a roller mixer (HIP-RMF40, Hyunel LAB-MATE, Korea) at 40 rpm for 60 minutes. pCK-HGF-X7 eluates were collected at 1, 5, 10, 20 and 40 min. At each time point, 0.8ml of solution was withdrawn for UV analysis and 0.8ml of fresh physiological saline was added to the vial to maintain the total volume of the solution. The vial was then placed on a roller mixer to continue the experiment. The concentration of the eluate extracted from pCK-HGF-X7 obtained at each time point was measured at 260nm using Ultraspec3000 (Amersham Pharmacia Biotech, Sweden). As a negative control, SS scaffolds without pCK-HGF-X7 were also tested using the above quantification method.

2. Results

The results are shown in tables 1 and 2. Approximately 78 μ g of pCK-HGF-X7 eluted from the non-polymer based SS scaffold at 1 minute, and 115 μ g at 40 minutes. Similarly, approximately 43 μ g of pCK-HGF-X7 eluted from the polymer-based SS scaffold at 60 minutes. These results indicate that the plasmid can be coated on both non-polymer-based and polymer-based SS scaffolds, and that the coated plasmid can be eluted from the SS scaffold. Thus, it was concluded that plasmid-eluting SS scaffolds were successfully prepared.

Table 1: quantification of pCK-HGF-X7 eluted from non-polymer-based SS scaffolds

Table 2: quantification of pCK-HGF-X7 eluted from Polymer-based SS scaffolds

B. Cobalt chromium scaffolds producing eluted plasmids

1. Materials and methods

(1) Cobalt chromium stent

Cobalt chromium stents (Co-Cr stent, ARTHOSPico, 2.75 mm. times.12 mm) were purchased from AMGinterationary (Germany).

(2) Generating Co-Cr scaffolds eluted from pCK-HGF-X7

a. Production of non-Polymer based pCK-HGF-X7 eluting Co-Cr scaffolds

To wash the surface of the SS scaffold struts, the scaffolds were sonicated in ethanol 3 times for 3 minutes each, and dried at 37 ℃ for 30 minutes. The scaffold was then immersed 1 time for 5 minutes in 5mg/ml pCK-HGF-X7 and dried for 30 minutes at 37 ℃.

b. pCK-HGF-X7-eluting Co-Cr scaffolds to produce polymers

To prepare a polymer-based Co-Cr scaffold, the scaffold was immersed 1 time in 5mg/ml Phosphatidylcholine (PC) polymer (CM5208, Vertellus specificities inc., UK) in ethanol. The PC polymer-based scaffold was then immersed 1 time 5 minutes in 5mg/ml pCK-HGF-X7 and dried 10 minutes at 37 ℃.

(3) Quantitative elution of pCK-HGF-X7 from Co-Cr scaffolds

To analyze the amount of pCK-HGF-X7 loaded on the Co-Cr scaffold, removal and re-replenishment of the bulk solution (0.8 ml out of 1 ml) was chosen to quantify the pCK-HGF-X7 eluate in a rapid and efficient manner.

The Co-Cr scaffolds coated with pCK-HGF-X7 were immersed in a freezing tube containing 1ml of physiological saline, respectively. The tube with the stent was placed on a roller mixer (HIP-RMF40, Hyunel LAB-MATE, Korea) at 40 rpm for 60 minutes. The pCK-HGF-X7 eluate was collected at 10, 20, 30, 40, 50 and 60 minutes. At each time point, 0.8ml of solution was withdrawn for UV analysis and 0.8ml of fresh physiological saline was added to the vial to maintain the total volume of the solution. The vial was then placed on a roller mixer to continue the experiment. The concentration of the eluate extracted from pCK-HGF-X7 obtained at each time point was measured at 260nm using Ultraspec 3000. As a negative control, the Co-Cr scaffold without pCK-HGF-X7 was also tested using the above quantification method.

2. Results

The results are shown in tables 3 and 4. Approximately 60 μ g and 85 μ g of pCK-HGF-X7 eluted from the non-polymer based Co-Cr scaffold at 1 minute and 20 minutes, respectively, and similarly approximately 43 μ g of pCK-HGF-X7 eluted from the polymer based Co-Cr scaffold at 60 minutes. These results indicate that the plasmids can be coated on non-polymer-based and polymer-based Co-Cr scaffolds, and that the coated plasmids can be eluted from Co-Cr scaffolds, although the amount of plasmids eluted from Co-Cr scaffolds was less than that of SS scaffolds. Therefore, the conclusion was that plasmid-eluting Co-Cr scaffolds were successfully prepared.

Table 3: quantification of pCK-HGF-X7 eluted from non-polymer based Co-Cr scaffolds

Table 4: quantification of pCK-HGF-X7 eluted from Polymer-based Co-Cr scaffolds

Example 8: efficacy evaluation of HGF-X7 eluting scaffold in rabbit balloon exfoliation model

The objective of this study was to evaluate the accelerating effect of HGF-X7 eluting scaffold on re-endothelialization in a rabbit balloon exfoliation model.

1. Materials and methods

(1) Animal(s) production

Upon arrival, ten new zealand white rabbits (male, 3.5-4.0kg, Doo-Yeol Biotech, Korea) were provided with feed and water ad libitum and allowed to rest for 7 days before stenting.

(2) Rabbit balloon denudation model and stent implantation

Rabbits were anesthetized by intramuscular injection of xylazine (5mg/kg) followed by ketamine (50 mg/kg). After sterilization with 95% alcohol and iodine, the neck was covered with sterile surgical gauze, exposing only the incision area, thus providing completely sterile conditions for the operation. After surgical exposure of the external carotid artery, a 5F guide cannula (Cordis, USA) was inserted into the external carotid artery. After insertion of a 1.4F guidewire (Terumo, Japan) into the femoral artery using standard fluoroscopy, a 2.8F microcatheter was inserted into the proximal portion of the external iliac artery. 1000U heparin and 0.1mg nitroglycerin were administered.

Balloon ablation of the external iliac artery was performed as follows: a 2.5 x 8mm balloon catheter (GoodMan, Japan) was inserted into the external iliac artery over a guidewire and the microcatheter was removed from the rabbit. After balloon catheter inflation (10atm), the external iliac artery endothelium denuded by a distance of about 1.0cm was interrupted by 10 consecutive withdrawals. The balloon catheter for iliac denudation was removed from the rabbit and a new balloon catheter fixed with pCK-HGF-X7 eluting SS stent (PES) or Bare Metal Stent (BMS) was inserted into the denudated external iliac artery. Stent implantation was performed for 15 seconds under balloon inflation at 12 atm. PES (n ═ 10) and BMS (n ═ 10) were implanted bilaterally.

(3) Optical Coherence Tomography (OCT) analysis

For Optical Coherence Tomography (OCT) analysis, a Helios occlusion balloon catheter (LightLab, USA) was inserted through a guidewire into the proximal portion of the external iliac artery, and the guidewire was then removed from the rabbit. An OCT imaging line (LightLab, USA) was then placed at a distance of 1.5cm from the distal end of the implanted stent. OCT images were obtained using a 10ml saline flush. After all devices were removed from the rabbits, the external iliac artery was ligated with 3-0 silk suture. The incision is then examined for the presence of bleeding. After bleeding control, the incised muscles, fascia and skin were sutured. Gentamicin (3 mg/kg/day) was administered intramuscularly for 3 days to prevent infection. Also, 32.5mg of clopidogrel (Sanofi-Aventis, France) and 25mg of aspirin (Bayer, Germany) were administered daily.

(4) Scanning Electron Microscope (SEM)

On days 14 and 28 after stent implantation, animals were sacrificed and vessels harvested and fixed with 2.5% glutaraldehyde solution for 2 hours. The fixed vessels were washed 3 times with Phosphate Buffered Saline (PBS) and 1% OsO₄The solution was post-fixed. The fixed vessels were washed 3 times with PBS and dehydrated continuously with 60% -95% ethanol. Finally, the dehydrated blood vessels were gold plated.

(5) Statistics of

Results are expressed as mean ± SEM and analyzed with SPSS (version 10.0, SPSS inc., Chicago, IL, USA). Statistical analysis of the data was performed using Student's t-test. P values less than 0.05 were considered significant.

2. Results and discussion

To evaluate whether pCK-HGF-X7 eluting scaffolds could accelerate the re-endothelialization process, changes in the size of the inner membrane and the nature of the cells covering the scaffold were examined by OCT and SEM, respectively.

Implant branchOCT images were obtained on days 0, 14 and 28 after the shelf. From each stent, 9 cross-sectional images were obtained. The baseline and subsequent data for OCT are shown in figure 14. The post-interventional results were similar for both groups (FIG. 14A; day 0). However, on day 14 after stent implantation, the cross-sectional area of the intimal dimension of the PES group (ID-CSA, mm) compared to the BMS group²) Significantly increased (fig. 14B; PES vs BMS, 0.23. + -. 0.05mm²Comparison is made at 0.48. + -. 0.09mm²And p is 0.03). ID-CSA similarity between the two groups at day 28 after stenting (FIG. 14B; PES vs BMS, 0.91. + -. 0.08 mm)²Comparison is made at 0.98. + -. 0.09mm²P is 0.76). These results indicate that pCK-HGF-X7 eluting stents promoted the growth of cells on the surface of the stent compared to bare metal stents.

Next, the cell types proliferating on the scaffold were determined by SEM analysis. SEM was performed on days 14 and 28. Fig. 15 shows that endothelial cells (see black arrows) uniformly covered the stent surface in the PES group, while a neointimal mixture consisting of smooth muscle cells (see white arrows) and endothelial cells was observed in the BMS group.

These results indicate that pCK-HGF-X7 eluting stents can accelerate re-endothelialization and are therefore useful tools for treating obstructed blood vessels.

The above results indicate that the presence of two isoforms of HGF (HGF and dHGF) can induce the growth and migration of endothelial cells more efficiently in vitro than HGF or dHGF alone, and that the transfer expression of the nucleotide sequences of the two isoforms of HGF (HGF and dHGF) in vivo can accelerate the re-endothelialization process of blood vessels. These results indicate that two isoforms of HGF can inhibit restenosis more effectively by rapid re-endothelialization activity than one isoform of HGF.

The entire disclosures of all publications (including patents, patent applications, journal articles, laboratory manuals, books, or other documents) cited herein are hereby incorporated by reference.

Claims

1. Use of a composition comprising two or more isoforms of HGF or one or more polynucleotides encoding the isoforms for the manufacture of a medicament for treating or preventing a cardiac disorder in a subject.

2. The use of claim 1, wherein the treatment or prevention of the cardiac disorder is by increasing perfusion or capillary density of cardiac tissue in the subject.

3. The use of claim 2, wherein the cardiac tissue is ischemic cardiac tissue.

4. The use of claim 1, wherein the treatment or prevention of the cardiac disorder is by enhancing endothelial repair at a site of vascular injury or diseased blood vessels in the subject.

5. Use of a composition comprising two or more isoforms of HGF or one or more polynucleotides encoding the isoforms for the preparation of a medicament for promoting endothelial cell growth in a blood vessel.

6. The use of claim 5, wherein the blood vessel is damaged.

7. The use of claim 6, wherein re-endothelialization of said blood vessel is promoted or accelerated.

8. The use of claim 5, wherein the blood vessel is a blood vessel in a subject in need of prevention or treatment of restenosis.

9. The use of any one of claims 1 to 8, wherein said two or more isoforms of HGF are administered as polynucleotides encoding said isoforms.

10. The use of any one of claims 1 to 4, wherein the medicament is administered by injection.

11. Use according to any one of claims 1 to 8, wherein the composition is administered by use of a delivery device.

12. The use of claim 11, wherein the delivery device is a stent.

13. The use according to claim 12, wherein the stent is selected from the group consisting of a non-polymer based stainless steel stent, a non-polymer based cobalt chromium stent and a polymer based cobalt chromium stent.

14. The use of claim 12, wherein the composition is eluted from the scaffold.

15. The use of claim 1, wherein said two or more isoforms of HGF comprise full length HGF (flHGF) and deletion variant HGF (dHGF).

16. The use of claim 15, wherein said two or more isoforms of HGF further comprise NK 1.

17. The use of claim 15, wherein said two or more isoforms of HGF consist of flHGF and dHGF.

18. The use of claim 15, wherein said flHGF and dHGF are each at least 80% identical to the wild-type sequence of human flHGF and human dHGF.

19. The use of claim 18, wherein said flHGF and dHGF are each at least 90% identical to the wild-type sequence of human flHGF and human dHGF.

20. The use of claim 19, wherein said flHGF and dHGF are each at least 95% identical to the wild-type sequence of human flHGF and human dHGF.

21. The use of claim 20, wherein said flHGF and dHGF are human flHGF and human dHGF.

22. The use of claim 15, wherein said flHGF and said dHGF are encoded by separate polynucleotides.

23. The use of claim 15, wherein said flHGF and said dHGF are encoded by the same polynucleotide.

24. The use of claim 1, wherein the one or more polynucleotides are operably linked to a promoter.

25. The use of claim 24, wherein the promoter is a constitutive promoter.

26. The use of claim 1, wherein the one or more polynucleotides are on different vectors.

27. The use of claim 1, wherein the one or more polynucleotides are on the same vector.

28. The use of claim 27, wherein the vector is a plasmid vector.

29. The use of claim 28, wherein the plasmid vector is a pCK vector.

30. The use of claim 27, wherein the vector is a viral vector.

31. The use of claim 15, wherein said flHGF and said dHGF are encoded by a hybrid HGF construct comprising HGF exons 1-18 or degenerates thereof which do not alter the encoded amino acid sequence, and further comprising an intron between exons 4 and 5, wherein said construct does not comprise other introns between exons in addition to said intron between exons 4 and 5.

32. The use of claim 31, wherein the intron is an inherent intron.

33. The use of claim 32, wherein said hybrid HGF construct comprises SEQ id no: 7.

34. The use of claim 31, wherein the intron is a fragment of an inherent intron.

35. The use of claim 34, wherein said hybrid HGF construct comprises SEQ id no: 8. 9 or 10.

36. The use of claim 1, wherein said subject is human and said isoforms of HGF are administered at a dose of about 1 μ g to about 100mg each.

37. The use of claim 1, wherein the subject is a human and the polynucleotides are administered at a dose of about 1 μ g to about 10mg each.

38. A composition for treating or preventing a cardiac disorder in a subject comprising two or more isoforms of HGF or one or more polynucleotides encoding the isoforms as active ingredients.

39. The composition of claim 38, wherein the treating or preventing the cardiac disorder is performed by increasing perfusion of ischemic cardiac tissue in the subject.

40. A composition for promoting endothelial cell growth in a blood vessel of a subject comprising two or more isoforms of HGF or one or more polynucleotides encoding the isoforms as active ingredients.