EA008538B1 - Techniques and compositions for treating cardiovascular disease by in vivo gene dilivery - Google Patents

Abstract

Methods are provided for treating patients with cardiovascular disease, including heart disease and peripheral vascular disease. The preferred methods of the present invention involve in vivo delivery of genes, encoding angiogenic proteins or peptides, to the myocardium or to peripheral ischemic tissue, by introduction of a vector containing the gene into a blood vessel supplying the heart or into a peripheral ischemic tissue.

Description

Government Funded Study Statement

A certain portion of the study described here was supported in part by grants from the United States Government with grant numbers UA-H0281201, NY768218 and 1P50NB53773.01 awarded by the National Institutes of Health. The United States Government may have certain rights in relation to this invention.

Cross reference to related applications

This application is a partial continuation of the United States application with registration number 09/609080, filed June 30, 2000, which is a partial continuation of the United States application with registration number 09/435156, filed November 5, 1999, which is a partial continuation of the United States application with registration number 08/722271 filed February 27, 1996 (currently receiving a patent), which is a partial continuation of the United States application with registration number 08/485472 filed June 7, 1995 (currently Time to get a patent I8 RA ex! number 5792453), which was a continuation of the United States application Serial No. 08/396207, filed February 28, 1995 g .; and this application is a partial continuation of the international application PCT / I899 / 02702, filed February 9, 1999, which is a partial continuation of the application of the United States with registration number 09/021773, filed February 11, 1998, which is a partial continuation of the application of the United States with registration number 08/485472, filed June 7, 1995 (currently received the patent I8 Ra! ei! No. 5792453); and this application is a partial continuation of the United States application with registration number 09/068102 filed April 30, 1998, which is a partial continuation of the United States application with registration number 08/852779 filed May 6, 1997 and is a partial continuation of the United States application with registration number 09/132167, filed August 10, 1998. All of the above patent applications are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to methods and compositions for the treatment of cardiovascular disease with gene therapy ίη νίνο. More specifically, this invention relates to methods and polynucleotide constructs for treating heart disease and / or for treating peripheral vascular disease by delivering ίη νίνο angiogenic transgenes.

State of the art

The American Heart Association (1995 statistical appendix) has reported that about 60 million adults in the United States suffer from cardiovascular disease. Cardiovascular disease is responsible for nearly a million deaths annually in the United States, accounting for over 40% of all deaths. Each year in the United States, there are approximately 350,000 new cases of angina pectoris (angina pectoris, aptum suture). general condition of the coronary artery, characterized by temporary periods of myocardial ischemia, leading to chest pain. Similarly, each year, approximately 400,000 patients are diagnosed as having congestive heart failure CHE, another manifestation of heart disease that is the most common indiscriminate cause of hospitalization in the United States. In 1996, an estimated 725,000 people suffered from peripheral vascular disease, of which more than 100,000 needed limb amputations.

Myocardial ischemia is an aspect of heart dysfunction that occurs when the heart muscle (myocardium) does not receive sufficient blood supply and, therefore, is deprived of the necessary levels of oxygen and nutrients. Myocardial ischemia can lead to various heart diseases, for example, angina pectoris, heart attack and / or congestive heart failure. The most common cause of myocardial ischemia is atherosclerosis (also called coronary artery disease (CAO), (i.e., coronary heart disease or CHD), which causes blockages in the coronary arteries, blood vessels that provide blood flow to the heart muscle. Existing treatments for myocardial ischemia includes pharmacological therapies, surgical bypass of the coronary arteries and percutaneous revascularization (restoration of blood supply to the limb) using methods such as allone angioplasty: Standard pharmacological therapy is based on strategies that include either increasing the blood supply to the heart muscle or decreasing the heart muscle's need for oxygen and nutrients, for example, increased blood supply to the myocardium can be achieved using agents such as calcium channel blockers or nitroglycerin: These agents are believed to increase the diameter of diseased arteries by relaxing smooth muscle in the walls of the arteries. Reducing the oxygen and nutrient demand of the heart muscle can be accomplished either by agents that reduce the hemodynamic load on the heart, such as arterial vasodilators, or agents that reduce the contractile response of the heart to a specific hemodynamic load, such as beta-adrenergic receptor antagonists. Surgical treatment of coronary heart disease usually involves shunting of diseased arterial segments with strategically placed shunts.

- 1 008538 transplants (usually transplantation of the saphenous vein or internal mammary artery). Percutaneous revascularization is usually based on the use of catheters to reduce constriction in affected coronary arteries. All these strategies are used to reduce the number, or to eliminate ischemic attacks, but they all have various disadvantages, some of which are discussed below.

Many patients with heart disease, including many of those whose severe myocardial ischemia led to a heart attack, are diagnosed as having congestive heart failure. Congestive heart failure is defined as abnormal cardiac function leading to insufficient cardiac output (cardiac output) to meet metabolic needs (Vtaip ^ a1b, E. (eb.), Ιη: Neai Ogosahs. Ν.Β. Zaipbegk, РЫ1абе1рЫа Rad 426, 1988). An estimated 5 million people in the United States suffer from congestive heart failure. Once the symptoms of CHE become moderately severe, the prognosis is worse than with most cancers, due to the fact that only half of these patients are expected to live more than 2 years , ν.Β. Zaipbegk, Rl1abe1rLa, Rada 471-485, 1988). Drug therapy can initially relieve symptoms of CHE (e.g., edema, exercise intolerance, shortness of breath) and, in some cases, prolong life. However, the prognosis of this disease, even with drug treatment, remains bleak, and the frequency of occurrence of CHE increases (see, for example, Vaidytap, K., Satbyuodu Οίπίοδ 13: 27-34, 1995). Symptoms of CHE include shortness of breath, fatigue, weakness, swollen legs, and exercise intolerance. On physical examination, patients with heart failure tend to increase heart rate and respiratory rate (a sign of fluid in the lungs), edema, expansion of the jugular veins and, usually, an enlarged heart. The most common cause of CHE is atherosclerosis, which, as discussed above, causes blockages in the coronary arteries that supply blood to the heart muscle. Thus, congestive heart failure is most often associated with coronary artery disease, which is so severe in its scale or suddenness that it leads to the development of chronic or acute heart failure. In such patients, extensive and / or sudden occlusion of one or more coronary arteries prevents sufficient blood flow to the myocardium, leading to severe ischemia and, in some cases, myocardial infarction or death of the heart muscle. Subsequent myocardial necrosis tends to be accompanied by progressive chronic heart failure or an acute state of low cardiac output - both of which are associated with high mortality.

Most patients with congestive heart failure tend to develop an enlarged, slightly contracting heart, a condition called dilated cardiomyopathy (or OSM, as it is called in this application). OSM is a condition of the heart that is usually diagnosed by detecting a dilated hypocontractile left and / or right ventricle. Again, in most cases, congestive heart failure associated with an enlarged heart is the result of coronary artery disease, often so serious that it can cause one or more myocardial infarction. However, in a very small number of cases, OSM can occur in the absence of characteristics of a coronary artery disease (for example, atherosclerosis). In some cases in which dilated cardiomyopathy is not associated with coronary heart disease (atherosclerosis), the cause of OSM is known or suspected. Examples include familial cardiomyopathy (e.g., associated with progressive muscular dystrophy, myotonic muscular dystrophy, Friedreich's ataxia, and hereditary dilated cardiomyopathy), infections leading to myocardial inflammation (e.g. infections with various viruses, bacteria, and other parasites), non-infectious inflammations (e.g. due to autoimmune diseases, reprititis cardiomyopathy, allergic reactions or transplant rejection), metabolic disorders that cause myocardium arditis (including dietary, endocrinological and abnormal electrolytes) and exposure to toxic agents that cause myocarditis (including alcohol, as well as certain chemotherapeutic drugs and catecholamines). However, in most cases of dilated cardiomyopathy (OSM) not associated with atherosclerosis, the cause of the disease remains unknown, and this condition is therefore called “idiopathic dilated cardiomyopathy” (or SCM). Despite the potential differences in the underlying causes of the disease, most patients with severe CHE have enlarged, thin-walled heart (i.e., OSM) and most of these patients exhibit myocardial ischemia (even though some may not have visible atherosclerosis). In addition, patients with OSM may experience angina pectoris attacks (angina pectoris), although they may not have severe coronary artery disease.

The emergence of CHE poses several major therapeutic problems, including progressive myocardial damage, hemodynamic insufficiency associated with dilated heart, the threat of systemic emboli (blood clots), and the risk of ventricular arrhythmias. Conventional revascularization is not preferred for the treatment of non-coronary heart disease (CHD) OSM, since occlusive (clogging) coronary disease is not

- 2 008538 primary problem. Even for patients for whom the cause of ESM is known or suspected, damage is usually not easy to reverse. For example, in the case of adriamycin-induced cardiotoxicity, cardiomyopathy is usually irreversible and leads to the death of more than 60% of affected patients. For some patients with OSM, the cause itself is unknown. As a result, there are no commonly used treatments for OSM. Doctors have traditionally focused on alleviating the symptoms present in a patient exhibiting dilated cardiomyopathy (OSM) (e.g., weakening fluid retention by diuretics and / or reducing the need for heart muscle oxygen and nutrients by angiotensin converting enzyme inhibitors). As a result, approximately 50% of patients with dilated cardiomyopathy (OSM) die within two years of diagnosis, often from sudden cardiac arrest associated with ventricular (ventricular) arrhythmias. Ventricular remodeling is an aspect of heart disease that often occurs after myocardial infarction and often leads to further damage to the ventricular function. In many cases, after healing of myocardial infarction, ongoing ischemia in the border region between the healed heart attack and normal tissue and other factors lead to dilatation and / or remodeling of the remaining heart tissue. These dilatation or remodeling, although initially adaptive in nature, often lead to further impairment of ventricular function. Whole heart dilatation occurs in approximately 50% of patients who have such heart attacks, and remodeling usually develops within a few months after myocardial infarction, although it can also occur early, in the range of 1-2 weeks after a heart attack. Weak left ventricular function is the most accurate and only prognostic factor for an adverse outcome after myocardial infarction. Thus, the prevention of ventricular remodeling after myocardial infarction would be a favorable factor. One approach to attempting to prevent ventricular remodeling is to treat a patient suffering from myocardial infarction with an angiotensin converting enzyme (ACE) inhibitor (see, for example, McEopaD, K.M., Tgaik. Accos. At. Ρΐινκίααηκ 103: 229-235, 1990 ; Juices, 1. Syi. Sagyu1. 18 (8irr1. IV)! U-4-GU-12, 1995). However, these agents are only marginally effective in preventing harmful ventricular remodeling, and new therapies are required.

Current treatments for CHE include pharmacological therapies, coronary revascularization procedures, and heart transplantation. Pharmacological therapies for CHE have been aimed at increasing the power of heart contraction (through the use of inotropic agents such as digitalis (digitalis) and beta-adrenergic receptor agonists), reducing the accumulation of fluid in the lungs and elsewhere (using diuretics) and reducing the load on the heart (by using agents that reduce systemic vascular resistance, such as angiotensin converting enzyme inhibitors). Beta-adrenergic receptor antagonists have also been tested. Although such pharmacological agents can improve symptoms and potentially prolong life, the prognosis in most cases remains bleak.

Some patients with heart failure due to associated coronary artery disease may experience improvement, at least temporarily, as a result of revascularization procedures such as surgical bypass surgery and coronary artery angioplasty. Such procedures have potential benefits when the heart muscle is not dead, but may be dysfunctional due to insufficient blood flow. If normal coronary blood flow is restored, previously a dysfunctional myocardium may contract more normally, and heart function may improve. However, if the patient has an insufficient microvascular (capillary) bed (for example, as can be found in more severe patients with CHE), revascularization will rarely restore cardiac function to normal or almost normal levels, even if mild improvements are sometimes noted. In addition, the occurrence of unsuccessful bypass vascular shunts and restenosis after angioplasty poses additional risks for patients being treated with these methods. Heart transplantation may be the preferred choice for patients with CHE who do not have other concomitant diseases and are relatively young, but this choice is suitable only for a small number of such patients and only at high cost. In general, it can be seen that CHE has a very weak prognosis and weakly responds to current therapies.

A further complication of physiological conditions associated with CHE are various natural adaptations that tend to appear in patients with a dysfunctional heart. Although these natural reactions may initially improve heart function, they often lead to other problems that can aggravate the disease, lead to deadlock in treatment, and have adverse effects on survival. There are three such adaptive reactions commonly observed in patients with congestive heart failure (CHE): (ί) volume retention (retention), induced by changes in sodium reabsorption, which expands plasma volume and initially improves cardiac output; (ίί) an increase in heart (due to dilatation and hypertrophy), which can increase the stroke volume of blood ejected by the ventricle in one contraction, while maintaining

- 3 008538 normal tension of the walls; and (ίίί) increased release of norepinephrine from adrenergic nerve endings colliding with the heart, which, by interacting with cardiac beta-adrenergic receptors, seeks to increase the heart rate, thereby increasing cardiac output. However, each of these three natural adaptations tends to ultimately lead to failure for various reasons. In particular, fluid retention tends to lead to edema, to fluid retention in the lungs, which disturbs respiration. An enlarged heart can lead to harmful remodeling of the left ventricle, followed by severe dilatation and increased wall tension, thereby exacerbating heart failure. Finally, prolonged exposure of the heart to norepinephrine tends to make the heart insensitive to adrenergic stimulation and is associated with a poor prognosis.

Disorders of the peripheral vasculature, like heart disease, often occur due to reduced blood flow to tissue (for example, skeletal muscle), which (like heart disease) becomes ischemic (anemic), in particular, with an increase in metabolic needs (for example, during physical exertion) . So, atherosclerosis present in the peripheral vessel can cause ischemia in the tissue supplied with the affected vessel. This problem, known as peripheral arterial arteriosclerotic occlusion disease (ΡΑΟΌ), most commonly affects the lower limbs of patients. As with other forms of cardiovascular disease, this condition, or at least some of its symptoms, can be treated with medications such as aspirin or other agents that lower blood viscosity, or surgery, such as transplantation of arteries, surgical removal deposits of fatty plaques or endovascular treatments, such as angioplasty. Although symptoms can be improved, the effectiveness of such treatments is usually insufficient, for reasons similar to those described above.

Recently, studies of methods for treating cardiovascular disease have turned to therapeutic agents associated with angiogenesis. Angiogenesis is usually called the development and differentiation of blood vessels. It is known that a number of proteins, commonly referred to as “angiogenic proteins,” stimulate angiogenesis. Such angiogenic proteins include members of the family of fibroblast growth factor (POP), family of vascular endothelial growth factor (TAR), platelet growth factor (REOR) and insulin-like growth factor (UR), and others (as described in more detail below and in the literature in this areas). For example, members of the POP and UBOR families were recognized as regulators of angiogenesis during growth and development. Their role in stimulating angiogenesis in adult animals has recently been investigated (as discussed below). The angiogenic activity of the POP and UEOR families was investigated. For example, it was shown that the acidic protein POP (aOPOP) in a collagen-coated matrix, when placed in the peritoneal cavity of adult rats, led to a well-vascularized and normally perfused structure (Tyotrkop e! A1., Prg. Ia! 1. Asab. 8εί. I8A, 86: 7928-7932, 1989). Injection of the main protein POP (LOPP) into the coronary arteries of adult rabbits during coronary occlusion resulted in reduced myocardial dysfunction, less damage to the myocardium and an increase in blood vessels in the risky bed (Uapashka \\ 'a-M | \ ha e! A1., 8c1epse, 257: 1401-1403, 1992). Similar results were reported in animal myocardial ischemia models using the LOBP protein (Nagaba e! A1., 1. C11p. 1 batch1. 94: 623-630, 1994; Ipdeg e! A1., At. 1. RukyuB, 266: H1588-H1595 , 1994). An increase in collateral blood flow has been shown in dogs treated with the UEOR protein (Wapb e! A1., Spsi1a1yup 89: 2183-2189, 1994).

However, the difficulties associated with the potential use of infusions of such proteins to stimulate cardiac angiogenesis include achieving the correct localization for a sufficient period of time and ensuring that this protein is and remains in the correct form and concentration necessary to absorb and stimulate the angiogenic effect in myocardial cells. Protein concentration, which is initially high (for example, after a bolus injection), but then drops rapidly (through clearance by the body), can be both toxic and ineffective. Another difficulty is the need for repeated infusions or injections of this protein.

Some publications have postulated the use of gene transfer to treat or prevent a disease, including some heart diseases. See e.g. No. 11yatk, Rgokres! Gog Oepe Tyegaru OG 1x11e1shs Neag! Egaeake. Atepsap 1oit1 oG Meb1sa1 8aepsek 306: 129-136, 1993; 8sype1beg apb Rgepsy, Thieu AbgeSH OG Abepouyik: Oepe Tyegaru Gog Sagbyuaksi1ag Egaeake. Sisi1a1yup 88: 1937-1942, 1993; and Mahig e! A1., Sogopagu Kek! ekop1k apb Oepe Tyegaru, Mo1eci1ag apb Ce11i1ag Ryagtasoduod, 21: 104-111, 1994. In addition, some groups proposed the transfer of wywo genes to the myocardium using plasmids, retrovirus, adenovirus and other vectors (see ., for example, Wagg e! a1., 8pir1etep1 II, Sps1a! yup, 84 (4): Alk1gas! 1673, 1991; Wagg e! a1., Oepe Tieg., 1: 51-58, 1994; Rzeps e! a1 ., Sisi1a1yup, 90 (5): 2402-2413, 1994; Rzepsy e! A1., S1gsi1abop, 90 (5): 2414-2424, 1994; Rzhepsy e! A1., S1gsi1aup, 90: 1517 Alk1gas! Io 2785, 1994; Be1bep, e! A1., No. 094/11506 (26 Mau 1994); Sixtap e! A1., C1gs. Kek., 73 (6): 1202-1207, 1993; Kakk-E1k1eg e! A1 ., Proc. Yab. Asab. 8sg I8A, 90: 1149 8-11502, 1993; MiYyayekeg e! A1., Nit. Oepe Tyeg., 6: 1457-1465, 1995; MiYyayekeg e! A1., C1g. Kek. 77 (6): 1077-1086, 1995; and Combapb e ! a1., At.Tyogas. 8igd., 60 (3): 721-728, 1995.

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However, in general, these reports provide little more than suggestions or suggestions regarding potential therapies. Of the messages providing data obtained for animals, the majority do not use models that are quite similar to the real states of ίη νίνο. In addition, tried ίη νίνο methods usually suffer from one or more of the following disadvantages: insufficient transduction and transgene expression efficiency; a noticeable immune response to the vectors used, including inflammation and tissue necrosis; and, importantly, the relative inability to target transduction and transgene expression for the organ of interest (e.g., gene transfer targeted to the heart led to a transgene also delivered to non-heart sites such as the liver, kidneys, lungs, brain and testes of test animals). As an example, the insertion of a transgene into a population of rapidly dividing cells will lead to a significantly reduced duration of transgene expression. Examples of such cells include endothelial cells, which create the inner layer of all blood vessels, and fibroblasts, which are dispersed throughout the heart. Targeting a transgene in such a way that only desired cells will receive and express this transgene, and in such a way that this transgene will not be distributed systemically, is also a critical consideration. If this is not achieved, the result will be systemic transgene expression and the problems associated with it. For example, inflammatory infiltrates have been documented after adenovirus-mediated gene transfer to the liver (Wapd e! A1., Propos. No. 111. Asab. 8c1. I8L, 91: 4407, 1994). In addition, inflammatory infiltrates have been documented in the heart after direct injection into the myocardium through a needle inserted into the myocardial wall (Egepsy e! A1., C1gci1abop, 90 (5): 2414-2424, 1994).

A method for treating a number of forms of congestive heart failure associated with beta-adrenergic signaling has recently been demonstrated by Nashshopb e! A1., in PCT publication \ νϋ 98/10085, published March 12, 1998. This method includes delivering genes encoding elements of a beta adrenergic signaling pathway to a patient’s heart with a heart disease associated with a decrease in beta adrenergic signaling.

SUMMARY OF THE INVENTION

This invention relates to methods and compositions for treating a cardiovascular disease, comprising delivering a transgene encoding an angiogenic protein or peptide to an affected tissue by introducing a vector containing this transgene into said tissue where the transgene is expressed and the symptoms of the disease are alleviated. For example, contractile (contractile) function and / or blood flow in the median can be increased by introducing a transgene-containing vector into at least one coronary artery of a patient, this transgene being delivered to and expressed in the myocardium. Methods are also provided for use in peripheral vascular diseases, for example, arteriosclerotic occlusion disease of peripheral arteries (ΡΑΟΌ). As described and illustrated here, these methods are therefore applicable to the treatment of heart disease, peripheral vascular disease, and the like.

The present invention provides a method for increasing blood flow in an ischemic (anemic) tissue of a patient, comprising delivering an angiogenic protein or peptide to an ischemic region of said tissue by introducing a vector containing a transgene to this tissue, as a result of which this transgene is expressed in the tissue and blood flow in this tissue increases. In one aspect, a vector containing a transgene encoding an angiogenic protein or peptide is introduced into ischemic skeletal muscle, where the angiogenic protein or peptide is expressed and causes an increase in blood flow and a decrease in ischemia in the tissue. Alternatively, the vector is inserted into a blood vessel supplying blood to the ischemic tissue (for example, by introducing into the coronary artery supplying the myocardium, or into the peripheral artery, such as the femoral artery supplying blood to the skeletal muscle). The vectors used in this invention may be a plasmid or preferably a viral vector, for example, defective in replication with adenovirus. Various aspects and therapeutic applications of the present invention are described and illustrated below.

In one aspect, the invention provides a method for increasing a contractile (contractile) function of a patient’s heart, comprising delivering a transgene encoding an angiogenic protein or peptide to the patient’s myocardium by introducing a vector containing this transgene to the myocardium (preferably delivery to one or more coronary arteries), where the transgene is delivered to the myocardium and expressed, and the contractile function of the heart increases. The transgene can be introduced, for example, by intracoronary injection into one or more coronary arteries or transplants of the saphenous vein or internal mammary arteries that supply blood to the myocardium. The transgene preferably encodes at least one angiogenic protein or peptide. The vectors used in this invention may be a plasmid or preferably a viral vector, including, by way of example, replication-defective adenovirus. By injection of a stock solution of a viral vector (preferably containing a small amount or not containing wild-type virus), deep (at least about 1 cm) into the lumen of one or both coronary arteries or grafts (preferably both in the right and left coronary arteries or grafts ) and preferably in an amount of 107-1013 viral particles, as determined by optical densitometry

- 5 008538 (more preferably 109-1011 viral particles), it is possible to locally transfect the desired number of cells, in particular cardiac myocytes, into the myocardium-encoding genes encoding an angiogenic protein or peptide, thereby maximizing the therapeutic efficiency of gene transfer and minimizing unwanted angiogenesis in extracardial regions (areas outside the heart) and the possibility of an inflammatory reaction to viral proteins. When using a cardiomyocyte-specific promoter, expression can be further limited by cardiac myocytes so that the potentially harmful effects of angiogenesis in non-cardiac tissues, such as the retina, are further reduced.

Kits and compositions are also provided that can be used in accordance with these therapeutic methods.

Brief Description of the Drawings

FIG. 1 graphically shows the percent wall thickening during (electro) pacemaking in a pig model of congestive heart failure. Percent wall thickening was evaluated sequentially in the interventricular septum and lateral wall before pacing (day 0) and every 7 days as heart failure progressed (as described in example 1). Symbols represent averages; error bars define one standard deviation (1 8Ό). Two-way analysis of variance ΑΝΟνΑ (repeated measurements) showed that the percentage thickening of the wall is affected by the duration of pacing (P <0.001) and the area (P = 0.001). In addition, the picture of changes in wall thickening was different between the two regions (P <0.0001). The average values for the percentage thickening of the wall at each time point were tested for differences between these two regions of ro81 braids (after that) according to the Tukey method; P values for these analyzes are shown below the error bars.

FIG. 2A and 2B represent graphically subendocardial blood flow during electrical pacemaking in the pig model of congestive heart failure described in Example 1.

For FIG. 2A, subendocardial (endo) blood flow was evaluated in the interventricular septum and lateral wall under the conditions listed along the x axis. The day refers to the day of prolonged pacing, in which these measurements were received (0, the beginning of pacing; 14, 14 days; 21-28, from day 21 to day 28). PACE (electrical pacemaker) indicates whether blood flow determinations were obtained with an activated pacemaker (+) or with an inactive (0). The pacemaker frequency was 225 beats per minute (Lmf) (See Table 3 here for digital values). Symbols indicate average values; error bars define one standard deviation (1 8Ό). Two-way analysis of variance ΑΝΟνΑ (repeatable measurements) showed that subendocardial blood flow is affected by the duration (P = 0.0001) and the region (P = 0.017) of pacing. In addition, the picture of changes in subendocardial blood flow was different between the two areas (P <0.006). The average values for subendocardial blood flow at each time point were tested for differences between the two regions of the ros braids according to the Tukey method; P values for these analyzes are shown below the error bars.

For FIG. 2B, subendocardial blood flow per stroke was evaluated in the interventricular septum and lateral wall under the conditions listed along the x axis. Symbols and conditions correspond to those described in FIG. 2A. (See table 4 here for numerical values). Two-way analysis of variance ΑΝΟνΑ (repeated measurements) showed that subendocardial blood flow per stroke is affected by duration (P = 0.0001) and the area of pacemaker (P = 0.0198).

FIG. 3A graphically shows the meridial wall tension at the end of the systole, evaluated sequentially in the interventricular septum and lateral wall before pacing (day 0) and every 7 days as heart failure progresses (as described in Example 1). Two-way analysis of variance ΑΝΟνΑ (repeated measurements) showed that the duration of cardiac stimulation affects the systolic tension of the wall (P <0.0001). However, the pattern of systolic wall tension was similar in both areas. Measurements were performed with an inactive pacemaker.

FIG. 3B graphically illustrates coronary vascular resistance during pacing in the pig congestive heart failure model described in Example 1. Coronary vascular resistance index was evaluated sequentially in the interventricular septum and lateral wall under the conditions listed along the x axis. The symbols and conditions are the same as those described in FIG. 2. Two-way analysis of variance ΑΝΟνΑ (repeated measurements) showed that duration of the coronary vessels (P = 0.0001) and the area of cardiac pacing (P = 0.013) influence the resistance of the coronary vessels. In addition, the pattern of changes in coronary vascular resistance was different between the two areas (P = 0.0012). The average values for the resistance of the coronary vessels at each time point were tested for differences between the two regions of the brachial braid (after that) according to Tukey's analysis. This analysis showed that the resistance of the coronary vessels was higher in the lateral wall than with the septum immediately after the start of pacing (P value is indicated under the error column).

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FIG. 4 shows schematically the construction of an exemplary replication defective recombinant adenoviral vector useful for gene transfer, as described in the examples below.

FIG. 5 is a schematic diagram that shows a rescue recombination construct encoding an adenovirus transgene.

FIG. 6A and 6B show diagrammatically the contractile function of the treated animals described in Example 5. FIG. 6A shows the results in animals examined 2 weeks after gene transfer, and FIG. 6B shows the results 12 weeks after gene transfer.

FIG. 7A, 7B and 7C show diagrams corresponding to contrast myocardial echocardiography. White areas indicate contrast enhancement (greater blood flow), and dark areas indicate reduced blood flow). FIG. 7A illustrates acute occlusion of bCx in a normal pig. FIG. 7B illustrates the difference in contrast enhancement between the interventricular septum (1U8 = MJP) and the LBx bed 14 days after gene transfer from ΙαοΖ. which indicates a different blood flow in two areas during atrial pacemaking (200 beats per minute (Lp)). In FIG. 7C, contrast enhancement seems equivalent in the MJP bed (1U8) and the LCx bed 14 days after gene transfer from PCP-5, which indicates similar blood flow in these two areas during atrial pacing. These results are described in Example 5.

FIG. Figure 8 shows the maximum contrast ratio (correlating with blood flow), expressed as the ratio of the maximum intensity of the video image in the ischemic region (LCx bed) divided by the maximum intensity of the video image in the interventricular septum (1U8), measured from video images using a computer-based video analysis program during atrial pacemaker (200 beats per minute) before gene transfer and 14 ± 1 days after gene transfer with 1; · ιοΖ (control gene) and with PCP-5, and in 5 animals, 12 weeks after the transfer of the PCP-5 gene (described in example 5). The blood flow to the ischemic bed remained 50% of normal after gene transfer with the control gene, but increased 2 times higher than normal after gene transfer with PCP-5 (p = 0.0018), and this effect persisted for at least 12 weeks.

FIG. 9 shows the number of vessels quantified by microscopic analysis in ischemic and non-ischemic regions after gene transfer from PCP-5 and from 1; · ιοΖ (described in Example 5). An increased number of capillaries surrounding each fiber was observed in the ischemic and non-ischemic regions of animals that received the transferred PCP-5 gene (p <0.038), compared with animals that received the ΙαοΖ gene.

FIG. 10A, 10B and 10C are obtained from gels documenting the expression of DNA, mRNA and protein after gene transfer of an angiogenic transgene to the myocardium in accordance with this invention (as described in Example 5). FIG. 10Ό was obtained from the gel after PCR amplification, showing the absence of any gene transfer to the retina, liver or skeletal muscle of the treated animals (as described in example 5).

FIG. 11 shows a comparison of wall thickening achieved with ίη νίνο gene transfer using angiogenic gene constructs, PCP-4, PCP-5 and PCP-2Y +/- §p (i.e., POP-2Y plus or minus the secretory signal peptide), as described in examples 6 and 7.

FIG. 12 shows that improved function in the ischemic region after transfer of the PCP-4 gene (as indicated by thickening of the wall) was associated with improved regional perfusion.

FIG. 13 shows a comparison of perfusion (blood flow) obtained from an injection of PCP-4, PCP-5 and PCP2Y +/- §p (= PCP-2Y plus or minus the secretory signal peptide), as described in examples 6 and 7.

FIG. 14 shows a comparison of wall thickening resulting from gene transfer with PCP-2 plus (PCP2Y + §p) or minus the secretory signal peptide (PCP-2b1 -§p) described in Example 7.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS Definitions "Heart Disease" refers to acute and / or chronic cardiac dysfunctions. Heart disease is often associated with a decrease in contractile (contractile) function of the heart and may be associated with an observed decrease in blood flow to the myocardium (for example, as a result of coronary artery disease). Manifestations of heart disease include myocardial ischemia (anemia), which may result from angina pectoris, heart attack, and / or congestive heart failure.

"Myocardial ischemia" is a condition in which the heart muscle does not receive sufficient levels of oxygen and nutrients, which is usually due to insufficient blood supply to the myocardium (for example, as a result of coronary artery disease).

“Heart failure” is defined clinically as a condition in which the heart does not provide sufficient blood flow to the body to satisfy metabolic needs. Symptoms include shortness of breath, fatigue, weakness, swollen legs, and exercise intolerance. On physical examination, patients with heart failure tend to increase heart rate and respiratory rate, wheezing (as a sign of fluid in the lungs), swelling, extensibility of the jugular veins, and, in many cases, an enlarged heart. Patients with severe heart disease

- 7 008538 deficiency suffer from high mortality; typically 50% of patients die within two years of developing this condition. In some cases, heart failure is associated with severe coronary artery disease ("IHD". SLE). usually leading to myocardial infarction and or to progressive chronic heart failure. or an acute condition with a low minute cardiac output, as described here and in the literature in this field. In other cases, heart failure is associated with dilated cardiomyopathy without associated severe coronary artery disease.

"Peripheral vascular disease" refers to acute or chronic dysfunction of the peripheral (i.e. non-cardiac) vasculature and / or tissues. supplied with peripheral vasculature. As with heart disease. peripheral vascular disease usually leads to insufficient blood flow to the tissues. supplied with blood by this vasculature. moreover, this lack of blood supply can lead. eg. to ischemia or. in some cases. to the death of tissue cells. Aspects of peripheral vascular disease include. without limitation. arteriosclerotic occlusive disease of peripheral vessels (ΡΑΟΌ) and peripheral muscle ischemia. Often, symptoms of peripheral vascular disease manifest in the patient's limbs. especially in the legs.

In application here. the terms “having a therapeutic effect” and “successful treatment” have essentially the same meaning. In particular. a patient. a person suffering from heart disease is essentially “successfully treated” in relation to this condition. if this patient detects an observed and / or measurable decrease or absence of one or more symptoms of heart disease after receiving the transgenic angiogenic factor in accordance with the methods of the present invention. A reduction in these signs or symptoms may also be felt by the patient. So. Success rates for treating heart disease include a patient showing or feeling that any of the symptoms of angina pectoris (angina pectoris) have decreased. fatigue. weaknesses. shortness of breath. swelling of the legs. wheezing. heart rate or respiratory rate. swelling or distensibility of the jugular veins. The patient may also exhibit higher exercise tolerance. having a smaller heart with improved ventricular function and cardiac function and usually need fewer hospital visits for heart conditions. Improvements in cardiovascular function may be sufficient to meet the patient's metabolic needs. and the patient may not detect symptoms with mild exertion or at rest. Many of these signs and symptoms can be easily observed with the naked eye and / or they can be measured by routine procedures. known to the doctor. Signs of improved cardiovascular function include increased blood flow and / or improved contractile function in the treated tissues. As described below. blood flow in a patient can be measured by visualization with thallium (as described by VGaiit16 in Neag! e! a1. Blood flow before and after angiogenic gene transfer can be compared using these methods. Improved heart function is associated with reduced signs and symptoms. as noted above. In addition to echocardiography. it is possible to measure the ejection fraction (left ventricle. b) of nuclear (non-invasive) methods. known in the art. Blood flow and contractile function can also be measured in peripheral tissues. treated in accordance with this invention.

"Angiogenic protein or peptide" means any protein or peptide. able to stimulate angiogenesis or angiogenic activity. i.e., the development of blood vessels.

"Polynucleotide" refers to the polymeric form of nucleotides of any length. or ribonucleotides. or deoxyribonucleotides. or their analogues. This term denotes the primary structure of this molecule and. Consequently. includes double-stranded and single-stranded DNA. as well as double-stranded and single-stranded RNA. It also includes modified polynucleotides. eg. methylated and / or capped polynucleotides.

Recombinant. as applied to polynucleotide. denotes. that this polynucleotide is the product of various combinations of cloning steps. restriction and / or ligation and other procedures. which lead to the design. which is different from a polynucleotide. detectable in nature.

A gene or transgene refers to a polynucleotide or part of a polynucleotide. containing sequence. which encodes a protein. In most situations. desirable. so that the gene also contains a promoter. functionally linked to a coding sequence for efficiently promoting transcription. Enhancers may also be included. repressors and other regulatory sequences for modulating gene activity. as is well known in this field (see .. for example. references cited below).

The terms polypeptide. the peptide and protein are used interchangeably to refer to polymers of amino acids of any length. These terms also include proteins. which are post-translationally modified through reactions. which include glycosylation

- 8 008538, acetylation and phosphorylation.

A heterologous component refers to a component that is introduced into a different object (organism) or which is produced in a different object (organism) than the one in which it is naturally localized. For example, a polynucleotide obtained from one organism and introduced by genetic engineering into a different organism is a heterologous polynucleotide that, when expressed, can encode a heterologous polypeptide. Similarly, a promoter or enhancer that is isolated from its native coding sequence and is operably linked to a different coding sequence is a heterologous promoter or enhancer.

A promoter, as used herein, refers to a polynucleotide sequence that regulates the transcription of a gene or coding sequence with which it is functionally linked. A large number of promoters, including constitutive, inducible and repressed promoters, from a large number of different sources, are well known in this field (and are identified in databases, for example, in SepWap) and are available as or inside cloned polynucleotide sequences (for example, from depositories such as ATCC, as well as other commercial or individual sources).

An enhancer, as used herein, refers to a polynucleotide sequence that enhances the transcription of a gene or coding sequence with which it is functionally linked. A large number of enhancers, from a large number of different sources, are well known in this field (and identified in databases, for example, in SepWap) and are available as or within cloned polynucleotide sequences (for example, from such depositories as ATCC, as well as other commercial or individual sources). A number of polynucleotides containing promoter sequences (for example, the well-known promoter of SMU) also contain enhancer sequences.

“Functionally (operatively) connected” means the immediate proximity of two or more components, where the components described in this way are in a relationship that allows them to function in their own way. A promoter is operably linked to a gene or coding sequence if this promoter regulates the transcription of this gene or this coding sequence. Although a functionally linked promoter is usually located upstream of the transcription (left) of the coding sequence, it is not necessarily adjacent to it. An enhancer is operably linked to a coding sequence if this enhancer enhances the transcription of the coding sequence. Functionally related enhancers may be located upstream of the transcription (to the left) of the coding sequences, inside or to the right of the coding sequences. A polyadenylation sequence is operably linked to a coding sequence if it is located at the right end (in the course of transcription) of the coding sequence, so that transcription passes through the coding sequence to the polyadenylation sequence.

A replicon refers to a polynucleotide containing a replication origin site (origin) that enables replication of a polynucleotide in a suitable host cell. Examples include the chromosomes of the target cell into which heterologous nucleic acid (e.g., nuclear and mitochondrial chromosomes) can be integrated, as well as extrachromosomal replicons (e.g., replicating plasmids and episomes).

Gene delivery, gene transfer, and the like, as used herein, refers to the introduction of an exogenous polynucleotide (sometimes called a transgene) into a host cell, regardless of the method used for administration. Such methods include a wide variety of well-known methods, such as vector-mediated gene transfer (e.g., viral infection / transfection or various protein or lipid-based gene delivery complexes), as well as methods that facilitate the delivery of naked polynucleotides (e.g., electroporation , gene gun delivery, and various other methods used to administer polynucleotides). The introduced polynucleotide may be stable or may be temporarily stored in the host cell. Stable preservation usually requires that the introduced polynucleotide either contain a replication origin compatible with the host cell or integrates into the replicon of the host cell, for example, an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome. A number of vectors are known that can mediate gene transfer into mammalian cells, as is known in the art and described herein.

Η νίνο gene delivery, gene transfer, gene therapy (gene therapy) and the like, as used herein, means the administration of a vector containing an exogenous polynucleotide directly into the body of an organism, such as a human or non-human mammal, as a result of which this exogenous polynucleotide is introduced into the cell of such an organism ίη νίνο.

A vector (sometimes called a carrier for gene delivery or gene transfer) refers to a macromolecule or complex of molecules containing a polynucleotide to be delivered to a host cell, ίη νίΐτο or ίη νίνο. The polynucleotide to be delivered may contain a coding sequence of interest in gene therapy.

- 9 008538

The vasculature or vasculature are terms related to the system of vessels carrying blood (as well as lymphatic fluid) through the body of mammals.

A blood vessel refers to any vessel in the mammalian vasculature, including arteries, arterioles, capillaries, venules, veins, sinuses, and blood vessels. In preferred aspects of the present invention, for the treatment of heart disease, vectors containing angiogenic transgenes are inserted directly into the vascular channel supplying blood to the myocardium, such vascular channels include coronary (coronary) arteries, as well as vessels such as saphenous veins or internal grafts arteries of the mammary gland.

Artery means a blood vessel through which blood passes from the heart. Coronary arteries supply blood to the tissues of the heart itself, while other arteries supply blood to the remaining organs of the body. The usual structure of an artery consists of a lumen surrounded by a multilayer arterial wall.

The individual or patient refers to a mammal, preferably a large mammal, most preferably a human.

Treatment (treatment) or therapy, as used herein, refers to the administration to an individual patient of agents that are capable of causing a prophylactic, curative or other beneficial effect on a given individual.

Gene therapy, as used herein, means administering vectors containing a therapeutic gene or genes to an individual patient.

A therapeutic polynucleotide or therapeutic gene refers to a nucleotide sequence that is capable, when transferred to an individual, of causing a prophylactic, therapeutic or other beneficial effect on that individual.

References

The practice of this invention will apply, unless otherwise indicated, generally accepted methods of molecular biology and the like that are within the skill of the art. Such methods are explained in the literature. See, for example, Mo1eci1ag C1occp. A aota OSU Mapia1 (8attook e a1, So1b 8rppd Nagog aota OSU, So1b 8rppd Nagog, Ν.Υ., 1989!.!)!; Siggep! Rgo! Osn ίη Mo1esi1ag Vyuodu (R. Aikibe1 e! A1. Ebk., 1987 apb irba! Eb); Ekkepba1 Mo1esi1ag Vyuodu (T. Vgsshp eb., 1Kb Rgekk 1991); Oepe Ehrtekkup Tesbpoodu (Ooebbe1 eb., Asabetu Rgekk 1991); Me! Bobk cat S1opshd apb Apa1uk1k ok Eisatuobs Sepek (A. Wo! Bete11 e! A1. Eb., Wah! 1e !! Pb1. 1990); Oepe Tgapkkeg apb Exrtekkup (M. Kpedeg 8! Osc! Op Rgekk 1990); KesotYpap! ΩΝΛ Me! 1yub1odu (K. Ai e! A1. Eb., Asabetu Rgekk 1989); RSK .: And Rgasbsa1 Arrgoasb (M. MsRbegkop e! A1., 1Kb Rgekk a! Ohogkog Ishuegkyu Rgekk 1991); Ce11 Sibige kog Vyusbesh1k! To (K. Abatk eb., E1ke \ tag 8s1epse RybbNegk 1990); Sepe Tgapkkeg Wes! Ogkog Mattabap Se11k (1. Mb1eg apb M. Sa1ok ebk., 1987); Mattabap Ce11 Vyu esbpo1odu (M. Vibeg eb., 1991); Ashta1 Ce11 Sibig (1. Ro11agb e! A1. Eb., Nitapa Rgekk 1990); Sibige ok Ashta1 Se11k, 2pb Eb. (K. Rgekbpeu e! A1. Ebk., A1ap K. b1kk 1987); E1o \\ 'Su! Ee! Gu apb 8otbpd (M. Me1ateb e! A1. Ebk., Abeu-Shk 1990); Mebubk w Ephutoodu series (Asabetyu Rgekk, 1ps.); Tesbshdiek W 1tiposbet1k! Gu, (S. Vi11osk apb R. Reg! Gikh ebk., Asabetyu Rgekk 1982, 1983, 1985, 1989); Forget ok! Extrept! A1 1tipo1odu, (Ό. Aeb apb s B1asket11, ebk.); Ce11i1ag apb Mo1eci1ag 1tipodoodus (A. Albac e! A1., A.V. 8aipbegk Co., 1991, 1994); Shtep! Rgo! Oso1k sh 1typoodu (1. Sobdap e! A1.! Bk. 1991); series Appia1 Ke \ te \ y ok 1tipo1odu; Series Abuapsek sh 1ttypo1odu; About 11profile 8up! Back1k (M. Sab eb., 1984); Ashta1 Ce11 Sibig (K. Rgekbpeu eb., 1Kb Rgekk 1987); Abegyuks1egok1k series, Tbtotoyk apb Uaksi1ag Vyuodu (b1rrsho !!, Abbatk apb Abkshk ribkbegk kog! be Atepsap Neag! Akkoshabop); series C1gci1abop (b1rr1pso !!, Abbot apb Abkshk ribkbegk kog! be Atepsap Neag! Akkoaa! yup); and a series of Sbsi1a! yup Kekeagsb (b1rrtso !!, Abbot apb Abkshk ribkbegk kog! be Atepsap Neag! Akkoshabop).

Additional links describing the delivery and surgical support that can be used in the methods of the present invention include the following links: Toro1, E1 (eb.), TBE Tech! (A.V. 8aipbetk Co. 1994); Ki! Runbokg, KV, Uaksi1ag 8itdegu, 3gb Eb. (A.V. 8aipbetco Co. 1989); Tbe Sesb Tekh! Bok ok Mebushe, 19! B Fuck. (A.V. 1992); and 8aL1k! op, Ό, Tbe Tekh! Loo ok ok 8itdegu, 14! b Eb. (A.V. 1991). Additional references describing the types of cells found in blood vessels and the types of cells of the vasculature that can be used in the methods of this invention include the following link: A. B1oot apb Ό. Raetseb, A Tech! Bw ok ok H1k! O1od (U.V. 8aipbet. Co. 1975).

Various publications have reported the use of gene transfer to prevent disease, including heart disease. See, for example, Me! Bob w Uboduod, Uo1. 7: Sepe Tgapkkeg apb Exrtekkup Rgo! Os1k, Mittau, E. (eb.), Ae1kk, Sbyop, Ν.Ι, 1991; Mahig e! A1., Mo1eci1ag apb Ce11i1ag Vu1odu, 21: 104-111, 1994; Rgepsb, No / 18: 222-229, 1993; Abbat, 1oitpa1 ok Mebka1 8s1epsek 306: 129-136, 1993; and 8sbpe1bet, Sbsi1abop 88: 1937-1942, 1993.

The references cited in the above section are hereby incorporated by reference herein to the extent that these references describe methods that are used in the practice of this invention.

- 10 008538

Inclusion as a reference

All documents cited in this application, including patents, patent applications and other publications, are hereby incorporated by reference.

Detailed description of various preferred options

Various preferred aspects of the present invention are summarized below and further described and illustrated in the following detailed descriptions and figures.

This invention relates to methods and compositions for the treatment of cardiovascular diseases, including myocardial ischemia, heart failure and peripheral vascular disease.

In a method for treating heart failure, a vector construct containing a gene encoding an angiogenic protein or peptide is targeted to the patient's heart, whereby this exogenous angiogenic protein is expressed in the myocardium, thereby facilitating cardiac dysfunction by improving blood flow and / or improving the contractile function of the heart. Improved heart function ultimately leads to a decrease or disappearance of one or more symptoms of heart disease or heart failure and prolongs life beyond the expected death.

Similarly, in the treatment of peripheral vascular disease in accordance with this invention, a vector construct containing a transgene encoding at least one angiogenic protein or peptide is targeted at the affected tissue, for example, ischemic skeletal muscle, as a result of which the synthesis of exogenous angiogenic protein facilitates or cures symptoms of peripheral vascular disease, for example, by increasing blood flow to the affected (for example, ischemic) tissue site, and / or in the muscle, by improving to ntraktilnoy function of the affected muscles.

Thus, in a preferred aspect, the present invention provides a method for treating a heart disease of a patient having myocardial ischemia, comprising delivering a vector with an integrated transgene to the patient’s myocardium by intracoronary injection, preferably by injecting the vector directly into one or both coronary arteries (or grafts), whereby the transgene is expressed and blood flow and / or contractile function are improved. By way of illustration, when using a vector containing a transgene encoding an angiogenic protein or peptide, such as, for example, ECE-5, ECE-4, aECE, BECE and / or UECE, the vector being delivered to the heart where the protein or peptide is It is produced to a therapeutically significant degree in the myocardium continuously for extended periods of time, angiogenesis can be stimulated in the affected area (region) of the myocardium. Other transgenes, such as transgenes encoding beta-adrenergic signaling proteins or other heart or cardiac muscle enhancing proteins, can also be used as described below in combination with the use of an angiogenic transgene. The vectors used in this invention may be a plasmid or preferably a viral vector, for example, a replication defective adenovirus or adeno-associated virus (ALU). Injection of an initial solution of a viral vector, for example, a solution that contains a relatively small amount of wild-type virus or does not contain wild-type virus, deep into the lumen of one or both coronary arteries (or grafts), preferably both in the right and left coronary arteries (or grafts) and preferably in an amount of 10 ⁷ -10 ¹³ viral particles as determined by optical densitometry (more preferably 10 ⁹ to 10 ¹¹ viral particles) can be locally transfect a desired number of cells in the pores Gennes myocardium encoding an angiogenic protein or peptide genes, thereby maximizing therapeutic efficacy of gene transfer, and minimizing undesirable angiogenesis in non-cardiac regions (areas outside the heart) and the possibility of an inflammatory response to viral proteins.

In another preferred aspect, the invention can also be used to treat a patient suffering from congestive heart failure by delivering a vector with an integrated transgene to the heart of said patient, the vector containing a transgene encoding an angiogenic protein or peptide, whereby this transgene is expressed in the myocardium , leading to increased blood flow and increased function in the heart. Among these patients suffering from congestive heart failure are patients who find dilated cardiomyopathy and patients who have severe myocardial infarction, usually associated with severe or occlusive coronary artery disease. The vector is preferably introduced into a blood vessel supplying blood to the myocardium of the heart so as to deliver the vector to the myocardium. Preferably, the vector is inserted into the lumen of the coronary artery, a saphenous vein graft or an internal mammary artery transplant; most preferably, the vector is introduced into the lumen of both the left and right coronary arteries. Intracoronary injection is preferably performed as a single injection, relatively deep into each selected artery (s) (for example, preferably at least 1 cm into the lumen of this vessel (s)).

The methods of the present invention are also applicable to prevent or ameliorate harmful ventricular remodeling in a patient who is suffering (or may suffer) from myocardial infarction

- 11 008538 card. Again, a vector containing a transgene encoding an angiogenic protein or peptide, preferably operably linked to a promoter for expressing this gene, is delivered to the patient’s heart where this transgene is expressed and harmful ventricular remodeling is attenuated.

Transgenes encoding angiogenic proteins and peptides

One or more transgenes encoding an angiogenic protein or peptide factor that can enhance blood flow and / or contractile function can be used in this invention. Any protein or peptide exhibiting angiogenic activity, as measured by the methods described here and in this field, can potentially be used in connection with this invention. A number of such angiogenic proteins are known in the art and new forms are identified routinely. Examples of suitable angiogenic proteins or peptides are members of the family of fibroblast growth factors (PCP), vascular endothelial growth factors (slaughter), platelet growth factors (ROSE), insulin-like growth factors (1CP) and others. Members of the PCP family include, but are not limited to, ECE (PCP-1), ECE (PCP-2), PCP-4 (also known as Y8! / K83), ECE-5, ECE-6. It has been shown that UESE is expressed by cardiac myocytes in response to июη νίτο and ίη νίνο; it is a regulator of angiogenesis under physiological conditions, as well as during an adaptive reaction to pathological conditions (Your e! a1. ΟΐΌΐι1ηΙίοη 89: 2183-2189, 1994). The UESE family includes, but is not limited to, members of the UESE-A subfamily (e.g., UESE-121, UESE-145, UESE-165, UESE-189, and UESE-206), as well as members of the UESE-B subfamily (e.g. UESE-167 and UESE-186) and the UESE-S subfamily. ROSE includes, for example, ROSE A and ROSE B, and 1CE includes, for example, 1SE-1. Other angiogenic proteins and peptides are known in the art and new ones are regularly identified. The nucleotide sequences of the genes encoding these and other proteins, and the corresponding amino acid sequences are also known in the art (see, for example, CeηBaηk sequence database).

Angiogenic proteins and peptides include precursors of peptides that are post-translationally processed into active peptides, and derivatives and functional equivalents of angiogenic proteins and peptides. Derivatives of an angiogenic protein or peptide are peptides having a similar amino acid sequence and preserving, to some extent, one or more activities of a related angiogenic protein or peptide. As is well known to those skilled in the art, applicable derivatives typically have significant sequence similarity (at the amino acid level) in regions or domains of a protein associated with angiogenic activity. Similarly, those skilled in the art will understand that a functional equivalent is a protein or peptide that has an activity that can replace one or more activities of a particular angiogenic protein or peptide. Preferred functional equivalents retain all the activities of a particular angiogenic protein or peptide; however, a functional equivalent may have activity that, when quantified, is stronger or weaker than the activity of a peptide or wild-type protein.

For details regarding the ECE family, see, for example, Wigdes, Αηη. Ν.Υ. Asab. δα. 638: 89-97, 1991; Vigdezz e! a1. Appy. Not. Vusyet. 58: 575-606, 1989; MiYaizeg e! A1., Nit. Sene TKeg. 6: 1457-1465, 1995; / Cap e! A1., Μο1. Ce11. Vu1., 8: 3487, 1988; δοάάοη e! A1., App. Ν.Υ. Asab. δα. 638: 98-108, 1991. In relation to person s1 / K83 (ie ECE-4), see Tapa e! A1., Proc. No.! 1. Asab. δα. I8A 84: 2980-2984, 1087. For the UESE-A protein of a person, see, for example, Tlzieg e! a1. 1. Βίο1. Sket. 206: 11947-11954, 1991, and references therein; MiYaizeg e! A1., ATP. Kez. 77: 1077-1086, 1995; and # and £ e1b e! A1., AO 98/10071 (12 Magsy 1998). Other variants of known angiogenic proteins have also been described; for example, variants of UESE proteins and proteins related to UESE, see, for example, Wapb e! A1., AO 99/40197, (12 Aidiz! 1999); and ΒοΙιΚη e! A1., AO 98/493300, (5 Shkheshg 1998). Combinations of angiogenic proteins and gene delivery vectors encoding such combinations are described in Caο! a1. υδδΝ 09/607766, P1eb 30 More than 2000, entitled Eia1 Do not! Seme TKegaru Sotrostz ^! ^ Οηz aib Mebubz ο £ ye, included thereby the link in full. As also understood by those skilled in the art, angiogenic proteins can stimulate angiogenesis by enhancing the expression, stability, or functionality of other angiogenic proteins. Examples of such angiogenic proteins and peptides include, for example, regulatory factors that induce a response to hypoxia (for example, hypoxia-induced factors such as H1E-1, H1E-2, etc.; see, for example, Ashchd e! A1., Proc. No.! 1. Asab. δα. and 8A 90 (9): 4304-8, 1993; Eo ^ zu еye e! a1., Ms1. Ce11. 1ο1. 16 (9): 4604-13, 1996; δαιιαι / а е! а1., К ^ еу Ιη!., 51 (2): 553-5, 1997; and ΟΚουΤ ^ e! a1., Οικο1. Kez., 9 (6-7): 327-32, 1997; as well as other regulatory factors, such as, for example, factors that are induced by physiological conditions associated with cardiovascular disease, such as cessation (e.g., inducible nitric oxide synthase (такжеδ), as well as its constitutive copy, cNΟδ; see, for example, ζοзЫζит ^ е! а1., C1gs. Kez., 73 (1): 205-9, 1993; SCabgat e ! a1., I. 1ο1. Sket., 269 (9): 6765-72, 1994; Rarare! ^ Ooroi1oz e! a1., At. I. Ra! ; and Pa1teg, e! a1., At. I. Ryuzyu1., 274 (2 P! 1): B212-9, 1998. Additional examples of such angiogenic proteins include some insulin-like growth factors (for example, 1CE-1) and angiopoetins (App), which have been reported to promote and / or stimulate the expression and / or activity of other angiogenic proteins, such as UESE (see, for example, ^ ab, e! A1., Eηbos ^^ ηο1οду, 137 (6):

- 12 008538

2262-68 (1996); Shggei, e1 a1., 1. Vu1. Sket., 271 (46): 29483-88 (1996); Rii ^ a, e1 a1., P1aBe1ek 46 (10): 161926 (1997); and Akakaga, e1 a1., C1gs. Kek. 83 (3): 233-40 (1998) and Vegtoy! e1 a1., Guy !. 1. Saiseg 85: 117-123, 2000). Similarly, hepatocyte growth factor (also called Sketter factor), which was reported to induce the formation of blood vessels w νίνο (see, for example, Pack! E! A1., Proc. No. 111. Asab. 8c1. I8A 90: 1937- 1941, 1993), increased, as reported, the expression of νΕΟΕ (see, for example, \ ¥ о | 1а е! А1., БаЬ. РкекР 79: 427-438, 1999). Additional examples of angiogenic polypeptides include natural and synthetic regulatory peptides (angiogenic polypeptide regulators) that act as promoters of endogenous angiogenic genes. Native angiogenic polypeptide regulators can be obtained from inducers of endogenous angiogenic genes. Ηί £, described above, is one illustrative example of such an angiogenic gene that has been reported to promote angiogenesis by inducing the expression of other angiogenic genes. Synthetic angiogenic polypeptide regulators can be constructed, for example, by the production of proteins that bind multiple zinc finger proteins that specifically bind to sequences located to the left of the coding regions of endogenous angiogenic genes, and which can be used to induce the expression of such endogenous genes. The study of numerous genes led to the development of rules for the construction of such DNA-binding proteins containing zinc fingers (see, for example, Kkobek aib K1id, 8s1ey11Ps Atepsai, EgGiagu 1993, pp 56-65; Scoo aib K1id, Prog. No. 111. Asab. δα. I8A, 91 (23): 11163-7, 1994; KeBag aib Rao, 8aeise, 263 (5147): 671-3, 1994; Scoo! a1., 1. Mo1. Vu1., 273 (3): 525-32, 1997; Rotegap / e! A1., 8aise 267: 93-96, 1995; and Ls e! A1., Proc. No. 111. Asab. Δα. I8A, 94: 5525-5530, 1997. How will it is clear to a specialist with qualifications in this field, numerous additional genes encoding proteins and peptides with A feature of directly or indirectly promoting angiogenesis is regularly identified, and new genes will be identified based on similarities with the known angiogenic protein or peptide encoding genes or with the detectable ability of such genes to encode proteins or peptides that promote angiogenesis. Sequence information for such genes and encoded polypeptides easy to obtain from sequence databases such as SeiVaik or EMB. Polynucleotides encoding such proteins can also be obtained from gene libraries, for example, using PCR or hybridization methods that are routine in the art.

Preferably, the angiogenic protein coding transgene is operably linked to a promoter that regulates the transcription and expression of this gene in a mammalian cell, such as a cell in the heart or in skeletal muscle. One currently preferred promoter is the CMV promoter. In another preferred embodiment, discussed further below, the promoter is a tissue-specific promoter, such as a heart-specific promoter (for example, a cardiomyocyte-specific promoter). Preferably, the gene encoding the angiogenic factor is also operably linked to a polyadenylation signal.

The success of the gene transfer approach requires both synthesis of the gene product and secretion from the transfected cell. Thus, angiogenic proteins and peptides include proteins and peptides that are naturally secreted or have been modified in such a way as to allow secretion, for example, by functional binding to a signal peptide. From this point of view, a gene encoding a secreted angiogenic protein, for example, ECE-4, ECE-5, or ECE-6, is preferable since these proteins contain functional secretory signal sequences and are easily secreted from cells. Many, if not all, human νΈΌΕ proteins (including, but not limited to, νΈΌΕ-121 and νΈΌΕ-165) are also easily secreted and diffuse after secretion. Thus, upon expression, these angiogenic proteins easily reach cardiac interstitium and induce angiogenesis. Blood vessels that develop in angiogenesis include capillaries, which are blood vessels of the smallest caliber, having a diameter of about 8 microns, and larger blood vessels, which have a diameter of at least about 10 microns. Angiogenic activity can be determined by measuring blood flow, increasing the function of the treated tissue, or the presence of blood vessels, using procedures known in the art or described herein. For example, the number or density of capillaries can be quantified in an animal visually or by microscopic analysis of a tissue site (see Example 5).

In the case of other angiogenic proteins, such as aECE (ECE-1) and BECE (ECE-2), which do not have a natural secretory signal sequence, fusion proteins having secretory signal sequences can be recombinantly obtained using a standard recombinant DNA methodology known specialist in this field. It is believed that both aECE and BECE are naturally secreted to some extent; however, the inclusion of an additional secretion signal sequence can be used to enhance the secretion of this protein. The secretory signal sequence is usually located at the Ν-end of the desired protein, but can be placed in any position suitable for secreting secretion of the angiogenic factor. For example, a polynucleotide containing a suitable signal sequence may be fused 5 ′ (left) relative to the first codon of a selected angiogenic protein gene. Suitable secretory signal

- 13 008538 sequences include signal sequences of the ECE-4, ECE-5, ECE-6 genes or the signal sequence of a different secreted protein, for example, 1L-1-beta. Example 7 below gives an example of one type of modification of an angiogenic protein for attaching a signal sequence from another protein, a modification achieved by replacing residues in an angiogenic protein with residues that control the secretion of this secreted second protein. A signal sequence derived from a protein that is normally secreted from cardiac myocytes can be used. Angiogenic genes can also provide additional functions that can improve, for example, the function of heart cells. For example, ECE can provide heart-enhancing and / or ischemic protective actions that may be independent of their ability to stimulate angiogenesis. Thus, angiogenic genes can be used to enhance cardiac function through mechanisms that are complementary or replace stimulation of angiogenesis regex. As an additional example, 1CE, which can promote angiogenesis, can also enhance muscle cell function (see, for example, Misago e1 a1., No. SC.1ge 400: 581-585, 1999); and also exhibit anti-apoptotic effects (see, for example, Leo e1 a1. Encoopodod 140: 4831-4840, 1999). Other proteins that enhance muscle cell function may also be used in accordance with the methods of the present invention.

As noted above, genes encoding one or more angiogenic proteins or peptides can be used in conjunction with this invention. Thus, a gene or genes encoding a combination of angiogenic proteins or peptides can be delivered using one or more vectors in accordance with the methods described herein. The families of angiogenic genes described herein and in the literature in this field contain numerous examples of such genes. Preferably, using such a combination, the genes can be obtained from different families of angiogenic factors (for example, a combination selected from two or more different members of the group consisting of ECE, VΕCE, ROSE and 1CE). To separately illustrate such a combination, a vector containing the ECE gene and the VΕCE gene can be used. As an illustrative example, the authors used a combination of the ECE gene (ECE-4 fragment 140) (see, for example, the ECE-4 gene and its variants described by Wassco E1 a1., In US patent No. 5459250, issued October 17, 1995 ., and related cases), and a variant of the VΕCE gene (mutein 2 of VΕCE-145) (see, for example, the VΕCE-145 gene and its variants described by Nu £ eI e1 a1., AO 98/10071, published March 12, 1998 g., and related cases). Such combinations may exhibit additive and / or synergistic effects. Numerous other combinations will be apparent to those skilled in the art based on these descriptions. Vectors containing angiogenic genes or combinations of angiogenic genes in accordance with this invention may also include one or more other genes that can be used to further enhance tissue blood flow and / or contractile function. In the heart, for example, genes encoding beta-ΑδΡ (described by Nattoib e1 a1., Pending simultaneous application AO 98/10085, published March 12, 1998), can be used in combination with one or more genes encoding angiogenic proteins or peptides. Other cardiac or muscle cell enhancing proteins may be similarly incorporated into the compositions and methods of the present invention.

Combinations of genes that can be used in accordance with this invention can be provided in a single vector (for example, as separate genes, each of which is under the control of a promoter, or as a single transcriptionally or translationally fused gene). Combinations of genes can also be provided as combinations of vectors (which can be produced from the same or different vectors, such as a combination of adenovirus vectors or a combination of adenovirus vector and adeno-associated (ΑΑν) vector); which can be administered to the patient simultaneously or sequentially. In the case of adenovirus (Αά) and adeno-associated virus (ΑΑν), the presence of Αά, which is usually a helper virus for ΑΑν, can enhance the ability of ΑΑν to mediate gene transfer. Thus, the Αά-vector can be introduced simultaneously with the introduction of the ΑΑν-vector or before the introduction of the ΑΑν-vector, in accordance with this invention. In addition to the efficiency of transfection, the desired duration of transgene expression also affects the choice of vector. To illustrate, since many angiogenic genes can cause long-term effects without the need for long-term expression (for example, by initiating or facilitating the process of angiogenesis, which leads to increased vascularization of the tissue), angiogenic genes can be introduced using adenovirus (or another vector that does not usually integrate into Host DNA), which can be used before the introduction of the ΑΑν vector, or in combination with the introduction of the ΑΑν vector carrying the transgene for which more is desired its long-term expression (eg, transgene beta-ΑδΡ). Other combinations of transgenes and / or vectors will be apparent to those skilled in the art based on the explanations and illustrations of the present invention.

For the treatment of humans, genes encoding angiogenic proteins of human origin are preferred, although angiogenic proteins originating from other mammals that exhibit cross interspecific activity, i.e., have an angiogenic asset, can also be used.

- 14 008538 nost in people.

Vectors for gene delivery ΐη νίνο

Typically, a gene of interest is transferred to the heart or peripheral vasculature ίη νίνο and it controls the production of the encoded protein. Preferably, such production is constitutive (although inducible expression systems may also be used).

The vectors useful in this invention include viral vectors, lipid-based vectors (e.g. liposomes), and other vectors that are capable of delivering DNA to non-dividing ίη νίνο cells. Currently preferred are viral vectors, in particular replication defective viral vectors, including, for example, replication defective adenoviral vectors and adeno-associated viral vectors. With respect to ease of preparation and use in the present invention, replication-defective adenoviral vectors are currently most preferred. Adenovirus effectively infects non-dividing cells and, therefore, is applicable for the expression of recombinant genes in the myocardium, due to the non-replicative nature of cardiac myocytes.

Various other vectors suitable for отерапη νίνο gene therapy can easily be used to deliver angiogenic protein transgenes for use in this invention. Such other vectors include other viral vectors (such as ALU), non-viral "delivery" platforms, as well as lipid-based vectors (such as liposomes, micelles, lipid-containing emulsions, and others that have been described in the art). As for AAU vectors, as is known in the art, they are preferably replication defective in humans, such as, for example, AAU vectors having deleted her and sar genes (the sequence of which must therefore be supplied with ίη 1gan5 for replication and packaging AAU vectors, usually in a packaging cell line), and the inserted transgene (including, for example, a promoter operably linked to it) is preferably flanked by inverted terminal repeats (ΙΤΚ) of the AAU.

Recombinant viral vectors contain one or more genes or sequences. Because many viral vectors exhibit size-related packaging limitations, and because replication-defective viral vectors are usually preferred for delivery of ίη νίνο, heterologous genes or sequences are typically introduced by replacing one or more parts of the viral genome. Such viruses can become defective in replication due to deletions, which requires that the deleted function (s) be provided by ίη 1gan5 during virus replication and encapsidation (using, for example, a helper virus or a packaging cell line carrying the genes necessary for replication and / or encapsidations) (see, for example, links and illustrations below). As stated above, modified AAU vectors in which transgenes are inserted instead of the ger and sar viral genes are also well known in the art. Similarly, modified viral vectors in which the delivered polynucleotide is located outside the viral particle have also been described (see, for example, Cype1, ΌΤ, e! A1. ΡΝΆδ 88: 88508854, 1991). Links describing these and other gene delivery vectors are known in the art, and a number of them are cited here.

As described above and in cited references, vectors may also contain other components or functional groups that further modulate gene delivery and / or gene expression, or which otherwise provide beneficial properties to target cells. Such other components include, for example, components that affect cell binding or targeting (including components that mediate cell type or tissue-specific binding); components that affect vector uptake by the cell; components that affect the processing and / or localization of the vector and its nucleic acid in the cell after absorption (such as agents that mediate intracellular processing and / or nuclear localization); and components that affect polynucleotide expression. Such components may also include markers, for example, detectable and / or selectable markers, which can be used to detect or select cells that have absorbed and express the nucleic acid delivered by this vector. Such components may be provided as a natural feature of the vector (for example, by the use of certain viral vectors that have components or functionalities mediating binding and uptake), or the vectors may be modified to provide such functionalities. The detectable marker gene allows specific detection of cells carrying the gene to be specifically detected (for example, makes it possible to distinguish from cells that do not carry the marker gene). One example of such a detectable marker gene is the 1ac2 gene encoding beta-galactosidase, which allows the detection of cells transduced by a vector carrying the 1ac2 gene using staining as described below. Breeding markers can be positive, negative or bifunctional. Positive breeding markers make it possible to select for cells that carry the marker, while negative breeding markers allow selective elimination of cells that carry the marker. A wide variety of such marker genes has been described, including bifunctional markers (i.e., positive / negative) (see, for example, BirSh.

- 15 008538 νθ 94/28143, RNHkkeb 8, Eth. 1994). Such marker genes may provide an additional control measure that may be beneficial in the context of gene therapy. A wide variety of such vectors are known in the art and are usually available (see, for example, various references cited above).

Works describing adenoviral vectors and other viral vectors that can be used in the methods of the present invention include the following references: IppuI /, M.Z., Abepousbay apb Tkey Kerysayop, w E1e1bk, B., e! A1., (ebk.) UyYodu, Uo1. 2, Kauep Rgekk No. Neu Wogk, pp. 16791721, 1990); Sgakat, E., e! A1., pp. 109-128 w Me! Kobk w Mo1esi1ag Wu1odu, Wo1. 7: Sepe TgapkGeg apb Exrgekkup Rgo! Os1k, Miggau, E. (eb.), Nitapa Rgekk, Syop, N.1. (1991); MSHeg, no., E! A1., EAZEV 1oit1 9: 190-199, 1995; 8skge1eg, N, Rkagtaseijsa As! And NeKekai 68: 145-159, 1994; 8skpe1beg apb Egepsk, Sisi1iop 88: 1937-1942, 1993; Sipe1 Ό.Τ., e! A1., Nitap Sepe Tkegaru 3: 147-154, 1992; Sgakat, E.b., e! A1., νθ 95/00655 (5th year 1995); Ea1sk-Rebegkep, E.Z., νθ 95/16772 (22, 1st type 1995); Oepeye, R. e! A1., νθ 95/23867 (8 Sperm et 1995); Nabbab, N. e! A1., νθ 94/26914 (24 No. Het 1994); Retsaibe !, M. e! A1., νθ 95/02697 (26 Apiage 1995); 2capd, ν., E! A1., νθ 95/25071 (12 Os. obeg 1995). Various adenoviral plasmids are also available from commercial sources, including, for example, Myung Vyukuk et al., OgIapo (see, for example, Mythophobia! Yogogakopak Zkee !: P1act1bk og Abepougik Ues! Og Sopkyisyop, 1996) . Various additional adenoviral vectors and methods for their preparation and purification are regularly identified.

Additional references describing AAU vectors that could be used in the methods of the present invention include the following: Sayeg, V., Napboook o аг Raguo1 gikek, yo1. 1, pp. 169-228, 1990; Vegpk, Uyoduod, rr. 1743-1764 (Cowep Rgeck 1990); Sayeg, V., Sigg. Orth. Vu! Eskpo1., 3: 533539, 1992; Mihuska, No., Siggep! Toryuk t М1сгоОо1оду apb 1tipo1od, 158: 92-129; E1o !! e, TK, e! A1., At. Sh. Kekr1g. Be11 Mo1. Vu1. 7: 349-356, 1992; Skye e! A1., App. ΝΥ Asab. 8sk, 770: 79-90, 1995; E1o !! e, TK, e! A1., νθ 95/13365 (18 Mau 1995); Tgretre, 1.R., e! A1., νθ 95/13392 (18 Mau 1995); Koip, K., Nitap Sepe Tkegaru, 5: 793-801, 1994; Coyp e! A1., νθ 98/11244 (19 Magsk 1998); Coyp e! A1., νθ 99/61601 (2 Essential 1999); E1o !! e, TK, e! A1., Sepe Tkegaru 2: 357-362, 1995; A11ep, 1.M., νθ 96/17947 (13 May 1996); and hey e! A1., Sepe Tkegar1e 3: 254261, 1996. Various additional AAU vectors and methods for their preparation and purification are regularly identified.

As described above and in the scientific literature, a number of retrovirus-derived systems have also been developed for use in gene delivery. As an illustration, the genus retrovirus lentivirus (for example, human immunodeficiency virus, feline immunodeficiency virus, etc.) can be modified in such a way that these viruses are able to transduce cells that are usually non-dividing (see, for example, Roexa e! A1. , PNA8 96: 11395-11399, 1996; No. a1byi e! A1., PNA8 96: 11382-11388, 1996; No. a1bY1 e! A1., Zyepse 272: 263-267, 1996; Zishuakakitag e! A1., 1. Uyo1. 71: 5841-5848, 1997; 2yeeu e! A1., No. 11. Vyu eskpo1. 15: 871-875, 1997; K1t e! A1., 1. U1go1. 72: 811-816, 1998; M1uokk1 e! a1.,

1. U1go1. 72: 8150-8157, 1998; see also Wiskskaskeg e! A1., B1ob 15: 2499-2504, 2000; see also Tke Za1k 1pk! iy! e, ν097 / 12622 (10 Arp1 1997)). Although HIV-based lentiviral vector systems have led to some degree of focus in this regard, other lentiviral systems have recently been developed, such as feline immunodeficiency virus lentiviral vector systems, which provide potential advantages over HIV-based systems (see, for example, Roeksia e! A1., No.a! Furniture 4: 354-357, 1998; 1kpk! Op e! A1., 1. U1go1. 73: 2491-2498, 1999; and 1okpk! Op e! A1., 1. U1go1. 73: 4991-5000, 1999; see also the review of Cotapo e! A1., Zet Cé11k 18: 19-39, 2000 and the references therein).

In addition to viral vectors, non-viral vectors are also known and continue to be developed that can be used as gene delivery vehicles. For example, non-viral protein-based delivery vehicles (platforms) have been described in the art, for example, macromolecular complexes containing a DNA-binding protein and a carrier or part of a molecule capable of mediating gene delivery, as well as lipid-based vectors (e.g. liposomes micelles, lipid-containing emulsions and others). References describing non-viral vectors that could be used in the methods of the present invention include the following: Lebe, EO, Nitap Sepe Tkegaru 6: 1129-1144, 1995; MSHeg, no., E! A1., EAZEV 1oit1 9: 190-199, 1995; Skopp, A., e! A1., Sigg. Orth. t vu! esk. 6: 698-708, 1995; 8skoye1b, KR., E! A1., VgKk Meb. Vi11. 51: 56-71, 1995; Vpdkat, K., e! A1., 1. Yrokote Kek. 3: 31-49, 1993; Vpdkat, K. L., VO 91/06309 (16 Mau 1991); Her1dpeg, R.E., e! A1., VO 91/17424 (14, no. 1991); 8o1obt e! A1., Wusket Eeyu 34: 13537-13544, 1995; VO 93/19768 (14 Oct. 1993, 1993); Eek e! A1., VO 93/125673; Her1dpeg, R.E., e! A1., I.8. Ra! Ep! 5,264,618 (Ref. 23, 1993); Erapb, K.M., e! A1., I.Z. Ra! Ep! 5,283,185 (Herbigu 1, 1994); Sao e! A1., VO 96/22765 (1 Aidik! 1996); SEEUJEKI e! A1., I.8. Ra! Ep! 5334761 (Aidik! 2, 1994); Her1dpeg, R.E., e! A1., I.8. Ra! Ep! 5459127 (Os! Obeg 17, 1995); Ouege11, Κ.ν., e! A1., VO 95/28494 (26 Oct. 1995, 1995); 1 krkee, VO 95/02698 (26th year 1995); Nasek apb Syusagope, VO 95/17373 (29 Ype 1995); B e! A1., VO 96/01840 (25 April 1996). Numerous additional lipid-mediated vein delivery vectors of genetics and vector delivery cofactors have been identified (see, for example, Co11ep e! A1., Nit. Sepe Tkeg. 10: 615-22, 1999; Coe e! A1., No.a !. Furniture 5: 387-391; Ea) as e! A1., Nit. Sepe Tkeg. 10: 395-406, 1999; Osuua e! A1., No.a !. Meb. 5: 707-710, 1999). Additionally was opi

- 16 008538, the development of systems that combine the components of viral and non-viral gene delivery systems can be used here (see, for example, RBSp e! A1., Mo1. Ce11 ΒίοΙ., 14: 2411-2418, 1994; see also Ωί No.co1a e! A1., Thread.Cepe (Neg. 10: 1875-1884, 1999). Various additional non-viral gene delivery vectors and methods for their preparation and purification are regularly identified.

As described above, the efficiency of gene delivery using a vector, such as a viral vector, can be increased by delivering this vector to a blood vessel such as an artery, or to tissue that is pre-infused and / or co-infused with a vasoactive agent, for example, histamine or a histamine agonist, or a protein of vascular endothelial growth factor (UESE), as described here and further illustrated in the pending application PCT \ νϋ 99/40945, published on August 19, 1999. Another example is The active agent that can be used to increase gene delivery efficiency is a nitric oxide donor such as sodium nitroprusside. Most preferably, the vasoactive agent is infused into a blood vessel or tissue simultaneously with the administration of the vector and / or several minutes before the administration of the vector. A vasoactive agent, as used herein, means a natural or synthetic substance that induces increased vascular permeability and / or enhances the transfer of macromolecules, such as gene delivery vectors, from blood vessels, for example, through the capillary endothelium. By increasing the permeability of the macromolecules or otherwise facilitating the transfer of the macromolecules to the artery perfused capillary bed, vasoactive agents can enhance the delivery of these vectors to the target areas and, therefore, enhance the overall expression of the transgene in the target tissues. The authors of this invention used histamine as a vasoactive agent, and it was found that it significantly enhances the delivery of the vector to the infusible region, such as the myocardium. Histamine derivatives and agonists, such as those that interact with histamine H receptors that can be used, include, for example, 2-methylhistamine, 2-pyridylethylamine, betahistine, and 2-thiazolylethylamine. These and other histamine agonists are described, for example, in Satcop 1C, Soaptap apb Sitap'k Thieu Ryagtasoodyuya 1 ΒοΟδ οί Tyegareibsk (8 ' ¹ ' Еb: Sbtap AS, Kai Τ.ν., Mek A..8., Tau1og R ., ebk) Regdatop Rgekk, 1990, pp 575-582 and other pharmacological scientific works. In addition to histamine and histamine agonists that can be used as vasoactive agents, vascular endothelial growth factors (UESE) and UESE agonists (as described here and in references cited) can also induce increased vascular permeability and, therefore, can be used as a vasoactive an agent for enhancing gene delivery in the context of the compositions and methods described herein. As with histamine, UESE is preferably infused into a blood vessel supplying blood to the target site for several minutes before the vector is infused. Nitric oxide donors, such as sodium nitroprusside (8 #), can also be used as vasoactive agents. Preferably, the nitric oxide donor (e.g., 8 #) is pre-infused into the target region (or into the blood vessel supplying the target tissue) several minutes before the moment of infusion of the vector composition and continues to be infused until the vector composition is infused. Administration may also continue during infusion of the vector composition.

An exemplary adenoviral vector that is helper independent and replication defective in humans

Typically, the gene of interest is transferred to the heart or to the peripheral vasculature, w1u0, and regulates the production of the encoded protein. Several different approaches to gene transfer are possible. At present, a helper independent replication defective system based on human adenovirus 5 (Ab5) is preferred. Using a single intracoronary injection (injection into the coronary vessels) of such a system based on recombinant Ab5, the inventors of this invention showed significant transfection of myocardial cells w1uo (Syugbaio apb Nashtopb, S1sh. Kek., 42: 123A, 1994). Non-replicative recombinant adenoviral vectors are particularly useful in transfection of coronary endothelium and cardiac myocytes, resulting in highly efficient transfection after intracoronary injection. Adenoviral vectors can also be used to transfect tissue supplied with a peripheral vasculature, for example, using intra-arterial or direct injection.

As shown here, the helper-independent replication-defective human adenovirus 5 system can be used to efficiently transfect a large percentage of myocardial cells W1U0 through a single intracoronary injection. The authors also showed that such a delivery method can be used to efficiently target the mammalian large heart myocardium vectors. Additional methods for targeting vectors to specific cells or tissue types are described below and in the literature in this field.

In the various illustrations described below, the recombinant adenovirus vectors used are based on human adenovirus 5 (described by McGogu ν.1. E! A1., Order 163: 614-617, 1988), which lacks the necessary early genes from the adenovirus genome (usually E1A / E1B) and, therefore, is unable to replicate if it does not grow in permissive cell lines, which provide the products of the missing genes w 1gai8. Instead of missing adenoviral genomic sequences, the transgene of interest can be cloned and expressed in tissue

- 17 008538 ni / cells infected with replication-defective adenovirus. Usually, adenovirus-based gene transfer does not lead to stable integration into the genome of the target cell. However, adenoviral vectors can multiply with a high titer and transfect non-replicating cells; and, although the transgene is not transmitted to daughter cells, it is suitable for gene transfer to differentiated (mature) cardiac myocytes that are not actively dividing. Retroviral vectors provide stable gene transfer, and high titers have now been obtained by pseudotyping of retroviruses (Vitk, e! A1., Prg. YaI. Asab. 8c1. I8A, 90: 8033-8037, 1993), but currently used retroviral the vectors are usually incapable of efficient transduction of non-replicating cells (e.g., cardiac myocytes). In addition, the potential dangers of transgene incorporation into host DNA are unjustified if short-term gene transfer is sufficient.

Indeed, the inventors have shown that a limited duration in the expression of angiogenic protein is sufficient to significantly improve blood flow and ischemic tissue function (see Example 5). Thus, transient gene transfer is therapeutically sufficient to treat such cardiovascular diseases. Within 14 days after the transfer of the POP-5 gene into the myocardium, the blood flow to the ischemic bed doubled and this effect persisted for at least 12 weeks (Example 5 and Fig. 8). Increased blood flow correlated with an increase in the number of capillaries in the heart (see example 5). The wall thickening also increased within two weeks after gene transfer and persisted for at least 12 weeks. Thus, an angiogenic factor gene must not be present in an infected cell for more than a few weeks to obtain a therapeutic effect. Once blood vessels developed, continued expression of exogenous angiogenic protein might not be required to maintain a new vascular structure and increased blood flow.

An advantage associated with non-dividing cells, such as monocytes, is that the viral vector is not easily diluted by dividing the host cells. However, if it is necessary or desirable to further increase the duration of transgene expression in the heart, various second-generation adenoviral vectors can be used, which have deletions of both E1 and E4, which can be used together with the introduction of cyclophosphamide (see, for example, ϋαί e ! a1., Proc. Ia. Asab. 8a. I8A, 92: 1401-1405, 1995).

293 human cells (Assecup, ATCC CBY573; WoskuShe, MO), which are human embryonic kidney cells transformed with E1A / E1B genes of adenovirus, are examples of useful permissive cell lines to produce such replication-defective vectors. However, other cell lines that allow replication-defective adenoviral vectors to multiply in them, for example, HeLa cells, can also be used.

Construction of a recombinant adenoviral vector

The adenoviral vectors used in this invention can be constructed by the “rescue” recombination method described in Otayat, Uyodod 163: 614-617, 1988. Briefly, a transgene of interest is cloned into a shuttle vector that contains a promoter, polylinker and partial flanking sequences adenovirus from which the E1A / E1B genes were deleted. As an example of a shuttle vector, the plasmid pAC1 (Upo1odu 163: 614-617, 1988) (or its analogue), which encodes parts of the left side of the human adenovirus 5 genome (U1to1odu 163: 614-617, 1988), minus the sequences E1A and Е1В, encoding the early protein, which are necessary for virus replication, and the ACMURURA plasmid (Сех-Ешх е! А1., 1. Вю1. Сет. 267: 25129-25134, 1992), which contains the polylinker, the SMU promoter and the 8У40 polyadenylation signal flanked by partial adenovirus sequences from which genes were deleted E1A / E1B. The use of the plasmid pAC1 or ACMURIA facilitates the cloning process. This shuttle vector is then co-transfected with a plasmid that contains the complete human adenovirus 5 genome with a length too large for encapsulation to 293. Cotransfection can be carried out by precipitation with calcium phosphate or lipofection (Ζ1ιηη§ е! А1., Вю1ес1шк | ек 15: 868-872 , 1993). Plasmid 1M17 encodes the complete human adenovirus 5 genome plus parts of the pBB322 vector, including the ampicillin resistance gene (4.3 kb). Although 1M17 encodes all the adenoviral proteins necessary to produce mature viral particles, it is too large for encapsulation (40 kb vs. 36 kb for the wild type). In a small subset of cotransfected cells, a “rescue” recombination between a transgene-containing shuttle vector, for example, plasmid pAC1, and a plasmid having the full adenovirus 5 gene, for example, plasmid p1M17, provides a recombinant gene that does not have E1A / E1B sequences and which contains interest the transgene, but secondarily loses an additional sequence, for example, pBB322 sequence, during recombination, thereby becoming small enough for encapsulation (see Fig. 1). Regarding the method described above, the authors reported successful results (Oyutbapo e! A1., S1tsiayop 88: 1-139, 1993 and Syugbapo apb Nattopb, S1sh. Vek. 42: 123A, 1994). The triggered SMU encoding β-galactosidase adenovirus NSΜU8PΠacΖ (C1sh Century 42: 123A, 1994) can be used to evaluate gene transfer efficiency using X-processing 0018538 §-1.

Targeted Vector Designs

Limited expression of an angiogenic transgene only in the heart or specific types of cells in the heart (e.g., cardiac myocytes) or in other target tissues, such as the peripheral vasculature, may provide certain benefits, discussed below.

The present invention contemplates the use of targeting not only by delivering a transgene, for example, to the coronary artery or other tissue-specific channel, but also using targeted vector constructs having features that tend to target gene delivery and / or gene expression in specific host cells or cell types the host (e.g., cardiac or other myocytes). Thus, such targeted vector constructs could include targeted delivery vectors and / or targeted vectors, described in more detail below and in publications in this field. Limiting delivery and / or expression may be beneficial as a means of further focusing the potential effects of gene therapy. The potential applicability of additional restriction of delivery / expression depends to a large extent on the type of vector used and the method and place of introduction of such a vector. As described herein, it has been observed that the delivery of viral vectors via intracoronary injection to the myocardium provides a highly targeted gene delivery in itself (see examples below). In addition, when using vectors that do not usually result in transgene integration into the replicon of the host cell (such as adenovirus and numerous other vectors), it is expected that cardiac myocytes will exhibit relatively long transgene expression since these cells usually do not replicate. In contrast, the expression of rapidly dividing cells, such as endothelial cells, tends to decrease due to cell division and renewal. However, other means of restricting delivery and / or expression may also be used, in addition to or instead of the illustrated delivery methods, as described herein.

Targeted delivery vectors include, for example, vectors (such as viruses, protein-based non-viral vectors, and lipid-based vectors) having surface components (such as a member of a ligand-receptor pair, the other half of which is detected on the host cell for targeting) or other traits that mediate preferential binding and / or preferential delivery of a gene to specific host cells or types of host cells. As is known in the art, a number of vectors of both viral and non-viral origin have their inherent properties of facilitating such preferential binding and / or have been modified to perform preferential targeting (see, for example, Eondctta e! A1., Ν; · ιΙ. Vu ! esk. 14: 1574-1578, 1996; Kakayaga, N. e! a1., 8c1epse 266: 1373-1376, 1994; Mi1eg, Ν., e! a1., EA8EB 1oigia1 9: 190-199, 1995; Skopi , A., e! A1., Sigg. Ορίη. Ίη Vyu! Esk. 6: 698-708, 1995; 8s1yuPe1b. 1P, e! A1., Βπΐίδΐι Meb. Vi. 51: 56-71, 1995; 8skge1eg, N, Rkagshaseibs As! A Nekebae 68: 145-159, 1994; Beb1eu, Ε.Ό., Nit. Sepe Tkeg. 6: 1129-1144, 1995; Sopagu, 1. T., e! A1., AO 95/34647 (21 keset 1995); Ouege 11, V.A., e! A1., AO 95/28494 (26 Os! Oeg 1995); and Thiopd, U.B. e ! A1., AO 96/00295 (4 April 1996)).

Targeted vectors include vectors (such as viruses, protein-based non-viral vectors, and lipid-based vectors), in which case delivery results in transgene expression, which is relatively limited to specific host cells or types of host cells. By way of illustration, angiogenic transgenes delivered in accordance with this invention can be operably linked to heterologous tissue-specific promoters, thereby limiting expression to only cells in a given specific tissue.

For example, tissue-specific transcriptional regulation sequences derived from a gene encoding a cardiomyocyte-specific myosin light chain promoter (MBS) or myosin heavy chain promoter can be fused to a transgene, for example, the EOE gene, in a vector such as the adenovirus constructs described above. Thus, the expression of this transgene can be relatively limited by cardiac myocytes (i.e., it will occur only in cardiac myocytes). Determined the efficiency of gene expression and the degree of specificity provided by cardiomyocyte-specific promoters MBC and MHC with 1; κΖ (using a recombinant adenovirus system, such as the one exemplified here); and heart-specific expression has been reported (see, for example, Bee e! a1., 1. Vu1. SN. 267: 15875-15885, 1992).

Since the MBS promoter can contain only about 250 bp, it is easily suitable even for vectors with a limited size, such as the adenovirus-5 packaging system, exemplified here. The myosin heavy chain promoter, which is known to be a strong transcription promoter, provides another alternative heart-specific promoter containing less than about 300 bp. Although other promoters, such as the troponin-C promoter, do not provide tissue specificity, they are small and highly effective.

Targeted Gene Expression

An unexpected discovery of the present invention is that recombinant adenovirus is very efficiently absorbed in the first vascular bed with which it occurs. Indeed, in the animal model of example 4, the absorption efficiency of this virus in the heart after intracoronary injection

- 19 008538 was 98%, i.e. 98% of this virus was removed during the first passage of the virus through the myocardial vascular bed. In addition, serum taken from these animals during the injection was unable to grow viral plaques (bhayat, νίτοίο ^ γ, 163: 614-617, 1988) before being diluted 200 times, which suggests the presence of a serum factor (or binding protein) which inhibits the reproduction of the virus. These two factors (the effective attachment of the first passage virus and the possibility of the presence of serum binding protein) can act together, limiting the expression of the first vascular bed encountered by the virus.

To further assess the extent to which gene transfer was limited by heart after intracoronary gene transfer, polymerase chain reaction (PCR) was used to observe whether there was evidence of extracardiac presence of viral DNA two weeks after gene transfer in two treated animals (example 4 below) . Animals showed the presence of viral DNA in their hearts, but not in their retinas, skeletal muscle, or liver. The sensitivity of PCR is such that one DNA sequence per 5,000,000 cells can be detected. Thus, these data showed that viral DNA was not present in extracardiac tissues two weeks after gene delivery. These results were further confirmed using other angiogenic proteins and derivatives, as described below. These findings are extremely important, as they confirm the concept of targeting a transgene to the heart (i.e., to ensure transgene expression in the heart, and not elsewhere). Localized delivery and transgene expression provides a safety advantage, further enhancing the usefulness of these methods in treating patients.

Propagation and purification of adenoviral vectors

Recombinant viral vectors, such as adenoviral vectors, can be plaque purified according to standard methods. As an illustration, the resulting recombinant adenoviral vectors can be propagated in 293 human cells (which provide the E1A and E1B functions ίη Ιηηδ) to titers in the preferred range of about 10 ¹⁰ -10 ¹² viral particles / ml. Propagation and purification methods have been described for various viral vectors, and they can be used with this invention. Adenoviral vectors are described as examples here, but other viral vectors, such as AAU, can also be used. For adenovirus, cells can be infected at 80% confluency and harvested after 48 hours. After 3 cycles of freezing and thawing of infected cells, cell debris (debris) is precipitated by centrifugation and the virus is purified by ultracentrifugation in the ΟδΟΙ gradient (double centrifugation in the ΟδΟΙ gradient is preferred). Before injecting ίη νίνο, stock solutions of viruses can be desalted (for example, by gel filtration through Sepharose columns such as Sephadex 6-25). The desalted stock virus solution can also be filtered through a 0.3 μm filter, if desired. Authors usually concentrate and purify the initial virus solution by double ultracentrifugation in the ΟδΟί gradient, followed by chromatography on Sephadex 625, with the columns equilibrated with phosphate buffered saline (PBS). The resulting virus stock solution typically has a final virus titer of at least about 10 ¹⁰ -10 ¹² virus particles / ml.

Preferably, the recombinant virus is highly purified and substantially free of wild type (potentially replicative) virus. For these reasons, propagation and purification can be carried out to eliminate impurities and wild-type virus, for example, by identifying a successful recombinant virus by PCR using suitable primers, performing two rounds of plaque purification and double ultracentrifugation in the ΟδΟί gradient.

Delivery of vectors carrying an angiogenic transgene

The agents and compositions that are used to deliver vectors carrying transgenes of angiogenic proteins depend on the particular vector used, as is well known in the art. However, typically the vector may be in the form of an injectable preparation containing a pharmaceutically acceptable carrier / diluent, such as, for example, phosphate buffered saline. Other pharmaceutical carriers, preparations and dosages are described below.

The currently preferred delivery method of ίη νίνο for heart disease (especially for vector constructs that are not otherwise targeted for delivery and / or expression, which is limited to the myocardium or other target tissue) is delivery by injection of the vector into a blood vessel or other channel, directly supplying blood to the myocardium or tissue, preferably by injection into one or both coronary arteries or other tissue-specific arteries (or by bolus injection into peripheral tissue). As an example for delivery to the myocardium, such an injection is preferably achieved by a catheter inserted to a significant extent (usually at least about 1 cm) in the lumen of one or both coronary arteries or one or more transplantation of the saphenous veins or internal mammary arteries or other channels delivering blood to the myocardium. Preferably, the injection is performed in both the left and right coronary arteries to provide a common distribution to all areas of the heart (for example, the left anterior descending artery (BAO), the left coronary artery (ΕΟχ) and the right). Injection prep

- 20 008538 rat adenoviral vector in accordance with this. optionally in combination with a vasoactive agent to enhance gene delivery. as described here. an efficient adenovirus-mediated angiogenic gene transfer can be performed to treat cardiovascular disease. for example, clinical myocardial ischemia or peripheral vascular disease. without any unwanted effects.

Vectors are delivered in quantity. enough for that. so that the transgene is expressed and provides therapeutic benefits. For viral vectors (such as adenovirus). the final titer of the virus in the injectable preparation is preferably in the range of about 10 ⁷ -10 ¹³ viral particles. making efficient gene transfer possible. Stock solution of adenovirus. preferably wild type free. can be injected deep into the lumen of one or both coronary arteries (or grafts). preferably to the right. and in the left coronary artery (or grafts). and preferably in an amount of 10 ⁹ -10 ³¹ viral particles. as determined by optical densitometry. Preferably, the vector is delivered as a single injection into each channel (e.g., into each coronary artery).

To further increase localized gene therapy vector delivery and to increase gene delivery efficiency. in accordance with this invention. a vasoactive agent can be infused. preferably a histamine or histamine agonist or vascular endothelial growth factor protein (UESE) or a nitric oxide donor (e.g. sodium nitroprusside). into the tissue to be treated. or simultaneously with the introduction of the angiogenic gene therapy vector. or a few minutes before the introduction of this vector.

By injecting the vector composition directly into the lumen of the coronary artery using coronary catheters. can target the gene quite efficiently and minimize the loss of recombinant vectors into the proximal aorta during injection. This type of injection enables local transfection of the desired number of cells. in particular. cardiac myocytes. in the affected myocardium encoding angiogenic protein or peptide genes. thereby maximizing the therapeutic efficacy of gene transfer and minimizing unwanted angiogenesis in extracardiac regions. For delivery to diseased tissues. supplied with blood by the peripheral vasculature. the vector may be inserted into one or more arteries. supplying such tissue with blood. or as a bolus injection into this tissue.

Vector designs. which specifically target the myocardium. such as vectors. including components of myocardial specific binding or absorption. and / or which include transgenes of angiogenic proteins. which are under the control of myocardial transcriptional regulatory regulatory sequences (e.g. cardiomyocyte-specific promoters). can be used instead of such directed injection methods or. preferably. together with such directed injection methods. as a way to further limit myocardial expression (e.g., ventricular myocytes). For vectors. which can cause an immune response. it is preferred to inject the vector directly into a blood vessel. supplying blood to the myocardium. as described above. although additional methods may be used to limit extracardiac delivery or otherwise reduce the potential for an immune response. Vectors may also be used. aimed at the fabric. supplied with blood by the peripheral vasculature. such as vectors. aimed at skeletal muscle. or promoters may also be used. specifically expressed in skeletal muscle.

As described in detail below. has been demonstrated. that using the methods of the present invention to deliver ίη νίνο viral vector. containing angiogenic transgene. expression of this transgene did not occur in hepatocytes. and viral RNA could not be detected in the urine at any time after intracoronary injection. Besides. no evidence of extracardiac expression in the eye could be found. liver or skeletal muscle by PCR two weeks after intracoronary transgene delivery in this way.

Various catheters and delivery routes can be used to achieve intracoronary delivery. as it is known in the art (see eg .. cited above references including:.. Τοροΐ EE (fucked) Tye Tech (ook about £ H1 (ez \ ^g en (yupa1 Sagbyu1odu 2pb Eb (\..... W.V. Baipbegk Co. 1994); KiSheyogb. K.V. Waxiag Bigdegu. 3GB Ub. (\ U.V. Baipbegk Co. 1989); Thieu Cec11 Tech (Looo o £ Mebkshe. 19 (11 Ub. ( \ U.V. 1992); and BaYk! Op. Ό .. Thieu Teh (Lauk o £ Bigdegu. 14 (11 Eb. (\ U.V. 1991)). Direct intracoronary (or into a transplanted) injection can be performed using a standard subcutaneous catheter based on the methods performed with fluoroscopic monitoring of the direction of the catheter. Any variety of coronary catheters or Stack perfusion catheters, for example, can be used in this invention, for example, various general purpose catheters, as well as modified catheters suitable for use in this invention, are available from commercial suppliers such as Α Α ν ν η еб еб С Бук Бук Бук Бук Beech. (etc (ASB). Tagde! Tyegareibsk. Wok (op BaepbPs and Sogb18. In the case where delivery to the myocardium is achieved by injection directly into the coronary artery (which is currently the most preferred nn). a number of approaches can also be used to insert a catheter into the coronary artery. as is known in the art. As

- 21 008538 example, a catheter can be conveniently inserted into the femoral artery and will pass retrogradely (backward) through the iliac artery and abdominal aorta and into the coronary artery. Alternatively, the catheter may first be inserted into the brachial artery or carotid artery and will pass retrograde into the coronary artery. The capillary bed of the myocardium can also be achieved by retrograde perfusion, for example, from a catheter placed in the coronary sinus. Such a catheter may also use a proximal balloon to prevent or reduce forward flow as a means of facilitating backward perfusion. For delivery to tissues supplied with blood by the peripheral vasculature, catheters can be inserted into arteries supplying blood to such tissues (for example, femoral arteries in the case of the leg), or can be introduced, for example, by bolus injection or infusion into the affected tissue.

Various combinations of vectors containing angiogenic genes and catheters or other devices for delivering ίη νίνο (for example, other devices capable of administering the pharmaceutical composition, usually in a buffered solution, into a blood vessel or muscle) can be included in the kits for use in accordance with this invention. Such kits may also contain one or more vasoactive agents for enhancing gene delivery and may further include instructions describing their use in accordance with any of the methods described herein.

Animal models

Important prerequisites for successful studies of cardiovascular gene therapy are (1) the genetic constitution of an animal model that is applicable for clinical cardiovascular disease, which can provide useful data on mechanisms for increased blood flow and / or contractile function, and (2) an accurate assessment of the effects of transfer genes. From this point of view, none of the earlier methods is satisfactory. Thus, the inventors used pork models that satisfy these assumptions. A pig is a particularly suitable model for studying human heart disease due to its proximity (relevance) to human physiology. The heart of a pig is similar to a human heart in the following ways. The pig has a natural coronary blood supply, very similar to human coronary blood supply, including the relative absence of coronary collateral vessels. Secondly, the heart size of a pig, as a percentage of the total body weight, is similar to the size of a person’s heart. In addition, the pig is a large animal model, which allows, therefore, a more accurate extrapolation of various parameters, such as effective doses of vectors, toxicity, etc. In contrast, the hearts of animals such as dogs and members of the murine family have a large number endogenous collateral vessels. In addition, with respect to the total body weight, the dog’s heart size is twice that of a person’s heart.

The animal model described here in Example 5 is an example of myocardial ischemia. (Since myocardial ischemia can also lead to congestive heart failure and / or occurs in connection with it, this particular model is additionally relevant for this situation). Using this model, it was demonstrated that vector-mediated delivery of a gene encoding an angiogenic protein attenuated myocardial ischemia and increased blood flow in the ischemic region. The development of collateral vessels also increased. By way of illustration, the inventors have successfully demonstrated gene transfer methods with several different angiogenic proteins, including both natural forms and muteins (as described in the examples below).

In this model, which imitates a clinical disease of the coronary arteries, the placement of an ameroid tourniquet around the left envelope of the artery (LСх) leads to a gradual complete closure (within 7 days of position) with minimal damage (1% of the left ventricle, 4 ± 1% of the LСх bed) ( !Ο! Η, е! А1., С1гси1абоп 82: 1778, 1990; ΚοίΗ, е! А1., At. 1. ΡΗγκίο1, 235: 1-11279, 1987; АЫ! Е, е! А1., С1гс. Кек. , 71: 1490, 1992; Nattoηά, e! A1., Sagby1., 23: 475, 1994; and Nattoηά, e! A1., 1. C1t. [Fart!., 92: 2644, 1993). Myocardial function and blood flow were normal at rest in the area previously perfused with a blocked artery (called the ischemic area), but the blood flow reserve is insufficient to prevent ischemia when myocardial oxygen demand increases, due to the limited development of endogenous collateral vessels. Thus, the Lcx bed is the subject of episodic ischemia, similar to clinical angina pectoris (angina pectoris). The relationships between the development of collateral vessels and blood flow function are stable within 21 days of application of the ameroid tourniquet and remain stable for four months (Κο! Η, е! А1., С1гси1а! Юп, 82: 1778, 1990; Κο! Η, е! А1 ., At. 1. ΡΗγκίο1, 235: H1279, 1987; A1che, e! A1., C1gs. Kek., 71: 1490, 1992). It was shown by telemetry that animals have periodic ischemic dysfunction in an endangered bed, during the day, associated with sharp increases in heart rate, during feeding, with interference from personnel, etc. Thus, this model had a bed with stable, but insufficient collateral vessels, and is the subject of periodic ischemia. Another clear advantage of this model is that there is a normally perfused and functioning area (LAO bed) adjacent to

- 22 008538 an abnormally perfused and dysfunctional area (bed Lcx), which provides a control bed in each animal.

Myocardial contrast echocardiography was used to approximate regional myocardial perfusion. The contrast medium consists of galactose microaggregates and increases the echogenicity (whiteness) of the image. Microaggregates are distributed into the coronary arteries and the myocardial wall in such a way that they are proportional to the blood flow (BkLa, s1 a1., S1gsioyoi, 90: 1513-1521, 1994). It was shown that the maximum contrast intensity was closely correlated with myocardial blood flow, as measured using microspheres (BkLaa, c1 a1., C1gci1a1yup, 90: 15131521, 1994). To document that the echographic images used in this invention accurately identified the Lcx bed and that myocardial contrast echocardiography can be used to evaluate myocardial blood flow, a hydraulic cuff blocker was placed around the proximal Lcx adjacent to the ameroid tourniquet.

In some aspects of the present invention, when animals were sacrificed, hearts were perfusion-fixed (glutaraldehyde, physiological pressures, ίΐη δίΐιι) to quantify capillary growth using microscopy. PCR was used to detect angiogenic protein DNA and mRNA in the myocardium from animals that underwent gene transfer. As described below, two weeks after gene transfer of myocardial samples from transduced 1ac2 animals, significant beta-galactosidase activity was detected by histological examination. Finally, using polyclonal antibodies to an angiogenic protein, expression of angiogenic protein in cells and myocardium from animals that received gene transfer was demonstrated.

As for demonstrating improved blood flow, various methods are known to those skilled in the art. For example, myocardial blood flow can be determined by the method using radioactive microspheres, as described in Po1y. e! A1., At. L Ryu81o1, 235: 1-H1279, 1987 or Po111. e! A1., C1gci1aBop, 82: 1778-1789, 1990. Myocardial blood flow can also be quantified, for example, by imaging using thallium, which involves perfusion of the heart with radioactive thallium, as described by Vgaiita I ίη Neai Bkeake, 4 (N e., pp. 276-311 (Baipjögk, P1i1aye1pYa, 1992). Cells in the heart are avid for thallium. Thallium uptake is positively correlated with blood flow. Thus, reduced uptake is a sign of decreased blood flow that occurs under ischemic conditions in which perfusion deficiency.In a conscious subject, angiogenic activity can be easily assessed by contrast echocardiography, as described in Examples 1 and 5 and Bype, Ό.Ρ, e! a1., C1gci1a1yup 58: 1072-1083, 1978. Improved myocardial function can be determined by measuring wall thickening, for example, transthoracic echocardiography.

The strategy for therapeutic research included timing of transgene delivery, a method and route of transgene administration, and selection of an angiogenic gene. In the ameroid model of myocardial ischemia, gene transfer was performed after a stable but insufficient development of collateral vessels. Earlier studies using the ameroid model included the delivery of angiogenic peptides during ameroid closure, before the development of ischemia and collateral vessels. However, this approach has not been used for several reasons. Firstly, such studies are not suitable for closely duplicating conditions that could be present in the treatment of clinical myocardial ischemia, in which gene transfer would be carried out under the conditions of existing myocardial ischemia; previous studies are similar to providing a peptide in the recurrence of ischemia and, therefore, they are less appropriate. Secondly, it was assumed, based on previous studies in cell culture, that an ischemic stimulus together with an angiogenic peptide would be the optimal medium for stimulating angiogenesis. This could be optimally achieved by transgene delivery at a time when heart disease is already present. The choice of method for achieving transgene delivery has been linked to these definitions. The limitation that this method should have been applicable for the subsequent treatment of patients with coronary artery disease made some approaches unsuitable (continuous infusion of the peptide into the coronary artery, direct injection of the plasmid into the heart, coating the heart with a resin containing the peptide to ensure slow, long-term release). Finally, the pork model provided an excellent way to track regional blood flow and function before and after gene delivery. The use of control animals that received the same vector (e.g., recombinant adenovirus), but with a reporter gene, provides control for these studies. The pig has a natural coronary circulation, very similar to the coronary circulation of people, including the relative absence of natural coronary collateral vessels. The porcine model also provided an excellent means for tracking regional blood flow and function before and after gene delivery. The use of control animals that received the same recombinant adenovirus construct but with a reporter gene provided control for these studies. Based on the foregoing and based on previous published studies, those skilled in the art will understand that it is expected that the results described below obtained in pigs are predictive of the results that will be obtained for humans.

- 23 008538

With regard to peripheral vascular disease, the delivery of angiogenic genes to the peripheral vasculature using the gene therapy vectors of the present invention can be tested using, for example, a model of peripheral ischemia with ligation of blood vessels of the hind limbs. See, for example, the femoral artery ligation model described by K. Tagscpd s1 a1. (see, for example, Wapd. s1 a1., C1gs. Kek., 79 (1): 62-9, 1996). As with the delivery of angiogenic genes to the ischemic myocardium, the delivery of angiogenic genes according to the invention to the peripheral vasculature and / or muscle associated with it can be used to overcome the effects of peripheral vascular disease.

Another animal model described here in Example 1 induces dilated cardiomyopathy, such as is observed in clinical congestive heart failure. In this model, continuous rapid electrical stimulation of the ventricle over a period of 3-4 weeks induces heart failure, which exhibits similar symptoms with many signs of clinical heart failure, including dilatation (expansion) of the left ventricle with impaired systolic function, similar to regional functional deviations from the norms observed in heart failure (including abnormalities associated with severe coronary artery disease and non-IHD (non-SAO) -ESM (dila th e cardiomyopathy) such as GOSM (idiopathic dilated cardiomyopathy)). Other models of congestive heart failure in animals include the induction of chronic ventricular dysfunction via intracoronary delivery of microspheres (see, for example, Laute e1 a1., 1. At. Co11. Sagby1. 18: 1794-1803 (1991); B1a81et e1 a1., At. 1. Sagby. Ra1. 5: 32-48 (1994); 8aLay e1 a1., At. 1. Rukyu. 260: H1379-H1384 (1991)). As an additional example of ventricular dysfunction, occlusion of the left coronary artery in the rat model can induce heart attacks, and then these animals can be examined and treated for the next days or weeks (see, for example, RGGGeg e1 a1., C1gs. Kek. 44: 503512 , 1979; RGGGeg e1 a1., At. 1. Ruku1. 260: H1406-1414, 1991).

Thus, these models can be used to determine whether delivery of a vector construct encoding an angiogenic peptide or protein is effective for alleviating cardiac dysfunctions associated with these conditions. These models are particularly useful in providing some of the parameters by which the effectiveness of отерапη νΐνο gene therapy for treating congestive heart failure and ventricular remodeling can be evaluated.

Therapeutic applications

The vectors of the present invention (e.g., replicatively defective adenovirus) enable highly efficient gene transfer of ίη νΐνο without significant necrosis or inflammation. Based on these results, some of which are described in detail in the examples below, it is seen that a sufficient degree of gene transfer ίη νΐνο is achieved for the implementation of functional changes ίη νΐνο. Gene transfer of an angiogenic protein, alone or in combination with another muscle-enhancing protein or peptide, will improve blood flow and enhance muscle function in the treated muscle. In addition, if desired, a vasoactive agent can be used together with these methods and compositions, as described herein, to further enhance gene delivery to the target region. Since a vasoactive agent (such as histamine, histamine agonist, nitric oxide donor or UEST protein) can be used to increase gene transfer efficiency at a vector dose of the gene, the inclusion of such an agent can be used to limit the amount of vector needed for administration to achieve a specific therapeutic effect .

In one aspect, the vectors and methods of the present invention can be used to treat dilated cardiomyopathy (OSM), a type of heart failure that is usually diagnosed by detecting dilated, hypocontractable left and / or right ventricle. As discussed above, OSM can occur in the absence of other characteristic forms of heart disease, such as coronary occlusion or myocardial infarction, in the history of the disease. Dilated cardiomyopathy (OSM) is associated with poor ventricular function and symptoms of heart failure. In these patients, expansion of the cavities of the heart and thinning of the wall usually leads to high stress on the wall of the left ventricle. Many patients exhibit symptoms even with weak physical effort or during rest and, therefore, are characterized as showing severe “type-ΙΙΙ” or “type-1U” heart failure, respectively (see, for example, the classification of heart failure of the New York Association of Cardiology ( NUNA)). As noted above, many patients with coronary artery disease can progress to dilated cardiomyopathy, often as the result of one or more heart attacks (myocardial infarction).

An additional application of the present invention is to prevent or at least mitigate destructive ventricular remodeling (also known as destructive remodeling, for short), which means expansion of the cavities after myocardial infarction, which can progress to severe heart failure. Even if ventricular remodeling has already begun, it is still advisable to stimulate an increase in blood flow, as it may still be effective in eliminating ventricular dysfunction. Similarly, stimulation of angiogenesis may be beneficial, since the development of a microvascular (capillary) bed may be

- 24 008538 also effective for eliminating ventricular dysfunction. Further, such angiogenic proteins or peptides may also have other enhancing effects. In a patient who had myocardial infarction, destructive ventricular remodeling is prevented if the patient does not have an expansion of the heart cavities and if symptoms of heart failure do not develop. Destructive ventricular remodeling is attenuated if there is any observable or measurable decrease in the existing symptom of heart failure. For example, a patient may find less shortness of breath and improved exercise tolerance. The methods for evaluating the improvement in heart function and the reduction of symptoms are essentially the same as those described above for IMS. It is expected that prevention or mitigation of destructive ventricular remodeling as a result of improved collateral blood flow and ventricular function and / or other mechanisms will be achieved within weeks after transfer of the angiogenic gene in a patient using the methods described herein.

In one example, this method for transferring an ίη νί \ Ό transgene encoding an angiogenic protein is used to demonstrate that gene transfer with a recombinant adenovirus expressing an angiogenic protein or peptide is effective in significantly reducing myocardial ischemia. In another example, this method of transferring an ίη νί \ Ό transgene encoding an angiogenic protein is used to treat a condition associated with congestive heart failure.

As the data below show, the expression of an exogenously provided angiogenic transgene leads to increased blood flow and / or improved function in the heart (or other tissue target). This increased blood flow and / or improved function will weaken one or more symptoms of a cardiovascular disease affecting the target tissue.

As described here, a number of different vectors can be used to deliver transgenes of angiogenic proteins ίη νί \ Ό in accordance with the methods of the present invention. By way of illustration, the replicatively-defective recombinant adenovirus vectors, exemplified herein, achieved highly efficient переносаη νί \ Ό gene transfer without cytopathic effect or inflammation in gene expression regions.

In the treatment of angina pectoris, which may be associated with coronary artery disease (ΓΑΩ), the transfer of the transgenic encoding angiogenic protein can be carried out at any time, but preferably it is carried out relatively soon after the occurrence of severe angina pectoris. In the treatment of the most congestive heart failure, the transfer of the gene encoding the angiogenic protein can be performed, for example, when the development of heart failure is probable or heart failure has been diagnosed. For the treatment of ventricular remodeling, gene transfer can be performed at any time after a patient has a heart attack, preferably within 30 days and even more preferably within 7-20 days after a heart attack.

As noted above, beta-adrenergic signaling proteins (beta-ΑδΡ) (including beta-adrenergic receptors (beta-ΑΚ), C-protein receptor kinase inhibitors (SSC inhibitors) and adenylyl cyclases (AS)) can also be used to enhance cardiac functions, as described and illustrated in detail in the United States Patent Application No. 08/924757 filed September 5, 1997 (based on application I8 60/048 933 filed June 16, 1997 and application I8 08/708 661 filed 5 September 1996), as well as PCT / I897 / 15610, filed September 5, 1997 and continuing with I8 turnout, registration number 09/008 097, filed January 16, 1998, and the continuation of business applications I8 with registration number 09/472667, filed December 27, 1999, each of which is incorporated herein by reference.

The compositions or products of this invention can easily be provided in the form of compositions suitable for administration to a patient in the bloodstream (for example, by intra-arterial injection or as a bolus infusion into tissue, such as skeletal muscle). A suitable administration form may be best determined by a medical practitioner. Suitable pharmaceutically acceptable carriers and their preparation are described in standard reference books for the preparation of medicines, for example, in Ket ^ ΐ ΐ Р 'Р Rkagtaseijs1 8c1epsez, E.A. MagTsp. See also Aand. W.E. ηηά Nunnov. Μ.Α., Räger Schücker o Р Prgo1etz ηηά Rärmez: 81aBn11u ηηά 81aB111 / erz, 1aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa, 10, 8irr 1. 42:28 (1988). The vectors of this invention should preferably be prepared in solution at a neutral pH, for example, about pH 6.5-8.5, more preferably about pH 7 to pH 8, with a filler to bring the solution to near isotonicity, for example 4.5% mannitol or 0.9% sodium chloride, pH buffered with buffers known in the art, such as sodium phosphate, which are generally considered safe, together with a common preservative such as metacresol (0.1-0.75%), more preferably with 0.150.4% metacresol. The desired isotonicity can be achieved using sodium chloride or other pharmaceutically acceptable agents, such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol or sorbitol), or other inorganic or organic solutes. Sodium chloride is preferred, in particular, for buffers containing sodium ions. If desired, solutions of the above compositions may also be prepared to increase shelf life and stability. Therapeutically useful compositions of this

- 25 008538 inventions are prepared by mixing the ingredients according to generally accepted procedures. For example, the selected components can be mixed to obtain a concentrated mixture, which can then be adjusted to a final concentration and viscosity by adding water and / or a pH control buffer or additional solute to control tonicity.

For use by a physician, the compositions must be provided in a dosage form containing an amount of a vector of the invention that will be effective in a single dose or in multiple doses to provide a therapeutic effect. As will be appreciated by those skilled in the art, an effective amount of a therapeutic agent will vary depending on many factors, including the patient’s age and weight, the patient’s physical condition and the level of angiogenesis and / or other action to be obtained, and other factors.

An effective dose of the viral vectors of the present invention will typically be in the range of about 10 ⁷ -10 ¹³ viral particles. As noted, the exact dose to be administered is determined by the attending physician, but preferably it is in 5 ml or less of a physiologically buffered solution (such as phosphate buffered saline), more preferably in 13 ml.

A preferred administration option is to administer by injection to one or more localized areas (for example, to one or both coronary arteries, in case of heart disease) using a suitable catheter or other device for delivering уπ у1уо.

The following examples are provided to further assist those of ordinary skill in the art. These examples are intended to illustrate and, therefore, should not be construed as limiting the invention. A number of exemplary modifications and variations are described herein, and other modifications and variations will be apparent to those skilled in the art. Such variations are believed to be within the scope of the invention described and claimed herein.

Examples

Example 1. Pork model of congestive heart failure and related myocardial ischemia

1-A. Animals and surgical procedure

Nine Yorkshire pigs (8 hrcg) weighing 40 ± 6 kg were anesthetized with ketamine (50 mg / kg intramuscularly 1M) and atropine sulfate (0.1 mg / kg intramuscularly 1M), followed by the administration of sodium amital (100 mg / kg intravenously 1U). After endotracheal intubation, halotane was delivered by a blower fan throughout the procedure. With left thoracotomy, catheters were placed in the aorta, pulmonary artery, and left atrium. The Konigsberg micromanometer was placed in the apex of the left ventricle and the epicardial monopolar abduction was placed 1.0 cm below the atrioventricular groove in the lateral wall of the left ventricle. A power generator (8reschach 5985; MebBhoshs, 1ps.) Was inserted into the subcutaneous pocket in the abdominal cavity. Four animals were equipped with a flow probe (Tgapkosh, 1ps.) Near the main pulmonary artery. The pericardium was loosely pulled together and the chest was closed. 7-10 days after thoracotomy, hemodynamic background, left ventricular function, and myocardial blood flow were measured. Then, ventricular electrical stimulation was initiated (220 ± 9 Lp (beats per minute) for 26 ± 4 days). The stimulus amplitude was

2.5 V, pulse duration 0.5 ms. Nine additional pigs (40 ± 7 kg) were used as controls; five pigs were thoracotomy and equipped with stimulation-free instrumentation and killed 30 ± 7 days after the initial thoracotomy. Data concerning the right and left ventricular masses were similar in the control animals, whether they were subjected to thoracotomy or not, so that their results were combined into a single control group.

1-B. Hemodynamic studies

Hemodynamic results were obtained from conscious vigilant animals after inactivation of the stimulator for at least 1 h and the animals were in the background. All data was obtained for each animal at intervals of 7 days. Pressure was obtained from the left atrium, pulmonary artery and aorta. Left ventricular b / b! obtained from high precision left ventricular blood pressure. Pulmonary arterial blood flow was recorded. Blood samples were taken from the aorta and lungs and were obtained to calculate the content of arteriovenous oxygen.

1-C. Echocardiographic examinations

Echocardiography is a method of measuring regional myocardial blood flow, which involves the injection of a contrast medium into an individual or animal. The contrast medium (galactose microaggregates) increases the echogenicity (“whiteness”) of the image after left atrial injection. These microaggregates are distributed into the coronary arteries and myocardial walls in proportion to the blood flow (8kua, e! A1., Sis1a! Yup, 90: 1513-1521, 1994). The maximum intensity of the contrast enhancement correlates with myocardial blood flow, as measured using microspheres (8kua, e! A1., Sis1a! Yup, 90: 1513-1521, 1994).

Two-dimensional and M-modal images were obtained with the HeMe visualization system !! Raskagb 8opok

- 26 008538

1500. Images were obtained from the near-sternal approximation at the level of the middle papillary muscle and recorded on a very high sensitivity CN 8 tape. The measurements were carried out in accordance with the criteria of the American Society of Echocardiography (8a1t Ό.Ι. e! A1., ^ι ^ ηΜοη, 58: 1072-1083, 1978). Due to the orientation along the midline of the interventricular septum (1U8) of the pig and the use of the right okrugrudnich type, M-modal measurements of the short axis were made through 1U8 and the anatomical lateral wall. All parameters, including terminal diastolic value (ΕΌΌ), terminal systolic value (Ε8Ό) and wall thickness, were measured on at least five random terminal expiratory strokes and the average was determined. The terminal diastolic value was obtained with the appearance of complex ΟΚ8. The end systolic value is taken at the moment of maximum lateral position 1U8 or at the end of the T-wave. Left ventricular systolic function was evaluated using fractional shortening, Ε8 - [(ΕΌΌΕ8Ό) / ΕΌΌ] χ100. Percent wall thickening (% \ νΤ1ι) was calculated as% \ νΤ1ι = | (Ε8 \ νΤ1ιΕΟ \ νΤ1ι) / ΕΌ \ νΤ1ι | χ 100. To demonstrate the reproducibility of echocardiographic measurements, animals were visualized on 2 consecutive days before the start of the electric cardiac pacing protocol. Data from individual demonstrations were highly reproducible (fractional shortening, Κ2 = 0.94, P = 0.006; thickening of the lateral wall, Κ2 = 0.90, P = 0.005). All these measurements were obtained with inactivated electrical stimulants.

1-Ό. Myocardial blood flow

Myocardial blood flow was determined by the method with radioactive microspheres, as described in detail earlier (Κοίΐι, Ό.Μ., е! А1., At. 1, РЬу8ю1. 253: Н1279-Н1288, 1987; Κοίΐι, Ό.Μ., е! А1. SyssynShup 82: 1778-1789, 1990). Transmural samples from the left ventricular lateral wall and interventricular septum were divided into the endocardial, middle-wall and epicardial third, and blood flow to each third of the transmural flow was determined. Transmural sections were made from areas in which echocardiographic measurements were made, so that blood flow and functional measurements corresponded in each bed. The microspheres were injected in the control state (not electrostimulated), with the initiation of ventricular electrical stimulation (225 Lp (beats per minute)), and then at 7-day intervals during ventricular electrical stimulation at 225 beats per minute; microspheres were also injected with inactivated electrical stimulators at 14 days (η = 4) and 21-28 days (η = 3). Myocardial blood flow per stroke was calculated by dividing the myocardial blood flow by the heart rate (recorded during the injection of microspheres) (Ιη6ο1ίϊ, C., е! А1., ΟϊΌνιΕιΟοη 80: 933-993, 1989). The mean left atrial and mean arterial pressures were recorded during the injection of the microspheres, so that an approximate value of the resistance of the coronary vessels could be calculated; coronary vascular resistance index is equal to mean arterial pressure minus mean left atrial pressure divided by transmural coronary blood flow.

1st. Systolic wall tension

Peripheral systolic wall tension could not be determined, since the authors could not obtain a suitable view for assessing the long axis of the left ventricle. Thus, the authors calculated the meridional terminal systolic wall stress (Sheskek, Ν., E! A1., ^ι ^ ι.ιΕιΙίοη 65: 99-108, 1982) using the equation: meridional terminal systolic wall stress (dyne) = (0,344χΡχΌ ) - [Ε (Ι-Ε / ϋ)], where P is the left ventricular end systolic end pressure in инах,, is the left ventricular end systolic diameter in cm, and E is the end systolic wall thickness. The meridional terminal systolic wall voltage was calculated both for the side wall and for 1U8 before initiating electrical stimulation and then sequentially at weekly intervals (with inactivated electrical stimulator).

1st. Terminal surgery

After 26 ± 2 days of continuous electrical pacing, the animals were anesthetized and intubated, and a sternotomy was performed along the midline of the sternum. Still contracting hearts were immersed in saline (4 ° C), coronary arteries were rapidly perfused with saline (4 ° C), the right ventricle and left ventricle (including the interventricular septum) were weighed, and transmural samples from each region were rapidly frozen in liquid nitrogen and stored at a temperature of -70 ° C.

1-C. Adenine nucleotides

ATP and ADP were measured in 1U8 transmural samples and the lateral wall in four animals with heart failure (electrically paced 28 days) and four control animals. Samples from animals with heart failure were obtained with inactivated electrical stimulants (60 min) on the day of killing the animals. Samples were obtained identically in all animals. ATP and ADP were measured in a high resolution liquid chromatograph ’^ a! Erz, as described previously (ζ1ζ, Κ.Β.,

1. Βίο1. Sows. 259: 2927-2935, 1984).

1-H. Statistical analysis

Data were expressed as mean ± standard deviation (8Ό). Specific measurements obtained in the control (before stimulation) state and at 1-week intervals during electrical pacemaking were compared with repeated measurements using ΑΝΟνΑ (SgiisM, Stylo 8oP \\ ags Co.). In some comparisons (lateral wall versus 1U8, for example, two-factor ΑΝΟνΑ was used. Roy 1us comparisons were performed using the "Tukey method" as described in the art. Nine animals survived 21 days of electrical pacing, six of them survived 28 days of electrical pacing. Results obtained for animals surviving 28 days were not statistically indistinguishable from animals surviving only 21 days. ΑΝΟνΑ was therefore performed on nine animals at four time points: control (lane stimulation d), 7 days, 14 days and 21-28 days. The null hypothesis is rejected when obtained P <0.05 (two-sided test).

results

1-1. Hemodynamic studies

Rapid ventricular electrical stimulation led to changes in hemodynamics that were significant after 7-14 days of electrical stimulation. At 7 days, the animals had increased left atrial and pulmonary arterial pressure. This pressure increased in relation to the norm with an increase in the duration of electrical stimulation (Table 1). Signs of circulatory stagnation (tachypnea, ascites and tachycardia) were evident 14-21 days. Pulmonary arterial blood flow (cardiac output per minute) decreased by 21 days of stimulation (control, 33 ± 0.1 L / min; 21 days, 1.9 ± 0.4 L / min; P <0.05).

Table 1. Hemodynamics and left ventricular function

η The control 7 d 14d 21-28 d R NN (LRS) nine 122 ± 16 136 ± 15 149 + 136 157 ± 15s, § 0,0004 MAR (mm Front) nine 103 ± 8 99 + 6 98 + 7 102 ± 14 0.52 RA (mm Nd) 7 24 ± 7 37 + 46 42 + 9s 48 ± 8s, d, ΐ 0.0001 BA (mm H§) 8 13 ± 3 25 + 5s 30 ± 7s 36 ± 6s, e 0.0001 AUO2O (ml / dl) 7 3.6 + 1.1 3.5 + 0.9 5.2 + 1.5 6.2 + 1.56.6 0,0005 EOO (cm) nine 3.9 ± 0.4 4.4 + 0.5 4.9 ± 0.6s 5.8 ± 0.6 s, £, 1 0.0001 P8 (%) nine 39 + 3 26 ± 5s 18 ± 6s, e 13 ± 4s, 1 0.0001 BU άΡ / άΐ 4 2849 ± 27 2408 ± 46 847 + 381s, units 1072 ± 123 s, e, 1 0.0001 (mm Nd / s) 8 0

Analysis of variance (repeated measurements) was used to determine whether the duration of electrical stimulation affected a particular variable; p-values from ΑΝΟνΑ are listed in the right column. Roy 1us test was performed according to the Tukey method: ar <0.05; Bp <0.01; avg <0.001 (against the control value for the same variable); c1p <0.05: ep <0.01; ίρΟΟ, ΟΟ 1 (compared to the previous week); dr <0.05; 1ιρ <0.01: ίρ <0.001 (compared with the value of 7 d); Roy Bos test by the Tukey method. The measurements were made with inactivated electrical stimulators (pacemakers), and the measurements represent an average of ± 8 Oe. 7 d: 7 days of stimulation; 14 d: 14 days of stimulation; 21-28 d: 21-28 days of stimulation. 1-1. Global left ventricular function

Left ventricular function was assessed using echocardiography and hemodynamic variables after the pacemakers were turned off. Fractional shortening (P8) progressively decreased with increasing duration of pacemaker (P = 0.0001; Table 1), reaching its lowest value at 21-28 days (control, 39 ± 3%; 21-28 days 13 ± 4% ; P <0.0001; table 1).

The magnitude of the left ventricular end diastolic pressure (Εϋϋ) progressively increased during electrical pacemaker * P <0.0001; tab. 1), reaching its maximum value on 21-28 days (control, 3.9 ± 4 cm; 21-28 days, 5.8 ± 0.6 cm; P = 0.0002). The left ventricular maximal positive LU άΡ / άΐ also decreased during the study (P = 0.0001; Table 1). A progressive decrease in maximum άΡ / άΐ was accompanied by an increase in left ventricular end diastolic pressure, confirming a decreased contractility of the left ventricle, since increased preload usually increases the left ventricular maximum άΡ / άΐ (MaYeg, G., εΐ a1., At. 1. SapNo1 . 35: 626-634.1975).

1 TO. Left ventricular regional function

-28008538

With an inactivated pacemaker, the regional left ventricular function was evaluated by measuring the percentage thickening of the wall, namely the left ventricular lateral wall and interventricular septum (1U8). Electrical pacing from the lateral wall caused a significant deterioration in the function of the lateral wall in comparison with 1U8 (P = 0.001; Fig. 1 and Table 2). This difference was significant on day 7 and increased additionally on day 21-28, as the lateral wall function worsened. 1U8 showed a slight decrease in wall thickening during the course of this study. The terminal diastolic wall thickness revealed progressive thinning during the study, which was more severe in the lateral wall (Table 2).

Table 2. Sequential thickening of the left ventricular wall

The control 7 d 14 d 21-28 d ρ (ΑΝθνΑ) 1U8 EAT 0.8 ± 0.1 0.7 + 0.1 0.7 + 0.1 0.6 + 0,] time: 0.0001 (cm) LAT ΕϋΊΊι 0.8 ± 0.1 0.7 + 0.1 0.6 ± 0.1 0.5 ± 0.1 area: 0,039 (cm) p (1U8 knot of NZ nz NZ Int .: 0.027 LAT) IV 8 Ψ11ι (%) 33 + 4 33 ± 5 28 ± 3 28 ± 6 time: 0.0001 LAT Τνώ (%) 35 ± 5 25 + 4 19 + 8 14 + 6 area: 0.001 p (1U8 knot nz 0.02 0.007 0.0001 Int .: 0.0001 LAT

Two-way analysis of variance (repeatable measurements) was used to determine whether terminal diastolic wall thickening (ET11) or% wall thickening (\ UTN) affected pacemaker duration (time) or region (lateral wall, LAT; or interventricular septum, 1U8), or whether the change in EET11 or \ UT11% is different between the two areas (inter.). The average values for ЕЭТ11 or \ УТ11% at each time point were tested for differences between the two regions ροδί 1yus using Tukey analyzes. Values represent mean ± 8E (standard deviation). 7 d: 7 days of stimulation; 14 d: 14 days of stimulation; 21-28 d: 21-28 days of stimulation. and = 9.

1st. Left ventricular regional blood flow

Subendocardial blood flow per minute increased more in 1U8 than in the lateral wall, at the beginning of electrical pacing (Fig. 2 and table. 3). This difference in regional blood flow during stimulation persisted over the duration of this study, and the picture of changes in blood flow was different between the two regions (P = 0.006). The picture of changes in blood flow per minute between these two areas during stimulation was consistent when measured in the endocardial (P = 0.006), mid-wall (P = 0.002), epicardial (P = 0.016) or transmural (parietal) (P = 0.003) departments (table . 3). In contrast, when the pacemaker was inactivated, the subendocardial blood flow showed no regional differences when measured in the control state, on day 14 or day 21-28 (Fig. 2 and Table 3).

-29008538

Table 3. Sequential myocardial blood flow

Day 0 Day 14 Day 21-28 ρ (ΑΝθνΑ)

Off incl. Off ON off incl. 1U8 ΕΝΟΟ 1.41 ± .2 1.964.38 1.684.22 2.354.4 1.884.1 2.674.3 time .0001 (ml / min / g) 6 6 ^{8 9} SAT 1.4ΟΓ.3 1.114.14 1.504.35 1.654.2 1.734.0 2.054.1 district .017 ΕΝΌΟ 3 5 5 6 (ml / min / g) p (IV 8 νϊ. No. .001 PP .002 out of .006 between 006 LAT) G / 5MGO 1.564.2 2.114.33 1.844.29 2.484.3 2.044.0 2.984.4 time (ml / iin / g) 0 one 9 6 .0001 LAT IGO 1.664.2 1.534.17 1.504.43 1.774.2 1.7b ± .3 2.124.0 area „019 (ml / min / g) 8 nine 9 6 p (GU5 te.Νχ .01 Π5 Pz. 001 between. .002 LAT) Β / 8ΕΡΙ 1.134.2 1.5SI.24 1.544.38 1.914.4 1.794.1 2.534.3 time (mp / msh / g) 7 8 4 8 .0001 ΕΑΤΕΡΪ 1.37 ± .2 1.48 ± .31 ] .24 ± 224 1.554.2 1.504.0 1.924.0 district: .17 (ml / min / g) 2 5 4 8 P (1U8 knot N3 Π5 P5 Π5 .049 pz LAT) between (kie G / 8 1.36 * .2 1.854.27 1.69 ± .3Ο 2.244.4 1.904.0 2.734.4 time ΤΚΑΝ8 1 6 9 0 .0001 (ml / min / g) LAT. 1.474.2 1.384: .22 1.414.33 1.654.2 1.664.0 2.034.0 district: .019 ΤΚΑΝ8 7 5 2 7 (ml / min / g) p (1U8 ν®. Ν® .001 P8 Π8 Pz .001 between .003 LAT) No. 1, z ± .z 1.32 ± .23 1.134.20 1.274.2 1.054.2 1.064.1 time .058 ΕΝΟΟ / 6 0 0 ΕΡΙ LAT 1.0) 4.0 0.774.10 1.214.06 1.074.1 1.154.0 1.074.1 district.:. 054 ΕΝϋΟ / 7 one twenty ΕΡΙ ρ (1У8 ν®. Νϊ .0002 P5 Π3 pz pz LAT) between.0008

Two-way analysis of variance (repeatable measurements) was used to determine whether subendocardial (ΕΝϋΟ) or transmural (ΤΚΑΝ8) blood flow affects pacemaker duration (time) or region (lateral wall, LAT; or interventricular septum, 1U8), or whether the pattern of changes in blood flow different between the two areas (between). Average values for blood flow at each time point were tested for differences between the two rock regions! Bos using Tukey analyzes. Values represent mean ± δϋ (standard deviation) of 5 animals. On: microspheres were injected during electrical stimulation of the ventricle (225 beats per minute). Off: The electrical stimulator is off. Day 0 = Control; Day 14: 14 days of stimulation; Day 21-28: 21-28 days of stimulation.

Endocardial blood flow: epicardial blood flow relationships did not change significantly as heart failure progressed (P = 0.058). However, with the onset of cardiac stimulation, the ratio of endocardial blood flow: epicardial blood flow was significantly lower in the lateral wall 30008538 than in the interventricular septum (1U8, 1.32 ± 0.23; lateral wall, 0.77 ± 0.10; P = 0.0002; tab. 3). Relations in both areas were> 1.0 throughout the rest of the study.

Endocardial blood flow per stroke (heart contraction) (Fig. 2 and Table 4) was similar in both areas before the start of cardiac stimulation (1U8, 0.013 ± 0.003 ml-min-1 g-1-stroke-1; lateral wall, 0.012 ± 0.004 ml-min-1 g-1 stroke-1; P = NC). Upon initiation of ventricular stimulation (225 beats per minute), there was a regional decrease in endocardial blood flow by one stroke (one reduction) in the lateral wall, but not in 1U8 (1U8, 0.009 ± 0.002 ml-min-1 g-1-beat-1; lateral wall, 0.005 ± 0.001 ml-min-1 g-1 beat-1; P = 0.001). At 14 days and 21-28 days, endocardial blood flow per stroke was less in the lateral wall than in the interventricular septum (1U8), during stimulation (Fig. 2 and Table 4). These data show that with the onset of stimulation myocardial hypoperfusion in the lateral wall began, and this relative ischemia persisted. However, endocardial blood flow per beat remained normal in both areas with the pacemaker turned off (Fig. 2 and Table 4).

Blood flow in both areas tended to increase during the last week of cardiac stimulation (Fig. 2 and Table 3). This picture was associated with a progressive decrease in the coefficient of resistance of the coronary vessels (Fig. 3), which suggests that changes in the structure and function of the coronary vessels may accompany remodeling of the left ventricle as heart failure progresses. The coefficient of resistance of the coronary vessels was significantly larger in the lateral wall than in 1U8 at the beginning of stimulation, and the picture of the change in the resistance of the coronary vessels was different between these two regions (P = 0.0012) (Fig. 3). These findings may indicate the effect of altered electrical activation on myocardial perfusion.

Table 4. Endocardial blood flow per heart beat (per beat)

Model Endocardial blood flow to stroke (ml / min / gram / stroke) PORK AMEROID ISCHEMIA (NK. 220 beats per minute; p-ch5) Non-ischemic bed 0.012 + 0.004 Ischemic bed 0.006 ± 0.002 p <0.001 * PORK INDUCED Stimulation of the left ventricle

SUSTAINABLE HEART INSUFFICIENCY (CHF) (TC 225 beats per minute; n = 5)

Stimulant

Stimulator on off

DAY 1U8 0.009 ± 0.002 0.013 + 0.003 (NK 122 bpm) Lateral wall 0.005 ± 0.001 0.012 ± 0.004 p = 0.001 nz DAY 14 1U8 0.010 + 0.003 0,0 And ± 0,002 (NK 149 bpm) Lateral wall 0.007 ± 0.001 0.010 + 0.003 p = 0.008 nz DAYS 21-28 1U8 0.012 + 0.002 0.013 ± 0.003 (NK 157 bpm) Lateral wall 0.009 + 0.001 0.012 ± 0.002 p = 0.024 nz

Data from the ameroid model of ischemia were published previously by the authors' laboratory (Napshyupb, NK apb MsKppap, Μ.ϋ., 1. At. Co11. Sagby1., 23: 475-82, 1994). Values represent mean ± δϋ (standard deviation). These data show that the collaterally dependent (ischemic) region of the ameroid model and the model of heart failure induced by left ventricular stimulation have similar defects in the endocardial blood flow by one heart beat (per beat)

-31 008538 in comparison with myocardial regions that have normal perfusion.

1M. End systolic tension of the left ventricular wall

There was a significant increase in the estimated meridional terminal systolic wall stress depending on the duration of stimulation (P <0.0001), but the nature of the change in wall stress was similar in the lateral wall and interventricular septum (1U8) (P = 33), and рοκ! The 1st test failed to show any regional differences in systolic wall tension at any particular time point (Fig. 3). The increase in the terminal systolic wall tension was approximately three times in the lateral wall (control, 168 ± 40 x 103 dyne; 28 days, 412 ± 143 x 103 dyne; P = 0.0001) and in 1U8 (control, 159 ± 35 x 103 dyne ; 28 days, 480 ± 225 x 103 dyne; P = 0.0001).

1-Ν. Autopsy

At autopsy, animals with heart failure had ascites (average, 1809 ml; range, 300-3500 ml) and dilated, thin-walled hearts, with all four cavities of the heart appearing to be greatly enlarged. The ratios of ventricular weight to body weight suggested hypertrophy of the right ventricle only, which confirmed the results from an earlier study using this model (Κο! 1, Ό.Α., е! А1., 1. С11п. [Пек1. 91: 939-949 , 1993). In comparison with the corresponding weight control animals, there was no change in the mass of the left ventricle in heart failure (control, 112 ± 10 g; heart failure, 114 ± 17 g); the ratios of left ventricular weight to body weight were also similar in both groups (control, 2.8 ± 0.3 g / kg; heart failure, 2.9 ± 0.3 g / kg). In contrast, heart failure was associated with increased right ventricular weight (control, 38 ± 3 g; heart failure, 52 ± 11 g, P = 0.003) and increased ratios of right ventricular weight to weight (control, 0.09 ± 0, 1 g / kg; heart failure, 1.3 ± 0.3 g / kg, P <0.003). Stimulated animals gave an increase in weight of 4 kg during the course of the study, an amount partially related to the accumulation of ascites. If the initial body weight was used to calculate the ratio of left ventricular weight to body weight, this ratio was still not significantly higher than the ratio of the corresponding weight control animals. These data confirm that there was no significant increase in left ventricular mass during this study.

1-O. Adenine nucleotides

Control animals found normal ATP / ADP ratios similar to those reported for a pig’s heart when taking biopsies with a drill followed by immediate immersion in liquid nitrogen (AU! E, RS apb Βοκκ, S., 1. Sagbuyuaks. Ra11u1. 3: 225-236, 1990), which confirms the fact that the sampling methods used were suitable. Animals with heart failure found a noticeable decrease in the ATP / ADP ratio in samples taken from the interventricular septum (1U8) (control, 14.8 ± 1.1; heart failure, 2.4 ± 0.3, P <0.0001, p = 4 for both groups) and from the lateral wall (control, 14.3 ± 4.0; heart failure, 2.4 ± 0.9, P = 0.0012, n = 4 for both groups). This confirms the imbalance between oxygen supply and oxygen demand in the myocardium.

1-p. Myocardial blood flow

Regional differences in myocardial blood flow, a direct consequence of rapid ventricular stimulation, may play a role in the pathogenesis of regional and global dysfunction in cardiac-induced heart failure. During pacing, a difference was found in myocardial blood flow per minute between the lateral wall of the left ventricle (adjacent to the site of stimulation) and the interventricular septum (1U8). Reduced blood flow was present in the lateral wall immediately upon initiation of pacemaker and persisted for 21-28 days. The lateral wall of the left ventricle, which receives less blood flow than 1U8, during pacemaking, found a progressive decrease in wall thickening (when the pacemaker is turned off) during 21-28 days of pacemaking. In contrast, 1U8, receiving a higher blood flow during pacemaking, maintained a relatively normal wall thickening for 21-28 days of pacing.

Since myocardial blood flow per minute does not allow easy assessment of relative myocardial ischemia, the inventors also expressed coronary blood flow in the form of endocardial blood flow per beat (one heart beat). The physiological basis for such an analysis lies in earlier experiments showing that regional subendocardial blood flow per minute (more than the outer wall of transmural blood flow) represents the primary determinant of regional myocardial contraction in conditions of progressive stenosis of the coronary arteries (61111ad, Κ.Ρ., e! a1., At. 1. Ρ1ινκίο1. 16: H727-H738, 1984) and that an increase in heart rate shifts this ratio of blood flow function down to a lower regional function at any level of sub endocardial blood flow (Oe1Laac, T., e! a1., 1. Ρ1ινκίο1. 477: 481-496, 1990). However, if the blood-to-function ratio is plotted as a function of regional function on endocardial blood flow per stroke to correct actions on the heart rate, then there is a single ratio at different heart rates, which indicates that endocardial blood flow primarily determines the level of function walls with a decrease in coronary blood flow

- 32 008538 (blood supply) (1pbo1y, C, e! A1., C1gcy1a! Ju 80: 933-993, 1989; Wokk, 1., C1gsy1ayop 83: 1076-1083, 1991). With the initiation of pacing, there was a> 50% decrease in endocardial blood flow per stroke in the lateral wall compared to 1U8 (P <0.001; Table 4).

In previous studies on a conscious pig, the inventors found that a 50% decrease in endocardial blood flow caused a 50% decrease in regional function and was associated with subendocardial blood flow by one reduction, similar to that in the lateral wall in these studies (Table. 4). The decrease in blood flow in the lateral wall during pacemaking continued throughout the study. These results provide evidence of myocardial ischemia in the lateral wall when ventricular pacing is initiated. In contrast, function and endocardial blood flow to one interventricular septum contraction (1U8) remained relatively normal. With the pacemaker turned off, subendocardial blood flow for one contraction remained normal in both areas throughout the study, while regional dysfunction persisted in the lateral wall, which is consistent with the presence of myocardial “shock” (“stunning”) in this area. Thus, the authors argue that prolonged ischemia of the lateral wall has a significant effect on the global (general) function of the heart during and after pacing.

Example 2. Obtaining illustrative constructs of gene delivery

2-A. Obtaining illustrative adenoviral constructs

A helper-independent, replicative-defective adenovirus-5 system was used as the initial gene delivery vector. As an initial illustration of vector constructs, the authors used genes encoding β-galactosidase and ECE-5. Recombinant adenoviruses encoding β-galactosidase or ECE-5 were constructed using full length cDNA. This system, used to produce recombinant adenoviruses, gave a packing limit of about 5 kb. for transgenic inserts. Each of the β-β; · ι1 and ECE-5 genes, functionally linked to the SMU promoter and to 8U40 polyadenylation sequences, had a size of less than 4 kb, quite within the bounds of packaging restrictions.

The full-length cDNA for human ECE-5 was isolated from the plasmid pBB122E (Ζΐιηη e! A1., Mo1. Ce11. Vu1., 8: 3487, 1988) in the form of an EcoB1 fragment of 1.1 kb, which includes 981 bp open reading frame of this gene, and cloned into the polylinker of the ACMURPA shuttle vector plasmid. The nucleotide and amino acid sequence of ECE-5 is described in FIG. 1 Ζΐιηη e! A1., Mo1. Ce11. Vu1., 8: 3487, 1988. RASMURURA is described in Sota /.- Ehh e! A1., 1. Vu1. Siet., 267: 25129-25134,

1992. RASMURRA contains the 5'-end of the adenovirus serotype 5 genome (mapping units 0-17), where the E1 region was replaced by the human cytomegalovirus promoter promoter (SMU promoter), followed by the multiple cloning site (polylinker) from pIS19 (plasmid, well known in the art), followed by a 8U40 polyadenylation signal. The ΕκΖ coding control adenovirus is based on a deletion of E1A / E1B from mapping unit 1 to 9.8. ECE-5 coding adenovirus (Ab.ECE-5) is based on a deletion of E1A / E1B from a mapping unit of 1.3 to 9.3. Both of these vectors eliminate the complete E1 A coding sequences and most of the E1 B coding sequences. Both vectors have transgene insertions cloned in an inverted orientation relative to adenovirus sequences. Thus, in the unlikely event of end-to-end reading (reading of the terminator), the adenovirus transcript would be antisense and would not express viral proteins.

The ECE-5 gene-containing plasmid was co-transfected (using calcium phosphate precipitation) into 293 cells with the DM17 plasmid (rDM17), which contains the entire human adenovirus 5 genome with an additional insert of 4.3 kb, which makes rDM17 too great for encapsidation into mature adenoviral virions. Cells are then covered with nutrient agarose. Infectious viral particles containing the angiogenic gene were obtained by rescue homologous recombination in 293 cells and isolated as individual plaques after 10-12 days. (Identification of a successful recombinant virus can also be carried out by co-transfection by lipofection and direct viewing of the cytopathic effect under a microscope, as described in Section 1, Section 11, 15 (5): 868-872, 1993). The obtained adenoviral vectors contain a transgene, but lack the E1 A / E1 B sequences and, therefore, are replication defective. The adenoviral vector carrying the ECE-5 gene is also referred to herein as Ab.ECE-5.

Although these recombinant adenoviruses were non-replicative in mammalian cells, they can multiply in 293 cells that were transformed with E1A / E1B and provided these essential gene products ίη 1taik. Recombinant virus from individual plaques was propagated in 293 cells and viral DNA was characterized by restriction analysis.

The successful recombinant virus was then subjected to two rounds of plaque purification using standard procedures. Viral stock cultures were propagated in 293 cells to titers in the range of 1010-1012 per milliliter (ml) as determined by optical densitometry. 293 human cells were infected at 80% confluency and the culture supernatant was harvested for 36-48 hours. After

- 33 008538 exposing the virus-containing supernatant to freeze-thaw cycles, cell debris (debris) was precipitated by standard centrifugation, and the virus was further purified by double ultracentrifugation in a gradient of cesium chloride (Ccc1) (stepwise gradient 1.33 / 1.45 Ccc1; Ccc1 was prepared in 5 mM Tris 1. pH 7.8); 90,000 x d (2 h). 105,000 x d (18 h)). Before injecting w νί \ Ό, the viral stock solutions were desalted by gel filtration through Sepharose columns (eg. C25 Sephadex. Balanced PBS). Final virus concentrations were about 1011 virus particles per milliliter (ml). as determined by optical densitometry. Viral stock solutions can conveniently be stored in cells in a medium at -70 ° C. For injection, the viral stock solutions are preferably resuspended in saline. The preparation of the adenovirus vector was highly purified and substantially free of wild-type virus (potentially replicative) (i.e. preferably containing less than about one (1) particle of replicatively competent adenovirus (JAS) per million. More preferably less than 1 in 109 and most preferably less than 1 in 1012). Thus. adenovirus infection and inflammatory infiltration in the heart were minimized.

Additional illustrations of adenoviral vector constructs are provided below and. in combination with other recommendations. secured here. other adenoviral vector constructs may be used. suitable for use in this invention. including constructions based on modified adenoviral vectors.

2-c. Additional illustrative vector and transgenic constructs

As described above. various viral and non-viral vectors can be used for gene delivery in accordance with this invention. As an illustrative example of another vector. can be obtained adeno-associated viral (AAU) vectors for delivery of w νί \ Ό in accordance with the methods of this invention. described above. As an illustrative example of another angiogenic gene. which can be used in the context of this invention. the authors received designs. containing the 1CE gene. as described above. as in adenovirus (Ab). and AAUvector designs.

Illustrative constructs contain the 1CE-1 gene under the control of a heterologous promoter (for the purposes of illustration, the SMU promoter was used) and are named hab / SE or gAAU / SE. Except them. constructions were designed. containing marker gene. eg. enhanced green fluorescent protein (ECEP). Constructs of hab / ju or gaau / ju can also be constructed to include a marker gene (eg. ECEP). Constructions. containing ecer. are commercially available (for example, from C1op (esk Pa1o A1 (about. SaShogsha).

To generate gab / 1CE. the 1CE-1 gene (available from ATCC) is subcloned into an adenoviral shuttle vector. such as RABKEYSHE-SMU. pAE5C1 and / or pAE (gas-SMU. The resulting 1CE-1 shuttle plasmid is subjected to recombination with a helper plasmid. pDM17. either in bacteria or in 293 cells depending on the shuttle vector used. The resulting gab / 1CE virus is checked for expression of 1CE protein -1 using RT-RCR and / or Western blotting.

As an illustration, the authors obtained constructs of GAB / 1CE using the shuttle vector and helper plasmid rDM17 in 293 cells essentially. as described and illustrated above for generating adenoviral vectors. containing the angiogenic ECE-5 gene. Adenoviral vectors. containing ecer. obtained as controls using similar methods.

The authors obtained the design of GAAU / 1CE using methods for producing recombinant AAU vectors. described in the art; see .. for example. links. related to obtaining AAU. quoted above. Although AAU vectors can be obtained using various methods. described in this field. the authors used a basic procedure with two transfections. mostly. as described, Bat1kk1 e (a1 .. 1. U1go1. 63: 3822-3828. 1989. Briefly, to obtain GAAU / 1CE, the 1CE-1 gene was subcloned into the DNA of the GAAU plasmid (so that the 1CE-1 gene was flanked by inverted repeats or 1THA AAU ) and this GAAU plasmid was then co-transfected into 293 cells with the AAU helper plasmid (to provide the ger and sarsh genes (hapc)). AAU infection was then initiated with helper adenovirus infection (the authors used adenovirus with deleted E1. known as b1312). Viral lysates are usually treated with heat to inactivate adenovirus and It is activated by pronase and DNAse according to standard methods (see e.g. ... Bati1kk1 f (a1 .. Kirga).

Various methods can be used to clean AAU vectors. For the purposes of this illustration, the authors used the standard procedure for ultracentrifugation in cesium chloride (SCC1) (two purifications using SCC1) for the initial separation of GAAU particles from impurities. mostly. as described in this field. After dialysis, the authors further purified the material using HPLC. In this example, the authors used an affinity chromatographic column. which is coated with heparin (ROYOB NOT. which is available from PE Vyukuyetk. Eok (eg Syu. SaShogsha) and was eluted with salt (1-2M No. 1C1). In this example, most AAU was eluted at approximately 0.7M NaCl. After dialysis against PBS ( pH 7.4) the vector was heat treated at 56 ° C for 60 min to break

- 34 008538 residual adenovirus. As in the case of adenovirus, the resulting stock solutions of the vector were usually titrated to DNase-resistant particles (ECR) and tested for the absence of a cytopathic effect. Expression of 1CE-1 in gab / 1CE and gAAU / 1CE was confirmed by Western blot analysis. Then they were additionally tested for the production of the functional protein 1CE-1 using the proliferation test on cultured MCE-7 cells. Briefly, 293 HEK cells (human embryonic kidney carcinomas) were transduced with gAO / SE or gAAU / 1CE on day 1 and cultured in serum-free medium. After 48 hours of incubation, serum-free medium was collected and placed on MCE-7 cells, which were cultured in serum-free medium. The proliferation of MCE-7 cells was monitored over the next 72 hours using a standard proliferation assay method (e.g., MTT analysis), mainly as described by Testaηη (see, for example, Testaηη, I. Ittiη1. Me111. 16: 55-63, 1983). An adenoviral or AAU vector carrying the gene for enhanced green fluorescent protein, gAB / ECEP or gAAU / ECEP, was used as a negative control, and human recombinant protein 1CE-1 was used as a positive control. The results from this MTT analysis showed that both the vector construct hAB / 1CE and the vector construct hAAU / 1CE were able to deliver the 1CE-1 transgene to human target cells (HEK 293) and that the medium of such target cells was then capable of induce cell proliferation of MCE-7 cells similarly to purified protein 1CE-1. No significant proliferation was observed using medium from cells transfected with negative controls (i.e., gab / ECEP or gAAU / ECEP). The authors also tested these vector constructs by direct transfection of MCC-7 cells and showed that 1CE-1 constructs (both in AAU and adenovirus) can be used to directly induce proliferation in transfected cells in a manner comparable to the introduction of 1CE-1 protein into these cells (at a concentration of about 3 μg / ml).

Additional tests to confirm the functionality of the 1CE-1 vector constructs can be performed using myocytes, in which one can observe the effects of 1CE-1 on the size and / or function of muscle cells. As an illustration, the effect of 1CE-1 on embryonic neonatal cardiomyocytes (ΝίΜ) or differentiated (mature) cardiomyocytes can be tested by various assays. For example, 1CE-1 can be delivered by adenoviral or AAU vectors for the induction of hypertrophy and cellular DNA synthesis in NСΜ. After transduction of NСΜ with a suitable multiplicity of infection (MO1), usually in the range of about 100-1000, cardiomyocytes are stained with crystalline green or neutral red stains. Cells are visualized under a microscope and the size of individual cells, including area, length and width, can be measured automatically (for example, using the software 1ade P1iz). The effect of 1CE-1 on cellular DNA synthesis can be quantified by the cellular incorporation of 3H-thymidine, by which cellular synthesis is observed on a 3H count after precipitation of TCA of cellular DNA.

Vectors containing angiogenic transgenes can be delivered to the heart via intracoronary delivery, as described and illustrated here. As an initial test of candidate vectors, before delivery to a model of a large animal such as a pig, the authors also used a rat model, in which the authors use indirect intracoronary delivery of the vector to the myocardium. In this model, delivery is achieved by introducing a solution containing the vector (for example, in phosphate buffered saline (PBS) or buffered HEPEδ saline) into the cavity of the left ventricle (namely, by introducing into the lumen of the cavity, opposite the ventricular wall) after pinching as pulmonary arteries and distal aorta. Thus, the blood flow from the cavity of this ventricle carries the material to be delivered to the coronary arteries, since alternative routes are temporarily blocked. The authors used the vascular constriction procedure to narrow the pulmonary artery and aorta (see, for example, Na_tsag, e! A1., Proc. No.! 1. Asab. Δα. ΥδΛ, 95: 5251-5256, 1998). The authors also used pretreatment with a vasoactive agent, as described above and in the corresponding pending application, cited above, to enhance gene transfer via intracoronary delivery. Authors usually use either histamine or sodium nitroprusside (δΝΓ) as a vasoactive agent. They can be used in the range of about 1-75 mg / ml. Typically, authors use about 25 mg / ml of histamine infused prior to delivery of the vector. In the case of δΝΓ, the authors usually use 50 mg / ml of this vasoactive agent with an infusion that begins a few minutes before the introduction of the vector and continues until the vector is completely injected. Using these procedures, the authors demonstrated very high levels of gene transfer to the myocardium through intracoronary delivery of both the adenoviral vector and the AAU vector. For example, using the GAAU / ECEP described above, delivered at a dose of about 1 x 1011 DNA-resistant particles, the authors can achieve transduction of the left ventricle (LU) at levels of about 30% of the cells (as measured by fluorescence microscopy, after fixing sections of LU in paraformaldehyde and cutting cryostat for slices of 8-10 microns and quantitative determination of the percentage of green area using the software 1tadeRgo P1iz). Gene expression in the myocardium was greatest in the epicardium, but significant expression was observed even in the endocardium. Additionally, the authors demonstrated relatively long-lived gene expression (with a slight, if any, decrease in expression levels between 30 days and 180 days after injection) after delivery of the AAU vector to the myocardium, as described. In addition, histological and pathological analyzes revealed

- 35 008538 a small inflammatory reaction or the absence of an inflammatory reaction in the heart and the absence of detectable gene expression in the liver or lung.

Example 3. Gene transfer to rat cardiomyocytes

3-A. Ab.v-da1 gene transfer and expression

Differentiated rat cardiomyocytes were obtained by Langendorff perfusion with perfusion solution containing collagenase in accordance with standard methods. Rod-shaped cells were cultured on laminin-coated plates and, at 24 hours, were injected with β-galactosidase-coding adenovirus obtained in Example 2 above with a 1: 1 infection multiplicity. After an additional 36-hour period, cells were fixed with glutaraldehyde and incubated with Xda1. Typically, 70-90% of differentiated myocytes expressed the β-galactosidase transgene after injection of recombinant adenovirus. With a multiplicity of infection of 1-2: 1, cytotoxicity was not observed.

3- B. Gene transfer and expression of GAAU / 1OP-1

To assess the effects of 1OP-1 expression in rat neonatal heart myocytes, 2 x 106 cells were plated on 10 cm diameter cell culture plates and 1 x 1010 DNA-resistant particles of GAAU / 1OP-1 or GAAU / EOPP were infected. Cells were cultured without serum in minimal medium and at normal oxygen levels at 37 ° C. Recombinant protein 1OR-1 (50 ng / ml) or phenylephrine (50 μM) was added to the culture as positive controls. Cells were visually evaluated 48 hours after treatment. Cells treated with gAAU / 1OP-1 showed significant hypertrophy (compared with hypertrophy obtained using phenylephrine) based on the morphological appearance, compared with untreated cells. The exogenously added protein 1OR-1, apparently, induced only weak hypertrophy in comparison with GAAU / 1OR-1. For the quantitative determination of hypertrophy, a stereological program was used, 1tade of Prgo P1ik (Meb1a SuBetBsk, Sag1kab, SaBGogsha). In short, the program 1tade Рgo Р1ик allows you to track individual cells and get their measurements. Cells were outlined in this program and their area sizes were calculated. Approximately 50-100 cells were counted per state and plots were plotted in the Rhtp statistical program. It was found that the cells treated with phenylephrine and infected with GAAU / 1OP-1 cells showed significant hypertrophy compared with untreated cells.

In addition to the study of hypertrophy, the level of 1OP-1 secretion into the medium after expression of GAAU / 1OP-1 was determined using the E8A analysis for the 1OP-1 protein. Briefly, protein expression was detected in the medium of infected GAAU / 1OP-1 cultures harvested for 48 hours at levels of approximately 0.1-1.0 ng / ml, representing a significant increase over the levels of 1OP-1 in control populations (untreated or infected with GAAU / EPP.

Example 4. The transfer of the gene W1UO in the pig myocardium

4- A. Gene transfer and expression Ab. yes1

The β-galactosidase-encoding adenoviral vector obtained in Example 2 was propagated in permissive cells 293 and purified by ultra-centrifugation in a Ccc1 gradient with a final viral titer of 1.5 x 105 virus particles based on the procedures of Example 2. Thoracotomy was anesthetized, ventilated pig weighing 40 kg. A 26-gauge needle (Caliber) was inserted into the middle left anterior descending (LAE) coronary artery and a vector (1.5 x 1010 viral particles) was injected in a volume of 2 ml in phosphate buffered saline. The chest was closed and the animal was allowed to exit anesthesia. On the fourth day after the injection, the animal was killed. The heart was fixed with glutaraldehyde, sections were made and incubated with X-da1 for 16.5 hours. After filling and preparation of sections, the tissue was stained with a contrasting dye eosin.

Microscopic analysis of tissue sections (transmural sections of the LAE bed 96 hours after the intracoronary injection of an adenovirus containing 1; . Myocardial zones remote from the LAE blood bed did not show X-da1 staining and served as a negative control, while diffuse gene expression was observed in myocytes and in endothelial cells. A significant proportion of myocytes showed β-galactosidase activity (blue staining), and, in subsequent studies using intracoronary injection of a closed chest, similar activity was present 14 days after gene transfer (n = 8). There was no evidence of inflammation or necrosis in the gene expression zones.

4-c. GAAU / EOPP gene transfer and expression

The adeno-associated viral vector encoding EORP was obtained, propagated and purified as described above in Example 2. Four pigs from the nursery (~ 30 kg each) were anesthetized, ventilated and subjected to midline neck venosection. A carotid (carotid) artery was isolated and a guidewire (special probe for catheter insertion) was introduced 5 PrzepsB 1n! The multipurpose angiocatheter 5 PrhepsB was placed in the left envelope artery (LXC) with the tip of the catheter placed at approximately

- 36 008538 exactly 1 cm into the lumen of the coronary artery. The syringe used to inject the gene was first washed with PBS and then the gene solution was poured into the syringe. Intracoronary histamine, 25 μg / min, was infused for 3 min in LXX before virus administration, followed by either 2.36 x 1013 gAAU / TOEP virus particles (n = 3) or 4.72 x 1013 gAAU / EOEP virus particles (and = 1). A total volume of 1.5 ml of the gene solution was injected into each pig at an infusion rate of 1 ml / 30 s. Then the angiocatheter and conductor were removed and the cervical incision was closed. Animals were allowed to recover from anesthesia and placed in a cage for their maintenance until the completion of this study.

At 6-8 weeks after injection of the gene, the pigs were sacrificed and tissues were collected. Hearts were dissected and placed in ice-cold saline. The coronary arteries were perfused in the cold and the tissue was collected and quickly frozen in liquid nitrogen. Other tissues were taken in a similar fashion as quickly as possible, and quickly frozen in liquid nitrogen. Both fluorescence microscopy and OPCR of tissue sections demonstrated successful delivery and expression of the ECEP gene by the GAAU vector using this direct intracoronary injection delivery in closed chest pigs. In particular, as shown below, the results from RT-PCR confirmed that the gene was successfully delivered and expressed in the bed supplied with blood by the injected artery (i.e. the LSC bed), in comparison with the bed of the left anterior descending coronary artery (LAO):

Pig No. 1 Pig No. 2 Pig No. 3 Pig No. 4

Slice 1 bcx + + + +

Slice 2 bcx + - ++

LAO ....

Example 5. Pork model of angiogenesis-mediated gene therapy (using the ECE-5 transgene)

In this pig model for myocardial ischemia and heart failure, animals were stressed by electrical stimulation of the atrium. The degree of stress-induced myocardial dysfunction and insufficient regional blood flow was quantified and then gene transfer was performed by intracoronary injection of an illustrative recombinant adenovirus expressing ECE-5. Gene transfer was performed after the development of stable but limited endogenous angiogenesis and detection of the presence of induced ischemia similar to angina pectoris (angina pectoris) in patients. Animals did not have ischemia at rest, but developed ischemia during atrial activity or electrical stimulation. Control animals received recombinant adenovirus expressing Ε1Ζ (β-yes!) To exclude the possibility that the adenovirus itself, regardless of ECE-5, stimulated the formation of new blood vessels. It was also a control for possible continued development of collateral vessels, not related to gene transfer. Two weeks after gene transfer, stress-induced cardiac dysfunction and regional blood flow were again measured.

Pigs receiving ΙηοΖ. showed a similar degree of cardiostimulation-induced dysfunction in the ischemic region before gene transfer and two weeks after gene transfer. In contrast, two weeks after receiving the ECE-5 gene, these animals found an increase in wall thickening and improved blood flow in the ischemic region during electrical pacing. These results showed that gene transfer of the angiogenic transgene (ECE-5) was effective in attenuating regional myocardial contractile dysfunction due to improved regional blood flow through newly formed blood vessels.

Ways

Animals and model

Used Yorkshire domestic pigs (§I8 xsgoGa. And = 27) with a weight of 47 ± 9 kg. Two animals were intracoronary injected with a recombinant adenovirus expressing 1; ιοΖ (1011 viral particles in 2.0 ml of saline) and were killed 3 or 5 days after injection. The remaining 25 animals had catheters placed in the left atrium, pulmonary artery, and aorta, which provided a means for measuring regional blood flow and monitoring blood pressure.

The wires were fixed with sutures on the left atrium for the possibility of ECG recording and atrial electrical stimulation. An ameroid tourniquet was placed around the proximal left envelope of the coronary artery. Ameroid material is hygroscopic and slowly swells, leading to complete closure of the artery 10 days after application, with minimal damage (<1% of the left ventricle) due to the development of collateral blood vessels. Myocardial function and blood flow are normal at rest in the area previously perfused with a clogged artery (ischemic area), but blood flow is insufficient to prevent ischemia when myocardial oxygen demand increases. The formation of collateral vessels is completed within 21 days of the placement of the ameroid and remains unchanged for at least 4 months (Κοΐΐι с1 a1., At. 1. Руу8ю1. 253: Н1279-Н1288, 1987). A hydraulic sleeve is also placed around

-37008538 arteries, adjacent but distal to the ameroid. These procedures are described in detail by Nuttop e1 a1., 1. At. Co11. Sagb1o1. 23: 475-482, 1994 and Nuttopb e1 a1., 1. C1t. 1 batch 1. 92: 2644-2652, 1993. Two animals died 5 and 7 days after the placement of the ameroid. 38 (± 2) days after the placement of the ameroid, when limited collateral circulation was developed and stabilized, the animals were studied to determine the regional function and blood flow induced by cardiac pacing, and then the animals received recombinant adenovirus expressing either 1; κΖ (n = 7, control animals), or ECE-5 (n = 16, treatment group) delivered by intracoronary injection. Then, after 14 ± 1 days, the studies were repeated to determine the regional function and blood flow induced by electric cardiac pacing. The next day, animals with Ab1; · κΖ (n = 7) and AbECE-5 (n = 11) were killed and tissues were collected. Five animals with AbECE-5 were examined 12 weeks after gene transfer and then killed. Recombinant adenovirus and transgene delivery.

The human helper independent replicatively defective adenovirus-5 system was obtained as described in Example 2 above.

For intracoronary transgene delivery, animals were anesthetized and the 5E arterial guide for the catheter was placed in the carotid (carotid) artery. A multipurpose (A2) 5E coronary catheter was inserted through the conductor and into the carotid arteries. Ameroid closure was confirmed in all animals by injection of a contrast medium into the left main coronary artery. Then the tip of the catheter was placed deep in the arterial lumen of the proximal aorta so that a minimal amount of material was lost during the injection. Four milliliters containing 2 x 1011 virus particles of recombinant adenovirus were delivered by slow injection of 2.0 ml to both the left and right coronary arteries.

Tests:

(ί) Regional contractile function and perfusion. Two-dimensional and M-modal images were obtained from the right periosternal approximation at the level of the middle papillary muscle using an ultrasound imaging system (He \\ '1e11 Raskagb 8opok 1000). Conscious animals were examined suspended in a comfortable supporting band to minimize body movement. Images were recorded on a CN8 tape using animals in the baseline (background) state and again during left atrial electrical stimulation (heart rate = 200 beats per minute). These studies were performed on day 1 before gene transfer and were repeated after 14 ± 1 days. Five animals were examined again 12 weeks after gene transfer (ECE-5) to determine whether the effect of improved function was maintained. The products heart rate pressure and left atrial pressure were similar in both groups before and after gene transfer, indicating conditions of similar oxygen demand and load. Echocardiographic measurements were performed using standardized criteria (8aEp e1 a1., Spsiabop 58: 1072-1083, 1978). To demonstrate the reproducibility of echocardiographic measurements, animals were visualized on two consecutive days. The data from these individual definitions were highly reproducible (thickening of the lateral wall: r2 = 0.90; P = 0.005). The percent decrease in function, measured by transthoracic echocardiography and ultrasound micrometry in this model, are similar (NattoNb e1 a1., 1. At. Co11. Sagby1. 23: 475-482, 1994 and Nattopb e1 a1., 1. C1t. : 2644-2652, 1993), which documents the accuracy of echocardiography for assessing ischemic dysfunction. The analysis was performed without knowledge of the treatment group.

The contrast medium (LeuM; galactose microaggregates) increases the echogenicity (whiteness) of the image after left atrial injection. Microaggregates are distributed in the coronary arteries and myocardial walls in proportion to blood flow. The maximum intensity of the contrast enhancement correlates with myocardial blood flow, as measured using microspheres (8kua e1 a1., Spi1aTsop 58: 1072-1083, 1978). Thirty-eight (± 2) days after the ameroid was placed, quite distant in time from the closure of the ameroid, but before the gene was transferred, contrast echocardiographic studies were performed during pacing (200 beats per minute) of the atria. Studies were repeated 14 ± 1 days after gene transfer and, in five animals, 2 weeks after gene transfer with ECE-5. The maximum contrast intensity was measured using video images using a computer program for analyzing video images (Soyogue II, Nova Mkokokhsa, 1pb1aparo11k, 1pb1apa), which provided objective measurement of the intensity of video images. The data were expressed as the ratio of the maximum intensity of the video image in the ischemic region (LCx bed) to the maximum intensity of the video image in the interventricular septum (1U8, the region receiving normal blood flow through the unclamped left anterior descending coronary artery). The differences in regional blood flow during atrial stimulation, as measured by contrast echocardiography, were similar to the differences measured by microspheres in the same model in the authors' laboratory, which confirms the accuracy of echocardiography for assessing regional myocardial blood flow. Contrast studies were analyzed without knowing which gene the animal received.

- 38 008538 (ίί) Assessment of angiogenesis.

The brachiocephalic artery was cannulated, and other large vessels were ligated. After an intravenous injection of heparin (10,000 IU), papaverine (60 mg) and then potassium chloride (to induce cardiac arrest in diastole), the aorta was pinched and the coronary vasculature was perfused. A solution of glutaraldehyde (6.25%, 0.1 M cacodylate buffer) was perfused at a pressure of 120 mm Hg; heart extracted; the bed was identified using color-coded dyes injected anterograde through the left anterior descending, left envelope and right coronary artery; and the ameroid was checked to confirm vascular closure. Samples taken from normally perfused and ischemic regions (endocardial third and epicardial third) were poured into plastic and prepared for microscopic analysis of the number of capillaries. Four cross sections with a thickness of 1 μm were taken from each sub-sample (endocardium and epicardium of each region), as described earlier (MaIKI-Sokk11o, Mkgotaks. Kek. 33: 98-117, 1987 and Poo1e apy MaIKI-SokKio, At. I. Rukyu1 259: H204-H210, 1990). The number of capillaries around each fiber and the cross-sectional area of the fiber in each of the eight fields of view in each sub-sample (randomly selected by system sampling) was measured by an image analyzer (Ukeoteik 150, Atepsap 1ppowak1op) at ΧΙ400. The number of capillaries around a total of 325 ± 18 fibers was measured. The density of capillaries (number per fiber cross-sectional area) was estimated by counting points of 15 ± 1 fields of view per sub-sample. The relative standard errors of the number of capillaries around the fiber, the cross-sectional area, and the density of the capillaries were

1.4, 4.1 and 4.2%, respectively. The capillary: fiber ratio was calculated as the product of the density of capillaries by the cross-sectional area. There was no significant difference in fiber cross-sectional area in myocardial samples from any group. Bromodeoxyuridine (50 mg / kg) was injected into the peritoneal cavity of five animals: a control animal (without an ameroid); two animals with ameroid blockers that received gene transfer 1; "Ζ two weeks before; and two animals with ameroid blockers that received ECE-5 gene transfer two weeks before. Thirty-six hours after the injection of bromodeoxyuridine (VKOI), the animals were killed and the tissue was dissected for analysis using the previously described methods (Kaliga e1 a1., ATP. Kek. 74: 383-400, 1994). Sections of the duodenum were used as positive controls.

(ΐϊϊ) Expression of DNA, mRNA and protein.

After gene transfer, left ventricular homogenates were tested to confirm the presence and expression of the transgene. The polymerase chain reaction (PCR) using a sense primer to the promoter SMU (SSASASSTSTSTTASTAAS; BEO ΙΌ N0: 1) and an antisense primer to the internal sequence ECE-5 (CAAAATSSSTASASATATSST; BEO ΙΌ N0: 2) amplified the expected fragment. Using a sense primer to the beginning of the ECE-5 sequence (ATSASSTSTSTSTSTSTSSTS; BEO ΙΌ N0: 3) and an antisense primer to the internal sequence of ECE-5 (i.e. BEO ΙΌ N0: 2), RT-PCR amplified the expected 400 bp fragment. Primers directed against the adenovirus DNA region were used to detect wild-type viral DNA or recombinant viral DNA in tissues (TSTSTTSTSASSASSTSTTS; BEO ΙΌ N0: 4) and (SATSTSAASTAAASSTSS; BEO ΙΌ N0: 5). Expected fragment 900 bp amplified from this recombinant virus. These studies were performed on tissue samples of 200 mg from the myocardium and other tissues. The sensitivity of detection of PCR was equal to 1 viral sequence per 5 million cells. A polyclonal antibody directed against ECE-5 (Kyaoka e1 a1., Shuya. 0p11a1to1. U1k. Bs1. 35: 3189-3198) was used in protein immunoblots from cultured rat fibroblasts of the heart 48 hours after ECE-5 or ΕκΖ gene transfer . The ECE-5 protein was detected in conditioned medium after the transfer of the ECE-5 gene, but not after the transfer of the gene Π · «Ζ. Methods for PCR and Western blotting have been described in detail in Nutty e1 a1., E C1t. Shuei. 92: 2644-2652, 1993, COL e! A1., I. C1t. Shuei. 91: 939-949, 1993 and Tk1 e! A1., At. I. MARK1. 267: H2079-H2085, 1994. To test the transgene for mitotic activity at ν 11th, differentiated (mature) rat heart fibroblasts were infected or not infected with adenovirus encoding ECE-5 or adenovirus encoding код Ζ. Media from these cell cultures were incubated with murine NIH 3T3 fibroblasts and the incorporation of tritiated thymidine was measured (Tk1 e1 a1., Epiospokdu 136: 38313838.1995). (ίν) Adenovirus release during intracoronary delivery.

Pulmonary arterial blood was taken continuously during intracoronary injection of recombinant adenovirus in three animals. Serum from each sample was used in a standard plaque assay. Undiluted serum (0.5 ml) was added to subconfluent H293 cells; after 10 days, plaques did not form. However, when 0.5 ml of serum was diluted 200-8000 times using BMEM (2% FCS), viral plaques formed on day 9. A single vascular bed (myocardial) separates the coronary and pulmonary arteries. If the virus does not attach in this bed after injection into the coronary artery, then the concentration of the virus in the pulmonary artery should reflect the dilution of coronary sinus blood with systemic venous blood throughout the injection time. Measurements from the authors' laboratory show that coronary blood flow represents 5% of pulmonary arterial blood flow. Using this dilution factor (2-fold), the duration of the coronary injection and the amount of virus injected, the authors calculated the amount of adenovirus,

- 39 008538 delivered to the pulmonary artery, with the assumption of the absence of escape or attachment of the virus. This estimate was compared with the measured amount and the difference was used as an estimate of the amount of virus excreted by the myocardial vascular bed.

(ν) Assessment of inflammation.

Hematoxylin / eosin and Mazzoy tricolor dyes were used to detect infiltrates of inflamed cells, cell necrosis and fibrosis. Murine ascites, pork monoclonal antibodies against CE4 and against SE8 (1.0 mg / ml; UMKE, 1is., Pu11tai, Aazidid) were used to detect markers of CE4 and SE8 on T-lymphocytes in frozen sections (6 μm) of the spleen (positive control ) and hearts. These studies were performed on transmural samples of the hearts of six animals that received ameroid blockers 50 days before killing: two animals that did not receive gene transfer, two animals that received ECE-5 gene transfer 2 weeks before killing, and two animals that received gene transfer 1; κΖ two weeks before. The analysis was performed without knowledge of the treatment group.

(νί) Statistical analysis.

Data were expressed as mean ± 1 standard error (standard error of the mean). The measurements made before and after the transfer of the ECE-5 and No. ген genes were compared using two-way analysis of variance (Sgisk4, Sgisk 8gr Sogrogayoi, Oak1aU, SaShogsha). Data from angiogenesis studies were also subjected to two-way analysis of variance. The null hypothesis was rejected when P <0.05.

Results using the PCP-5 transgene

Three measurements were used to assess whether ECE-5 gene transfer was effective in treating myocardial ischemia: regional contractile function and perfusion (measured before and after gene transfer) and the number of capillaries. All measurements were carried out without knowing which gene the animals received (ECE-5 vs. No. Ζ).

Regional contractile function and blood flow

Thirty-eight days after the placement of the ameroid, the animals showed a thickening of the wall during electrical atrial stimulation. Pigs receiving No. имели had a similar degree of electrical stimulation of dysfunction induced in the ischemic region before gene transfer and two weeks after gene transfer. In contrast, two weeks after ECE-5 gene transfer, there was a 2.7-fold increase in wall thickness in the ischemic region during electrical stimulation (P <0.0001; Fig. 6). Wall thickening in a normally perfused area (interventricular septum) was normal during electrical stimulation and was not affected by gene transfer (% wall thickening: No.Ζ: before gene transfer 53 ± 8%, after gene transfer 51 ± 6%; ECE-5 : before gene transfer 59 ± 4%, after gene transfer 59 ± 6%).

Improved regional blood flow has been associated with improved function in the ischemic region. Two weeks after the transfer of gene No. Ζ, there was a persistent deficiency of blood flow in the ischemic region during electrical stimulation (Fig. 8). However, animals receiving the ECE-5 gene showed a homogeneous contrast enhancement in two areas two weeks later, indicating improved blood flow in the ischemic region (P = 0.0001). To determine whether improved function and perfusion in the ischemic bed were long-term, five animals were tested again 12 weeks after ECE-5 gene transfer. Each animal showed persistent improvements in function (P = 0.005; FIG. 6) and perfusion (P = 0.001; FIG. 8).

Angiogenesis

Animals (without gene transfer) that were uninfected with ameroid constriction of blood vessels had physiological reactions identical to those of animals receiving coding No. Adenovirus, which indicated that vector No. Ζ did not adversely affect native angiogenesis. To assess angiogenesis, the number of myocardial capillaries was quantified using microscopic analysis of perfusion-fixed hearts (Fig. 9). The number of capillaries surrounding each myocardial fiber was greater in the endocardium of ischemic and non-ischemic regions in animals that received ECE-5 gene transfer, compared with the same animal heart regions that received gene 1 transfer; · κΖ (P = 0.038) . Thus, improved regional function and perfusion were associated with capillary angiogenesis two weeks after ECE-5 gene transfer. The increased number of capillaries around each fiber tends to increase in the epicardial part of the wall after ECE-5 gene transfer (P = 0.13). Other criteria for capillarity, such as the number of capillaries per fiber cross-sectional area and the number of capillaries per fiber number, did not change in the endocardium or epicardium.

Expression of DNA, mRNA and protein

After obtaining the established beneficial effects of ECE-5 gene transfer on function, perfusion, and the number of capillaries around each fiber, it was urgent to demonstrate the presence and expression of this transgene in the heart. Polymerase chain reaction (PCR) and reverse transcriptase coupled to PCR (RT-PCR) were used to detect transgene DNA and mRNA

- 40 008538 single E6E-5 in the myocardium from animals that received the transfer of the E6E-5 gene.

After gene transfer, left ventricular samples were tested to confirm transgene incorporation and expression. Briefly, 3 days after intracoronary transfer of the 1ac2 gene, the myocardium was treated with X-da1 and then stained with a contrast dye eosin X120. A study using standard histological methods revealed that most of the myocytes showed β galactosidase activity (blue staining). Activity was also visible 14 ± 1 days after gene transfer in all animals treated with 1ac2 gene transfer. A higher increase in activity in cells containing β-galactosidase activity confirms gene expression in myocytes. PCR analysis using a sense primer directed against the internal sequence of E6E-5 was performed to confirm the presence of recombinant adenoviral DNA encoding E6E-5 in the ischemic (ΕΟχ) and non-ischemic (LAO) regions of three animals that received the E6E-5 gene transfer . These results, shown in FIG. 10 A, confirmed the presence of the expected fragments of 500 bp Then, the expression of E6E-5 mRNA was examined 14 days after gene transfer. As shown in FIG. 10B, amplified RT-PCR fragment 400 bp was present in both areas from two animals, while control animals did not detect a signal. A polyclonal antibody directed against E6E-5 was used in protein immunoblots from cultured rat cardiac fibroblasts 48 hours after transfer of the E6E-5 or 1ac2 gene. As shown in FIG. 10C, the E6E-5 protein was detected after the transfer of the E6E-5 (E) gene, but not after the transfer of the 1ac2 (β) gene, which demonstrated the expression and extracellular secretion of the protein after the transfer of the E6E-5 gene. Finally, PCR using a set of primers directed against adenoviral DNA (E2 region) was performed to determine if adenoviral DNA was present in the retina, liver or skeletal muscle of two animals that received an intracoronary injection of adenovirus 14 days before. As shown in FIG. 10Ό, the expected amplified fragment of 900 bp was found only in the control track (+) containing recombinant adenovirus (as a positive control), but not in the tracks related to the retina (d), liver (1) or skeletal muscle (t) of the treated animals.

Successful gene transfer has been confirmed in both ischemic and non-ischemic regions. Immunoblotting showed the E6E-5 protein in the myocardium of animals that received E6E-5 gene transfer. In additional experiments using cultured fibroblasts, the authors confirmed that transfer of the E6E-5 gene gave these cells the ability to synthesize and secrete extracellular E6E-5. The medium from cultured cells infected with a recombinant adenovirus expressing E6E-5 showed a mitogenic response (14-fold increase compared to control; P = 0.005). Finally, two weeks after the gene transfer, myocardial samples (but not liver samples) from 1ac2 infected animals showed β-galactosidase activity during histological examination. These studies confirm gene transfer and expression of ίη νίνο and demonstrate the biological activity of the transgene product.

Two weeks after the intracoronary injection of recombinant adenovirus, the inventors were unable to detect viral DNA in the liver, retina or skeletal muscle by PCR, despite the presence of viral DNA in the myocardium. These experiments showed that intracoronary delivery of the adenoviral vector minimized the systemic arterial spread of the virus to a level below the detection thresholds of PCR methods. This method may be difficult to succeed in animals with smaller coronary arteries, such as rabbits.

To evaluate the effectiveness of myocardial uptake of adenovirus, the inventors measured the amount of adenovirus released from the heart by taking pulmonary arterial blood samples during intracoronary injection. Unexpectedly, 98.7% of the virus was excreted by the heart during the first passage. Undiluted serum obtained from the pulmonary artery during intracoronary delivery of the virus was unable to form viral plaques under suitable conditions. Thus, this invention provides an effective heart-specific gene delivery system.

Inflammation score

Microscopic examination of transmural sections of the hearts of animals that received recombinant adenovirus did not reveal infiltrates of inflamed cells, cell necrosis or increased fibrosis. As an additional assessment of the cytopathic effect induced by adenovirus, the authors conducted immunohistological studies to detect antigens ΟΌ4 and ΟΌ8, which would indicate the presence of cytotoxic T cells. These studies showed rare positive cells on transmural sections of the heart from uninfected animals (η = 2) or animals that received recombinant adenovirus (η = 4). The liver did not contain inflammation.

Example 6. Gene-mediated angiogenesis using the E6E-4 transgene

This experimental example demonstrated successful gene therapy using a gene encoding another angiogenic protein, E6E-4. The protocol for therapy with the E6E-4 gene was essentially as described in Example 5 for E6E-5.

The human E6E-4 gene was isolated from a cDNA library, which was constructed from mRNA of ΝΙΗ3Τ3 cells transformed with Kaposi sarcoma DNA. E6E-4 cDNA has a length of about 1.2 kb and cody

- 41 008538 constructs a polypeptide of 206 amino acids, including a signal polypeptide of 30 amino acids at the конце-terminus (Ee11 Βονί е! А1. Се11 50: 729-737, 1987; Ве ^ кй е! А1., 1. Се11 Βίο1. 121: 705-713, 1993). The inventors subcloned the PCP-4 cDNA in the form of a substantially full-sized 1.2 kb CI-KI fragment. to the EtoS website in the pACCMURpA8K adenoviral vector (for simplicity pAC8K). The 5'-start site was at a base pair of 243, and the 3'-end at a base pair of 863. Recombinant adenovirus encoding PCP-4 (also called Ab.PCP-4 here) was obtained as described in Example 2 to obtain PCP- 5 adenovirus.

The expression of PCP-4 in heart tissue (and the lack of expression in other tissues, including the liver, skeletal muscle and in the eye) was confirmed by Western blot analysis using anti-PCP-4 antibody for detection. The mitogenic effect of PCP-4 on the proliferation of endothelial cells w ν ш! Γο was also tested.

Forty-five days after the ameroid was placed, animals were subjected to studies to determine stress-induced regional function and blood flow, and then the resulting recombinant adenovirus expressing PCP-4 (n = 6 animals) was delivered by intracoronary injection. Thirteen days later, studies to determine stress-induced regional function and blood flow were repeated. The next day, animals were killed and tissues were collected.

Transgene Delivery

As in the case of PCP-5, gene transfer was performed after attenuation of endogenous angiogenesis and inducible myocardial ischemia similar to angina pectoris (angina pectoris) in patients. For intracoronary transgene delivery, the animals were anesthetized and the 5P arterial guide for the catheter was placed in the carotid (carotid) artery. A multipurpose (A2) 5P coronary catheter was inserted through the conductor and into the carotid arteries. Ameroid closure was confirmed in all animals by injection of a contrast medium into the left main coronary artery. Then the tip of the catheter was placed 1 cm into the arterial lumen so that the minimum amount of material was lost in the proximal aorta during injection. Five milliliters containing 1.5 x 1012 virus particles of recombinant adenovirus expressing PCP-4 were delivered by slow injection of 3.0 ml into the left and 2.0 ml into the right coronary artery.

ECE-4 Transgene Results Regional Function and Perfusion

Forty-five days after the placement of the ameroid, the animals showed impaired thickening of the wall during electrical atrial stimulation. In contrast, two weeks after the transfer of the PCP-4 gene, there was a 2.7-fold increase in wall thickness in the ischemic region during electrical stimulation (p <0.0001; FIGS. 11 and 12). Thickening of the wall in a normally perfused area (interventricular septum) was normal during stimulation and was not affected by gene transfer. The improvement in function after the transfer of the PCP-4 gene was statistically indistinguishable from the improvement obtained after the transfer of the PCP-5 or PCP-2Y gene + signal peptide (cr) (Fig. 11). Improved function in the ischemic region was associated with improved regional perfusion (Fig. 12). As shown in FIG. 12, prior to the transfer of the PCP-4 gene, there was insufficient blood flow in the ischemic region during electrical stimulation. Two weeks after the transfer of the PCP-4 gene, a uniform contrast enhancement was seen in both areas, indicating improved blood flow in the ischemic region (p = 0.0001). Results with PCP-4 were statistically indistinguishable from results obtained with PCP-5 and PCP-2b1 + cr (Fig. 13). PCP-2Y + kr is described in Example 7 and is PCP-2 containing a signal sequence.

Transgene Expression

Exclusive expression of PCP-4 in the heart was confirmed by PCR and RT-PCR using primers specific for the sequences encoding this transgene. PCP-4 DNA and mRNA were found in the heart, but they were absent in the eye, liver, and skeletal muscle. These data confirm the results obtained using Ab.PCP-5 (n = 2) and Ab.CPP-2b1 + cr (n = 1). Thus, exclusive expression of this transgene in the heart was confirmed in all four animals that received adenoviral vectors containing different genes encoding an angiogenic protein.

No myocardial inflammation

Investigated transmural myocardial biopsies from three animals that received Ab.SRP-4. Animals were killed 2 weeks after gene transfer. There was no evidence of infiltrates of inflamed cells, necrosis, or increased fibrosis in these sections compared to control ameroid animals that did not receive adenovirus. This took place both in the box LAO and in the box LCx. These microscopic preparations were examined by a pathologist who evaluated without knowing the variant (blindly) and expressed the opinion that there was no evidence of myocarditis in any section.

Example 7: Gene-mediated angiogenesis using mutein PCP-2

This experiment demonstrated successful gene therapy using a gene encoding a third angiogenic protein, PCP-2. This experiment also demonstrates how angiogenic protein can be modified to increase secretion and potentially improve potency.

- 42 008538 angiogenic gene therapy in enhancing blood flow and cardiac function in the heart. The protocol used for therapy with the human ECE-2 gene was virtually identical to the protocol used for ECE-5 and ECE-4 above.

Acidic ECE (aECE, ECE-1) and basic ECE (ECE-2) do not contain a native secretory sequence; although some protein secretion may take place. An alternative secretory pathway that does not include a Golgi apparatus has been described for acidic ECE. Two ECE-2 constructs (ECE-2L1 + 8p and ECE-2b1-8p) were obtained, one with a sequence encoding a signal peptide (ECE-2Y + §p) for the classical secretory pathway of the protein and one without a signal peptide peptide sequence (ECE- 2Y-8p), for testing the improved efficacy of ECE-2 having an added signal peptide compared to the same protein without an added signal peptide.

As shown below, ECE-2 has a loop structure of five residues, which extends from amino acid residue 118 to residue 122. This loop structure was replaced by cassette directed mutagenesis with the corresponding loop of five residues from interleukin-1B to obtain mutants with the replacement of the loop ECE- 2Y. Briefly, the gene encoding S1i3.5ESE-2 (^ 66οη e! A1., App. Ν.Υ. Asab. 8c1. 638: 98-108, 1991) was cloned into the expression vector T7 pET-3a (M13), produced from pET-3a (Κο ^ η ^ Γ§ e! a1., Sene 56: 125-135, 1987), between the restriction sites She1 and BatN1. Unique restriction sites, Βδ! ΒΙ and 8рI, were introduced into this gene in such a way as not to produce changes in the encoded amino acids (i.e. silent mutations), at positions that flank codons encoding the ^ Γ117-Τιρ123 ECE-2 segment.

Structural comparison of 1 loops β9-β10 in ECE-1, ECE-2 and ΙΌ-1 β.

110 115 120 125 130

ECE-1 ΕNНΥNΤΥI§ΚΚНΑΕΚНВЕУС ^ ΚΚNС (8ΕΟ Ш N0: 6)

110 115 120 125 130

ECE-2 8 \\ ¥ \ T ¥ K8KK ¥ ..T8U ¥ UAEKTS (8ΕΟ ΙΌ N0: 7)

110 115 120 125

ΙΌ-1β NNΚ ^ ΕЕΕ§Α ^ Е..ΡNVΥI8Τ§ ^ ΑΕ (8ΕΟ ΙΌ N0: 8)

The numbering for ECE-1 and ECE-2 comes from amino acid residue 1, decrypted from the cDNA sequence encoding the form of 155 residues (described in No. 66οη e! A1., At. Ν.Υ. Asab. 8cE 638: 98-108, 1991 ), and the numbering for EC-1 β comes from residue 1 of the mature polypeptide of 153 residues (1b.).

Replace the residues Agd 118-8ν 8119-Τν g 120-Τ11Γ121-8eg 122 ECE-5 with the human sequence A1aSk-Rke-Rgo-Ay! from the corresponding loop of the structural analogue of EC-1 β (115-119) was performed essentially as follows.

The plasmid DNA, pET-3a (M13), was digested with !δ! ΒΙ and 8pP, and the resulting larger DNA fragment was isolated by agarose gel electrophoresis. This DNA fragment was ligated using T4 DNA ligase with double-stranded DNA obtained by annealing two synthetic oligonucleotides: 5'-CCΑΑССΑΤΤССΑΑΤСΤΑΑΤΑ

ΑСΤΑСΑΑΤΑССΤΑСССССΤСΤ СССССАет ^ 'С СΤΑΑСΤССΤΑ ΤСΤССССΑСΤΤ ААСС-3' (8ΕΟ ΙΌ N0: 9) and 5'-СΤΑСССΤΤΑΑ СΤСССΑСΑΤΑ ^ АС ^ A ^ А ААССССССАС ΑССССΤΑССΤ А ^ ОТАС ^ А0 АСА ^ 8 АОАА 10), which contain ends compatible with the ends obtained by cleavage of Βδ! ΒΙ and 8p1E, the ligation product was used to transfect BxCepsea ^ 1ί cells (strain OH5o). The desired mutant plasmid (ECE-2Y) was selected based on sensitivity to cleavage at the newly introduced AI2 restriction site (underlined above).

ECE-2Ys with and without signal peptide were constructed using a polymerase chain reaction (PCR) method. To add the sequences of the ECE-4 signal peptide to the 5 'ECE-2Y and to ensure that the signal peptide will be cleaved from the ECE-2BE, a gene cassette used ΕοϊΌΐίβΙι Κ е! a1. to obtain secreted ECE-1. Using a primer (рЕ1В: 5'-ССССАТСССС ССАТСССССС ССССССССС CC-3 '(8ΕΟ ΙΌ N0: 11), mating with the 5'-part of the ECE-4 signal peptide, and a second primer (рЕ2К: 5'-СССΑΑΤΤСΤС ΤСΑΑССΤССΤ 6А'ПТ -3 '(8Ε0 ΙΌ N0: 12) to the 5'part of ECE-1, the inventors synthesized, using PCR, a DNA fragment containing a WatCh site at the 5'-end of the sequences of the ECE-4 signal peptide, followed by the first ten amino acids ECE-1 and the Ε ^ ΚΙ site at the 3'-end. Using a different pair of primers (pЕ3К: 5'-СССΑΑΤΤСΑΤ СССТСААССС СΑΑΑΤСΑСС-3 '(8Ε0 ΙΌ N0: 13) and рЕ4НА: 5'-ССΤСΤΑСΑΤΤ AC ^ OTaETS TSSSSACOTSS ΤΑΤSSCΤΑSS ΤСΤΤΑССΑСΑ СΑΤΤССΑΑСΑ ААААС-3 '(8Ε0 ΙΌ N0: 14)), mating with the sequences 5'- and 3'- ECE-2Ы, respectively, the authors obtained a second DNA fragment that has the site Ε ^ ΚΙ on 5'-end and influenza hemagglutinin (HA) label plus the Xa! Site at the 3'-end of ECE-2E1. Then, these two fragments were subcloned into the pcc ^ NΑ3 vector at the Bbc and Xba sites by ligation of three molecules. The plasmid pECE2 ^ I / c ^ NΑ3, which was similar to p8ΡECE-2 ^ I / s ^ NΑ3, except that it did not have a signal peptide, was subcloned in a similar manner. Then both plasmids were sequenced to confirm the correct insert. Then both fragments of ECE-2 B! isolated from pc ^ N3 by the splitting of WbH and Xa! and subcloned into RASCMURPAK (+) (for simplicity, pAC8K), which is a person

- 43 008538 night vector, to obtain a recombinant virus. The recombinant virus and the injected vector were prepared essentially as described in Example 2. Gene transfer was performed as described in Example 5 (using 8 animals for ECE-2Y cr + and 6 animals for ECE-2Y cr-, with the vector ΕκΖ serving as a control, all with 1011-1012 viral particles).

Results using PCP-2 muteins

Two weeks after the ECE-2L + kr gene transfer, the ratio of maximum contrast intensities (bCx / bY) during stress induced by electrical stimulation at 200 beats per minute of stress significantly improved in comparison with pregene transfer. FIG. 13 shows the results using intracoronary gene transfer by recombinant adenovirus expressing ΕκΖ, ECE-5, ECE-2Y + cr, ECE-2Y-cr and ECE-4 for comparison. The black column on the right side of FIG. 13 shows the normal blood flow ratio using this method. ECE-2Y + cr normalized the ratio of the maximum intensities of blood flow contrasts in these animals.

The percentage of wall thickening also improved two weeks after intracoronary delivery of recombinant adenovirus expressing ECE-2Y + cr. FIG. 11 shows the results using intracoronary gene transfer of a recombinant adenovirus expressing ΕκΖ, ECE-5, ECE-2Y + cr, ECE-2Y-cr and ECE-4 for comparison. The black column on the right side of FIG. 11 shows the normal percentage of wall thickening before induced stress stimulation. ECE-2Y + cr improved regional function to a degree that was statistically indistinguishable from the degree of improvement using ECE-5. Although a slight improvement was noted after ECE-2Ycr gene transfer, the improvement using the signal peptide-containing transgene was more significant (FIG. 13).

Claims (65)

CLAIM 1. A method of increasing blood flow in an ischemic tissue of a skeletal muscle of a patient, comprising delivering a transgene encoding an angiogenic protein or peptide to ischemic tissue of a skeletal muscle by injecting a solution containing a vector containing a transgene into said tissue, whereby this transgene is expressed in tissue where an angiogenic protein or peptide causes an increase in blood flow and a decrease in tissue ischemia, and where the angiogenic protein or peptide is selected from platelet growth factor, fibroblast growth factor, insulin like growth factor induced by factor hypoxia, angiopoietin, nitric oxide synthase, hepatocyte growth factor, zinc-binding protein containing many zinc finger motifs that induces the expression of an endogenous angiogenic gene. 2. The method according to claim 1, in which the injection is a bolus injection. 3. The method according to claim 1 or 2, in which the solution contains at least about 1 ml. 4. The method according to any one of the preceding paragraphs, where the specified angiogenic protein or peptide is a fibroblast growth factor. 5. The method of claim 4, wherein said fibroblast growth factor is aECE, BECE, ECE-4, ECE-5, or ECE-6. 6. The method according to any one of claims 1 to 3, where the specified angiogenic protein or peptide is an insulin-like growth factor. 7. The method according to claim 6, where the specified angiogenic protein or peptide is an insulin-like growth factor 1. 8. The method according to any one of claims 1 to 3, wherein said angiogenic protein or peptide is a hypoxia-induced factor. 9. The method according to any one of claims 1 to 3, where the specified angiogenic protein or peptide is angiopoietin. 10. The method according to any one of claims 1 to 3, where the specified angiogenic protein or peptide is a growth factor for hepatocytes. 11. The method according to any one of claims 1 to 3, wherein said angiogenic protein or peptide is a zinc binding protein containing a variety of zinc finger motifs that induces the expression of an endogenous angiogenic gene. 12. The method according to any one of claims 1 to 3, wherein said angiogenic protein or peptide is constitutive nitric oxide synthase. 13. The method according to any one of claims 1 to 12, wherein said vector further comprises a second transgene. 14. The method according to any one of claims 1 to 12, where the specified vector contains at least two transgenes, each of which independently encodes an angiogenic protein or peptide. 15. The method according to claim 6, where the second transgene encodes a protein that induces blood flow and / or contractile function. 16. The method according to any one of the preceding paragraphs, where the specified angiogenic protein or peptide contains a secretory signal sequence. 17. The method according to any one of the preceding paragraphs, where the patient has a peripheral disease - 44 008538 vessels. 18. The method according to any one of the preceding paragraphs, where the specified vector is an adenoviral vector or an adeno-associated viral vector. 19. The method of claim 18, wherein the vector is a replicatively defective adenoviral vector. 20. The method according to claim 19, where approximately 10 ⁹ -10 ¹² adenoviral particles are delivered w1uo. 21. The method according to any one of claims 1 to 17, where the vector is a non-viral vector. 22. The method according to item 21, where the vector is a delivery platform based on a non-viral protein or a lipid-based vector. 23. The method according to any one of the preceding paragraphs, where the vector is a targeted vector. 24. The method according to item 23, where the targeted vector includes half of the ligand-receptor pair, and the target cells in ischemic tissue include the other half of the ligand-receptor pair. 25. The method according to any one of the preceding paragraphs, where the expression of the transgene is triggered by a constitutive promoter. 26. The method according A.25, in which the constitutive promoter is a promoter of SMU. 27. The method according to any one of claims 1 to 24, where the expression of the transgene is triggered by a tissue-specific promoter. 28. The method according to any one of the preceding paragraphs, where the contractile function in ischemic tissue increases. 29. The method according to any one of the preceding paragraphs, where the delivery of the vector to the patient’s ischemic tissue coincides with or is preceded by several minutes of delivery of the vasoactive agent into the blood vessel supplying the ischemic tissue. 30. The method according to clause 29, in which the vasoactive agent comprises a nitric oxide donor. 31. The method according to clause 29, in which the vasoactive agent includes sodium nitroprusside. 32. The method according to p, in which sodium nitroprusside is at a concentration of about 1-75 mg / ml 33. The method of claim 32, wherein the sodium nitroprusside is at a concentration of about 50 mg / ml. 34. The composition for gene therapy for use in the method according to claim 1, comprising a vector containing a transgene encoding an angiogenic protein or peptide selected from platelet growth factor, fibroblast growth factor, insulin-like growth factor, hypoxia-induced factor, angiopoietin, synthase nitric oxide, hepatocyte growth factor, and a zinc-binding protein containing many zinc finger motifs that induces the expression of an endogenous angiogenic gene. 35. The composition according to clause 34, where the specified vector is a viral vector. 36. The composition of claim 35, wherein said vector is a replicatively defective viral vector. 37. The composition of claim 35, wherein said vector is an adenoviral vector. 38. The composition of claim 37, wherein said vector is a replicatively defective adenoviral vector. 39. The composition according to clause 37, containing approximately 10 ⁷ -10 ¹³ adenoviral vector particles. 40. The composition according to § 39, containing approximately 10 ⁹ -10 ¹² adenoviral vector particles. 41. The composition according to clause 34, where the expression of the specified transgene is triggered by the promoter of the SMU, which is contained in the vector. 42. The composition according to clause 34, where the expression of the specified transgene is triggered by a tissue-specific promoter, which is contained in the vector. 43. The composition of claim 34, wherein said angiogenic protein or peptide is a fibroblast growth factor. 44. The composition according to item 43, wherein said angiogenic protein or peptide is a fibroblast growth factor selected from the group consisting of aOPOP, LOPP, POP-4, POP-5 and POP-6. 45. The composition according to clause 34, in which the specified angiogenic protein or peptide is a platelet growth factor. 46. The composition according to item 45, in which the specified platelet growth factor is selected from the group consisting of RESP A and RESP B. 47. The composition of claim 34, wherein said angiogenic protein or peptide is an insulin-like growth factor. 48. The composition of claim 47, wherein said angiogenic protein or peptide is an insulin-like growth factor 1. 49. The composition of claim 34, wherein said angiogenic protein or peptide comprises a signal peptide. 50. The composition of claim 34, wherein said angiogenic protein or peptide is an angiogenic polypeptide regulator. 51. The composition of claim 34, wherein said vector further comprises a second transgene encoding an angiogenic protein or peptide. - 45 008538 52. The composition of claim 34, wherein said vector comprises a transgene or transgenes encoding at least two angiogenic proteins or peptides. 53. The composition of claim 52, wherein said angiogenic proteins or peptides are each independently selected from the group consisting of platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor, hypoxia-induced factor, angiopoietin, nitric oxide synthase, hepatocyte growth factor and a zinc-binding protein containing many zinc finger motifs that induces the expression of an endogenous angiogenic gene. 54. The composition of claim 52, wherein the first of these angiogenic proteins or peptides is selected from the group consisting of platelet-derived growth factor, fibroblast growth factor, insulin-like growth factor, hypoxia-induced factor, angiopoietin, nitric oxide synthase, hepatocyte growth factor, and zinc-binding protein containing many motifs of the type of "zinc finger", which induces the expression of an endogenous angiogenic gene, and in which the second of these angiogenic proteins or peptides is selected from another representative specified group. 55. The composition according to paragraph 52, in which the first of these angiogenic proteins or peptides is a fibroblast growth factor, and the second of these angiogenic proteins or peptides is an insulin-like growth factor. 56. The composition of claim 52, wherein said vector comprises a transgene or transgenes encoding a fibroblast growth factor and an insulin-like growth factor. 57. The composition according to any one of claims 34 to 56, further comprising a pharmaceutical excipient. 58. The kit for use in the method according to claim 1, comprising a vector containing a transgene encoding an angiogenic protein or peptide selected from a platelet growth factor, fibroblast growth factor, insulin-like growth factor, hypoxia-induced factor, angiopoietin, nitric oxide synthase, growth factor hepatocytes, and a zinc-binding protein containing many zinc finger motifs that induces the expression of an endogenous angiogenic gene. 59. The kit according to § 58, further comprising a device for introducing this composition into the tissue ίη νίνο. 60. The kit of claim 59, wherein the device is a catheter. 61. A kit according to any one of claims 58-60, further comprising a vasoactive agent. 62. The kit of claim 61, wherein the vasoactive agent contains a nitric oxide donor. 63. The kit of claim 61, wherein the vasoactive agent comprises sodium nitroprusside. 64. The kit according to item 63, in which sodium nitroprusside is at a concentration of about 1-75 mg / ml. 65. The kit of claim 63, wherein the sodium nitroprusside is at a concentration of about 50 mg / ml.