JP2002515065A - Techniques and compositions for treating heart failure and ventricular reconstitution by in vivo delivery of an angiogenic transgene - Google Patents

Abstract

(57) Abstract: Methods of treating patients with congestive heart failure (including dilated cardiomyopathy and congestive heart failure associated with severe coronary artery disease) and preventing harmful ventricular reconstitution after myocardial infarction Or provide a way to mitigate. A preferred method of the invention relates to the in vivo delivery of a gene encoding an angiogenic protein or peptide to the myocardium by directly injecting the vector containing the gene into the blood vessels filling the heart.

Description

DETAILED DESCRIPTION OF THE INVENTION                In vivo delivery of angiogenic transgene          Techniques and compositions for treating heart failure and ventricular reconstitution                     Presentation on government sponsored research   Certain inventions described herein are subject to grants approved by the National Institutes of Health. Partially supported by the U.S. Government under Kim No.VA-HL0281201 and No.IP50HL53773.01 It was done. The United States government may have certain rights in the invention.                                Field of the invention   The present invention relates to methods and compositions for treating cardiovascular disease. More details In particular, the present invention relates to heart failure and to in vivo delivery of angiogenic transgenes. Techniques and polynucleotide constructs for treating ventricular reconstitution.                                Background of the Invention   In the United States, three to four million adults have congestive heart failure (herein "CH F "); and the incidence of CHF has been reported to be increasing (eg See, for example, Baughman, K., Cardiology Clinics 13: 27-34, 1995). every Years, in hospitals in the United States, CHF is the most frequent non-selective hospitalization, and 500,000 Exclusion diagnosis for the patient. Once symptoms of heart failure become moderately severe, Prognosis is worse than most cancers, where 50% of cancer patients die within two years (Brau nwald, E. (eds.), Heart Disease, W.B.Saunders, Philadelphia, pp. 471-485, 198. 8). Medical treatment may include symptoms of heart failure (eg, edema, exercise intolerance and Shortness of breath) may be relieved first, and may prolong life, Even when used, the prognosis of this disease is severe (eg, Baughman, K., Cardiology Clini cs 13: 27-34, 1995).   CHF is defined as abnormal cardiac function resulting in insufficient cardiac output to meet metabolic demands (Braunwald, E. (eds.), Heart Disease, W.B.Saunders, Philadelphi a, p. 426, 1988). Symptoms include shortness of breath, fatigue, weakness, leg swelling, and exercise intolerance Including gender. Physical examination showed that patients with heart failure had elevated heart and respiratory rates, Indicator of fluid in the lungs), edema, jugular vein dilatation, and generally a tendency to have cardiac hypertrophy There is. The most common cause of CHF is blocking the coronary arteries that supply blood to the heart muscle. Atherosclerosis that occurs. Ultimately, such interruptions can Myocardial infarction (dead myocardium), subsequent loss of heart function and consequent heart failure Can cause. Other causes of CHF include valvular heart disease, high blood pressure, and viral heart Infections, alcohol, and diabetes. Some cases of heart failure It can occur without apparent etiology and is called idiopathic.   Current treatments for CHF include pharmacological treatment, coronary revascularization procedures (Eg, coronary artery bypass surgery and angioplasty), and heart transplants. I can do it. Pharmacological treatment increases cardiac contractility (digitalis and β -By use of cardiotonic agents such as adrenergic receptor agonists), lung And reducing fluid accumulation elsewhere (due to the use of diuretics); and And reduce the activity of the heart (like angiotensin converting enzyme inhibitors) Use of drugs that reduce severe systemic vascular resistance). β-Adrena Phosphoretic receptor antagonists have also been tested. Such pharmacology Drugs can improve symptoms and potentially prolong life, but in most cases prognosis Remains grim.   Some heart failure patients due to associated coronary artery disease, at least temporarily, Benefits from revascularization procedures such as coronary artery bypass surgery and angioplasty Can receive. Such a procedure works because the heart muscle is not dead and due to improper blood flow There are potential benefits if they can be deficient. When normal coronary blood flow returns Viable, dysfunctional myocardium can contract more normally, and cardiac function can be improved You. However, due to inadequate microvascular bed, revascularization is mildly improved Although attention is sometimes paid to the heart function of CHF patients, It is only rare to recover up to   Heart transplantation is an appropriate choice for relatively young patients without other confounding diseases. However, this is only an option for a small number of heart failure patients and is very costly. It takes. In summary, CHF has a very poor prognosis and current treatment It reacts poorly to the law.   Further complicating the physiological conditions involved in CHF are the dysfunctional hearts. There are various natural indications that tend to occur in patients who have. These natural responses are May initially improve cardiac function, but these may eventually exacerbate CHF and combine treatment. A problem arises that can be disturbed and have detrimental effects on survival. Common for CHF There are three such adaptive responses observed: (i) sodium reabsorption Volume retention induced by changes in blood pressure, which causes the plasma volume to expand and Improve output; (ii) increase stroke volume while maintaining relatively normal wall tension Adrenaline affecting the heart (from dilation and hypertrophy); Increased release of norepinephrine from phosphorus-acting nerve endings, Interacts with drainergic receptors to increase heart rate and contractility Tend to increase, which increases cardiac output. However, these three Each of the likely adaptations ultimately tends to fail for various reasons. In particular, Fluid retention tends to produce edema and fluid retained in the lungs that impair respiration There is harm; dilation of the heart is harmful with subsequent severe dilation and increased wall tension Left ventricular remodeling can occur, thus exacerbating CHF. Finally, norepinev Prolonged exposure of the heart to phosphorus causes the heart to respond to adrenergic stimuli. Unresponsive, and this leads to poor prognosis.   Angiogenesis generally refers to the development and differentiation of blood vessels. Typically, "angiogenesis Many proteins, known as proteins, are known to promote angiogenesis It is. Such angiogenic proteins include fibroblast growth factor (FGF) Millie, vascular endothelial growth factor (VEGF) family, platelet-derived growth factor (PDGF) family Family, the insulin-like growth factor (IGF) family and others (below and And those described in more detail in the art). For example, FGF and VEGF family members regulate angiogenesis during growth and development Recognized as a factor. The role of promoting angiogenesis in these adult animals Have recently been tested (described below). FGF and VEGF family pulses Tube forming activity is reasonably well established in the setting of protein injection . For example, a collagen-coated matrix when placed in the peritoneal cavity of an adult rat Acid FGF (“aFGF”) protein produces good vascularization and normal perfusion (Thompson et al., PNAS, 86: 7928-7932, 1989). Coronary artery Basicity in adult dog coronary arteries during sleep at risk during obstruction Injection of FGF ("bFGF") protein has been reportedly Led to decreased dysfunction, smaller myocardial infarction, and increased vascularity (Yanagi sawa-Miwa et al., Science, 257: 1401-1403, 1992). A similar result is the dynamics of myocardial ischemia. Product models have been reported using bFGF protein (Harada et al., J. Clin. Inves t., 94: 623-630, 1994; Unger et al., Am. J. Physiol., 266: H1588-H-1595, 1994). An increase in collateral blood flow was shown in dogs treated with VEGF protein ( Banai et al., Circulation 89: 2183-2189, 1994). This promotes cardiovascular formation The difficulties associated with the potential use of such protein injections include: Sharp position is achieved, and with the proper form of angiogenic effect in the cells of the heart muscle And be present and stay at the concentrations required for uptake and promotion. Another The difficulty is the need for repeated transfusions or infusions of protein.   Some groups include plasmids, retroviruses, and adenoviruses in vivo. Describes gene transfer into myocardium using viruses and other vectors (See, for example, Barr et al., Addendum II, Circulation, 84 (4): Abstract 1673, 1991; Ba rr et al., Gene Therapy, 1: 51-58, 1994; French et al., Circulation, 90 (5): 2414-24. 24 (1994); French et al., Circulation, 90: 1517 Abstract No. 2785 (1994); Giord ano et al., Clin. Res., 42: 123A (1994); Giordano et al., J. Investigative Med., addendum. 2, 43: 287A (1995); Guzman et al., Circulation Research, 73 (6): 1202-1207 (199). 77 (6): 1077-1086 (1995); French et al., Circulation, 90 (5): 2402-2413 (1994). Rowland et al., Am. Thorac. Surg., 60 (3): 721-728 (1995)). general In addition, these reported methods suffer from one or more of the following deficiencies: inappropriate transduction Entry efficiency and transgene expression; significant immune response to the vector used (Including inflammation and tissue necrosis); and importantly, transduction to the heart Relative inability to target transfection and transgene expression (ie, gene transfer is Delivery to non-heart sites (eg, test animal liver, kidney, lung, brain, and testes) Transgene reached). Direct by inserting needle into myocardial wall Inflammatory infiltration in the heart after extensive intramyocardial injection has been demonstrated (French et al., Ci. rculation, 90 (5): 2414-2424 (1994)).   Specific forms of congestive heart failure associated with beta-adrenergic signaling Methods for treating are described in Hammond et al. (Shared US patent application Ser. No. 08 / 708,661 (1). (Filed September 5, 996), which is incorporated herein in its entirety). More shown. This method is useful for reducing beta-adrenergic signaling. Β-adrenergic signaling to the heart of patients with associated heart disease And the delivery of genes encoding tract elements. Myocardial ischemia and peripheral vessels Methods for treating various forms of heart disease such as diseases are described in Hammond et al. (PCT Publication W096 / 2 6742 (published September 6, 1996)).   One form of congestive heart failure is widespread, resulting in the development of chronic or acute heart failure. It is associated with coronary artery disease ("CAD"), which is very severe or sudden. This In such patients, extensive and / or sudden occlusion of one or more coronary arteries Prevents sufficient blood flow to the muscles, causing severe ischemia and, in some cases, myocardial infarction or Causes myocardial death. The resulting myocardial necrosis is followed by a progressive chronic heart Insufficiency or acute hypovolemic conditions (both of which lead to high mortality) There is a direction.   Some forms of congestive heart failure may have an unknown cause or There is a particular problem with treatment, as is incurable. One such example is " Dilated cardiomyopathy "(ie, DCM herein). DCM is the other distinctive mind In the absence of a form of the disease (eg, coronary artery occlusion) or a history of myocardial infarction, For dilated, hypocontractile left and / or right ventricular findings More typically, the condition of the heart is diagnosed. Patients with DCM have severe coronary Even without arterial disease, angina can be experienced. Some of these cases In, the cause of DCM is known or suspected. Examples include familial cardiomyopathy (Eg, progressive muscular dystrophy, myotonic dystrophy, Friedreich Associated with ataxia (Freidrich's ataxia) and hereditary dilated cardiomyopathy ), Infections that cause myocardial inflammation (eg, various viruses, bacteria, and other parasites) Infections), non-infectious inflammation (eg, autoimmune diseases, obstetric cardiomyopathy, hypersensitivity Metabolic disorders that cause myocarditis (nutritional Abnormalities, including endocrine and electrolyte abnormalities), and the toxicity that causes myocarditis Exposure to factors (alcohol, and certain chemotherapeutics and catecholamines) Including). However, in many cases, about 80% have no cause for DCM. It remains known and therefore the condition is referred to as "spontaneous dilated cardiomyopathy". DCM occurrence Treatment of progressive myocardial injury, hemodynamic ineffectiveness associated with dilated heart, and systemic occlusion Causes serious major treatment problems, including placement and the risk of ventricular arrhythmias. In traditional revascularization, dilated myocardium is not a major problem because obstructive coronary artery disease is not a major problem. It is not an option for treating the disease. Even in patients with known or suspected causes of DCM, Wounds are typically not easily reversible. For example, adriamycin-induced cardiotoxin In gender, cardiomyopathy is generally irreversible and more than 60% of affected patients Bring death to others. In most patients with DCM, the cause itself is unknown. You. As a result, there is no general applicable treatment for DCM. Doctor is a tradition Focus on reducing the symptoms that appear in patients with DCM (eg, Reduced fluid retention and / or angiotensin converting enzyme inhibitors Reduce myocardial oxygen and nutritional needs). As a result, about 50 of the patients showing DCM % Within two years of diagnosis, often due to sudden cardiac arrest associated with ventricular arrhythmias And die.   "Ventricular reconstitution" often occurs after a myocardial infarction, resulting in poor ventricular function. After myocardial infarction healing, the heart tends to dilate and reconstitute. This extension or re- Configuration often causes additional ventricular dysfunction. Poor left ventricular function is The reverse outcome following a myocardial infarction is the easiest to predict. Therefore, after myocardial infarction Prevention of ventricular reconstitution is beneficial. One approach to prevention of ventricular remodeling is to Patients with myocardial infarction can be treated with angiotensin converting enzyme (“ACE”) inhibitors (Eg, McDonald, KM, Trans. Assoc. Am. Physici). ans 103: 229-235, 1990; Cohn. J. Clin. Cardiol. 18 (Appendix IV) IV-4-IV-12, See 1995). However, these drugs help prevent diastolic ventricular reconstitution It is only more effective, and new treatments are needed.                                Summary of the Invention   The present invention introduces a transgene encoding an angiogenic protein into the myocardium of a patient. For the treatment of congestive heart failure (dilated cardiomyopathy, as well as Including congestive heart failure associated with severe coronary artery disease), and adverse ventricular reconstitution And methods and compositions for the prevention or alleviation of Various aspects of the invention Includes:   A patient suffering from congestive heart failure, including the step of delivering a vector to the patient's heart A vector for treating a transgene, wherein the vector comprises a vector for transgene expression. Encodes an angiogenic protein or peptide operably linked to a promoter Including transgenes. Such patients with congestive heart failure have dilated Patient with dilated cardiomyopathy. Representative among other patients with congestive heart failure Demonstrating severe myocardial infarction associated with severe or obstructive coronary artery disease Patient. Synthesis of angiogenic proteins in the heart muscle promotes angiogenesis, And increase blood flow. The vector preferably delivers the vector to cardiomyocytes Is introduced into a blood vessel that supplies blood to the heart muscle. Preferably, Bek Is introduced into the lumen of a coronary artery, a saphenous vein graft, or an internal thoracic artery graft; Most preferably, the vector is introduced into the lumen of both the left and right coronary arteries Is done. Intracoronary infusion is preferably relatively deep within the artery, preferably the left coronary artery. It is made at least about 1 cm into the lumen of the pulse and right coronary artery. Intracoronary injection is also Saphenous vein graft and / or internal thoracic artery transfer, which can be done in addition to intracoronary infusion The implant lumen can be made at about 1 cm. The vector is a single injection into each of these vessels Delivery.   Suffered (or may suffer from) myocardial infarction, including delivery of the vector to the patient's heart. A method for preventing or mitigating adverse ventricular remodeling in a patient, the method comprising: Is an angiogenic protein operably linked to a promoter for gene expression. Includes genes encoding proteins or peptides. Angiogenic tamper in myocardium The synthesis of proteins induces angiogenesis, thus increasing blood flow and detrimental ventricular remodeling. Relax configuration.   A preferred embodiment for promoting angiogenesis in the treatment of heart failure according to one of the preceding embodiments. In a preferred method, the vectors are FGF (fibroblast growth factor), VEGF (vascular endothelium). Growth factor), PDGF (platelet-derived growth factor), or IGF (insulin-like growth factor) A small number encoding an angiogenic protein or peptide from the family Contains at least one gene. In certain embodiments, the vector is FGF (FGF-4 , FGF-5, FGF-6, aFGF (also known as FGF-1), and bFGF (also known as FGF-2) Known), but not limited thereto. Like New angiogenic proteins may be naturally secreted or enhance secretion And modified proteins. Of these angiogenic proteins Presently preferred are naturally secreted or modified to enhance secretion Are the forms of FGF-4, FGF-5, FGF-6, FGF-1, and FGF-2. Other embodiments In one embodiment, the gene is VFGF (a member of the VEGF-A family (eg, VEGF-121). , VEGF-145, VEGF-165, VEGF-189, and VEGF-206), members of the VEGF-B family. Bars (eg, VEGF-167 and VEGF-186), and members of the VEGF-C family Or encoding PDGF or IGF.   An angiogenic protein-encoding gene is a mammalian cell (preferably, a cardiomyocyte) Operably linked to a promoter that directs transcription and expression of the gene in You. One currently preferred promoter is the CMV promoter. Other favors In a preferred embodiment, the promoter is a tissue-specific promoter, preferably a cardiac promoter. It is a liver specific promoter, more preferably a ventricular myocyte specific promoter. Preferred examples of ventricular myocyte-specific promoters include ventricular myosin light chain-2 promoter. And the myosin heavy chain promoter. Preferably, the angiogenic factor The encoding gene is also operably linked to the polyadenylation signal.   The present invention treats heart failure (including DCM) or causes adverse ventricular remodeling. In a preferred method of alleviating growth (eg, after myocardial infarction), the vector comprises A viral vector or a lipid-based vector, preferably a viral vector. is there. Vectors include targeting vectors (particularly vectors that preferentially target ventricular myocytes). ). Currently preferred viral vectors include adenovirus (Ad) and Or adeno-associated virus (AAV). Both human and non-human will Preferably, the recombinant viral vector is human. And replication deficiency. If the vector is an adenovirus, the vector is preferably Alternatively, it encodes an FGF (eg, FGF-1, FGF-2, FGF-4, FGF-5, or FGF-6) A polynucleotide having a promoter operably linked to the gene And is replication defective in humans. Currently preferred replication-defective adenovirus vector Have a deletion to remove the E1A and E1B genes, or have E1A, E1B, And a deletion that removes the E4 gene. Preferably about 10⁶~Ten¹⁴Adenovirus Vector particles, more preferably about 10⁸~Ten¹²Vector particles are vascular (preferably Is introduced into the blood vessels that supply the myocardium. Most preferably, the angiogenic protein Adenovirus vector containing the chromosome gene is directly coronary in the lumen of both coronary arteries. It is introduced into the patient's heart by intravenous infusion.   For AAV vectors, the vector is preferably adjacent to the AAV inverted terminal repeat (ITR). Promoter operably linked to a gene encoding an angiogenic protein to be contacted And a polynucleotide having a promoter. Preferably, the AAV vector is human. And replication deficiency. A currently preferred replication-defective AAV vector is one or more AAV replication Has a deletion or capsid forming sequence that affects More preferably, AAVr Both the ep and cap genes are removed (hence the (known in the AAV art) As described above, the AAV vector is replicated and transmitted (typically in packaging cell lines). Must be supplied in a transformer for packaging).   The vector contains a lipid-based vector containing a gene encoding an angiogenic protein. It can be a tar. A variety of such lipid-based vectors are available, And emulsions containing lipids and have been described in the art.                             BRIEF DESCRIPTION OF THE FIGURES   Figure 1. Wall thickening during pacing in a porcine model of congestive heart failure The percentage of conversion is shown in the graph. Percentage of wall thickening advanced heart failure Before pacing (day 0) and every 7 days, the ventricular septum and Evaluation was performed continuously (described in Example 1). Symbols indicate mean values; error bars indicate 1SD Is shown. Two-Way ANOVA (repeat measurement) reduces the percentage of wall thickening Influenced by pacing duration (P <0.001) and area (P = 0.001) It has been shown. Moreover, the pattern of change in wall thickening is between the two areas Was different (P <0.0001). About the percentage of wall thickening at each time point Means were tested by Tukey's method for differences between the two regions after hoc; The P values for these analyzes are shown below the error bars.   FIGS. 2A and 2B show the porcine model of congestive heart failure as described in Example 1. The graph shows the subendocardial blood flow during the pacing period.   Referring to FIG. 2A, subendocardial (inner) blood flow in the ventricular septum and side wall is shown along the X-axis. Were evaluated continuously under the conditions listed above. The number of days is the pacing (0 days, pacing start date; 14, 14 days; 21-28, 21-28 days) ). PACE is obtained with a pacemaker whose blood flow measurement has been activated (+) or stopped (0). Or not. The pacemaker speed was 225 bpm (for numbers See Table 3 herein). Symbols represent mean values; error bars are 1 SD Is shown. Two-way ANOVA (repeated measurement) paces subendocardial blood flow Was affected by the period (P = 0.0001) and region (P = 0.017). Was. In addition, the pattern of change in subendocardial blood flow differs between the two regions. (P <0.006). The mean value for subendocardial blood flow at each time point was Tested for differences between regions by Turkey analysis; for these analyses. Are shown below the error bar.   For FIG. 2B, the subendocardial blood flow per heart beat in the ventricular septum and side wall is shown. , Continuously evaluated under the conditions listed along the X-axis. Symbols and conditions Same as 2A (see Table 4 herein for numerical values). Two-Way AN OVA (repeated measurement) reduces the subendocardial blood flow per heartbeat It was shown to be affected by duration (P <0.0001) and region (P = 0.0198).   FIG. 3A shows that if heart failure progresses, the ventricle will be released before pacing (day 0) and every 7 days. The end-systolic wall load when continuously evaluated at the septum and side wall is shown in the graph. (Described in Example 1). Two-Way ANOVA (repeat measurement) reduces the wall load of contraction Was shown to be affected by the duration of sourcing (P <0.0001). But, The pattern of shrink wall loading was similar in both regions. Page for which measurement has stopped Made using a maker.   FIG. 3B shows a page in a porcine model of congestive heart failure as described in Example 1. The coronary vascular tolerance during the singing period is shown in the graph. Indices of coronary vascular tolerance are listed on the X-axis Under the conditions listed, evaluations were performed continuously in the ventricular septum and lateral wall. Symbols and The conditions are the same as in FIG. Two-Way ANOVA (repeated measurement) Vascular tolerance index affected by pacing period (P = 0.0001) and region (P = 0.013) Was shown to have been affected. Furthermore, the pattern of change in coronary vascular tolerance is There was a difference between the two regions (P = 0.0012). Coronary vascular tolerance at each time point Were tested by Tukey analysis for the difference between the two regions after hoc. Was. This analysis shows that coronary vascular resistance is just as good in the septum immediately after pacing begins. The resistance on the side wall was higher than the resistance (P value was Shown below).   FIG. 4 is useful for gene transfer into cells and heart, as described in the Examples below. 1 shows a schematic diagram of the construction of an exemplary replication-defective recombinant adenovirus vector.   FIG. 5 shows the rescue of the recombinant construction of the transgene-encoded adenovirus. FIG.   6A and 6B illustrate the local contractile function of treated animals as described in Example 5. Shown in the figure. FIG. 6A shows the results of the animals tested two weeks after gene transfer. And FIG. 6B shows the results 12 weeks after gene transfer.   FIGS. 7A, 7B and 7C show a diagram corresponding to a contrast ultrasound cardiac examination of the myocardium. Show grams. White areas indicate contrast enhancement (more blood flow) and are darker The area shows a decrease in blood flow. FIG. 7A shows acute LCx occlusion in normal pigs. FIG. Shows the contrast between IVS and LCx bed 14 days after transfection with lacZ. 3 shows the difference in enhancement. This is two areas of atrial pacing (200 bpm). 5 shows different blood flows in the area. In FIG. 7C, contrast enhancement uses FGF-5. The IVS and LCx beds 14 days after the introgression appeared to be equivalent. this child Indicates that the blood flow was similar in the two regions during the atrial pacing. These results are described in Example 5.   FIG. 8 is an ischemic region divided by peak image intensity in the ventricular septum (IVS). Peak contrast ratio expressed as a percentage of the peak image intensity in the area (LCx floor) Shows the correlation (correlation with blood flow). This indicates that lacZ (control remains) was present in 5 animals. Before and 14 ± 1 before and after gene transfer using FGF-5 Days and 12 weeks after FGF-5 gene transfer, during the period of atrial pacing (200 bpm). Measure from image images using a computer-based image analysis program (Described in Example 5). Blood flow to the ischemic bed is controlled by a gene using a control gene. After transfer, 50% of normal remained, but after gene transfer using FGF-5, 2% of normal Increased more than double (P = 0.0018). This effect lasted for at least 12 weeks.   FIG. 9 shows ischemic activity after gene transfer using FGF-5 and gene transfer using lacZ. The number of vessels as quantified by microscopic analysis in the area and non-ischemic area (Shown in Example 5). FGF-5 gene transfer compared to animals receiving the lacZ gene Each fiber in the ischemic and non-ischemic areas of the animals (P <0.038) The number of surrounding capillaries has increased.   FIGS. 10A, 10B, and 10C show an example of a method for treating myocardium (as described in Example 5). DNA, mRNA, and protein expression after angiogenic transgene gene transfer Derived from a gel that proves the reality. FIG.10D shows retina, liver, or skeletal muscle of treated animals. Gel after PCR amplification to demonstrate that there is no detectable gene transfer into the gel (Described in Example 5).   FIG. 11 shows different angiogenic gene constructs as described in Examples 6 and 7. , FGF-4, FGF-5, and FGF-2L1 +/- sp (i.e., FGF-2L1 plus or minus Wall fertilization achieved by in vivo gene transfer using (secretory signal peptide) Show comparison of thickening   FIG. 12 shows the ischemic area after FGF-4 gene transfer (as indicated by thickening of the wall). 4 shows that the improvement in the functioning of the patient was related to the improvement of regional perfusion.   FIG. 13 shows that FGF-4, FGF-5, or FGF-2L, as described in Examples 6 and 7. 1 +/- sp (= FGF-2L1 plus or minus signal peptide) 3 shows a comparison of perfusion (blood flow).   FIG. 14 shows that FGF-2 plus (FGF-2L1 + sp) or FGF-2 + as described in Example 7. As a result of gene transfer using L1 minus secretion signal peptide (FGF-2L1-sp) 3 shows a comparison of the thickening of the wall of FIG.                       Detailed Description of the Preferred Embodiment Definition   "Heart failure" is a condition in which the heart does not provide adequate blood flow to meet metabolic demands Clinically defined as Symptoms include shortness of breath, fatigue, frailty, leg swelling, and And exercise intolerance. On physical examination, patients with heart failure Increased number and respiratory rate, rattle (an indicator of fluid in the lungs), edema, jugular vein dilation, And generally has a tendency to have cardiac hypertrophy. High mortality in patients with heart failure Typically, 50% of patients die within 2 years of developing this condition. In some cases, heart failure is associated with severe coronary artery disease (`` CAD ''). Typically, as described herein and in the art, myocardial infarction and This results in progressive chronic heart failure or an acute low cardiac output state. In other cases, Heart failure is not associated with severe coronary artery disease and has spread (as described below). Associated with dilated cardiomyopathy.   "Dilated cardiomyopathy" (or DCM as used herein) refers to coronary artery occlusion Or in the absence of other characteristic forms of heart disease, such as a history of myocardial infarction Typically diagnosed by dilated low-systolic left and / or right ventricular findings This is a type of heart failure that is severed. DCM is associated with poor ventricular function and heart failure Related to the shape. In these patients, room expansion and thin walls are common This results in high wall tension of the left ventricle. No typical obstructive coronary artery disease Nevertheless, these patients often have complaints of angina. Other symptoms These include shortness of breath, fatigue, weakness, leg swelling, and exercise intolerance. Many Patients exhibit these symptoms even during mild exertion or at rest, And characterized as severe (i.e., type III or type IV heart failure, respectively) . On physical examination, patients with DCM have increased heart rate and respiratory rate, Has sound (indicator of fluid in the lungs), edema, jugular vein dilation, and generally cardiac hypertrophy Be inclined There is a direction. Ultrasound cardiography is typically used in diagnosing DCM. DCM Patients with angina experience angina despite having no severe coronary artery disease obtain. In some cases, the cause of DCM is known or Suspected. Examples include familial cardiomyopathy (e.g., progressive muscular dystrophy, muscle tone). Dystrophy, Friedreich's ataxia, and hereditary dilated cardiomyopathy Linked cardiomyopathy), infections that produce myocardial inflammation, non-infectious inflammation (e.g., autoimmune diseases, Inflammation due to humiliating cardiomyopathy, hypersensitivity reaction, or graft rejection), metabolic disorders Harm (including nutritional, endocrine, and electrolyte abnormalities), and exposure to toxic drugs ( (E.g., alcohols, chemotherapeutic agents, and certain catecholamines). You. In most cases (about 80%), the cause of DCM is still unknown This condition is called "spontaneous dilated cardiomyopathy".   As used herein, the terms "have therapeutic effect" and "successfully "Treatment" has essentially the same meaning. In particular, (Severe CAD or DCM related Patients suffering from heart failure are identified as having an angiogenic condition according to the method of the invention. Reduction or elimination of one or more of the above symptoms after receiving a child transgene If it is shown and / or detected, Be treated. Reduction of these signs or symptoms may also be felt by the patient. Follow Indicators of successful treatment of such heart failure conditions include angina, fatigue, frailty Shortness of breath, leg swelling, rattle, heart rate or respiratory rate, edema, or jugular vein dilation Patients show and feel a reduction in any of the symptoms of Patients also have better exercise tolerance, with improved ventricular and cardiac function Required visits with a smaller heart and generally related to cardiac status Is reduced. Preferably, the improvement in cardiac function meets the metabolic need of the patient Is a requirement, and the patient does not show symptoms during mild exertion or at rest . Many of these signs and symptoms are tolerated by routine procedures familiar to physicians. It is easily visible and / or easily measurable. Heart (especially previous ischemic territory) One measure of improved cardiac function with increased blood flow in the But also. As described below, blood flow in the patient was measured using thallium imaging (Bra unwald, Heart Disease, 4th ed., pages 276-311 (Saunders, Philadelphia, 1992) Or by echocardiography (Examples 1 and 5, and Sahn, D. J. et al., Circulation. 58: 1072-1083, 1978). You. Blood flow before and after angiogenic gene transfer can be compared using these methods. Improvement in cardiac function is associated with reduced signs and symptoms, as described above. Ultrasound heart In addition to testing, the ejection fraction (LV) is measured by nuclear (non-invasive) techniques as is known in the art. Can be determined.   "Ventricular remodeling" often occurs after a myocardial infarction and typically includes the following: It is characterized as follows. After healing of myocardial infarction, between healed infarcted tissue and normal tissue Persistence of ischemia and other factors in the borderline region of the And / or lead to reconstitution (a condition previously referred to as infarct expansion). The expansion of the whole heart Occurs in about 50% of patients with such dilation. This extension, or reconstruction, Can cause further impairment of ventricular function. Reconstitution is usually 1-2 weeks after infarction Develops within months after myocardial infarction, although it can occur as quickly .   `` Adverse left ventricular reconstruction '' (or shortly `` adverse reconstruction '') refers to a chamber following a myocardial infarction. Expansion. This can progress to severe heart failure. The treatment method described in this specification By using the law, such harmful reconstitution can be prevented or at least It is expected that this can be substantially alleviated. That ventricular reconstitution had already begun Even so, it is still desirable to induce angiogenesis. Because of the development of the microvascular bed Increased raw and blood flow may still be effective to offset ventricular dysfunction This is because that. In patients with myocardial infarction, adverse ventricular remodeling If there is no dilatation of the chamber and symptoms of heart failure (as described in Heart Failure above) "Prevented" if not ill. Harmful ventricular remodeling can lead to existing heart failure symptoms In the absence of any observable or measurable reduction You. For example, patients may have reduced shortness of breath and exhibit improved exercise tolerance. Heart machine Methods for assessing improvement in function and reduction of symptoms are essentially the same for DCM. Similar to the method described above. As a result of improved collateral blood flow and ventricular function Prevention or amelioration of adverse ventricular remodeling may occur in patients using the methods described herein. Can be achieved within weeks after angiogenic gene transfer in vivo in is expected.   "Polynucleotide" refers to a ribonucleotide or deoxyribonucleotide. Of any length or nucleotides of any of their analogs -Refers to the form. This term refers to the primary structure of the molecule, and therefore double- and single-stranded. Includes DNA and double and single stranded RNA. Also methylated and / or Or modified polynucleotides such as capped polynucleotides.   "Recombinant" as applied to a polynucleotide means that the polynucleotide Various combinations of ringing, restriction and / or ligation steps, and found in nature Be the product of another procedure that produces a construct that is distinct from the polynucleotide means.   "Gene" or "transgene" includes a sequence that encodes a protein A polynucleotide or a portion of a polynucleotide. In most cases, A gene is operably linked to a coding sequence in order to effectively promote transcription. It is also desirable to include a motor. As is well known in the art, enhancers , Repressors, and other regulatory sequences also regulate the activity of the gene. (See, for example, the references cited below).   The terms "polypeptide", "peptide" and "protein" refer to amino acids of any length. It is used interchangeably to refer to a no-acid polymer. These terms also refer to glycos Proteins that are post-translationally modified by reactions involving acetylation, acetylation, and phosphorylation Including quality.   "Angiogenic peptide or protein" refers to angiogenic or angiogenic activity, That is, it refers to any protein or peptide that promotes vascular development. Vascular form As a blood vessel that develops in adulthood, it has a minimum vessel inner diameter of about 8 microns in diameter. Certain capillaries and larger inner diameter vessels having at least about 10 microns in diameter Is mentioned. Angiogenic activity is known in the art or as described herein. Measure blood flow, determine cardiac function or increase the presence of blood vessels using standard procedures. Can be determined. For example, the number or density of capillaries can be Can be quantified by microscopic analysis of the tissue site (see Example 5). Myocardial blood Flow is Roth, DM, et al., Am. J. Physiol. 253: H1279-H1288, 1987 or Roth, DM, et al., Ci. rculation 82: 1778-1789, 1990, radioactive microsphere technology Can be determined by Myocardial blood flow can also be measured, for example, by Braunwald in Heart Disease , 4th edition, pages 276-311 (Saunders, Philadelphia, 1992) Can be quantified by thallium imaging, including perfusion of the heart with. In the heart Cells have avidity for thallium. Thallium uptake, blood Positively correlates with flow. Thus, reduced uptake is an ischemic condition in which perfusion deficiency is present Shows a decrease in blood flow as occurs in In conscious individuals, angiogenic activity is Examples 1 and 5, and Sahn, DJ, et al., Circulation. 58: 1072-1083, 1978 It can be easily evaluated by contrast echocardiography as described above. Myocardial function Improvement in wall thickness can be measured by wall thickening, for example, as measured by transthoracic echocardiography. Can be determined. Angiogenesis was compared to that before transgene treatment. Increase in angiogenic activity in patient heart after receiving adult transgene If it can be measured, it is "accelerated." Active peptides include angiogenic peptides And peptide precursors that are post-translationally processed. Preferably vascular The forming protein or peptide is a member of the fibroblast growth factor (FGF) family (aFGF (F GF-1), bFGF (also known as FGF-2), FGF-4, FGF-5, and FGF-6 (Including but not limited to), the vascular endothelial cell growth factor (VEGF) family Members (VEGF-A family members (e.g., VEGF-121, VEGF-145, VEGF -165, VEGF-189, and VEGF-206), members of the VEGF-B family (e.g., VEGF -167, and VEGF-186), including but not limited to members of the VEGF-C family. Not); members of the platelet-derived growth factor (PDGF) family (eg, PDGF A or Is a member of the PDGF B), or insulin-like growth factor (IGF) family. However, for example, any that exhibits angiogenic activity that can be measured by the methods described above. A gene encoding a protein or peptide of the present invention is useful in the method of the present invention. It seems to be. Angiogenic peptides or proteins also include FGF, VEGF, PDG "Derivatives" and "functional equivalents" of angiogenic proteins such as F, and IGF including. Derivatives of such an angiogenic protein or peptide are similar amino acids. One or more of an angiogenic protein or peptide having a noic acid sequence and associated It is a peptide that retains the activity of the compound to some extent. Useful derivatives are generally In a region or domain of a protein that is associated with Has substantial sequence similarity. “Sequence similarity” refers to the origin of the peptide and Regardless of the amino acid sequence in two different proteins or peptides Refers to the similarity found between As is well known in the art, substituted amino The acid has a relatively similar (e.g., size and charge) There are many amino acid substitutions that are "conservative". Substantial sequence similarity is For linked proteins or peptides or subunits, typically At least 30% sequence identity (typically measured over stretches of 10 or more amino acids) ), Preferably 70%, more preferably 90%, and even more preferably Or 95% sequence similarity. As is known in the art, conservative additions or omissions Losses generally do not appear to be involved in activity when promoting angiogenesis. It may be located in a protein region or domain. "Functional equivalent" Or replace one or more activities of a particular angiogenic protein or peptide A protein or peptide having activity is meant. The preferred functional equivalent is Retains all of the activity of a particular angiogenic protein or peptide; however, Functional equivalents, when measured quantitatively, are measured in angiogenic assays Has stronger or weaker activity than the wild-type protein or peptide I can do it. Preferred functional equivalents are related angiogenic proteins or peptides. Alternatively, it may have an activity within 1% to 10,000% of the activity of the subunit, preferably 10% 10001000%, and more preferably within 50% -200%. As known in the art In addition, various techniques (including computer modeling, for example) Can be used to assess the potential impact of Vascular using the in vitro and / or in vivo assays described herein One can easily screen for retention of formation potential.   A "heterologous" component is introduced into an entity that is different from the naturally located entity. Or the components produced therein. For example, they are from one organism and Polynucleotides introduced into living organisms by genetic engineering techniques are heterologous polynucleotides. Otide, and may encode a heterologous polypeptide when expressed. Similarly, heaven A program that has been removed from the original coding sequence and is operably linked to another coding sequence. A promoter or enhancer is a heterologous promoter or enhancer. You.   As used herein, "promoter" refers to an operably linked gene. Or a polynucleotide sequence that controls the transcription of a coding sequence. Various different Numerous promoters from various sources (constitutive, inducible, and repressible promoters) ) Are well known in the art (identified in databases such as GenBank). Available) or as a cloned polynucleotide sequence. (Eg, from a depository such as the ATCC and other commercial or personal sources).   As used herein, an “enhancer” is an operably linked gene or gene. Refers to a polynucleotide sequence that enhances transcription of a coding sequence. Various different origins Numerous enhancers from sources are well known in the art (databases such as GenBank). Identified as a cloned polynucleotide sequence or Are available within a range (eg, depository institutions such as the ATCC and other commercial or From human sources). Includes promoter sequences (eg, a conventional CMV promoter) Many polynucleotides also include an enhancer sequence.   "Operably linked" means that each component so described is intended to Refers to the juxtaposition of two or more components that have a relationship capable of acting as described above. promoter Regulates the transcription of a gene or coding sequence. Operably linked. An operably linked promoter is usually a coding sequence. Located upstream of the row, but not necessarily adjacent to it. Enhancer Is operably linked to a coding sequence if it increases transcription of the coding sequence. . Operably linked enhancers are upstream, internal, or downstream of the coding sequence. Located in. Polyadenylation sequences allow transcription from the coding sequence to the polyadenylation sequence. If it is located at the downstream end of the coding sequence, Linked to Noh.   A “replicon” allows the polynucleotide to replicate in a suitable host cell Refers to a polynucleotide containing a replication origin. Examples include heterologous nucleic acids Target cell replicons (eg, nuclear and mitochondrial chromosomes) And extrachromosomal replicons (eg, replicating plasmids and episomes) included.   As used herein, “gene delivery”, “gene transfer” and the like are used for introduction. Regardless of the method used, exogenous polynucleotides into the host cell (sometimes referred to as (Also called "transgene"). Such methods include: Vector-mediated gene transfer (eg, viral infection / transfection, or Various other protein-based or lipid-based gene delivery complexes And techniques that facilitate the delivery of “naked” polynucleotides (eg, Various others used for perforation, "gene gun" delivery, and introduction of polynucleotides And various well-known techniques such as The introduced polynucleotide is Can be maintained stably or transiently in the host cell. For stable maintenance, typically The introduced polynucleotide contains an origin of replication compatible with the host cell, or is stained In vitro replicon (eg, plasmid) or nuclear or mitochondrial staining Either integration into the host cell's replicon, such as the body, is necessary. The As is known in the art and described herein, a number of vectors are It is known that it can mediate the transfer of a gene into a mammalian cell.   As used herein, "in vivo" gene delivery, gene transfer, gene therapy, etc. A vector containing an exogenous polynucleotide can be transferred to an organism (eg, human or human). Mammal), exogenous polynucleotides are introduced directly into the body of the mammal. The term refers to being introduced into cells of such an organism in vivo.   "Vector" (sometimes called a "vehicle" for gene delivery or transfer) Is a polynucleotide delivered to a host cell whether in vitro or in vivo. Refers to macromolecules or molecular complexes containing nucleotides. Polynucleotides delivered The tide may include a coding sequence of interest in gene therapy. For example, vectors For example, viral vectors (eg, adenovirus (“Ad”), adeno-associated virus (AAV), and retroviruses), liposomes and other lipid-containing complexes, And other macromolecular complexes that can mediate delivery of the polynucleotide to the host cell Coalescing is included. Vectors further regulate gene delivery and / or gene expression. Or other components that provide advantageous properties to the targeted cell. Or a function. Such other components include, for example, cells that bind or target Components that affect cell transformation (components that mediate cell-type or tissue-specific binding) Components that affect the uptake of vector nucleic acids by the cell; Components that affect the localization of polynucleotides in cells after Mediator); and components that affect polynucleotide expression . Such a component may also be a marker (eg, a nucleic acid delivered by the vector). Detectable cells that can be used for detection or selection of uptake and expressing cells And / or selectable markers). Such a component is (Eg, components that mediate binding and uptake) The use of certain viral vectors having specific or functional properties) or vectors Can be modified to provide such functionality. Very variety of such vectors Are well known in the art and are generally available (e.g., as described herein). See various references cited).   A “recombinant viral vector” is a virus containing one or more heterologous genes or sequences. Ruth vector. Many viral vectors have large packaging-related To indicate size limitations, heterologous genes or sequences are typically It is introduced by replacing one or more locations in the system. Such viruses replicate Because it can be defective, it is submitted in trans during viral replication and capsid formation. Requires the deletion function provided (eg, for replication and / or encapsidation) Use a helper virus or packaging cell line that has the necessary genes (See, for example, the following literature and description). Polynucus to be delivered Modified viral vectors in which leotide is carried outside of the viral particle have also been described. (See, for example, Curiel, DT et al., PNAS: 88: 8850-8854, 1991).   As used herein, viral "packaging" refers to the sum of viral vectors. Refers to a series of intracellular events that result in formation and assembly. Packaging is typically Is called a “proviral genome”, or a “vector plasmid”. Provectors (typically flanked by appropriate viral "packaging sequences") As a result, recombinant plasmids that can be packaged in a manner similar to the viral genome Replication), followed by encapsidation of the nucleic acid or another Include cover. Therefore, vector plasmids can be packaged under appropriate conditions. When incorporated into a cell line, they can replicate and assemble into viral particles. Many The "rep" and "cap" of the virus found in the virus genome code for replication, respectively. And a gene encoding a capsid forming protein. "Replication "Defective" or "non-replicable" viral vectors can be replicated and / or packaged. Deletion or alteration of one or more functions that require A virus that is unable to initiate viral replication after being taken up by cells Refers to a vector. Producing stocks of such replication-defective viral vectors In order for the viral or proviral nucleic acid to be delivered in trans, Introduced into a “packaging cell line” that has been modified to contain the encoding gene obtain. For example, such packaging genes are It can be stably incorporated into the pre-con. Alternatively, they can be Using a "rasmid" or a helper virus having a gene encoding a deletion function Transfection.   The “detectable marker gene” specifically detects cells having the gene ( For example, genetics that allow to be distinguished from cells that do not have a marker gene) I am a child. Many such marker genes are known in the art. these As a preferred example, a detectable marker encoding a protein that appears on the cell surface Gene, which results in simple and rapid detection and / or cell selection. Another is promoted. For example, the lacZ gene encoding β-galactosidase was detected It can be used as a possible marker. As a result, as described below, Transduction is carried out using a vector having the lacZ gene detected by staining.   “Selectable marker gene” means that a cell having the gene Can be specifically selected for or against its existence in the presence It is a gene to make. By way of example, an antibiotic resistance gene may Positive selectable markers that can be positively selected in the presence of different antibiotics Can be used as a gene. Selectable markers can be positive, negative or Is bifunctional. Positive selectable markers are those of cells that carry the marker. Enable selection. On the other hand, a negative selectable marker has a marker Allows cells to be selectively removed. A variety of such marker genes Described and contains bifunctional (ie positive / negative) markers (For example, International Publication WO 92/08796, published on May 29, 1992, and international publication No. WO 92/28143, published Dec. 8, 1994), which is incorporated herein by reference. You. Such marker genes may be advantageous in gene therapy settings. An additional control indicator may be provided.   "Vascular" or "vascular" carries blood (and lymph) in the mammalian body A term that refers to the vascular system.   "Vascular" includes arteries, arterioles, capillaries, venules, veins, sinuses, and vascular vessels. Any blood vessel of the mammalian vasculature. In a preferred aspect of the present invention, the vascular form The vector containing the adult transgene is directed directly into the vascular conduit that supplies blood to the heart muscle. Is entered. Such vascular conduits can be coronary and saphenous or internal thoracic artery transfer. Includes vessels such as implants.   "Artery" refers to a blood vessel from which blood exits the heart. The coronary arteries The other arteries supply the rest of the body, while supplying the tissues. artery The general structure consists of a lumen surrounded by multiple layers of arterial walls.   An "individual" or "patient" is a mammal, preferably a large mammal, most preferably Or human.   As used herein, "treatment" or "therapy" refers to an individual patient, in an individual. It refers to the administration of an agent that can elicit a prophylactic, therapeutic or other beneficial effect.   As used herein, “gene therapy” refers to a vector that contains a therapeutic gene for an individual patient. Administration. A "therapeutic polynucleotide" or "therapeutic gene" is an individual when transferred to an individual. Nucleotide sequences that can cause a prophylactic, therapeutic or other beneficial effect in the body Say.   The references cited in the above sections are those references used in the practice of the present invention. To the extent that it teaches certain techniques, it is incorporated herein by reference.Incorporated as reference   References cited in this application, including patents, published patent applications, and other publications, The literature is hereby incorporated by reference.Detailed description of various preferred embodiments   Various preferred aspects of the present invention are summarized below and further detailed below. It is described and illustrated in the description and drawings.   The present invention relates to dilated cardiomyopathy ("DCM") and coronary artery disease ("CAD") and To treat congestive heart failure, including severe heart failure associated with myocardial infarction, and By introducing a transgene encoding an angiogenic protein into the patient's myocardium Accordingly, the present invention relates to methods and compositions for preventing or alleviating malignant ventricular remodeling.   In a method of the present invention for treating congestive heart failure, an angiogenic protein is provided. Alternatively, the vector construct containing the gene encoding the peptide is Where the synthesis of myocardial exogenous gene angiogenic proteins is Promotes formation, and thus improves blood flow through the newly formed blood vessels, Dysfunction recovers. Improved heart function will ultimately reduce symptoms of heart failure or To disappear and prolong life beyond predicted mortality within 2 years of ongoing severe CHF Lead.   A preferred method is localization of angiogenic proteins, which is relatively restricted to the patient's myocardium A vector construct and / or delivery method that results in the expression to be expressed is used. Vessel Examples of forming proteins include FGF families (FGF-1, FGF-2, FGF-3, FGF-4, FGF -5 and members of FGF-6, more preferably FGF-2, FGF-4 and FGF-5); A member of the vascular endothelial growth factor (VEGF) family (this is the VEGF-A family (eg, For example, VEGF-121, VEGF-145, VEGF-165, VEGF-189 and VEGF-20 6), members of the VEGF-B family (eg, VEGF-167 and VEG F-186), and members of the VEGF-C family, including but not limited to Platelet-derived growth factor (PDGF) family; and insulin-like growth factor (IGF) family; Including members of the family.   Preferred vectors for use in the present invention include viral vectors, lipid-based vectors. And other vectors that can deliver DNA to non-dividing cells in vivo. It is. Viral vectors, particularly replication-defective virus vectors (eg, replication-defective Adenovirus (Ad) and adeno-associated virus (AAV) vectors Is currently preferred. Replication deficiency is required for easy production and use in the present invention. Adenovirus vectors are currently most preferred.   Currently preferred means for in vivo delivery, particularly delivery and delivery restricted to the myocardium And / or for vector constructs not otherwise labeled for expression) Injection of the vector into blood vessels that can directly supply the myocardium, preferably into the coronary arteries By injection. Such injections are preferably made in one or both sides of the lumen of the coronary artery. Or delivering blood to one or more saphenous vein or internal thoracic artery grafts or myocardium A catheter introduced quite deeply (typically at least about 1 cm) into another conduit Achieved using tell. Injections that are not introduced deep enough into the coronary artery May result in a systemic distribution of the arteries.   Stock viral vector stock (preferably, free of wild-type virus) Preferred for unilateral or bilateral coronary lumens (or grafts and other blood vessels) Is in both the left and right coronary arteries (or grafts and other blood vessels), and preferably 10 as determined by optical density measurement⁶~Ten¹⁴The amount of virus particles (more Preferably, 10⁸~Ten¹²Affected by deep injections of viral particles) Gene that encodes a protein that promotes angiogenesis in Number Of cells (particularly cardiomyocytes) can be transfected locally. This maximizes the therapeutic efficacy of gene transfer and favors extracardiac sites. Minimizes unwanted effects and possible inflammatory responses to viral proteins You. Specific to the myocardium, such as vectors that integrate myocardial specific binding or uptake components Targeted and / or myocardial-specific transcriptional regulatory sequences (eg, ventricular myocyte-specific Vector incorporating an angiogenic protein transgene under the control of a heterologous promoter) As a means for constructs to further restrict expression to cardiac muscle (particularly ventricular myocytes) It can be used instead of, or preferably with, such directional injection techniques. For vectors capable of eliciting an immune response, as described above, the vascular Direct injection of the receptor is preferred, but limits the potential for expression outside the heart Additional techniques for can also be used.   Patients can be administered an angiogenic gene treatment at any time in the case of CHF, More preferably, within about one to four weeks after the occurrence of myocardial infarction. Dosing is a single (one time) crown Preferably via intra-arterial injection (eg, to each coronary artery) but more than one injection May be needed from time to time.   As described in detail below, we have identified angiogenic protein transgenes. The use of such a technique with a vector having an offspring is And effectively show no observed effects on non-cardiac tissue Function in the myocardium of a large mammalian heart without producing an inflammatory response It was shown that it could be enhanced. Inflammatory cell wetting, cell necrosis or increased fibrosis No deviation was detected. Myocardial blood flow improved 2 weeks after angiogenic gene transfer Thus, ischemia was alleviated, and thus myocardial function was enhanced. These beneficial effects are small It lasted for at least 12 weeks. By way of example, we have used a single intracoronary injection ( About 10¹¹Vector particles), which are pulsed to produce therapeutic benefits without toxicity. It has been observed to provide proper expression of tube forming proteins. For enhancing myocardial blood flow Thus, our analysis shows that high yields of more than 60% of cardiomyocytes in vivo It has been shown that mass transduction can be achieved using this procedure.   Adenovirus-mediated gene transfer generally involves stable transfer of the transgene into the host genome. No incorporation occurs. Therefore (vectors have been shown to exist for up to 5 months However, transgene expression is relatively transient. However, because of the heart condition mentioned above In the treatment method of the present invention, the transient expression of the angiogenic transgene Appropriate to achieve the above goals. In fact, for the purposes of the present invention, Presently, they are generally more desirable than stable incorporation in terms of safety. Because the inn This is because destruction of the main genome can be avoided. As the experimental example proved below, Transgene expression can enable simultaneous recovery of collateral vessel development and normal cardiac function It was maintained long enough. Thus, the angiogenic factor gene produces a therapeutic effect. Need not be present in transfected cells for more than a few weeks to . Once new blood vessels are formed, the subsequent expression of exogenous angiogenic proteins May not be needed to maintain new vasculature and increased blood flow .   Thus, the technology of in vivo angiogenic transgene delivery provided by the present invention Effectively avoids the need for multiple injections or infusions of angiogenic proteins. The local transduction and transgene expression achieved using the treatment methods of the present invention are still Also, avoid the potentially deleterious effects of angiogenesis in non-myocardial tissue such as the retina.   Another aspect of the invention is to prevent or alleviate ventricular remodeling in a patient after myocardial infarction. This is the method. This method is used to promote angiogenesis to the patient's heart so as to promote angiogenesis. In vivo integration of vector constructs containing a gene encoding a synthetic protein or peptide Includes targeting in body delivery. Augmented blood vessels achieved using the method of the invention Distribution and blood flow tend to reduce ischemia, and therefore adverse ventricular remodeling Slow progress to progress. To correct these disease states, the patient preferably Immediately after the infarction occurs, preferably within one month after the infarction and even more preferably Or within one to three weeks of infarction. The present invention also provides a It is used in patients who are likely to present infarcts such as Unless otherwise indicated Targeted genetics in the heart of patients to prevent and alleviate ventricular remodeling Techniques and vector compositions to affect child transfer are essentially the same as in treating DCM. The same. Animal model   Key requirements for successful research in gene therapy are (a) clinical depression Applicable to ischemic heart failure, and provides information about angiogenesis in the setting of CHF. The composition of animal models that can provide useful data on canism, and (b) It is an accurate assessment of the effect of gene transfer. In the present invention, the pig model Meet the required conditions. Pigs have been linked to human heart disease due to their relevance to human physiology. Is a particularly suitable model for studying. Pig hearts can be infected in the following manner. Resembles a heart. Pigs are human, including the absence of native coronary collateral vessels. It has a native coronary circulation very similar to that of. Second, the whole The size of the pig heart as a percentage of weight is similar to that of the human heart. further Pigs are large animal models. Therefore, effective vector dosage, toxicity, etc. It allows more accurate extrapolation for such various parameters. In contrast, Animal hearts, such as members of the nu and mouse families, have many endogenous collaterals. Has blood vessels. Compared to total body weight, the size of a dog heart is two times the size of a human heart. It is twice.   Two pig models were used in the present invention. Described herein in Example 1. The first model performed mimics clinical congestive heart failure. In this model Continuous and rapid ventricular pacing over a period of three to four weeks Similar to many features, including DCM and heart failure associated with severe coronary artery disease Induces sexual heart failure. Such pacing is similar to that seen in heart failure , Local functional abnormalities, including severe coronary artery disease (CAD) or abnormalities associated with DCM ) Produces left ventricular hypertrophy with impairment of systolic function similar to that of (d). Therefore, this model Delivery of a vector construct encoding an angiogenic peptide or protein, Determine if it is effective to alleviate cardiac dysfunction associated with these conditions Can be used to This model is useful for treating congestive heart failure and ventricular remodeling. Provide several parameters to evaluate the efficacy of in vivo gene therapy for It is particularly useful in providing.   A second model (a model of myocardial ischemia) is described herein in Example 5. You. Using this second model, the gene encoding the angiogenic protein was identified. Denovirus-mediated delivery stimulates side branch vascular development in the ischemic area and reduces blood flow Have been shown to alleviate myocardial ischemia. For illustration , We have identified three different veins, including both native forms and muteins. These gene transfer techniques with tube forming proteins have been successfully demonstrated (see below). As described in detail in the examples).   In a second model, the ameloid contractor (am) around the left circumflex (LCx) coronary artery The placement of the eroid constrictor is the smallest infarction (1% of left ventricle, 4 ± 1% of LCx floor) Resulting in a gradual complete closure (within 7 days of deployment) (Roth et al., Circulation, 82: 1778, 1990; Roth et al., Am. J. Physiol. 235: 1-11279, 1987; White et al., Circ. Res. Chem., 71: 1490, 1992; Hammond et al., Cardiol. 23: 475, 1994; and Hammond et al., J. . Clin. Invest. , 92: 2644, 1993). Myocardial function and blood flow are due to occluded arteries. In the previously perfused area (called the ischemic area), there is limited endogenous collateral blood Blood flow is usually at rest due to vessel development, but securing blood flow depends on myocardial oxygen demand. If is increased, it is insufficient to prevent ischemia. Therefore, LCx beds are clinically Subject to incidental ischemia resembling angina. The relationship between collateral vessel development and flow function is It is stable within 21 days of Lloyd placement and remains unchanged for 4 months ( Roth et al., Circulation, 82: 1778, 1990; Roth et al., Am. J. Physiol. , 235: H1279, 1 987; White et al., Circ. Res. , 71: 1490, 1992). Animals feed, occupy and work throughout the day Dangerous conditions related to sudden increases in heart rate, such as during personnel Has been demonstrated by telemetry to have ischemic dysfunction for a period of time. Therefore This model has a stable but improper side branch vessel Get blood. Another distinct advantage of this model is that it is abnormally perfused and functions. Adjacent to the area (LCx floor) is a normally perfused and functional area (LAD floor) Thereby providing a control floor within each animal.   Myocardial ultrasonography echocardiography was used to predict regional myocardial perfusion. Construction The contrast material is composed of microaggregates of galactose, and the echogenicity of the image (white Increase). Microaggregates are coronary and myocardial in a manner proportional to blood flow. It is distributed on walls (Skyba et al., Circulation, 90: 1513-1521, 1994). Contrast Peak intensity is closely correlated with myocardial blood flow, as measured by microparticles (Skyba et al., Circulation, 90: 1513-1521, 1994). Used in the present invention That the echocardiographic images obtained accurately identify the LCx floor, and that the myocardial ultrasound To demonstrate that shadow cardiography can be used to assess myocardial blood flow, Hydraulic cuff occluder around proximal LCx adjacent to ameloid It was installed in.   In this study, when animals were sacrificed, capillary growth was quantified by microscopy. Perfusion fixation of the heart (glutaraldehyde, physiological pressure, in situ) Was). PCR was performed on angiogenesis in cardiac muscle from animals that received the gene transfer. Used to detect protein DNA and mRNA. As described below, Two weeks post-transfection, myocardial sumps from all five lacZ transduced animals Showed substantial β-galactosidase activity on histological examination. Finally , Angiogenic proteins in cells and myocardium from animals that have received the gene transfer Expression was demonstrated using polyclonal antibodies to angiogenic proteins .   Strategies for therapeutic research include transgene delivery opportunities, transgene routes of administration, And selection of angiogenic genes. Amyloid model of myocardial ischemia Gene transfer was performed after the development of stable but inadequate side branch vessels. A Previous studies using meloid models have shown that American ischemic and Included delivery of an angiogenic peptide during closure of the Lloyd. But this strategy does not Not used for several reasons. First, previous studies have shown that clinical myocardial ischemia (Where gene transfer is provided in an ongoing myocardial ischemia setting) Not appropriate to closely overlap existing conditions; previous studies have Similar to, and therefore less relevant to, providing peptides in Secondly, Based on previous studies in cell culture, ischemic stimulation in combination with angiogenic peptides It was estimated to be the optimal environment for stimulation of angiogenesis. Angiogenesis is Total onset (associated with DCM or severe coronary artery disease) or ventricular reconstitution If present, it can be optimally achieved by simultaneous transgene delivery. These decisions Relevant to the determination was the choice of how to achieve transgene delivery.   The porcine model is a superior hand with local blood flow and function before and after gene delivery. Dan provided. Same recombinant adenovirus construct but with reporter gene The use of control animals that have received Provided a role. Based on the foregoing and previously published studies, those skilled in the art will: Results described below in pigs are expected to predict outcomes in humans Understand that it will be. Transgenes encoding angiogenic proteins and peptides   In the present invention, an angiogenic protein capable of promoting angiogenic activity in the heart Alternatively, one or more transgenes encoding peptide factors can be used. Honcho "Vascular shape" which can be measured by the methods described in the textbook and methods in the art. Any protein or peptide that exhibits "active activity" (defined above) It can potentially be used in connection with lighting. Appropriate angiogenic protein or peptide Are fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor ( PDGF), insulin-like growth factor (IGF), and other family members Is exemplified. The members of the FGF family are aFGF (FGF-1), bFGF (FGF-2), and FGF- 4 (also known as "hst / KS3"), including but not limited to FGF-5 and FGF-6 Absent. VEGF is stimulated by cardiomyocytes in response to ischemia in vitro and in vivo. Has been shown to be expressed under physiological conditions and in pathological conditions. Are regulators of angiogenesis during the adaptive response to ((Banai et al., Circulation 89: 218 3-2189, 1994). The VEGF family is a member of the VEGF-A subfamily (e.g., VEGF-121, VEGF-145, VEGF-165, VECF-189 and VEGF-206), and VEGF-B Subfamilies (e.g., VEGF-167 and VEGF-186) and the VEGF-C subfamily Including, but not limited to, PDGF includes PDGF A and PDGF B No. The nucleotide sequences of the genes encoding these proteins and the corresponding Amino acid sequences are known in the art (e.g., these sequences and other sequences). See the GENBANK sequence database for columns). About the FGF family Is, for example, Burgess, Ann. N. Y. Acad. Sci. 638: 89-97, 1991; Burgess et al., Annu. Re v. Biochem 58: 575-606, 1989: Muhlhauser et al., Hum. Gene Therapy 6: 1457-1465, 1 995; Zhan et al., Mol. Cell. Biol. , 8: 3487, 1988; Seddon et al., Ann. N. Y. Acad. Sci. 638: See 98-108, 1991. For human hst / KS3 (i.e., FGF-4), for example, , Taira et al., Proc. Natl. Acad. Sci. USA 84: 2980-2984, 1987. Human VE Regarding GF, Tischer et al., J. Biol. Chem. 206: 1 1947-11954, 1991, and references therein; Muhlhauser et al., Cir. Res. 77: 1077-10 86, 1995). Such genes and encoded polypeptides Sequence information is readily available from sequence databases such as GenBank or EMBL. It is possible. The polynucleotides encoding these proteins are also Child libraries (eg, PCR or hybridization routine in the art) By using application technology).   Successful gene transfer approaches rely on gene product synthesis and transfection Requires both secretion from the expressed cells. From this perspective, FGF-4, FGF-5 or FG The gene encoding F-6 is preferred. Because these proteins are functional It contains a secretory signal sequence and is easily secreted from cells. For the most part But not many human VEGF proteins (these include VEGF-121 and VEGF-165 (But not limited to) are also readily secreted and may diffuse after secretion You. Thus, when expressed, these angiogenic proteins are readily found in the myocardial stroma. And can induce angiogenesis.   Other angiogenic proteins lacking the native secretory signal sequence (eg, aFGF (FGF-1) and bFGF (FGF-2)) using a fusion protein having a secretory signal sequence. The protein is produced recombinantly using standard recombinant DNA methodology well known to those skilled in the art. Can be It is believed that both aFGF and bFGF can to some extent be naturally secreted. However, the inclusion of additional secretory signal sequences may enhance protein secretion. Can be used for Secretory signal sequences are typically those of the desired protein. Located at the N-terminus, but any position appropriate to allow secretion of angiogenic factors Can be placed in different locations. For example, a polynucleotide containing the appropriate signal sequence may be selected It may be fused 5 'of the first codon of the selected angiogenic protein gene. Appropriate The secretory signal sequence is the signal sequence of FGF-4, FGF-5, FGF-6 gene, or IL- Includes signal sequences for different secreted proteins such as 1β. Example 7 below One type of angiogenic protein to include a signal sequence from another protein Modification (this modification is due to residues in the angiogenic protein Which are modifications achieved by substitutions with residues that direct secretion of proteins. . A signal sequence derived from a protein normally secreted from cardiomyocytes is used. Profit You.   For human treatment, genes encoding angiogenic proteins of human origin are preferred. Preferably, angiogenic proteins of other mammalian origin that exhibit cross-species activity (ie, , Which have angiogenic activity in humans) can also be used. Vectors for gene delivery in vivo   Generally, the gene of interest is transferred into the heart (including cardiomyocytes) in vivo and is Direct the production of the encoded protein. Preferably, the production is constitutive (But an inducible expression system can also be used).   Vectors useful in the present invention include viral vectors, lipid-based vectors ( For example, liposomes and other vectors, which transfer DNA in vivo Non-dividing cells). Currently, virus vectors, In addition, replication-defective virus vectors (for example, replication-defective adenovirus vectors And adeno-associated virus (AAV) vectors. In the present invention Replication-deficient adenovirus vectors are the most preferable. In comparison to retroviruses, adenoviruses generally have Does not require replication of the host cell. Because integration is an adenovirus It is not a component of the life cycle. Adenovirus can efficiently detect non-dividing cells Staining, and therefore due to the non-replicating nature of cardiomyocytes, It is useful for expressing   A variety of other vectors suitable for in vivo gene therapy are available for use in the present invention. It can be easily used to deliver angiogenic protein transgenes. This Other vectors, such as, for example, other viral vectors (eg, AAV), non-viral Includes a protein-based delivery platform, and a lipid-based vector. References describing these and other gene delivery vectors are known in the art. Yes, some of which are incorporated herein.   As described above and in the cited references, the vector may also contain other components or May include functionality, which may further regulate gene delivery and / or gene expression Can provide or otherwise provide beneficial properties to the targeted cells. this Other components such as, for example, components that affect binding or targeting to cells (cell types Containing components that mediate specific or tissue-specific binding); Influencing the uptake of the target nucleic acid; polynucleotide in the cell after uptake (Eg, agents that mediate nuclear localization); and polynucleases Contains components that affect leotide expression. Such components may also include a marker ( These include cells that express the nucleic acid utilized and delivered by the vector. Detectable and / or selectable markers used to detect or select Is a car). Such components are the natural features of the vector (eg, For example, certain viruses that have components or functionality that mediate binding and uptake Vector utilization), or the vector provides such a function. It can be modified to provide sex. Selectable markers are positive, negative It can be active or bifunctional. The positive selectable marker is the marker Allows selection of cells harboring the To allow cells carrying the key to be selectively eliminated. Various such ma Marker genes have been described, and these are bifunctional (ie, positive Active / negative markers (eg, Lupton, S. et al. , WO 92 / 08796,199 Published May 29, 2; and Lupton, S. ; See WO 94/28143, published on December 8, 1994. thing). Such marker genes may be advantageous in the context of gene therapy Additional control means may be provided. A wide variety of such vectors are available Known in the field and generally available (eg, the species cited above). See various references).   Adenovirus vectors and other viruses that can be used in the methods of the invention References describing subvectors include: Horwitz, M .; S. , Adenoviridae an d Their Replication, Fields, B. Et al. (Eds.), Virology, Volume 2, Raven Press N ew York, 1679-1721 (1990); Graham, F. Et al., Pp. 109-128, Methods in Molecular  Biology, Volume 7: Gene Transfer and Expression Protocols, Murray, E. (Edit) , Humana Press, Clifton, N. J. (1991); Miller, N. Et al., FASEB Journal 9: 190-19 9, 1995; Schreier, H. Pharmaceutica Acta Helvetiae 68: 145-159, 1994; Schne ider and French, Circulation 88: 1937-1942, 1993; Curiel D. T. Et al., Human Gene Therapy 3: 147-154, 1992; Graham, F .; L. Et al., WO 95/00655 (1995 January 5, 2009); Falck-Pedersen, E. S. , WO 95/16772 (June 22, 1995); Denefle, P. Haddada, H. et al., WO 95/23867 (September 8, 1995); Et al., WO 94/26914 (November 2, 1994) 4th); Perricaudet, M. Et al., WO 95/02697 (January 26, 1995); Zhang, W. et al. Et al., WO 95/25071 (October 12, 1995). Various adenovirus plasmids are also commercially available. Available from sources (eg, Microbix Biosystems of Toronto, Ontari o (for example, Microbix Product Information Sheet: Plasmids for Adenovirus Ve ctor Construction, 1996)).   Further references describing AAV vectors that can be utilized in the methods of the invention Include: Carter, B .; Handbook of Parvoviruses, Volume I, pages 169-128, 1990; Berns, Virology, 1743-1764 (Raven Press 1990); Carter, B. Curr. Op in. Biotechnol. 3: 533-539, 1992; Muzyczka, N. . Current Topics in Microbiolog y and Imnlunology, 158: 92-129, 1992; Flotte, T. R. Am. J. Respir. Cell Mol . Biol. 7: 349-356, 1992; Chatterjee et al., Ann. NY Acad. Sci. , 770: 79-90, 1995; Fl otte, T. R. Et al., WO 95/13365 (May 18, 1995); Trempe, J. et al. P. Et al., WO 95/13392 ( May 18, 1995); Kotin, R. , Human Gene Therapy 5: 793-801, 1994; Flotte, T. R. Gene Therapy 2: 357-362, 1995; Allen, J. et al. M. , WO 96/17947 (June 1, 1996) 3)); and Du et al., Gene Therapy 3: 254261, 1996.   Additional references describing non-viral vectors that can be utilized in the methods of the invention. References include: Ledley, FD, Human Gene Therapy 6: 1129-1144, 1995; Mil. ler, N. Et al., FASEB Journal 9: 190-199, 1995; Chonn, A. Curr. Opin. in Biote ch. 6: 698-708, 1995; Schofield, JP. Et al., British Med. Bull. 51: 56-71, 1995; Brig ham, K. L. J. Liposome Res. 3: 3149, 1993; Brigham, K. L. , WO 91/06309 (1991 May 16); Felgner, P. L. Et al., WO 91/17424 (November 14, 1991); Solodin et al., B iochemistry 34: 13537-13544, 1995; WO 93/19768 (October 14, 1993); Debs et al. WO 93125673; Felgner, P. L. Et al., US Patent No. 5,264,618 (November 23, 1993); Ep. and, R. M. U.S. Patent No. 5,283,185 (February 1, 1994); Gebeyehu et al. No. 5,334,761 (August 2, 1994); Felgner, P. L. Et al., U.S. Patent No. 5,459,127 Issue (October 17, 1995); Overell, R. W. Et al., WO 95/2849 4 (October 26, 1995); Jessee, WO 95/02698 (January 26, 1995); Haces and Ci ccarone, WO 95/17373 (June 29, 1995); Lin et al., WO 96/01840 (Jan. 25, 1996). Day). Exemplary adenovirus vectors that are helper-independent and replication-defective in humans Tar   Generally, the gene of interest is transferred in vivo to the heart, including cardiomyocytes, And direct the production of the encoded protein. Several different introgressions An approach is possible. Helper-independent replication based on human adenovirus 5 Defective systems are presently preferred. Using this system, we provide a single intracoronary injection (Giordano and Hammond, Clin. Res. , 42: 123A, 1994). It was demonstrated that transfection into more than 0% cardiomyocytes had occurred. Non-duplicate Recombinant adenovirus vector provides highly efficient transfer after intracoronary injection. For transfection of coronary endothelium and cardiomyocytes And is particularly useful.   As demonstrated herein, a helper-independent replication-deficient human adenotype 5 Virus transfers a large proportion of cardiomyocytes in vivo by a single coronary injection Could be used in transfecting effectively. The present inventors have proposed such a transmission. Technology can be used to efficiently target vectors to the myocardium of a large mammalian heart Has also shown. Further targeting vectors to specific cells or tissue types Means are described herein below and in the art.   In various examples shown below, the recombinant adenovirus vector used Lacks the essential early genes (usually E1A / E1B) from the adenovirus genome And therefore increase in permissive cell lines that provide the missing gene product in trans. Human adenovirus 5 that cannot replicate unless propagated (McGrory WJ et al., Viro 163: 614-617, 1988). Adenovirus genome missing If the transgene of interest is replaced by a replication-defective adenovirus Can be cloned and expressed on tissues / cells. Adenovirus-based gene transfer The transfer generally does not result in stable integration of the transgene into the host genome (0. Less than 1% of adenovirus-mediated transfection results in the assembly of the gene into host DNA However, adenovirus vectors are grown at high titers and Can transfect replicating cells; and the transgene is passaged into daughter cells. However, it is suitable for gene transfer into adult cardiac myocytes (not actively dividing). It is off. Retroviral vectors provide stable gene transfer and are currently High titers can be obtained by retroviral pseudotyping (Burns et al.). Proc Natl Acad Sci (USA) 90: 8033-8037, 1993). However, recent retro Ils vectors generally efficiently transform non-replicating cells (eg, cardiomyocytes). Cannot be introduced. In addition, the potential danger of transgene integration into host DNA is short-term. If early stage gene transfer is sufficient, it is not guaranteed.   Indeed, we have found that the limited duration of angiogenic protein expression is Demonstrated to be sufficient for qualitative angiogenesis (see Example 5). Therefore Transient gene transfer is therapeutically appropriate for the treatment of such cardiovascular conditions It is. Within 14 days after gene transfer of FGF-5 into the myocardium, ischemic bed blood flow , Increased by a factor of 2 and the effect lasted for at least 12 weeks (Example 5 and FIG. 8). Increased blood flow correlated with an increase in the number of heart capillaries (see Example 5). ). Wall thickening also increased within two weeks after gene transfer and was at least 12 Lasted a week. Thus, the angiogenic factor gene is sensitive in order to produce a therapeutic effect. It is not necessary for the stained cells to be present for more than a few weeks. Once the blood vessels As it occurs, continued expression of exogenous angiogenic proteins is responsible for maintaining new vasculature. May not be necessary for retention and increased blood flow.   The advantage associated with non-dividing cells, such as muscle cells, is that It is not easily "diluted" by fission. But in the heart It is necessary or desirable to further enhance the duration of transgene expression Different second generation adenovirus vectors carrying both E1 and E4 deletions -This can be used in combination with cyclophosphamide administration. Are also possible (eg, Dai et al., Proc. Nat'l. Acad. Sci. USA 92: 1401-1 405, 1995). Additional injections to further increase the degree of initial gene transfer Alternatively, injection into the isolated coronary circulation may also be used.   Human 2, a human embryonic kidney cell transformed with the adenovirus E1A / E1B gene 93 cells (Accession number, ATCC CRL1573; Rockville, MD) Representative of useful permissive cell lines for the production of protein. However, replication-defective Ade Other cell lines that allow the propagation of the viral vector in cells may also be used. (Eg, HeLa cells). Construction of recombinant virus vector   The adenovirus vector used in the present invention is McGrory W. J. (Viro 163: 614-617, 1988). . Briefly, the transgene of interest comprises a promoter, a polylinker, and Contains adenovirus sequences containing deletions of the E1A / E1B gene essential for viral and viral replication Cloned into a shuttle vector. The transgene is located within the polylinker It can be cloned into convenient restriction sites. A suitable shuttle vector , Plasmid pAC1 (McGrory et al., Virology, 163: 614-617, 1988) (or analog (This encodes the left terminal portion of the human adenovirus type 5 genome, but (Vi rology 163: 614-617, 1988), where the E1A and E1B sequences encoding the early proteins Deleted), and plasmid ACCMVpLpA (Gomez-Foix et al., J. Am. Biol. Chem. , 2 67: 25129-25134, 1992). pACCMVpLpA is adenovirus serotype 5 Contains the 5 'end of the genome (map units 0-17), where the E1 region is Replaced with the gallovirus enhancer-promoter (CMV promoter), pUC1 Multiple cloning site (polylinker) from 9 (plasmid well known in the art) ), Followed by the SV40 polyadenylation signal. Use of plasmid pAC1 or ACCMVPLA Can facilitate the cloning process.   The shuttle vector then has a length that is too large to be capsidized. Together with a plasmid containing the entire genome of human adenovirus type 5 Vesicles can be co-transfected. Co-transfection is well known in the art. Any means commonly used (eg, calcium phosphate precipitation or lipofer (See, for example, Biotechniques 15: 868-872, 1993). See).   As an example of a plasmid for co-transfection, the plasmid "JM17 "Is the entire genome of human adenovirus type 5 and the ampicillin resistance gene ( Four. 3 kb). JM17 is a mature virion Encodes all of the adenovirus proteins required to produce Too encapsidated in mature virions (36 kb vs. 40 kb for wild type).   In a small subset of co-transfected cells, "Recombinant" refers to a transgene-containing shuttle vector (eg, plasmid pACl) And a recombinant genome containing the transgene of interest in place of the deleted E1A / E1B sequence Has the entire genome of adenovirus type 5 (eg, plasmid pJM17) With additional plasmids, and secondarily during recombination (eg, For example, the pBR322 sequence) is deleted, thereby making it small enough to form a capsid. (FIGS. 4 and 5; Giordano et al., Nature Medicine 2 (5): 534-538, 1996; Giord ano et al., Circulation, 88: 1-139, 1993; and Giordano and Hammond, Clin. R es. , 42: 123A, 1994). Adenovirus HCMVS using X-gal treatment CMV promoter driven by β-galactosidase gene in PllacZ (Clin . Res. 42: 123A, 1994) can be used to assess the efficiency of gene transfer.   Illustrative examples illustrating the preparation and use of such vectors are provided below. . The advantage of these vectors is their ability to achieve highly efficient gene transfer (in High titer virus (> 60% of target organ cells transfected in vivo) The ease of obtaining the stock and the ability to disrupt cells such as cardiomyocytes that do not divide Including the ability to achieve gene transfer of these vectors. Targeted angiogenic protein vector constructs   The heart of an angiogenic transgene, or a particular cell type within the heart (eg, myocardium Expression restricted to (cells) may provide certain advantages as described below.   The invention not only depends on the delivery of the transgene to the coronary arteries, but also, for example, In addition, gene transfer to specific host cells or host cell types (eg, cardiomyocytes) Targeting vectors that have a tendency to target expression and / or gene expression The use of cell targeting by the use of a target construct is contemplated. Described in more detail below And targeting such vectors, as described in publications in the art. The targeting construct comprises a targeted delivery vector and / or a targeting vector. Sending And / or limiting expression may further increase the potential efficacy of gene therapy It can be useful as a means of focusing. Of further limiting delivery / expression Most of the potential utility depends on the type of vector used and the It depends on the method and position of introducing the vector. As indicated herein, Delivery of viral vectors by intracoronary injection into the myocardium is inherently highly targeted Has been observed to provide optimized gene delivery (see Examples below). When). In addition, a vector that does not result in integration of the transgene into the host cell replicon. With vectors (eg, adenovirus and numerous other vectors), Cells are expected to show transgene expression for a relatively long time. Because Vesicles do not usually replicate. In contrast, rapidly dividing, like endothelial cells Expression in cells tends to decrease due to cell division and turnover. However, as indicated herein, other means of limiting delivery and / or expression Steps can also be added to or used in place of the illustrated delivery method.   Targeted delivery vectors include, for example, surface components (eg, ligand-receptor pairs). (The other half of this pair is found in the targeted host cell) Or preferential binding to a particular host cell or host cell type and / or Vectors with other features that mediate gene delivery (eg, viral, non-viral) Protein-based vectors and lipid-based vectors). Public in the field As we know, numerous vectors of both viral and non-viral origin have Have unique properties to promote preferential binding and / or preferential targeting (Eg, Douglas et al., Nature Biotechnolog y 14: 1574-1578, 1996; Kasahara, N. Et al., Science 266: 1373-1376, 1994; Miller, N. . Et al., FASEB Journal 9: 190-199, 1995; Chonn, A. Curr. Opin. in Biotech. 6: 6 98-708, 1995; Schofield, JP et al., British Med. Bull. 51: 56-71, 1995; Schreier H . Pharmaceutica Acta Helvetiae 68: 145-159, 1994; Ledley, F. D. , Human Gene Therapy 6: 1129-1144, 1995; Conary, J. T. Et al., WO 95/34647 (1 December 21, 995); Overell, R. W. Et al., WO 95/28494 (October 26, 1995); and Truo ng, V. L. Et al., WO 96/00295 (January 4, 1996)).   Targeting vectors are relatively limited in delivery to a particular host cell or host cell type. Vectors that produce expression of a transgene (viral, non-viral protein vectors) Source vectors and lipid-based vectors). For example, according to the present invention, The angiogenic transgene achieved can operate on a heterologous tissue-specific promoter To thereby restrict expression to cells in that particular tissue You.   For example, encoding left ventricular myosin light chain-2 (MLC2V) or myosin heavy chain (MHC) Tissue-specific transcription control sequences derived from the gene Into a transgene (eg, FGF gene) in a suitable vector. It Thus, the expression of the transgene can be relatively restricted to ventricular cardiomyocytes. lacZ MLC with_2VAnd the efficacy of gene expression provided by the MHC promoter and The degree of specificity was determined using a recombinant adenovirus system as exemplified herein. T) has been determined. And, heart-specific expression has been reported (eg, Lee Et al., J Biol Chem 267: 15875-15885, 1992).   Since the MLC2V promoter contains only about 250 bp, the addenda exemplified herein Within delivery vectors that are limited in size, such as the novirus type 5 packaging system Even fits easily. Myosin heavy chain pro known as a strong transcription promoter The motor provides another alternative heart-specific promoter and has less than 300 bp Including. Other promoters, such as the troponin-C promoter, are highly effective. But small enough to provide no such tissue specificity.   Coronary arteries using high-titer vectors in the method for the treatment of heart disease Targeting the gene transfer to the heart by intra-injection is presently preferred. Adenovirus vector propagation and purification   Recombinant viral vectors, such as adenoviral vectors, are prepared according to standard methods. Plaques can be purified. For example, the resulting recombinant adenovirus virus vector Has a titer of about 10^Ten-Ten¹²Human 293 cells in the preferred range of virus particles / ml La (Providing E1A and E1B function to the host). Growth and purification techniques Describes various viral vectors that can be used in combination with the present invention. ing. Adenovirus vectors are exemplified herein, but may be other, such as AAV. May also be used. For adenovirus, cells are 80% It can be harvested 48 hours after infection with confluence. Three freezes of infected cells -After the thawing cycle, pellet the cell debris by centrifugation and run over a CsCl gradient. Virus was purified by centrifugation (preferably double CsCl gradient ultracentrifugation). In vivo Prior to injection of the virus, a stock of virus (eg, Sepharos such as Sephadex G25) desalting (by gel filtration through an e-column). Desalted virus stock Also, if desired, it can be filtered through a 0.3 micron filter. The inventor Typically use double CsCl ultracentrifugation followed by phosphate buffered saline (PBS) Column storage on Sephadex G25 The solution is concentrated and purified. The resulting virus stock is typically Luss titer at least about 10^Ten~Ten¹²Virus particles / ml.   Preferably, the recombinant adenovirus is highly purified and substantially wild-type Contains no (potentially replicable) virus. For these reasons, proliferation and And purification can be performed, for example, by PCR using appropriate primers. Identification, performing two plaque purifications, and dual CsCl gradients Ultracentrifugation can be performed to remove contaminants and wild-type virus. Delivery of an angiogenic transgene-bearing vector   Hands used to deliver angiogenic protein transgene carrying vectors The steps and compositions will depend on the particular vector used, as is well known in the art. You. Typically, however, the vector is, for example, a phosphate-buffered saline solution. In the form of injectable preparations containing various pharmaceutically acceptable carriers / diluents. Can get. Other pharmaceutical carriers, formulations and dosages are described below.   Currently preferred means of in vivo delivery (especially limited to otherwise myocardial For vector constructs that are not targeted for delivery and / or expression) By injecting the vector directly into the blood vessel supplying the myocardium, preferably one or both By injection into one of the coronary arteries. Such an injection is preferably one or both. Lumen of one coronary artery, or lumen of one or more saphenous veins, or internal breast Inside the lumen of an arterial graft or other conduit that delivers blood to the myocardium By introducing a catheter substantially (typically at least about 1 cm) Achieved. Preferably, the injection is made in both coronary arteries and all heart Distributed throughout the site.   The vector is delivered in an amount sufficient for the transgene to be expressed, and Provide points. Injectable in viral vectors (eg, adenovirus) The final titer of virus in a working preparation is preferably about 10⁶~Ten¹⁴Will Particles, which allows for efficient gene transfer. Preferably, the field Adenovirus vector stocks containing no live virus may contain one or both It can be injected deep into the lumen of the coronary artery (or graft), preferably the right and left It can be injected into both coronary arteries (or grafts), and preferably is an optical densitometer. 10 to be decided⁸~Ten¹²It can be injected in the amount of virus particles. Preferred Alternatively, the vector is administered in a single injection within each conduit (eg, within each coronary artery). Delivered.   Inject the vector composition directly into the lumen of the coronary artery via a coronary catheter Target the gene more efficiently, and In contrast, it is possible to minimize the loss of the recombinant vector. Notes of this type Engraftment involves angiogenesis protein in a desired number of cells of the affected myocardium, especially myocardial cells. Or allows local transfection of the gene encoding the peptide, This maximizes the therapeutic effects of gene transfer and Minimize unwanted angiogenesis.   Vector constructs specifically targeted to the myocardium (eg, myocardial-specific binding or Uptake components and / or myocardium-specific transcriptional regulatory sequences (eg, ventricular muscle-specific pro Vector that incorporates an angiogenic protein transgene under the control of a motor) Can be used as a means to further restrict expression to the heart muscle (especially ventricular myocytes). Can be used instead of, or preferably in combination with, directed injection techniques . For vectors that can elicit an immune response, supply the vector to myocardium as described above You Direct injection into the blood vessels, but to limit the potential for non-myocardial expression Additional techniques may also be used.   As described below, the import of viral vectors containing an angiogenic transgene The use of such techniques for in vivo delivery has led to transgene expression occurring in hepatocytes. And viral RNA is found in urine at any time after coronary artery injection. Not proved. In addition, extracardiac in the eye, liver, or skeletal muscle Evidence of gene expression is based on two weeks of delivery of this mode of transgene into the coronary artery. Later, it could not be detected by PCR.   A variety of catheters and delivery routes can be routed into the coronary arteries, as is known in the art. Can be used to achieve delivery. Direct intracoronary (or graft vascular Infusion is based on standard percutaneous catheters under fluoroscopic guidance Can be performed using the method. Any of a variety of coronary catheters or e.g. stac A k perfusion catheter can be used in the present invention. For example, various general purposes Catheters, as well as modified catheters, are suitable for use in the present invention, e.g., , Advanced Cardiovasucular Systems (ACS), Target Therapeutics, Boston Sci Commercially available from entific and Cordis. In addition, delivery to the myocardium If achieved by direct injection, which is currently the most preferred, Is used to introduce a catheter into the coronary artery, as is known in the art. Can be done. For illustration, a catheter can be conveniently introduced into the femoral artery and Through the iliac and abdominal aorta and into the coronary artery in the opposite direction. Or The catheter is first introduced into the brachial or carotid artery and then into the coronary artery Passed through. Detailed descriptions of these and other technologies can be found in the art. (Eg, Topol, EJ (ed.), The Textbook of Interventional Cardiolo) gy, 2nd edition (W.B.Saunders Co. 1994); Rutherford, RB, Vascular Surgery, ed. Third edition (W.B.Saunders Co. 1989); Wyngaarden JB et al. (Eds.), The Cecil Textbook of Medicine, 19th edition (W.B.Saunders, 1992); and Sabiston, D, The Textbo References cited above, including ok of Surgery, 14th edition (W.B.Saunders Co. 1991) See literature). Targeted gene expression   An unexpected finding of the present invention is that recombinant adenovirus In the first vascular bed encountered by Ilus, it is very efficiently taken up. And Indeed, in the animal model of Example 4, the heart after injection into the coronary artery The efficiency of virus uptake in the gut is 98% (ie 98% of the virus is (Removed in the first pass of the virus through the myocardial vascular bed). Further The serum obtained from the animals during the infection is Growth was not possible (Graham, Virology, 163: 614-617, 1988). Suggests the presence of a serum factor (or binding protein) that inhibits ruth growth. this And two factors (effective attachment of virus to the first channel and serum binding protein). Quality potential) are both gene expression for the first vascular bed encountered by the virus Restrict.   Increase the degree to which gene transfer into the heart following gene transfer into the coronary arteries is limited. The polymerase chain reaction (PCR) was used to evaluate two treated animals Evidence of extracardiac presence of viral DNA two weeks after gene transfer It was determined whether or not to perform (Example 4 below). The animal, in its heart, But not in the retina, skeletal muscle or liver. The feeling of PCR The degree is such that a single DNA sequence is detectable per 5,000,000 cells. Therefore, These data indicate that two weeks after gene delivery, in extramyocardial tissue, viral DNA Demonstrated that it does not exist. These results are in addition to other angiogenic proteins And derivatives were used as described below. These findings are very important is there. Because they target myocardial transgenes (ie, That provide expression in the heart of the mouse but do not provide expression elsewhere) To check the Localized transgene delivery and expression is safe To further enhance the use of the methods of the invention in treating patients. Therapeutic application   The present method of in vivo transfer of a transgene encoding an angiogenic protein comprises: Treatment of congestive heart failure (deficits related to dilated cardiomyopathy (DCM), and some Prevention of coronary artery disease (CAD) in children) and prevention of ventricular remodeling Can be applied for mitigation. As the data below shows, Expression of an angiogenic transgene provided to the rat increases blood flow and cardiac ischemia And promotes angiogenesis resulting in function in non-ischemic areas. This increased blood vessel Distribution alleviates congestive heart failure and reduces ventricular dysfunction associated with ventricular reconstitution cancel.   As described herein, the methods of the present invention allow for many different vectors. Can be used to deliver an angiogenic protein transgene in vivo. As an example Thus, the replication-defective recombinant adenovirus vector exemplified herein is Extreme in vivo without cytopathic effects or inflammation in the area of gene expression And achieved efficient gene transfer.   Transgenes encoding angiogenic proteins in the treatment of congestive heart failure Gene transfer, for example, after a diagnosis of the likelihood of developing heart disease or heart failure. Can be. For treatment of ventricular reconstruction, gene transfer occurs after the patient has suffered an infarction. At any time, preferably within 30 days, more preferably 7-20 days after infarction Within can be done.   The compositions or products of the present invention can be administered to the bloodstream (eg, by intracoronary infusion). ) Can be conveniently provided in the form of a suitable formulation. The appropriate dosing format is Can be optimally determined for each patient individually by the physician. Suitable pharmaceutically acceptable Carriers and their formulations are described in standard prescribing papers (eg, by E.W. Remington's Pharmaceuticals Sciences). Wang, Y.J. and Ha nson, M.A. "Parental Formulations of Proteins and Peptide: Stability and Stabilizers '', Journals of Parental Sciences and Technology, Technical R eport No. 10, see also Addendum 42: 2S (1988). The vector of the present invention is preferably Preferably, a neutral pH (eg, about pH 6.5 to about pH 8.5, more preferably about pH 7 to 8) An excipient that makes the solution nearly isotonic (eg, 4.5% mannitol or 0% (9% sodium chloride). pH is an approved preservative (E.g., 0.1% to 0.75%, more preferably 0.15% to 0.4% Resole) together with a known buffer solution (eg, Sodium sulfate). The desired isotonicity is Or other pharmaceutically acceptable agents (eg, dextrose, boric acid, tartar) Like sodium acidate, propylene glycol, mannitol and sorbitol (A suitable polyol or other inorganic or organic solute). salt Sodium chloride is particularly preferred for buffers containing sodium ions. Desired place If so, a solution of the composition may also be prepared to enhance shelf life and stability. You. Therapeutically useful compositions of the present invention comprise a mixture of ingredients after generally accepted procedures. It is prepared by combining. For example, mix the selected ingredients and concentrate Product, which in turn uses water and / or buffers to control pH. Or by adding additional solutes to control tonicity And can be adjusted to final viscosity.   For use by a physician, the composition provides a therapeutic effect of the vector of the invention. That is effective in one or more doses to induce angiogenesis sufficient to May be provided. As will be appreciated by those skilled in the art, The effective amount depends on many factors, including the age and weight of the patient, the physical condition of the patient, (Including the level of angiogenesis available as well as other factors).   An effective dose of a compound of the invention is typically about 10⁶~Ten¹⁴The range of virus particles Box, preferably about 10⁸~Ten¹²Virus particles. The number of virus particles is 1 0¹⁴But preferably 10¹⁴Not exceed. Administered as described The exact dose to be determined is determined by the attending clinician, but is preferably In a physiological buffered solution (eg, phosphate buffered saline), more preferably Is in 1-3 ml.   The current preferred mode of administration in the case of heart disease is a suitable coronary catheter Into one or both coronary arteries (or if one or more saphenous veins Or internal mammary artery graft) by intracoronary injection.   The following examples are provided to further assist those skilled in the art. Such an embodiment Is intended to be illustrative, and thus should be regarded as limiting the invention is not. Many exemplary modifications and alterations are described in this application and Modifications and alterations will be apparent to those skilled in the art. Such changes are noted herein. It is considered within the scope of the claimed and claimed invention.                                  Example   A preferred method of the present invention is for treating patients with heart failure and / or ventricular remodeling. Involved in the installation.   Example 1 demonstrates that long-term (over 3-4 weeks) rapid ventricular pacing. A model of induced heart failure is described. Examples are given in animal models of heart failure. If the left ventricle is dilated, left ventricular myocardial blood flow is abnormally low and signs of heart failure Demonstrate that the weather was present. In this model, myocardial requirements are often Negates muscle blood flow (oxygen supply), resulting in myocardial ischemia.   Example 2 shows the construction of an adenovirus vector used in the present invention. You.   Example 3 describes a preliminary study to establish the efficacy of the adenovirus vector of the invention. A typical in vitro experiment is described. Control (encoding β-galactosidase) Adenovirus vector (AdlacZ) was also tested. The results are Recombinant adenovirus vector can actually infect adult cardiomyocytes and has high efficiency Showed that it could provide transgene expression.   Example 4 describes an adenovirus vector encoding β-galactosidase (Adla Infection of porcine myocardium in vivo using cZ) and β-gal in myocardium Demonstrate expression.   Example 5 demonstrates the in vivo infusion of FGF-5 angiogenic transgene into myocardium. Ruth-mediated delivery can improve local blood flow and local myocardial contractile function Demonstrate that failure can be remedied.   Example 6 demonstrates in vivo in vivo infusion of FGF-4 angiogenic transgene into myocardium. Ruth-mediated delivery can improve local blood flow and local myocardial contractile function Demonstrate that failure can be remedied.   Example 7 demonstrates the in vivo infusion of FGF-2 angiogenic transgene into myocardium. Ruth-mediated delivery can improve local blood flow and local myocardial contractile function Demonstrate that failure can be remedied. Example 1: Porcine model of congestive heart failure and concomitant myocardial ischemia 1-A. Animal and surgical procedures   Nine Yorkshlre pigs (Sus scrofa) weighing 40 ± 6 kg were treated with ketamine (50 mg / kg, IM) and And atropine sulfate (0.1 mg / kg, IM), followed by sodium amital (100 mg / kg, IV) Anesthetized. After endotracheal intubation, halothane (0.5-1.5%) is pressured throughout the procedure. It was delivered by a circulating ventilator. At the time of left thoracotomy, connect the catheter to the aorta, pulmonary artery and It was placed in the left atrium. A Konigsberg micromanometer is placed at the apical left ventricle, The lead was placed 1.0 cm below the atrioventricular groove on the left ventricular side wall. Generator (Spectrax 5985; Med tronic, Inc.) was inserted into a subcutaneous pocket in the abdomen. Around the main pulmonary aorta of four animals The box was equipped with a flow probe (Transonic, Inc.). Pericardium We joined together and closed the chest. Seven to ten days after thoracotomy, hemodynamics and left ventricular function Baseline measurements were made to create myocardial blood flow. Next, ventricular pacing is opened. (220 ± 9 bpm (heart rate per minute) 26 ± 4 days). The stimulus amplitude is 2.5V The pulse duration was 0.5 ms. Nine additional pigs (40 ± 7 kg) Used as a troll; for 5 animals, thoracotomy without pacing Surgery and instrumentation were performed and sacrificed 30 ± 7 days after the first thoracotomy. Equipment use Data on right and left ventricular weights, whether or not Since these animals were similar to the roll animals, these data were used as one control group. And pooled. 1-B. Hemodynamic research   Non-sedated conscious after inactivating the pacemaker for at least 1 hour Hemodynamic data was obtained from the animals, and the animals were in a basal state. All day Data were obtained at 7 day intervals in each animal. Left atrial, pulmonary and aortic pressures obtained . Left ventricular dP / dt was obtained from high fidelity left ventricular pressure. Pulmonary artery flow was recorded. Aorta And pulmonary blood samples were obtained for calculation of differences in anterior venous oxygen content. 1-C. Echocardiographic study   Echocardiography is a method of measuring regional myocardial blood flow, which involves the use of individual contrast agents. Injection into the body or animal. The contrast agent (micro-aggregation of galactose) Increases the echogenicity ("whiteness") of the image after injection into the left atrium. Fine Microaggregation is distributed in the coronary arteries and myocardial wall in a manner proportional to blood flow (Skyba et al. Circulation, 90: 1513-1521, 1994). The peak intensity of contrast enhancement Correlate with myocardial blood flow as measured by cross spheres (Skyba et al., Circulation, 90 : 1513-1521, 1994).   Two-dimensional and M-mode using the Hewlett Packard Sonos 1500 imaging system Image was obtained. At the level of the central papillary muscle, an image was obtained by a right parasternal approach, and VHS Recorded on tape. American Society of Echocardiography (Sahn DJ et al., Circul ation 58: 1072-1083, 1978). Porcine ventricular septum (IVS ), And the use of the right parasternal viewpoint, the short-axis M-mode measurement is based on IVS and And through the anatomical side wall. End-diastolic dimensions (EDD) and end-systolic dimensions (ES D), as well as wall thickening, should be measured in at least five random end-tidal beats. And averaged. End diastolic dimensions were obtained at the beginning of the QRS complex. End-systolic dimension, IVS At the end of the outermost or end of the T-wave. Left ventricular contraction function shortened Evaluated using fractional shortening FS = [(EDD-ESD) / EDD] × 100. Pa -Thickening of the centrifuge wall (% WTh) is calculated as% WTh = [(ESWTh-EDWTh) / EDWTh] x 100. Calculated. A pacing protocol was developed to demonstrate the repeatability of echocardiographic measurements. Before starting, the animals were imaged for two consecutive days. Data from different measurements is highly Reproducibility (shortening rate, R^Two= 0.94, P = 0.006; side wall thickening, R^Two= 0.90, P = 0.005 ). All these measurements were obtained with the pacemaker inactivated. 1-D. Myocardial blood flow   Myocardial blood flow is increased by radioactive microsphere technology as described in detail previously. (Roth, DM et al., Am. J. Physiol. 253: H1279-H1288, 1987; Roth, DM Et al., Circulation 82: 1778-1789, 1990). Transmural sample of left ventricular side wall and IVS Is divided into three parts: endocardium, midwall, and epicardium, and blood into each of the three. The flow and transmural flow were measured. Transmural part, blood flow and function measurement Were taken from the area where the echocardiogram was measured so that they corresponded to each bed. Miku Open the ventricular pacing (225 bpm) in a controlled state (no pacing). Inject at the beginning, then again at 7-day intervals during ventricular pacing at 225 bpm During this period, the pacemaker was inactive on days 14 (n = 4) and 21-28 (n = 3). It has become. Myocardial blood flow per beat, myocardial blood flow to heart rate (during microsphere infusion) Calculated by dividing by (recorded) (Indolfi, C et al., Circulation 80: 933-99). 3 (1989)). Mean left and atrial pressures were recorded during microsphere infusion. , An estimate of coronary vascular resistance could be calculated; coronary vascular resistance index = mean atrial pressure-mean left Atrial pressure / transmural coronary blood flow. 1-E. Systolic wall stress   Peri-systolic wall stress is used by the present inventors to assess the long axis of the left ventricle. Could not be measured because no appropriate opinion could be obtained. Therefore, we have , The meridional end-systolic wall stress (Riechek, N. et al., Circulation 65: 99-108 (1982)). Calculated using the following formula: end-systolic wall stress (dyne) = (0.334 × P × D) ÷ [h ( Ih / D), where P is the left ventricular end systolic stress (dyne) and D is the left ventricular contraction End-diameter (cm) and h is end-systolic wall thickness. The meridional end-systolic wall stress is Before pacing and at weekly intervals thereafter (pacemaker off), Calculated for both wall and IVS. 1-F. Terminal surgery   26 ± 2 days after continuation of pacing, animals were anesthetized, intubated, and midline sternotomy Opened. Immerse the still beating heart in saline (4 ° C) and remove the coronary artery immediately. The locus is perfused with saline (4 ° C.) and the right and left ventricles (including IVS) are weighed, Then the transmural samples in each area are quickly frozen in liquid nitrogen and at a temperature of -70 ° C. saved. 1-G. Adenine nucleotide   ATP and ADP were administered to four animals with heart failure (pacing for 28 days) and four animals. Measurements were taken in IVS and lateral transmural samples of control animals. heart failure On the day the animals were sacrificed, the pacemaker was turned off (6 0 min). Samples were obtained in all animals as well. ATP and ADP, as described above Waters high performance liquid chromatography (Pilz, R.B., et al., J. Biol. C hem.259: 2927-2935 (1984)). 1-H. Statistical analysis   Data are expressed as mean ± standard deviation (SD). Control (before pacing) Unique measurements taken at weekly intervals during pacing and pacing were repeated ANOVA ( Crunch4, Crunch Software Corp.). Some comparisons (for example, Binary ANOVA was used in sidewall vs. IVS). Post hoc comparison, Performed using the "Tukey method" as described in the art. 9 animals 21 days of pacing survived; 6 of them survived 28 days of pacing. 28th day The data for animals that survived for a period of time were statistically indistinguishable from data for animals that survived only on day 21. Noh. Therefore, ANOVA was performed on 9 animals at 4 time points: (Before pacing), days 7, 14, and 21-28. Null hypothesis, P <0. Rejected in case of 05 (two-tailed).                                   result 1-I. Hemodynamic studies   Rapid ventricular pacing reduces hemodynamics that were significant 7-14 days after pacing Changes have occurred. On day 7, the animals had increased mean left atrial and pulmonary artery pressure. These pressures became increasingly abnormal with an additional few weeks of pacing (Table 1). Signs of circulatory congestion (tachypnea, ascites, and tachycardia) are evident by day 14-21 Met. Pulmonary artery flow (cardiac output) decreased by day 21 of pacing (control , 3.3 ± 0.1 L / min; Day 21, 1.9 ± 0.4 L / min; P <0.05).                      Table 1. Hemodynamics and left ventricular function   Using an analysis of variance (with repeated measurements), the duration of pacing can be influenced by specific variables. The p-value from ANOVA is shown in the right column. Post hoc trial The test was performed by the Tukey method;^ap <0.05;^bp <0.01;^cp <0.001 (vs. the same variable Control value);^dp <0.05;^ep <0.01;^fp, 0.001 (vs. previous week);^gp <0 .05;^hp <0.01;ⁱp <0.001 (vs. day 7); post hoc test by Tukey's method . Measurements were made using an inactivated pacemaker and expressed as mean ± SD. 7d: P Pacing day 7; 14d: pacing day 14; 21-28d: pacing days 21-28. 1-J. Global left ventricular function   Left ventricular function, hemodynamics after echocardiography and pacemaker inactivation Was evaluated according to the statistical variables. Fractional shortening is called pacing And gradually decreased with the period of aging (P = 0.0001; Table 1), and reached its minimum on days 21-28. Values reached (control, 39 ± 3%; days 21-28, 13 ± 4%; P <0.0002). left End ventricular end diastolic dimensions gradually increased during pacing (P <0.0001; Table 1) and ranged from 21 to It reached its maximum on day 28 (control, 3.9 ± 0.4 cm; days 21-28, 5.8 ± 0. 6 cm; P = 0.0002). Left ventricular peak positive dP / dt is also seen throughout this study. (P = 0.0001; Table 1). Normal increase in preload increases left ventricular peak dP / dt As such, a gradual decrease in peak dP / dt is due to an increase in left ventricular end-diastolic pressure. Concomitantly, this demonstrated a decrease in left ventricular contractility. (Mahler, F et al., Am. J. Cardio l. 35: 626-634 (1975)) 1-K. Left ventricular local function   When the pacemaker is not activated, local left ventricular function is The IVS was evaluated by measuring the percentage of wall thickening. Ventricular pacing from the side wall Caused a significant deterioration in the function of the side wall as compared to IVS (P = 0.001; And Table 2). This difference is significant at day 7 and as sidewall function worsens On day 21 and day 28. Over the course of the experiment, IVS It showed an insignificant reduction in thickness. End-diastolic wall thickness progresses during the experiment Thinning, indicating that it was more severe in the sidewall (Table 2). ).                      Table 2. Left Ventricular Wall Thickening Over Time  End-diastolic wall thickness (EDTh) or wall fertilization using a two-way analysis of variance (repeated measures) % Thickening (WTh) is the pacing period (hours) or area (sidewall, LAT; or heart Affected by septum, IVS, or in EDTh or WTh% It was determined if the changes were different between the two regions (inter). ED at each time The mean values for Th and WTh% were post hoc in two regions by Tukey analysis. Were tested for differences between them. Values represent means ± SD. 7d: 7th day of pacing; 14d Pacing day 14; 21-28d: pacing days 21-28. n = 9. I-L Left ventricular regional blood flow   Subendocardial blood flow per minute at the start of pacing There was an increase over the side wall (FIG. 2 and Table 3). This during pacing Regional blood flow differences persisted throughout the experiment, and the pattern of change in blood flow was 2 (P = 0.006). One minute between two areas during pacing The pattern of change in blood flow is endocardial (P = 0.006), mid-wall (P = 0.002), Either measured on adventitia (P = 0.016) or transmural (P = 0.003) sections (Table 3). In contrast, if the pacemaker is not working, 14 On either day or measured in control condition on days 21-28 Again, subendocardial blood flow showed no local differences (FIG. 2 and Table 3).                          Table 3. Myocardial blood flow over time  Subendocardial blood flow (ENDO) or transmural using a two-way analysis of variance (repeated measures) (TRANS) Blood flow is determined by the duration (hours) or area of pacing (sidewall, LAT; Venous septum, IVS) or the pattern of blood flow changes It was determined whether the two regions differed (inter). The blood flow at each point All mean values were tested for differences between the two area post-hocks by Tukey analysis. Tested. Values represent the mean ± SD for 5 animals. On: Ventricular pacing (22 Microspheres were injected during 5 bpm). Off: Pacemaker is not working No. Day 0 = control; day 14; pacing day 14; days 21-28: pace 21-28 days of ling.   Endocardial to epicardial blood flow ratio did not change significantly as heart failure progressed (P = 0.058). However, with the start of pacing, the endocardial to epicardial ratio And was substantially lower than in IVS (IVS, 1.32 ± 0.23; 77 ± 0.10; P = 0.0002; Table 3). Percentage in both areas throughout the rest of the experiment It was larger than 1.0. Endocardial blood flow per beat (Figure 2 and Table 4) Before the start of the priming, they were similar in both regions (IVS, 0.013 ± 0.003 mL x min)^-1× g^-1× beat^-1; Side wall, 0.012 ± 0.004mL × min^-1× g^-1× beat^-1P = NS). Ventricular page At the beginning of the sing (225 bpm), the heart per beat on the side wall is not IVS There was a local defect in intimal blood flow (IVS, 0.009 ± 0.002 mL x min)^-1× g^-1× beat Movement^-1; Side wall, 0.005 ± 0.001mL x min^-1× g^-1× beat^-1P = 0.001). Day 14 and 2 On days 1-28, the endocardial flow per beat was evident during the pacing and IVS It was lower on the side wall than on the front (FIG. 2 and Table 4). These data are Show that myocardial hypoperfusion in mice begins with the onset of pacing, and this phase Contrasting ischemia persisted. However, the endocardial blood flow per beat is When off, it remained normal in both regions (Figure 2 and Table 4).   Blood flow in both areas tended to increase during the last week of pacing (FIG. 2 and Table 3). This pattern shows a progressive decrease in the coronary resistance index (FIG. 3), which indicates that as heart failure progresses, coronary vasculature and This suggested that degeneration in function could be accompanied by left ventricular reconstitution. Coronary vascular resistance index Is significantly larger on the side wall than on IVS at the beginning of pacing And the pattern of change in coronary vascular resistance was different in the two regions (P = 0.00 12) (Fig. 3). These findings indicate the effects of altered electrical activation on myocardial perfusion. I can do it.                     Table 4. Endocardial blood flow per beatData from the ameroid ischemia model have been previously released from our lab. (Hammond, H.K. and McKiman, M.D., J. Am. Coll. Cardiol., 23: 475-82 (199 Four)). Values represent the mean ± 1 SD. These data are the collateral branches of the ameloid model. The side walls of the dependent (ischemic) and left ventricular pacing-induced heart failure models Similar defects in endocardial blood flow per beat compared to the constantly perfused myocardial region To have. 1-M. Left ventricular end systolic wall stress   Estimated meridional end systolic wall stress for pacing period There was a significant increase (P <0.0001), but the pattern of wall stress And IVS (P = 33), and post-hoc testing was performed at any particular time. No local differences in shrinkage wall stress were also noted at the point. Ventricular end systolic wall The increase in stress at the sidewall (control, 168 ± 40 × 10^Threedyne; day 28 , 412 ± 143 × 10^Threedyne; P = 0.0001) and IVS (control, 159 ± 35 × 10^Threedy ne; Day 28, 480 ± 225 × 10^Threedyne; P = 0.0001). 1-N. Autopsy   At necropsy, animals with heart failure had ascites (mean volume, 1809 mL; range, 300-3500 mL) and dilated thin-walled heart (all four tufts were significantly enlarged) It had. The ratio of ventricular weight to body weight suggests right ventricular hypertrophy and this model was used. (Roth, D.A., et al., J. Clin. Invest. 91: 939-94). 9 (1993)). Left associated with heart failure as compared to weight-matched control animals Ventricular mass did not change (control, 112 ± 10 g; heart failure, 114 ± 17 g); left The ventricular weight to body weight ratio was also similar in both groups (control, 2.8 ± 0.3 g / kg; heart failure, 2.9 ± 0.3 g / kg). In contrast, heart failure has increased right ventricular weight Vs. weight (control, 38 ± 3 g; heart failure, 52 ± 11 g; P = 0.003) and right ventricular weight Body weight ratio (control, 0.09 ± 0.1 g / kg; heart failure, 1.3 ± 0.3 g / kg; P <0.003) is connected with. The paced animals gained 4 kg body weight over the course of the experiment, Partly due to ascites accumulation. Initial weight is used to determine the ratio of left ventricular weight to weight When calculated, the ratio is still more significant than the ratio from a control animal of matching weight Not high. These data indicate a substantial increase in left ventricular weight during the course of the experiment. Confirm that there is no. 1-0. Adenine nucleotide   Control animals showed normal ATP / ADP ratios, which were collected by drill biopsy. Similar to that reported immediately after in pig hearts immersed in liquid nitrogen (Wh ite, F.C., and Boss, G., J. Cardiovasc. Pathol. 3: 225-236 (1990)). Means that the sampling technique used is appropriate. Motion with heart failure object Indicates a sample collected from IVS (control, 14.8 ± 1.1; heart failure, 2.4 ± 0.3; P <0.0001, n = 4 (both groups) and samples taken from the side wall (control , 14.3 ± 4.0; heart failure, 2.4 ± 0.9; P = 0.012, n = 4 (both groups), AT It showed a significant decrease in P / ADP ratio. This will increase the demand and supply of oxygen in the myocardium. I was convinced of the imbalance between pay. 1-P. Myocardial blood flow   Local differences in myocardial blood flow, immediate results of rapid ventricular pacing Play a role in the pathogenesis of local and global dysfunction in induced heart failure Can play. During pacing, between the left ventricular side wall (adjacent to the stimulation site) and the IVS There was a difference in myocardial blood flow per minute. Reduced blood flow starts pacing It soon appeared on the side wall and lasted 21-28 days. Left ventricular side wall (this is Accepts less blood flow than IVS during pacing) between 21 and 28 days of pacing Show a gradual decrease in wall thickening (pacing off). In contrast, IVS ( It accepts more blood flow during pacing), 21-28 days of pacing During this time, maintain relatively normal wall thickening.   Myocardial perfusion per minute does not readily allow assessment of relative myocardial ischemia The present inventors have also expressed coronary blood flow as endocardial blood flow per beat. The physiological basis for such an analysis lies in previous experiments, which Localized subendocardial blood flow (rather than the outer wall of transmural flow) It is a major determinant of regional myocardial contraction under stenotic conditions (Gallagher, K.P. et al. , Am. J. Physiol. 16: H727-H738 (1984)), and increased heart rate This flow-function relationship is shifted down, with lower local functions at any level. (Delbaas, T. et al., J. Physiol 477: 481-496 (1990)). However, if the flow-function relationship is such that local function vs. one beat to correct for heart rate effects A single relationship at different heart rates when plotted as pericardial perfusion This means that when coronary blood flow is reduced, endocardial blood flow per beat is a level of wall function. (Indolfi, C. et al., Circulation 80: 933-993 (1 989); Ross, J., Circulation 83: 1076-1083 (1991)). With the start of pacing , > 50% reduction in endocardial blood flow per beat in the side wall compared to IVS (P <0.001; Table 4).   In previous studies in conscious pigs, we found that endocardial blood flow A 50% decrease causes a 50% decrease in local function, and an intracardiac per beat It is associated with the membrane downstream, similar to that seen on the side wall of this study (Table 4). Prove that. During pacing, decreased blood flow in the side wall was observed during the study It lasted. These data indicate that at the onset of ventricular pacing, Provides evidence of myocardial ischemia. In contrast, IVS function and inner pericardial flow per beat Remained relatively normal. When the pacemaker is turned off, the inner heart per beat Submembrane blood flow remained normal in both areas during the study period, but local dysfunction Followed on the side wall, consistent with the development of stunned myocardium in that area. Therefore, The authors noted that persistent ischemia of the sidewall significantly affected overall function during and after pacing. It is assumed that there is a significant effect. Example 2: Preparation of adenovirus construct   A helper-independent replication-deficient human adenovirus-5 line was used. Vector construction As in the first description of the architecture, we used β-galactosidase and FGF-5. The coding gene was used. pairs encoding β-galactosidase or FGF-5 Recombinant adenovirus was constructed using full length cDNAs. Recombinant adenow The system used to generate the virus was based on transgene inserts. A packing limit of about 5 kb has been imposed. Each CMV promoter and SV- Each of the β-gal and FGF-5 genes operably linked to the 40 polyadenylation sequence Less than 4 kb, well within the scope of packaging construction.   The full length cDNA for human FGF-5 was obtained from plasmid pLTR122E (Zhan et al., Mol. Cell. Bi ol., 8: 3487, 1988) as a 1.1 kb ECOR1 fragment. Contains a 981 bp open reading frame gene and is a shuttle vector. Cloned into the polylinker of the plasmid ACCMVpLpA. FGF-5 Otide and amino acid sequences are disclosed in FIG. 1 of Zhan et al., Mol. Cell. Biol., 8: 3487, 1988. Is done. pACCMVpLpA is described in Gomez-Foix et al., J. Biol. Chem., 267: 25129-25134, 1992. It is listed. pACCMVpLpA contains the 5 'end of the adenovirus serotype 5 genome (Map unit 0-17), where the E1 region is the human cytomegalovirus enhancer Promoter (CMV promoter) and replaced with pUC19 (plus ) Followed by multiple cloning sites (polylinker), followed by the SV40 A denylation signal follows. The control adenovirus encoding lacZ is Based on the E1A / E1B deletion from the top unit 1-9.8. Adenoid encoding FGF-5 Ils (Ad.FGF-5) is based on an EIA / EIB deletion from map units 1.3-9.3. This Both of these vectors contain the entire E1A coding sequence and most of the E1B coding sequence. Remove the throat. Both of these vectors provide anti-sense to adenovirus sequences. With the transgene insert cloned in the sense orientation. Therefore, If one read-through transcript is present, the adenovirus transcript will It is antisense and does not express viral proteins.   Plasmid containing FGF-5 gene was transferred to 293 cells using plasmid JM17 (pJM17). Co-transfected (using a calcium phosphate precipitate) Contains the entire human adenovirus 5 genome with an additional 4.3 kb insert, which Makes pJM17 very large, so that the capsid form into mature adenovirus virions I can't do it. The cells are then overlaid on nutrient agarose. Angiogenesis Infectious viral particles containing the gene are transformed by homologous rescue recombination in 293 cells. And isolated 10 to 12 days later as a single plaque. (Success The identification of recombinant viruses that have been And direct microscopic examination of cytopathic effects (Zh ang et al., Biotechniques 15 (5): 868-872, 1993). Adenovirus obtained The vector contains the transgene but lacks the E1A / E1B sequence and, therefore, It becomes replication deficient. Adenovirus vectors carrying the FGF-5 gene are also Called Ad.FGF-5 in the textbook.   These recombinant adenoviruses are non-replicating in mammalian cells, but They were transformed with E1A / E1B and transduced these essential gene products in trans. Proliferate in the provided 293 cells. Recombinant virus from individual plaques contains 293 cells Proliferate in the vesicle and the viral DNA is characterized by restriction analysis.   The successful recombinant virus was then subjected to two rounds using standard procedures. Plaque purification was performed. Optical density determination of virus stock in 293 cells When measuring by standard, 10 per milliliter (ml)^Ten~Ten¹²The range of virus particles Grow to titer in box. Infect human 293 cells at 80% confluence, and Culture supernatants were collected at 36-48 hours. The virus-containing supernatant is subjected to a freeze-thaw cycle. After that, the cell debris is pelleted by standard centrifugation and the virus is Further purified by two cesium chloride (CsCl) gradient ultracentrifugations, (discontinuous 1. 33 / 1.45 CsCl gradient; CsCl prepared in 5 mM Tris, 1 mM EDTA (pH 7.8); 90,000 × g ( 2hr), 105,000 × g (18hr)). Prior to in vivo injection, the virus stock was Gel filtration through a pharose column (eg, G25Sephadex equilibrated with PBS) It was desalted. When measured by optical densitometry, the final virus concentration is 1 millimeter. About 10 per liter (ml)¹¹Virus particles. Virus stocks It is conveniently stored in cells in medium at minus 70 degrees Celsius. Purified for injection The virus is preferably resuspended in saline. Adenovirus vector Preparation is highly pure and wild-type virus (potentially replicative) is present. Did not. Thus, adenovirus infection and inflammatory invasion in the heart Minimized. Example 3: Gene transfer in adult rat cardiomyocytes   Adult rat cardiomyocytes are harvested using collagenase-containing perfusate according to standard methods. Prepared by Langerdorf perfusion. Laminin-coated plate of rod cells For 24 hours, and encodes the β-galactosidase obtained in Example 2 above. Adenovirus at a multiplicity of infection of 1: 1. After another 36 hours, cells Was fixed with glutaraldehyde and incubated with X-gal. one Persistently, 70-90% of adult cardiomyocytes undergo β-galacterial infection after recombinant adenovirus infection. The tosidase transgene was expressed. At a multiplicity of infection of 1-2: 1, cytotoxicity No sex was observed. Example 4: In vivo gene transfer into porcine myocardium   The adenovirus vector encoding β-galactosidase obtained in Example 2 was , Growing in permissive 293 cells, 1.5 × 10 5 based on the procedure of Example 2.^TenWill The particles were purified by CsCl gradient ultracentrifugation using the final virus titer. 40 kg pigs were anesthetized, ventilated and thoracotomy performed. 26 gauge butterfly injection A needle is inserted into the center of the left interventricular anterior coronary artery (LAD) and the vector (1.5 × 10^TenC Irs particles) were injected in 2 ml volume in phosphate buffered saline. Close the chest, Animals were allowed to recover. Four days after the injection, the animals were sacrificed. Glutar ardhi heart Were fixed, sectioned and incubated with X-gal for 16.5 hours. Parcel After embedding and sectioning, the tissues were counterstained with eosin.   Tissue sections (transmural sections of LAD beds 96 hours after intracoronary injection of adenovirus containing lacZ) The cells that stained positive for β-galactosidase by microscopic analysis Appears in coronary LAD beds with many tissue sections, demonstrating qualitative proportions Significant magnitude of gene transfer was revealed. Myocardial region distal to the LAD circulating bed Areas did not show X-gal staining and served as a negative control. But, Sporadic expression of the gene was observed in muscle and endothelial cells. Closed chest In further studies using intracoronary injection, a significant proportion of myocytes were Showed galactosidase activity (blue staining) and 14 days after gene transfer (n = 8) Similar activity was present. In the area of gene expression, evidence of inflammation or necrosis Did not. Example 5: Pigs for Angiogenesis-Mediated Gene Therapy (Using FGF-5 Transgene) Dell   In this porcine model for myocardial ischemia and heart failure, animals were Subjected to loading by stimulation (pacing). Myocardial dysfunction induced by load And quantifies the extent of inadequate local blood flow, then transfection expresses FGF-5 Performed by intracoronary injection of an exemplary recombinant adenovirus. Stable but After limited endogenous angiogenesis has developed, gene transfer is performed and induced There was blood (similar to angina in the patient). This animal is not ischemic at rest Although not, ischemia developed during activity or during atrial pacing. Control Animals receive recombinant adenovirus expressing lacZ (β-gal) and receive FGF-5 and Independently ruled out that adenovirus itself may have stimulated neovascularization. This This also means that the possibility of sustaining side branch vascular development not associated with gene transfer is controlled. Became a roll. Two weeks after gene transfer, load-induced cardiac dysfunction and local The blood flow was again measured.   Pigs receiving lacZ should be sensitized in the ischemic area before and two weeks after gene transfer. Showed similar degrees of sourcing-induced dysfunction. In contrast, receiving the FGF-5 gene Two weeks later, the animals developed a thickened wall in the ischemic area during the pacing period. Increased blood flow and improved blood flow. This results in an angiogenic transgy (FGF-5) gene transfer is due to local blood flow improvement by newly formed blood vessels. Demonstrated that it was effective in ameliorating local myocardial contractile dysfunction Was. Method Animals and models   Livestock Yorkshire pigs weighing 47 ± 9 kg (probable gland disease, n = 27) were used. Was. Recombinant adenovirus expressing lacZ in two animals (2.0 ml in saline, Ten¹¹Virus particles). And 3 or 5 days after injection Later slaughtered. The remaining 25 animals had catheters in the left atrium, pulmonary artery, and aorta. Devices to measure local blood flow and monitor blood pressure. Offered. Suture wire to left atrium for ECG recording and atrial pacing Functioned. Place an ameroid constrictor around the proximal circumflex of the left coronary artery Was. Ameloid substances are hygroscopic and swell slowly, so after 10 days of placement With minimal infarction (<1% for left ventricle) due to collateral vessel development, gradually Leading to complete arterial occlusion. Myocardial function and blood flow can affect arterial occlusion (ischemic area). Thus, the previously perfused area is normal at rest. But blood flow If myocardial oxygen demand increases, it is insufficient to prevent ischemia. Collateral vessels Development is complete within 21 days of ameloid placement and for at least 4 months It remains unchanged (Roth et al., Am. J. Physiol. 253: H1279-H1288, 1978). Hydraulic A cuff was also placed around the artery (proximal if not distal to the ameloid). these hand The order is described in detail elsewhere (Hammond et al., J. Am. Coll. Cardiol. 23: 475-482, 1). 994 and Hammond et al., J. Clin. Invest. 92: 2644-2652, 1993). Two animals are amelo He died 5 and 7 days after id placement. 38 (± 2) days after ameloid placement, limited If a proper collateral circulation develops and stabilizes, the animal is used for research and pacing induction Sexual local function and blood flow, then lacZ (n = 7, control animals) or Is a recombinant adenovirus expressing any of FGF-5 (n = 16, treatment group) Delivered by entry. Then 14 ± 1 days later, pacing-induced local function and The study defining blood flow and blood flow was repeated. At a later date, AdlacZ (n = 7) and AdFGF-5 (n = 11) The object was sacrificed and the tissue was collected. Five AdFGF-5 animals were studied for 12 weeks after gene transfer. And then sacrificed. Recombinant adenovirus and transgene delivery   The helper-independent replication-defective human adenovirus 5 system was described in Example 2 above. It was prepared as follows.   Animals are anesthetized for intracoronary delivery of the transgene and 5F arterial sheath Was placed in the carotid artery. 5F multipurpose (A2) coronary catheter through sheath It was inserted into the coronary artery. Ameroid closure in all animals , Confirmed by injection of contrast agent into the left main coronary artery. Then the tip of the catheter The end should be cut into the arterial lumen to minimize material loss to the proximal aorta during injection. I put it deep inside. 2 × 10 of recombinant adenovirus¹¹Virus particles Inject 4 ml containing 2.0 ml slowly into left and right coronary artery Delivered by: Assay: (I) Local contractile function and perfusion. 2D and M-mode images from Hewlett Packard Papillary muscle level using an ultrasound imaging system (Hewlett-Packard Sonos 1000) Obtained from the right sternum approach. Hang the awake animal with a painless hanging bandage Researched with minimal body movements. Images using animals in the ground state Record on VHS tape and again during left atrial rhythm (heart rate = 200 beats / min) I Was. These studies were performed one day prior to gene transfer and repeated 14 ± 1 days . Five animals were retested 12 weeks after transfection with FGF-5 and improved It was determined whether the effect on Noh was sustained. Heart rate x blood pressure and left atrial pressure Similar before and after gene transfer in both groups. This is a similar mind The muscle oxygen demand and the load condition are shown. Echocardiographic measurements are standardized standards (Sahn et al., Circulation 58: 1072-1083, 1978). Echocardiogram Animals (n = 5) were imaged on two consecutive days to demonstrate the reproducibility of did. Data from separate measurements were highly reproducible (sidewall thickening: r^Two= 0.90; P = 0.005). Transthoracic echocardiography and sonomicrometry in this model The percent reduction in function measured by the same method was similar (Hammond et al., J. Mol. , Am. Coll. Cardiol. 23: 475-482, 1994 and Hammond et al., J. Clin. Invest. 92: 2644. -2652, 1993). This is the accuracy of the echocardiography for the assessment of ischemic dysfunction Record The analysis was performed without knowledge of the treatment groups.   Contrast agent (Levovist; microaggregate of galactose) is present in the image after left atrial injection. Increases echogenicity (whiteness). Microaggregates form blood in the coronary arteries and myocardial wall. Distributed in a manner proportional to flow. The peak intensity of contrast enhancement Myocardial blood flow as measured by airflow (Skyba et al., Circulation 58: 1072-1083, 1978) Is correlated with 38 (± 2) days after placement of ameloid (10 days after ameloid closure) Minutes later, but prior to gene transfer), conduct an ultrasound-enhanced cardiac study with atrial rhythm (200 bpm) Went between. The study was repeated 14 ± 1 days post-transfection and 5 animals This was repeated 12 weeks after gene transfer using FGF-5. Peak contrast Intensity is measured using a computer-based video analysis program (Color Vue II, Nova Mic rosonics, Indianapolis, Indiana) from video images, An objective measure of video intensity was provided. Data were collected from the ventricular septum (IVS, In the area receiving normal blood flow through the left anterior descending coronary artery) Expressed as the ratio of peak video intensity in the ischemic area (LCx bed) divided by the degree. Super Differences in local blood flow between atrial rhythms, as measured by sonography, In our lab, the difference measured by microspheres in this same model Was similar. This is an ultrasound-enhanced cardiac exam for the assessment of regional myocardial blood flow Record the accuracy of Contrast studies are knowledge of which genes an animal has received Was analyzed without. (ii) Evaluation of angiogenesis   The brachiocephalic artery was cannulated and the other large blood vessel was ligated. Hepari (10,000 IU), papaverine (60 mg), and then potassium chloride (which induces diastolic cardiac arrest). After intravenous injection), the aorta was cross-clamped and coronary blood The tubing was perfused. Glutaraldehyde solution (6.25%, 0.1 M cacodylate buffer) Perfused at 120 mm Hg pressure; heart removed; floor left anterior descending artery, left circumflex artery , And identified using color-coded dyes injected antegrade through the right coronary artery Ameloid was then examined to confirm closure. Normally perfused emptiness Samples collected from blood areas (endocardium (1/3) and epicardium (1/3)) And prepared for microscopic analysis of capillary number. Four 1μm thick Cross sections were obtained as previously described (Mathieu-Costello, Microvasc. Res. 33: 98- 117, 1987 and Poole & Mathieu-Costello, Am. J. Physiol. 259: H204-H210,19 90), from each subsample (endocardium and epicardium in each region). Each sub 8 fields in the sample (randomly selected by systematic sampling ), The number of capillaries around each fiber and the cross-sectional area of the fiber in each The measurement was carried out using an X1400 (Videometric 150, American Innovision). Total 32 The number of capillaries around 5 ± 18 fibers was measured. Capillary density (fiber cross-sectional area Number of samples) by point counting for 15 ± 1 fields per subsample. Estimated. The number of capillaries around the fiber, the cross-sectional area of the fiber, and the capillary density The relative standard errors were 1.4, 4.1 and 4.2%, respectively. Capillary versus fiber Was calculated as the product of capillary density and fiber cross-sectional area. From any group There was no significant difference in the cross-sectional area in myocardial samples. Bromodeoxyuridine ( 50 mg / kg) with 5 animals: control animals (no ameloid); Two animals with ameloid occlusion that received the cZ gene transfer; and FGF two weeks prior Two animals with ameloid occlusion that received the -5 gene transfer were injected intraperitoneally. BR At 36 hours after DU injection, animals are sacrificed and tissues are treated as previously described. (Kajstura et al., Circ. Res. 74: 383-400, 1994). . Duodenal sections were used as a positive control. (iii) DNA, mRNA, and protein expression   Following gene transfer, the left ventricular homogenate determines the presence and expression of the transgene. Received a study to record. Sense primer for CMV promoter (GCA GAGCTCGTTTAGTGAAC; SEQ ID NO: 1) and antisense against internal FGF-5 sequence Polymerase chain reaction using a primer (GAAAATGGGTAGAGATATGCT; SEQ ID NO: 2) The expected 500 bp fragment was amplified by PCR. Beginning of FGF-5 sequence Sense primer (ATGAGCTTGTCCTTCCTCCTC; SEQ ID NO: 3) and internal FGF -5 sequence using an antisense primer (ie, SEQ ID NO: 2) RT-PCR amplified the expected 400 bp fragment. Adenovirus DNA E2 Wild-type or recombinant virus in tissue using primers for the region DNA was detected (TCGTTTCTCAGCAGCTGTTG; SEQ ID NO: 4) and (CATCTGAACTCAAAG CGTGG; SEQ ID NO: 5). Anticipated 900 bp fragment from recombinant virus Amplified. These studies were performed on 200 mg tissue samples from myocardium and other tissues. I went in. The PCR detection sensitivity was one viral sequence per 5 million cells. FGF-5 Polyclonal antibody (Kitaoka et al., Invest. Ophthalmol. Vis. Sci. 35: 3189-3 198, 1994), and cultured rat hearts 48 hours after FGF-5 or lacZ gene transfer. Used in immunoblotting of proteins from the media of visceral fibroblasts. FGF-5 The protein was found in conditioned media after FGF-5 gene transfer, but Not found after transfer of the gene. Methods for PCR and Western Blot Has been described in detail elsewhere (Hammond et al., J. Clin. Invest. 92: 264). 4-2652, 1993, Roth et al. Clin. Invest. 91: 939-949, 1993, and Tsai et al., A m. J. Physiol. 267: H2079-H2085, 1994). In vitro for mitogenic activity To test the transgene, adult rat cardiac fibroblasts were FGF-5 coded Infected with an adenovirus encoding lacZ or an adenovirus encoding lacZ, or Did not infect. Culture medium from these cell cultures is used in NIH 3T3 mouse fibroblasts. Incubated with the cells and the tritiated thymidine incorporation was measured. (Tsai et al., Endocrinology 136: 3831-3838, 1995). (Iv) Adenovirus release during intracoronary delivery   Pulmonary artery blood was injected during intracoronary injection of recombinant adenovirus in three animals To remove continuously. Serum from each sample is subjected to a standard plaque assay Used. Undiluted serum (0.5 ml) with sub-confluent H29 Added to 3 cells; after 10 days, plaques had not formed. But 0.5ml Was diluted 200- to 8000-fold with DMEM (2% FBS), virus plaques Formed by day. Single vascular bed (myocardium) separates coronary and pulmonary arteries I do. If no virus attaches to this bed after injection into the coronary arteries, Pulmonary artery concentration reflects dilution of coronary sinus blood by systemic venous blood over the time of injection Should be. Measurements from our laboratory indicate that coronary artery flow is 5% is indicated. This dilution factor (20x), the duration of the coronary injection, and Using the amount of adenovirus injected and injected, we delivered the pulmonary artery The amount of adenovirus that has been Calculated. This estimate is compared to the measured amount and differences are cleared by the myocardial vascular bed. It was used as an estimate of the amount of virus that was aborted. (V) Evaluation of inflammation   Use hematoxylin / eosin staining and Masson trichrome staining to Vesicular infiltration, cell necrosis, and fibrosis were detected. Mouse ascites, porcine anti-CD4 and anti-C Using D8 monoclonal antibody (1.0 mg / ml; VMRD, Inc., Pullman, Washington) T lymphocytes in spleen (positive control) and frozen section of heart (6 μm) CD4 and CD8 markers on the sphere were detected. These studies were conducted 50 days before sacrifice. Performed on transmural samples of the hearts of six animals that received meloid occlusion: two animals Received no transfection, two received FGF-5 transfection two weeks ago, and two Received the lacZ gene transfer two weeks before. The analysis was performed without knowledge of the treatment groups. (Vi) Statistical analysis   Data are expressed as mean ± 1 s.e.m. Before transfection with FGF-5 and lacZ And later measurements were compared using a two-way analysis of variance (Crunch4, Crunch Sof tware Corporation, Oakland, California). Data from angiogenesis studies are also A two-way analysis of variance was performed. The null hypothesis was rejected when P <0.05. Results using FGF-5 transgene   Is FGF-5 Gene Transfer Effective in Treating Myocardial Ischemia Using Three Measurements? Not evaluated: local contractile function and perfusion (evaluated before and after gene transfer) And the number of capillaries. Perform all measurements without knowing which gene the animal has received Performed (FGF-5 vs. lacZ). Local contractile function and blood flow. Thirty-eight days after ameloid placement, animals receive electrical atrial stimulation ( (Rhythm) showed impaired wall thickening. Pigs receiving lacZ are transfected Showed similar degree of rhythm-induced dysfunction in ischemic area before and two weeks after . In contrast, 2 weeks after FGF-5 gene transfer, the wall thickening of the ischemic area during rhythm was 2.7 times Increased (p <0.0001; FIG. 6). Wall thickening in the normal perfusion area (ventricular septum) Normal during rhythm and unaffected by gene transfer (% wall thickening) : LacZ: 53 ± 8% before gene transfer, 51 ± 6% after gene transfer; FGF-5; 59 before gene transfer ± 4%, 59 ± 6% after gene transfer).   There was improved local blood flow associated with improved function in the ischemic area. la Two weeks after cZ gene transfer, sustained flow deficiency in ischemic area during rhythm (FIG. 8). However, animals receiving the FGF-5 gene transfer had two regions two weeks later. Exhibited uniform contrast enhancement. It improved in the ischemic area Shows blood flow (p = 0.0001). Improved function and perfusion in the ischemic bed last longer Five animals were tested again 12 weeks after FGF-5 gene transfer to determine if did. Each animal had function (P = 0.005; FIG. 6) and perfusion (P = 0.001; FIG. 8). Sustained improvement in Angiogenesis. Uninfected ameloid-stenotic animals (not transfected) Is the same physiological response as animals receiving the adenovirus encoding lacZ. Had the answer. This suggests that the lacZ vector does not adversely affect natural angiogenesis. Indicates that To assess angiogenesis, count the number of myocardial capillaries Quantification was performed using microscopic analysis of the glands (FIG. 9). Of the capillaries surrounding each myocardial fiber The number is FGF- when compared to the same region of the heart of an animal transfected with lacZ. 5 in the endocardium in the ischemic and non-ischemic areas of Was larger (P = 0.038). Therefore, improved local function and perfusion is not It was associated with capillary angiogenesis two weeks after gene transfer. Growth around each fiber The number of isolated capillaries increases in the epicardial portion of the wall following FGF-5 gene transfer. (P = 0.13). Number of capillaries per cross section of fiber and per number of fibers Other measurements of capillaries such as the number of capillaries in the pericardium or epicardium Did not change. DNA, mRNA, and protein expression. Function, perfusion, and each of FGF-5 gene transfer Having established a beneficial effect on the number of capillaries around the fibers, Demonstrating the presence and expression of the transgene was inevitable. Polymer Using reverse transcriptase (RT-PCR) combined with the polymerase chain reaction (PCR) and PCR, Transgene FGF-5 DNA and myocardium in cardiac muscle from animals that have received GF-5 gene transfer And mRNA were detected.   Following gene transfer, the left ventricular sample is tested and tested for transgene uptake and And the expression was recorded. Briefly, three days after transfection of lacZ into the coronary artery, the myocardium was treated with gal and then counterstained with Eosin X120. Uses standard histological techniques Test showed that most muscle cells showed β-galactosidase activity (blue staining) It revealed that. Activity was also observed in all animals that had received the lacZ gene transfer. Was observed 14 ± 1 days after gene transfer. Higher magnification indicates β-galactosidase activity Crossed striations are shown in the containing cells. This confirms gene expression in muscle cells. I accepted. Sense primer for CMV promoter and for internal FGF-5 sequence PCR analysis using antisense primers was performed in three animals that had received the FGF-5 gene transfer. Encodes FGF-5 in ischemic (LCx) and non-ischemic (LAD) regions of animals Performed to confirm the presence of recombinant adenovirus DNA. The results shown in FIG. The presence of the expected 500 bp fragment was confirmed. Next, FGF-5 mRNA was released. Currently tested 14 days after gene transfer. As shown in FIG. 10B, RT-PCR The amplified 400 bp fragment was present in both regions of the two animals. In contrast Control animals showed no signal. Polycloth for FGF-5 Null antibodies were isolated from cultured rat 48 hours after FGF-5 or lacZ gene transfer. Used in immunoblotting of proteins from cardiac fibroblast medium. Figure As shown in 10C, FGF-5 protein was found after FGF-5 (F) gene transfer. However, it was not found after lacZ (β) gene transfer. This is the FGF-5 gene Figure 2 shows protein expression and extracellular secretion after transfer. Finally, PCR, Performed using a set of primers for irs DNA (E2 region) Irs DNA was obtained from two animals that received an intracoronary injection of adenovirus 14 days ago. It was determined whether it was present in the retina, liver, or skeletal muscle. As shown in FIG.10D In addition, the expected 900 bp amplified fragment was used to transform the recombinant adenovirus ( Found only in the containing control lane (+) (as a positive control) Rays derived from retina (r), liver (l), or skeletal muscle (m) of treated animals Was not found in the   Successful gene transfer was recorded in both ischemic and non-ischemic areas. Immunity Blotting was performed on FGF-5 proteins in myocardium from animals that had received the FGF-5 gene transfer. Parkin showed. In further experiments using cultured fibroblasts, the invention The authors conclude that gene transfer of FGF-5 synthesizes FGF-5 and secretes it extracellularly. It was noted that the cells had been given the capacity. Recombinant adenovirus expressing FGF-5 Medium from cultured cells infected with A. showed a mitogenic response (control 14-fold increase; P = 0.005). Finally, two weeks after gene transfer, Their myocardial samples (not liver samples) showed β-galactoscopy on histological examination. It showed tosidase activity. These studies have demonstrated successful in vivo gene transfer and Confirm expression and demonstrate the biological activity of the transgene product.   Two weeks after intracoronary injection of recombinant adenovirus, we used PCR Thus, despite the presence of viral DNA in the heart muscle, the liver, retina, or bone No viral DNA could be detected in the skeletal muscle. In addition, viral DNA is No detectable in urine 2-24 hours after intravenous injection. These experiments are Intracoronary delivery of viral vectors allows for the detection of systemic arterial distribution of the virus by PCR. It was shown to have been minimized to a level below the exit limit. This technology allows for smaller crowns Difficult to achieve in animals with aortic size (eg rabbits) May be.   In order to evaluate the efficiency of adenovirus uptake of myocardium, we Adenoids released from the heart by sampling pulmonary artery blood during intra-arterial injection The amount of irs was measured. Surprisingly, 98.7% of the viruses Removed by the heart during the first pass). During intracoronary delivery of virus Undiluted serum from the pulmonary artery can Was impossible to form. Thus, the present invention provides a cardiac-specific gene delivery system. Provide stem effectively. Assessment of inflammation. Microscopic examination of transmural sections of hearts of animals receiving recombinant adenovirus Showed no inflammatory cell infiltration, cell necrosis, or increased fibrosis. Adeno As a further assessment of the virus-induced cytopathic effect, we used cells Immunohistological study to detect CD4 and CD8 antigens indicating the presence of cytotoxic T cells Was done. These studies were performed in uninfected animals (n = 2) or recombinant adenovirus. Very rare positive cells were shown on transmural sections of heart from animals receiving luz (n = 4). The liver also had no inflammation. Example 6: Gene-mediated angiogenesis using FGF-4 transgene   This example uses FGF-4, a gene encoding a different angiogenic protein. Had demonstrated successful gene therapy. The protocol for FGF-4 gene therapy is Essentially as described in Example 5 above for FGF-5.   Human FGF-4 gene was constructed from mRNA of Kaposi's sarcoma DNA-transformed NIH3T3 cells Isolated from cDNA library. This FGF-4 cDNA is approximately 1.2 kb in length, and A 206 amino acid polypeptide containing a 30 amino acid signal peptide at the N-terminus. (Dell Bovi et al., Cell 50: 729-737, 1987; Bellosta et al., J. Cell. Biol. 121: 705-713, 1993). We have essentially constructed an EcoR1 fragment 1.2 kb in total length. All FGF-4 cDNAs were transferred to the adenovirus vector pACCMVpLpASR (for simplicity, pACS R) at the EcoR1 site. 5 'start site at 243 base pairs Present and the 3 'end was at 863 base pairs. Recombinant Ade encoding FGF-4 Virus (also referred to herein as Ad.FGF-4) and FGF-5 adenovirus Fabricated as described in Example 2 for fabrication.   Expression of FGF-4 in heart tissue (and other sources including liver, skeletal muscle, and eyes) Loss of expression in tissues) by Western blot using anti-FGF-4 antibody for detection. Confirmed by lot analysis. Fraction of FGF-4 on endothelial cell proliferation in vitro The crack promoting effect was also tested.   Forty-five days after ameroid placement, the animals were placed in a stress induced station. The animals were subjected to studies to define local function and blood flow, and then the animals were Received recombinant adenovirus expressing FGF-4, delivered by intravenous infusion (N = 6 animals). Thirteen days later, define stress-induced local function and blood flow The study to do was repeated. The next day, the animals were killed and the tissues were collected. Transgene delivery   As with FGF-5, endogenous angiogenesis is quiescent and induced myocardial ischemia Gene transfer was performed after the appearance of angina pectoris in the elderly). Transgy The animal is anesthetized and a 5F arterial sheath is placed in the carotid artery for intracoronary delivery of Was placed. 5F multipurpose coronary catheter was inserted into the coronary artery through the sheath . Ameloid closure in all animals by contrast injection into the left main coronary artery It was confirmed. The catheter tip was then placed 1 cm into the arterial lumen and during the injection The least material was lost to the proximal aorta. FGF-4 expressing recombinant Denovirus 1.5 × 10¹²5 ml containing virus particles and 3.0 ml into the left coronary artery. And delivered by slow infusion of 2.0 ml into the right coronary artery. Results using FGF-4 transgene Local function and perfusion   Forty-five days after ameloid placement, animals showed impaired wall thickening during atrial rhythm. versus In contrast, two weeks after FGF-4 gene transfer, the wall thickness in the ischemic area increased 2.7 times during the rhythm. Increased (p <0.0001, FIGS. 11 and 12). In the normally perfused area (ventricular septum) Wall thickening is normal during rhythm and is not affected by gene transfer Was. Improvement of function after FGF-4 gene transfer was achieved by FGF-5 or FGF-2LI + signal peptide. Statistically indistinguishable from the gains obtained after gene transfer using “sp” Noh (Fig. 11). The improved function in the ischemic area is due to improved local irrigation. Related to flow (Figure 12). As shown in FIG. 12, the rhythm was adjusted prior to FGF-4 gene transfer. During the period there was a lack of blood flow in the ischemic area. 2 weeks of gene transfer using FGF-4 After a while, a uniform contrast enhancement was observed in the two areas. This is in the ischemic area Improved blood flow (p = 0.0001). Results using FGF-4 were And FGF-2LI + sp were statistically indistinguishable from the results obtained with FIG. ). FGF-2LI + sp is described in Example 7, which refers to FGF-2 containing a signal sequence. To taste. Transgene expression   Exclusive expression of FGF-4 in the heart depends on the sequence encoding this transgene. Confirmed by performing PCR and RT-PCR using primers specific for . FGF-4 DNA and mRNA have been found in the heart but have been found in the eyes, liver, and skeletal muscle. Was not found. These data indicate that Ad.FGF-5 (n = 2) and Ad.FGF-2LI + sp (n = 1) to confirm the derived data. Therefore, this tiger in the heart Exclusive expression of transgenes transduces genes encoding different angiogenic proteins This was confirmed in all four animals that received the containing adenovirus vector. Absence of myocardial inflammation   Transmural myocardial biopsy from three consecutive animals receiving Ad.FGF-4 Was inspected. Animals were sacrificed two weeks after gene transfer. Accepts adenovirus These sections contained inflammatory cells compared to control ameloid animals that did not. There was no evidence of increased vesicle infiltration, necrosis, or fibrosis. This means LA This was true for both D and LCx beds. These slides were Myocarditis was reviewed by a pathologist who made the assessment and in any section It was commented that there was no evidence of this. Example 7: Gene-mediated angiogenesis using FGF-2 mutein   This example uses a third angiogenic protein encoding gene, FGF-2, Demonstrated successful gene therapy. This experiment also demonstrates how angiogenic proteins A pulse that can modify quality to increase secretion and enhance blood flow and function in the heart It demonstrates that the efficacy of tube forming gene therapy can potentially be improved. Human FGF-2 gene The protocol used for treatment is for FGF-5 and FGF-4 above Is substantially the same as what is done.   Acidic FGF (aFGF, FGF-1) and basic FGF (FGF-2) are natural secretory signal sequences But some protein secretion can occur. Alternative secretory pathways (Golgi ) Are described for acidic FGF. Two FGF-2 constructs (FGF-2L I + sp and FGF-2LI-sp) were created; the same without additional signal peptide Improved FGF-2 with additional signal peptide over one protein To test for potency, one would consider a signal for the classical protein secretion pathway. Has a sequence encoding a null peptide (FGF-2LI + sp) and one is a signal No peptide coding sequence (FGF-2LI-sp).   As shown below, FGF-2 has a loop structure of 5 residues, To residue 122. This loop construct was used to generate FGF-2LI loop substitution mutants. Construction is performed by cassette-directed mutagenesis to generate the corresponding 5-residue Is replaced with a loop. Easy, human Glu^3,5Gene encoding FGF-2 (Seddon et al. , Ann.N.Y.Acad.Sci.638: 98-108, 1991), and the T7 expression vector pET-3a (M13) (pET -3a derivative (Rosenberg et al., Gene 56: 125-135, 1987)) And BamHI. Unique restriction endonuclease site , BstBI and SplI, in such a way that the amino acids encoded do not change Encodes FGF-2 segment Ser117-Trp123 by method (ie, silent mutation) You The gene was introduced into the gene at a position adjacent to the codon. Structural alignment of β9-β10 loops in FGF-1, FGF-2 and IL-1β¹ 1 Numbering for FGF-1 and FGF-2 is based on the cDNA sequence encoding the 155 residue form. Deduced from amino acid residue 1 (Seddon et al., Ann. NY Acad. Sci. 638: 98 -108, 1991), and that the numbering of IL-1β is residue 1 of the mature 153 residue polypeptide? (Id.)   Structural analog IL-1β of residues Arg118-Lys119-Tyr120-Thr121-Ser122 of FGF-2 With the human sequence Ala-Gln-Phe-Pro-Asn (115-119) from the corresponding loop of Essentially it was done as follows:   Plasmid DNA, pET-3a (M13) was subjected to BstB1 and Spl1 digestion and the resulting The larger DNA fragment was isolated using agarose gel electrophoresis. D The NA fragment was ligated to the following two synthetic oligonucleotides using T4 DNA ligase: Ligated with double stranded DNA obtained by annealing the otide: 5 ''-CGAAC GATTG GAATCTAATA ACTACAATAC GTACCGGTCTGCGCAGTTTC CTAACTGGTA TGTGGCACTT A AG C-3 '(SEQ ID NO: 9) and 5'-GTACGCTTAA GTGCCACATA CCAGTTAGGA AACTGCGCAG ACCGGTACGT ATTGTAGTTA TTAGATTCCA ATCGTT-3 '(SEQ ID NO: 10), these are BstB1 And ends compatible with ends generated by Spl1 digestion. Lighesi The product is used to transfer to Escherichia coli (DH5α strain) cells. Desired change Replace the heterologous plasmid (FGF-2LI) with the newly introduced Af12 restriction site (underlined above ) Were selected based on their susceptibility to cleavage.   FGF-2LI with and without signal peptide, polymerase chain reaction It was constructed by using a (PCR) based method. FGF-4 signal peptide arrangement The sequence was added to the 5 'of FGF-2LI and the signal peptide was added to the FGF-2LI protein. Used by Forough R. et al. To ensure that they are cut from the substrate The gene cassette was used to obtain secreted FGF-1. 5 'of FGF-4 signal peptide Primer (pF1B: 5'-CGGGATCCGC CCATGGCGGG GCCCGGGACG GC-3 ') (SEQ ID NO: 11), and a second primer for the 5' portion of FGF-1 (pF2R: 5'-CGGAATTCTG TGAAGGTGGT GATTTCCC-3 ') (SEQ ID NO: 12) They used PCR to add a BamHI site at the 5 'end of the FGF-4 signal peptide sequence. DNA fragment containing the first 10 amino acids of FGF-1 and an EcoRI site at the 3 'end. Was synthesized. Another pair, matching the 5 'and 3' sequences of FGF-2LI, respectively Primer (pF3R: 5'-CGGAATTCAT GGCTGAAGGG GAAATCACC-3 '(SEQ ID NO: 13) And pF4HA: 5'-GCTCTAGATT AGGCGTAGTC TGGGACGTCG TATGGGTAGC TCTTAGCAGA C ATTGGAAGA AAAAG-3 '(SEQ ID NO: 14)), we used the second DNA fragment. I got a gment. This has an EcoRI site at the 5 'end and influenza red blood cells It has an agglutinin (HA) tag and an XbaI site at the 3 'end of FGF-2LI. Then this These two fragments were ligated to BamHI and Xba by ligation of the three molecules. At the I site, it was subcloned into the pcDNA3 vector. Plasmid pFGF-2LI / cDNA 3 (similar to pSPFGF-2LI / cDNA3 except that it has no signal peptide Was subcloned in a similar manner. Then both plasmids Were sequenced to confirm the correction of the insert. Then both FG The F-2LI fragment was released from pcDNA3 by digestion with BamHI and XbaI and PACCMVpLpASR (+) (pACSR for simplicity) (this creates a recombinant virus Which is a shuttle vector for the synthesis of Recombinant virus And injection vectors were prepared essentially as described in Example 2. Gene transfer was performed as described in Example 5 (8 animals for FGF-2LIsp +). And 6 animals for FGF-2LIsp- All 10¹¹~Ten¹²With virus particles). Results using FGF-2 mutein   Two weeks after gene transfer with FGF-2LI + sp, the peak during rhythmic stress at 200 bpm The contrast ratio (LCx / LV) was significantly improved compared to before gene transfer. Figure 13 express lacZ, FGF-5, FGF-2LI + sp, FGF-2LI-sp and FGF-4 for comparison. Figure 4 shows the results of using the resulting recombinant adenovirus intracoronary gene transfer. Figure The black bar graph on the right in 13 shows a normal flow ratio using this method. FGF-2LI + sp normalizes peak contrast flow ratio in these animals .   The percentage of wall thickening was also determined by coronary movement of recombinant adenovirus expressing FGF-2LI + sp. It improved 2 weeks after intravenous delivery. FIG. 11 shows lacZ, FGF-5, FGF-2 for comparison. Intracoronary of recombinant adenovirus expressing LI + sp, FGF-2LI-sp and FGF-4 Figure 3 shows the results using gene transfer. The black bar graph on the right side of FIG. Shows normal rate of wall thickening before tress. Local function is FGF-5 by FGF-2LI + sp And improved to the point of being statistically indistinguishable. After gene transfer with FGF-2LI-sp Although there are some improvements that are recognized by The improvement with the peptide was better (Figure 13).

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated country EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, L S, MW, SD, SZ, UG, ZW), EA (AM, AZ , BY, KG, KZ, MD, RU, TJ, TM), AL , AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, E E, ES, FI, GB, GE, GH, GM, GW, HU , ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, M D, MG, MK, MN, MW, MX, NO, NZ, PL , PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, U Z, VN, YU, ZW (72) Inventor Hammond, H. Kirk             United States California 92037,             La Jolla, Waverly Avenue             5770 (72) Inventors Kelly and Tamsin L.             United States California 92037,             La Jolla, Waverly Avenue             5770

Claims (1)

[Claims] 1. A method of treating a patient suffering from congestive heart failure, the method comprising delivering a vector to the patient's heart, wherein the vector is operable with a promoter for expression of the gene. A method comprising a linked gene encoding an angiogenic protein or peptide. 2. 2. The method of claim 1, wherein said congestive heart failure is associated with severe coronary artery disease and myocardial ischemia. 3. 2. The method of claim 1, wherein said congestive heart failure is associated with dilated cardiomyopathy. 4. 2. The method of claim 1, wherein the vector is delivered to a blood vessel supplying blood to the heart's myocardium. 5. 5. The method of claim 4, wherein the blood vessel supplying blood to the myocardium is a coronary artery, a saphenous vein graft, or an internal thoracic artery graft. 6. 5. The method of claim 4, wherein the vector is delivered directly to the left and / or right coronary artery by intracoronary infusion. 7. 7. The method of claim 6, wherein the intracoronary infusion is performed into an approximately 1 cm lumen of the left and / or right coronary artery. 8. 2. The method of claim 1, wherein the angiogenic protein or peptide is a member of the fibroblast growth factor (FGF) family. 9. The method according to claim 8, wherein the angiogenic protein or peptide is FGF-4, FGF-5, FGF-6, FGF-1, or FGF-2. 10. 10. The method of claim 9, wherein said angiogenic protein is FGF-4. 11. 10. The method of claim 9, wherein said angiogenic protein is FGF-5. 12. 11. The method of claim 10, wherein said angiogenic protein is FGF-1. 13. 11. The method of claim 10, wherein said angiogenic protein is FGF-2. 14． 2. The method of claim 1, wherein said angiogenic protein comprises a secretory signal sequence. 15. 15. The method of claim 14, wherein said angiogenic protein is FGF-2 modified by inclusion of a heterologous secretory signal sequence. 16. 2. The method of claim 1, wherein the angiogenic protein or peptide is a member of the vascular endothelial growth factor (VEGF) family. 17． The method according to claim 16, wherein the angiogenic protein or peptide is VEGF-121, VEGF-145, VEGF-165, VEGF-189, VEGF-206, VEGF-167, VEGF-186, or VEGF-C. . 18. 2. The method of claim 1, wherein said angiogenic protein or peptide is a member of the platelet derived growth factor (PDGF) family. 19. 19. The method of claim 18, wherein said angiogenic protein or peptide is PDGF-A or PDGF-B. 20. 2. The method of claim 1, wherein the angiogenic protein or peptide is a member of the insulin-like growth factor (IGF) family. 21. 2. The method of claim 1, wherein said promoter is a CMV promoter. 22. 2. The method of claim 1, wherein said promoter is tissue-specific for cardiomyocytes. 23. 23. The method of claim 22, wherein said tissue-specific promoter is a ventricular cell muscle-specific promoter. 24. 24. The method of claim 23, wherein the ventricular myocyte-specific promoter is a ventricular myosin light chain-2 promoter or a ventricular myosin heavy chain promoter. 25. The method according to claim 1, wherein the vector is a viral vector or a lipid-based vector. 26. 26. The method of claim 25, wherein said vector is a viral particle. 27. 27. The method of claim 26, wherein said viral particle is a replication-defective adenovirus (Ad). 28. 27. The method of claim 26, wherein said viral particle is a replication-defective adeno-associated virus (AAV). 29. About 10 ⁶ to 10 ¹⁴ viral vector particles are delivered by injection, The method of claim 2 6. 30. Adenoviral vector particles of about 10 ⁸ to 10 ¹² is delivered by infusion method of claim 27. 31. A method for preventing and alleviating ventricular remodeling in a patient suffering from myocardial infarction, the method comprising delivering a vector to the patient's heart, wherein the vector comprises a promoter for gene expression. A method comprising an operably linked gene encoding an angiogenic protein or peptide, thereby increasing myocardial blood flow and mitigating adverse ventricular remodeling. 32. 32. The method of claim 31, wherein the vector is delivered to a blood vessel supplying blood to the heart's myocardium. 33. 33. The method of claim 32, wherein the blood vessel supplying the myocardium is a coronary artery, a saphenous vein graft, or an internal thoracic artery graft. 34. 33. The method of claim 32, wherein the vector is delivered directly to the left and / or right coronary artery by intracoronary infusion. 35. 35. The method of claim 34, wherein the intracoronary infusion is performed into an approximately 1 cm lumen of the left and / or right coronary artery. 36. 32. The method of claim 31, wherein said angiogenic protein or peptide is a member of the fibroblast growth factor (FGF) family. 37. 37. The method of claim 36, wherein the angiogenic protein or peptide is FGF-4, FGF-5, FGF-6, FGF-1, or FGF-2. 38. 38. The method of claim 37, wherein said angiogenic protein is FGF-4. 39. 38. The method of claim 37, wherein said angiogenic protein is FGF-5. 40. 40. The method of claim 39, wherein said angiogenic protein is FGF-1. 41. 40. The method of claim 39, wherein said angiogenic protein is FGF-2. 42. 32. The method of claim 31, wherein said angiogenic protein comprises a secretory signal sequence. 43. 43. The method of claim 42, wherein said angiogenic protein is FGF-2 modified by inclusion of a heterologous secretory signal sequence. 44. 32. The method of claim 31, wherein said angiogenic protein or peptide is a member of the vascular endothelial growth factor (VEGF) family. 45. The method of claim 44, wherein the angiogenic protein or peptide is VEGF-121, VEGF-145, VEGF-165, VEGF-189, VEGF-206, VEGF-167, VEGF-186, or VEGF-C. . 46. 32. The method of claim 31, wherein said angiogenic protein or peptide is a member of the platelet derived growth factor (PDGF) family. 47. 47. The method of claim 46, wherein the angiogenic protein or peptide is PDGF-A or PDGF-B. 48. 32. The method of claim 31, wherein said angiogenic protein or peptide is a member of the insulin-like growth factor (IGF) family. 49. 32. The method of claim 31, wherein said promoter is a CMV promoter. 50. 32. The method of claim 31, wherein said promoter is tissue-specific for cardiomyocytes. 51. 51. The method of claim 50, wherein said tissue-specific promoter is a ventricular myocyte-specific promoter. 52. 52. The method of claim 51, wherein the ventricular myocyte-specific promoter is a ventricular myosin light chain-2 promoter or a ventricular myosin heavy chain promoter. 53. 32. The method according to claim 31, wherein said vector is a viral vector or a lipid-based vector. 54. 54. The method of claim 53, wherein said vector is a viral particle. 55. 55. The method of claim 54, wherein said viral particle is a replication-defective adenovirus (Ad). 56. 55. The method of claim 54, wherein said viral particle is a replication-defective adeno-associated virus (AAV). 57. About 10 ⁶ to 10 ¹⁴ viral vector particles are delivered by injection, The method of claim 5 4. 58. Adenoviral vector particles of about 10 ⁸ to 10 ¹² is delivered by infusion method of claim 55.