EA031308B1 - Sdf-1 delivery for treating ischemic tissue - Google Patents

Abstract

Provided are methods of treating a cardiomyopathy in a subject by administering directly to, or expressing locally in, a weakened, ischemic, and/or peri-infarct region of myocardial tissue of the subject an amount of stromal-cell derived factor-1 (SDF-1) effective to cause functional improvement in at least one of the following parameters: left ventricular volume, left ventricular area, left ventricular dimension, cardiac function, 6-minute walk test, or New York Heart Association (NYHA) functional classification. Also provided are methods of treating critical limb ischemia in a subject by administering a DNA plasmid encoding human SDF-1 by direct injection into the affected limb.

Description

Scope of invention

This application relates to methods of delivering SDF-1 and compositions for treating cardiomyopathy, as well as the use of methods of delivering SDF-1 and compositions for treating ischemic cardiomyopathy. This application further relates to methods of delivering SDF-1 and compositions for the treatment of critical limb ischemia.

Background of the invention

Ischemia is a condition in which blood flow is completely obstructed or significantly reduced in certain parts of the body, which leads to a lack of oxygen, a decrease in the supply of substances and accumulation of metabolites. Although the degree of ischemia depends on the severity of the obstruction of the vessel, its duration, tissue sensitivity and the degree of development of collateral vessels, dysfunctions usually occur in ischemic organs and tissues, while prolonged ischemia leads to atrophy, denaturation, apoptosis and necrosis of the affected tissues.

In ischemic cardiomyopathies, which are diseases affecting the coronary arteries and causing myocardial ischemia, the degree of ischemic damage to myocardial cells is caused by a transition from reversible cell damage to irreversible cell damage as the duration of obstruction of the coronary artery increases.

Critical limb ischemia (CIC) is the latest stage of peripheral vascular disease (BPS) of the lower limbs of atherosclerotic nature and is associated with high morbidity and mortality from both cardiovascular diseases and large amputation.

The estimated frequency of CICs in the United States ranges from 125,000 to 250,000 patients per year, and it is assumed that it increases as patients age. The prevalence of BPS increases dramatically with age, the disease affects about 20% of Americans aged 65 years and older. The current standard of care for people with CIC includes revascularization of the lower extremities either through open and endovascular surgical interventions on peripheral vessels, or by amputation of the extremity (if revascularization was unsuccessful or impossible). One-year mortality of patients with CIC is 25% and can reach 45% among those who underwent amputation. Despite the advanced techniques of vascular and surgical interventions, a significant proportion of patients with CIC are not subject to revascularization. Among these patients, only from 20% to 30% of patients with CIC undergo treatment, for 30% more amputation will be required, and 23% will die within 3 months.

Gene therapy strategies for opening closed vessels or stimulating angiogenesis are being actively studied. In a clinical study involving 6 patients with CICs who were shown to have a large amputation, patients were evaluated on receiving NV1FGF, a non-viral gene therapy sequence expressing fibroblast growth factor 1 (FGF1), within 3-5 days prior to amputation. NV1FGF was administered intramuscularly at 8 sites in doses of 0.4 to 4 mg. In this clinical study, it was documented that, within 3 cm from the injection site of all doses, expression of transgenic FGF1 was observed, and that plasmids that had entered the blood vessel were quickly cleaved. A phase II double-blind, placebo-controlled, multicenter clinical study conducted in the United States (TALISMAN202) was evaluated by 71 patients treated with placebo CIC or NV1FGF as part of 1 of 5 treatment regimens in a dose of 2 to 16 mg administered by 8 intramuscular injections affected leg. This clinical study showed that intramuscular administration in doses up to 16 mg NV1FGF was safe and well tolerated. The primary end point was transcutaneous oxygen partial pressure, which increased in comparison with baseline values in the NV1FGF and placebo treatment groups, but healing of the ulcers improved in the NV1FGF treatment group. Similarly, in a double-blind, randomized, placebo-controlled study of 125 patients Nikol et al. showed that the introduction of NV1FGF significantly reduced (twice) the risk of all amputations, including large limb amputations, in addition, there was a tendency to reduce the risk of death. The obtained clinical studies have shown that the safety window in the treatment of free plasmid DNA is similar to that in the clinical study proposed by the applicants.

Summary of the Invention

This application relates to a method of treating cardiomyopathy in a patient. Cardiomyopathy may be, for example, cardiomyopathies associated with lung embolism, venous thrombosis, myocardial infarction, transient ischemic attack, peripheral vascular disease, atherosclerosis, and / or other myocardial damage or vascular disease. The method includes direct administration or local expression in a weakened, ischemic and / or peri-infarctional area of the patient's myocardial tissue, an amount of SDF-1 effective to obtain a functional improvement of at least one of the following parameters: the volume of the left ventricle, the area le

- 1,031,308 ventricle, left ventricular size, heart function, six-minute walk test result (6MWT), or assessment by the New York Heart Association (NYHA) functional classification.

In an aspect of this application, the amount of SDF-1 injected into a weakened, ischemic and / or peri-infarction region of myocardial tissue is effective for obtaining a functional improvement of at least one of the following parameters: left-ventricular end-systolic volume, left ventricular ejection fraction, local violation index myocardial contractility, end-diastolic length of the left ventricle, end-systolic length of the left ventricle, end-diastolic area of the left ventricle, end-systolic area Adi left ventricle, left ventricular end-diastolic volume, six-minute walk test result (6MWT), or assessment by the New York Heart Association (NYHA) functional classification. In another aspect of this application, the amount of SDF-1 injected into a weakened, ischemic and / or peri-infarction region is effective for improving the left ventricular end-systolic volume. In an additional aspect of the present application, the amount of SDF-1 introduced into the weakened, ischemic and / or peri-infarction region is effective for improving the left ventricular ejection fraction.

In some aspects of this application, the amount of SDF-1 injected into a weakened, ischemic and / or peri-infarction region is effective in improving the left ventricular end-systolic volume by at least about 10%. In other aspects of this application, the amount of SDF-1 administered to a weakened, ischemic, and / or peri-infarct area is effective in improving the left ventricular end-systolic volume by at least about 15%. In still some further aspects of the present application, the amount of SDF-1 introduced into the weakened, ischemic and / or peri-infarction area is effective in improving the left ventricular finite systolic volume by at least about 10%, improving the left ventricular ejection fraction by at least about 10% , an improvement in the index of impaired local myocardial contractility by at least approximately 5%, an improvement in the distance when tested with six-minute walking at least 30 m and an improvement in the NYHA class by less measure at 1 class. In an additional aspect of the present application, the amount of SDF-1 introduced into the weakened, ischemic and / or peri-infarction region is effective in improving the left ventricular ejection fraction by at least about 10%.

In another aspect of the present application, the amount of SDF-1 administered to the weakened, ischemic and / or peri-infarction region is substantially effective in improving vasculogenesis in the weakened, ischemic and / or peri-infarct region by at least about 20%, determined by vascular density or measured by visualizing myocardial perfusion (for example, using single-photon emission computed tomography or positron emission tomography), while improving the total quiescent count, total stress score and / or su mmar account difference of at least about 10%. SDF-1 can be introduced by injecting a solution containing a plasmid expressing SDF-1 into a weakened, ischemic and / or peri-infarct region and expressing SDF-1 in a weakened, ischemic and / or peri-infarct region. SDF-1 can be expressed in a weakened, ischemic, and / or peri-infarct region in an amount effective to improve the left ventricular end-systolic volume.

In an aspect of the present application, the plasmid SDF-1 can be injected into a weakened, ischemic and / or peri-infarction region by repeated injections of the solution, with a solution containing from about 0.33 mg / ml to about 5 mg / ml plasmid SDF-1 being injected with each injection . In one example, the plasmid SDF-1 may be injected into a weakened, ischemic, and / or peri-infarct region during at least about 10 injections. Each injection carried out in a weakened, ischemic and / or peri-infarct area may have a volume of at least about 0.2 ml. SDF-1 can be expressed in a weakened, ischemic, and / or peri-infarct region for more than three days.

In the sample application, each injection of a solution containing plasmids expressing SDF-1 can be carried out in a volume of at least about 0.2 ml and at a concentration of plasmid SDF-1 from about 0.33 mg / ml to about 5 mg / ml per injection . In another aspect of the present application, at least one functional parameter of the heart can be improved by injecting a plasmid SDF-1 injected into a weakened, ischemic and / or peri-infarct region of the heart with an injection volume of at least one injection site, 2 ml, at least about 10 injection sites with a plasmid SDF-1 concentration from about 0.33 mg / ml to about 5 mg / ml per injection.

In an additional example, the amount of plasmid SDF-1 introduced into a weakened, ischemic and / or peri-infarction region, which can improve at least one of the functional parameters of the heart, is more than approximately 4 mg. The volume of the plasmid SDF-1 solution introduced into the weakened, ischemic and / or peri-infarction region, which can improve at least one of the functional parameters of the heart, is

- 2 031308 at least approximately 10 ml.

In another aspect of this application, a creature that is administered SDF-1 may be a mammal, such as a human or a pig. Plasmid SDF-1 can be administered to a patient by catheterization, such as intracoronary catheterization or intraventricular catheterization. Patient's myocardial tissue can be visualized to determine the area of the weakened, ischemic and / or peri-infarct area before the introduction of the plasmid SDF-1, and the plasmid SDF-1 can be introduced into the weakened, ischemic and / or peri-infarction area identified during imaging. Visualization may include at least one of echocardiography, magnetic resonance imaging, coronary artery angiography, electroanatomical mapping, or fluoroscopy.

The application also relates to a method for treating myocardial infarction in a large mammal by injecting plasmid SDF-1 into the peri-infarction region of the mammalian myocardium using catheterization, such as intracoronary catheterization or intraventricular catheterization. SDF-1 catheterization can be expressed by the peri-infarction region in an amount effective to obtain a functional improvement of at least one of the following parameters: left ventricular volume, left ventricular area, left ventricular size, heart function, test result with a six-minute walk (6MWT) or assessments by the New York Heart Association's functional classification (NYHA).

In the aspect of the present application, the amount of SDF-1 injected into the peri-infarction region is effective for obtaining functional improvement of at least one of the indicators: left ventricular finite systolic volume, left ventricular ejection fraction, local myocardial contractility index, left ventricular finite diastolic length, finite systolic index the length of the left ventricle, the end-diastolic area of the left ventricle, the left ventricular end-systolic area, the left-ventricular final-systolic volume A test result with a six-minute walking (6MWT), or evaluation of the functional classification of New York Heart Association (NYHA).

In another aspect of the present application, the amount of SDF-1 administered to the peri-infarct region is effective for improving the left ventricular end-systolic volume. In an additional aspect of the present application, the amount of SDF-1 introduced into the weakened, ischemic and / or peri-infarction region is effective for improving the left ventricular ejection fraction.

In some aspects of the present application, the amount of SDF-1 administered to the peri-infarct region is effective in improving the left ventricular end-systolic volume by at least about 10%. In other aspects of this application, the amount of SDF-1 injected into the peri-infarction region is effective in improving the left ventricular end-systolic volume by at least about 15%. In additional aspects of this application, the amount of SDF-1 injected into the peri-infarct region is effective in improving the left ventricular end-systolic volume by at least about 10%, improving the left ventricular ejection fraction by at least about 10%, improving the impaired local contractility index myocardium by about 5%, improvement in distance when tested with six-minute walking at least 30 m, or improvement in the NYHA class by at least 1 class. In an additional aspect of the present application, the amount of SDF-1 introduced into the weakened, ischemic and / or peri-infarction region is effective in improving the left ventricular ejection fraction by at least about 10%.

In another aspect of the present application, the amount of SDF-1 administered in the peri-infarct area is substantially effective in improving vasculogenesis in the peri-infarction area by at least about 20%, as measured by vascular density.

In an aspect of the present application, the plasmid SDF-1 can be injected into a weakened, ischemic and / or peri-infarction region by repeated injections of the solution, with a solution containing from about 0.33 mg / ml to about 5 mg / ml plasmid SDF-1 being injected with each injection . In one example, the plasmid SDF-1 may be injected into a weakened, ischemic, and / or peri-infarct region during at least about 10 injections. Each injection carried out in a weakened, ischemic and / or peri-infarct area may have a volume of at least about 0.2 ml. SDF-1 can be expressed in a weakened, ischemic, and / or peri-infarct region for more than three days.

In the sample application, each injection of a solution containing plasmids expressing SDF-1 can be carried out in a volume of at least about 0.2 ml and at a concentration of plasmid SDF-1 from about 0.33 mg / ml to about 5 mg / ml per injection . In another aspect of the present application, at least one functional parameter of the heart can be improved by injecting a plasmid SDF-1 injected into a weakened, ischemic and / or peri-infarct region of the heart with an injection volume of at least one injection site, 2 ml, at least about 10 injection sites with a plasmid SDF-1 concentration from about 0.33 mg / ml to about 5 mg / ml per injection.

- 3 031308

In an additional example, the amount of plasmid SDF-1 introduced into a weakened, ischemic and / or peri-infarction region, which can improve at least one of the functional parameters of the heart, is more than approximately 4 mg. The volume of the plasmid SDF-1 solution introduced into the weakened, ischemic and / or peri-infarction region, which can improve at least one of the functional parameters of the heart, is at least about 10 ml.

The application further relates to a method for improving the left ventricular end-systolic volume in a large mammal after myocardial infarction. The method includes the introduction of plasmid SDF-1 in the peri-infarction region by intraventricular catheterization. SDF-1 can be expressed in the peri-infarction region in an amount effective to obtain a functional improvement in the left ventricular end-systolic volume.

In some aspects of the present application, the amount of SDF-1 administered to the peri-infarct region is effective in improving the left ventricular end-systolic volume by at least about 10%. In other aspects of this application, the amount of SDF-1 injected into the peri-infarction region is effective in improving the left ventricular end-systolic volume by at least about 15%. In additional aspects of this application, the amount of SDF-1 injected into the peri-infarct region is effective in improving the left ventricular end-systolic volume by at least about 10%, improving the left ventricular ejection fraction by at least about 10%, improving the impaired local contractility index myocardium by about 5%, improvement in distance when tested with six-minute walking at least 30 m, or improvement in the NYHA class by at least 1 class.

In an aspect of the present application, the plasmid SDF-1 can be injected into a weakened, ischemic and / or peri-infarction region by repeated injections of the solution, with a solution containing from about 0.33 mg / ml to about 5 mg / ml plasmid SDF-1 being injected with each injection . In one example, the plasmid SDF-1 may be injected into a weakened, ischemic, and / or peri-infarct region during at least about 10 injections. Each injection carried out in a weakened, ischemic and / or peri-infarct area may have a volume of at least about 0.2 ml. SDF-1 can be expressed in a weakened, ischemic, and / or peri-infarct region for more than three days.

In the sample application, each injection of a solution containing plasmids expressing SDF-1 may have a volume of at least about 0.2 ml, and the concentration of plasmid SDF-1 is from about 0.33 mg / ml to about 5 mg / ml per injection . In another aspect of the present application, the endosystolic volume of the left ventricle of the heart can be improved by at least about 10% by injecting the plasmid SDF-1 into a weakened, ischemic and / or peri-infarct region of the heart with an injection volume of at least one injection site. 0.2 ml, at least about 10 places for injection and at a concentration of plasmid SDF-1 from about 0.33 mg / ml to about 5 mg / ml per injection.

In an additional example, the amount of plasmid SDF-1 introduced into a weakened, ischemic, and / or peri-infarct region and effective to improve the left ventricular end-systolic volume is more than about 4 mg. The volume of the plasmid SDF-1 solution introduced into the weakened, ischemic and / or peri-infarction region and effective for improving the left ventricular end-systolic volume of the heart is at least about 10 ml.

This application further relates to a method for treating critical limb ischemia in a patient. The method includes the introduction of ACRX-100 (also known as JVS-100), which is a sterile biological product (consisting of a plasmid containing the nucleotide sequence of SEQ ID NO: 6, free plasmid DNA encoding human SDF-1 cDNA and 5% dextrose ), by direct injection into the ischemic limb. Preferably, injections are made directly into muscle tissue, for example, the upper leg (quadriceps muscle of the thigh) and / or lower leg (preferably the gastrocnemius muscle), using multiple injection sites. The sequence of this plasmid is shown below.

The sequence of the plasmid used in the product ACRX-100 (also known as JVS100)

- 4 031308

AGATCTCCTAGGGAGTCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAAC GACSSSCGSSCATTGACGTCAATAATGACGTATGTTSSCATAGTAACGSCAATAGGGACTTTC CATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTAT CATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCC CAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATT ACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGA TTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGAC TTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG GAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGC TGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATT GGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACC CCCTTGGCTTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACACCCCCGCTTCCTC ATGTTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTC CCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCTT TATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACAGGA TGGGGTCTCATTTATTATTTACAAATTCACAT ATACAACACCACCGTCCCCAGTGCCCGCAGT TTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTC TTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTCCAGCGACTCATGG TCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACGATGCCCACC ACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGGGAG CGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGC TGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAG GGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAG ACTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTGCCATCGGTGACCA CTAGTGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGG TTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTA agtttaaagctcaggtcgagaccgggcctttgtccggcgctcccttggagcctacctagactc AGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCT GTTCTGCGCCGTTACAGATCGGTACCAAGCTTGCCACCACCATGAACGCCAAGGTCGTGGTCG TGCTGGTCCTCGTGCTGACCGCGCTCTGCCTCAGCGACGGGAAGCCCGTCAGCCTGAGCTACA GATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCAACGTCAAGCATCTCAAAATTC T SAACA CSSSAAAC TGTGCCCTT CAGAT TGTAGCCCGGCT GAAGAACAACAACAGACAAGT GT GCATTGACCCGAAGCTAAAGTGGATTCAGGAGTACCTGGAGAAAGCCTTAAACAAGTAATCTA GAGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCT TCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCC ACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCAT TCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGG CATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGGGCCGCGGTGGCC ATCATGACCAAAATSSSTTAACGTGAGTTTTCGTTSSACTGAGCGTCAGACSSCGTAGAAAAG ATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAA CCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTA ACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCAC CACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCT GCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAG GCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTAC ACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAG GCGGACAGGTATCCGGTAAGCGGCAGGGTCG GAACAGGAGAGCGCACGAGGGAGCTTCCAGGG GGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTT TTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGG TTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGAATTCAGAAGAACTCGTCAAGAAGGCGATA GAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCA TTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGC CACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGG CAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCT GGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAG ACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCA GGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGC AGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCT TCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGA TAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAG AACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTG TGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATC TTGTTCA ATCATGCGAAACGATCCTCATCCTGTCTCTTGATC (SEQ ID N0: 6)

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Brief description of the figures

The above and other features of the application will become apparent to those skilled in the art to which this invention pertains upon reading the following description with reference to the accompanying figures.

FIG. 1 is a diagram depicting the expression of luciferase at different amounts and volumes of cDNA in a model in pigs.

FIG. 2 is a diagram depicting the percentage change in left-ventricular end-systolic volume with different amounts of plasmid SDF-1, determined using a model of congestive heart failure in pigs 30 days after injection of SDF-1.

FIG. 3 is a diagram depicting the percentage change in the left ventricular ejection fraction with varying amounts of plasmid SDF-1, determined using the congestive heart failure model in pigs 30 days after injection of SDF-1.

FIG. 4 is a diagram depicting the percentage change in the index of impaired local myocardial contractility with different amounts of plasmid SDF-1, determined using the model of congestive heart failure in pigs 30 days after injection of SDF-1.

FIG. 5 is a diagram depicting the percent change in left ventricular end-systolic volume with different amounts of plasmid SDF-1, determined using a model of congestive heart failure in pigs 90 days after injection of SDF-1.

FIG. 6 is a diagram depicting the percentage change in vascular density with different amounts of plasmid SDF-1, determined using a model of congestive heart failure in pigs 30 days after injection of SDF-1.

FIG. 7 is a schematic representation of the SDF-1 plasmid vector.

FIG. 8 shows an image showing plasmid expression in a significant part of the pig's heart.

FIG. 9 shows a diagram depicting the end-systolic volume of the left ventricle at the initial time and 30 days after the first injection. In all groups, a similar increase in the left ventricular end-systolic volume was shown after 30 days. N = 3 for all points on the graph. Data are presented as mean ± standard error of the mean (SOS).

FIG. 10 is a diagram depicting the left ventricular ejection fraction at the initial time and 30 days after the first injection. In all groups, there was no improvement in the left ventricular ejection fraction. N = 3 for all points on the graph. Data are presented as mean ± SOS.

FIG. Figure 11 shows an image of luciferase expression in a rat ischemic leg 3 days after injection (A) and a schema for the duration of the ACRX-100 vector when expressed in the model of hind limb ischemia (IZK) in rodents (B).

FIG. 12 shows the image of bioluminescence in the muscle of the rabbit hind limb 3 days after the injection of the cDNA plasmid ACL-01110L containing the luciferase gene.

FIG. 13 is a diagram of the dosing parameters of ACL-01110L when introduced into the rabbit's hind limb.

FIG. 14 shows an example of angiograms and evaluation of the rabbit's hind limb at the initial time point (A and C) and 30 days after the injection of ACRX-100 (B and D).

FIG. Figure 15 shows a chart of the percentage change in angiographic assessment 30 and 60 days after injection of ACRX-100, which was normalized to control groups.

FIG. 16 shows the biodistribution scheme of ACRX-100 after injection into the heart muscle.

FIG. 17 shows the relationship between the expression of SDF-1 and CXCR4 after ischemic injury. CXCR4 is the main receptor for the SDF-1 protein.

Detailed description

Unless otherwise indicated, all technical and scientific terms used herein have a generally accepted meaning, known to the person skilled in the art to which the application (s) belongs. All patents, patent applications, published applications and publications, sequences from the Genbank database, websites and other published materials referred to throughout the disclosure of the present invention, unless otherwise indicated, are incorporated herein in their entirety by reference. . If there is a set of values for the term specified in this document, the value specified in this section takes precedence. Unless otherwise indicated, all technical terms used in this document have a generally accepted meaning, known to the person skilled in the art to which this application relates. The well-known meanings of terms from the field of molecular biology can be found, for example, in Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, SpringerVerlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.

This document describes methods involving conventional techniques used in the field of molecular biology. Such techniques are commonly known in this field and are described in detail in methodological manuals such as Molecular Cloning: A Laboratory Manual, 2nd edition, v.1-3,

- 6,031,308 edited by Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22: 1859-1862, 1981 and Matteucci et al., J. Am. Chern. Soc. 103: 3185, 1981. Chemical synthesis of nucleic acids can be carried out, for example, using commercially available automatic oligonucleotide synthesizers. Immunological methods (eg, production of antigen-specific antibodies, immunoprecipitation and immunoblotting) are described, for example, in Current Protocols in Immunology, ed. Coligan et al., John Wiley & Sons, New York, 1991 and Methods of Immunological Analysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992. The generally accepted methods of gene transfer and gene therapy have also been adapted for use in this application (see, for example, Gene Therapy: Principles and Applications, edited by T. Blackenstein, Springer Verlag , 1999; Gene Therapy Protocols (Methods in Molecular Medicine), edited by R. Robbins, Humana Press, 1997 and Retro-vectors for the Human Gene Therapy, edited by S. R. Hodgson, Springer Verlag, 1996.

If the link is given to a uniform resource locator or other similar pointer or address, it should be understood that such pointers may change, and specific information on the Internet may appear and disappear, but equivalent information can be found on the Internet during a search. In addition, the link indicates the availability and wide dissemination of such information.

As used herein, the abbreviation ACRX-100 refers to a sterile biological product consisting of a plasmid containing the nucleotide sequence of SEQ ID NO: 6, free plasmid DNA encoding human SDF-1 cDNA, and 5% dextrose (in this application ACRX -100 may also be called JVS-100).

The term “nucleic acid” as used herein refers to a polynucleotide containing at least two covalently linked nucleotides or nucleotide analogs. The nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analogue of DNA or RNA. Nucleotide analogs are commercially available, and methods for producing polynucleotides containing similar nucleotide analogs are also known (Lin et al. (1994) Nucl. Acids Res. 22: 5220-5234; Jellinek et al. (1995) Biochemistry 34: 11363-11372; Pagratis et al. (1997) Nature Biotechnol. 15: 68-73). The nucleic acid may be single-stranded, double-stranded, or a mixture thereof. In this document, unless otherwise indicated or obvious from the context, the nucleic acid is double-stranded.

The term DNA is intended to include all types and sizes of DNA molecules, including EDNA, plasmids and DNA, containing modified nucleotides and analogs of nucleotides.

As used herein, the term nucleotides includes mono-, di- and triphosphates of nucleosides. The nucleotides also include modified nucleotides, non-limiting examples of which include thiophosphate nucleotides, desapurine nucleotides, and other nucleotide analogs.

As used herein, the term “subject or patient” refers to animals in whose body large DNA molecules must be introduced. It includes higher organisms such as mammals and birds, including humans, primates, rodents, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, cats, dogs, horses, chickens, etc.

The term “large mammal” as used in this document means a mammal whose normal weight in adulthood is at least 10 kg. Such large mammals may include, for example, humans, primates, dogs, pigs and cattle, meaning that mammals of a smaller size, such as mice, rats, guinea pigs and other rodents, are excluded from consideration.

As used herein, the term “administering to a patient” means a procedure by which one or more delivery vehicles and / or large nucleic acid molecules, together or separately, are administered or applied to the patient in such a way that the target cells that the patient ultimately interacts with. and / or large nucleic acid molecules.

Used in this document, the term delivery, interchangeable with the term transduction, refers to the process by which exogenous nucleic acid molecules are transferred into the cell so that they are inside the cell. The delivery of nucleic acids is a process that differs from the expression of nucleic acids.

As used herein, the term Multiple Cloning Patch (CMP) is a section of a nucleic acid in a plasmid that contains a number of sections cleaved by a restriction enzyme, any of which can be used together with a standard recombination technique for splitting a vector. The term enzyme digestion by a restriction enzyme means the catalytic breakdown of a nucleic acid molecule by an enzyme that acts only on certain parts of the nucleic acid molecule. Many of these restriction enzymes

- 7 031308 commercially available. The use of such enzymes is well known to those skilled in the art. Often, the vector is linearized or fragmented by a restriction enzyme that cleaves the molecule within the CMP to ensure that exogenous sequences are linked to the vector.

As used in this document, the term "origin of replication" (often called THP) means the specific nucleic acid sequence with which replication begins. Alternatively, a stand-alone repeating sequence (ANN) may be used. if the host cell belongs to yeast.

As used herein, the term selectable or selectable markers means the introduction of detectable changes into the cell, allowing easy identification of cells containing the expression vector. In general, a selectable marker is a marker that gives a property that provides detection. A positive selectable marker is a marker. in the presence of which detection is ensured, while the negative marker selectable is the marker. in whose presence identification is impossible. An example of a positive selectable marker is the drug resistance marker. Normally turning on the marker. selected by sensitivity to the drug. contributes to the cloning and identification of transformants. for example genes. which are responsible for neomycin resistance. puromycin. hygromycin. dihydrofolate reductase. alanine aminotransferase. zeocin and histidinol. as well as useful selectable markers. In addition to the markers. responsible for the phenotype. providing the definition of transformants on the basis of giving them some properties. other types of markers are also provided. including selected markers. such as green fluorescent protein. determination of which is based on calorimetric analysis. Alternatively, selectable enzymes can be used. such as thymidine kinase (TK) of herpes simplex virus or chloramphenicol acetyltransferase (CAT). The person skilled in the art should also be aware of the use of immunological markers. possibly. in combination with the sorting of cells with activated fluorescence. The type of marker used is not considered important until then. as long as it can be expressed simultaneously with the nucleic acid. gene encoding product. Additional examples of selectable and selectable markers are well known to the person skilled in the art.

Used in this document, the term transfection is used to refer to the absorption of a cell of foreign DNA. A cell is considered transfected. if exogenous DNA was introduced through the cell membrane. Many transfection techniques are well known in the art (see, for example, Graham et al. Virology 52: 456 (1973); Sambrook et al. Molecular Cloning: A Laboratory Manual (1989); Davis et al. Basic Methods in Molecular Biology (1986); Chu et al., Gene 13: 197 (1981)). Similar techniques can be used to introduce one or more exogenous DNA compounds. such as nucleotide integration vector and other nucleic acid molecules. in suitable cell owners. This term covers chemical. electrical and viral transfection procedures.

Used in this document, the term expression means a process. during which the nucleic acid is translated to form peptides or transcribed to form RNA. which eg. can be translated to form peptides. polypeptides or proteins. If the nucleic acid is derived from genomic DNA. belonging to the corresponding eukaryotic host cell or eukaryotic organism. expression may involve splicing of mRNA. In the case of expression of a heterologous nucleic acid in a host cell, this substance must first be delivered to the cell. and then. when it will be inside the cage. ultimately stay inside the core.

The term gene therapy as used herein includes the transfer of heterologous DNA into mammalian cells. in particular man. suffering from a disorder or disease. against which are directed treatment or diagnosis. DNA is introduced into selected target cells in this way. so that heterologous DNA is expressed and the drug coded in it is produced. Alternatively, heterologous DNA may in some way cause the expression of DNA. which encodes a therapeutic drug; she can encode the product. such as peptide or RNA. which in some way causes. directly or indirectly. expression of the drug. Gene therapy can also be used to deliver nucleic acid. gene encoding product. in order to replace a defective gene or add a gene product. produced by the body of a mammal or a cell. into which the nucleic acid was introduced. The nucleic acid introduced may encode a therapeutically active compound. such as growth factor or its inhibitor. tumor necrosis factor or its inhibitor. as well as the receptor. which is not normally produced in the host mammal, or which is not produced in therapeutically effective amounts or over time. sufficient to provide a therapeutic effect. This heterologous DNA. coding drug. may be modified prior to introduction into the cells of the affected host organism in order to enhance or otherwise alter the product or to express it.

The term heterologous nucleic acid sequence as used herein.

- 8,031,308 slots usually refer to DNA encoding RNA and proteins that are not normally produced in vivo in the cell in which they are to be expressed, as well as causing or encoding mediators that alter the expression of endogenous DNA by affecting transcription, translation or other regulated biochemical processes. This heterologous nucleic acid sequence may also be referred to as foreign DNA. Any DNA that can be considered or determined by a person skilled in the art as heterologous or foreign to the cell in which it is to be expressed, in this document, refers to heterologous DNA. Examples of heterologous DNA include unlimited DNA that encodes detectable protein markers, such as proteins for drug resistance, DNA that encodes therapeutically effective substances, such as anti-cancer drugs, enzymes and hormones, and DNA that encodes other types of proteins, such as antibodies. Antibodies encoded in heterologous DNA can be secreted or expressed on the surface of the cell into which the heterologous DNA has been introduced.

As used herein, the term cardiomyopathy refers to the disruption of the functioning of the myocardium (i.e., cardiac muscle) caused by any cause. Patients with cardiomyopathy are often at risk for arrhythmias, sudden cardiac death, and hospitalization or death due to heart failure.

The term ischemic cardiomyopathy, as used herein, means weakness of the heart muscle caused by insufficient oxygen supply to the myocardium, the most common cause of which is coronary insufficiency.

The term coronary heart disease, as used herein, refers to any condition in which the heart muscle is affected or ineffective due to complete or relative insufficiency of the blood supply; Atherosclerosis is the most common cause. Coronary artery disease, acute myocardial infarction, chronic ischemic heart disease and sudden cardiac death are referred to coronary heart disease.

As used in this document, the term myocardial infarction refers to the damage or death of a portion of the heart muscle (myocardium) due to the inability of the blood supply to this portion.

The term six-minute walk test, or 6MWT, as used in this document means a test that measures the distance a patient can quickly travel on a flat solid surface for 6 minutes (6MWD). It assesses the general and joint reactions of all systems involved in the exercise, including the respiratory and cardiovascular systems, systemic circulation, peripheral circulation, blood, neuromuscular units, and muscle metabolism. It does not provide specific information about the functioning of each individual organ and system involved in the exercise, or the mechanism for limiting exercise performance, which is possible during cardiopulmonary tests with maximum exercise. When conducting a 6MWT performed at an arbitrary pace, the submaximal level of functionality is assessed (see examples in AM J Respir Crit Care Med, vol. 166, p. 111-117 (2002)).

The term Functional Classification of the New York Heart Association (NYHA), as used herein, refers to the classification of degrees of heart failure. With it, patients fall into one of four categories based on how limited they are in their physical activity; limitations / symptoms relate to normal breathing, varying degrees of dyspnea, and heart pain.

NYHA class Symptoms I There are no symptoms, as well as restrictions in daily physical activity, such as shortness of breath when walking, climbing stairs, etc. II Minor symptoms (mild dyspnea and / or angina) and slight limitations of daily activity. III Noticeable activity limitations due to symptoms, even during activity with an intensity below the average, for example, when walking for short distances (20-100 m). Comfort only at rest. IV Serious restrictions. Symptoms occur even at rest. It is mainly found in patients who are bedridden.

This application relates to compositions and methods for treating cardiomyopathy in a patient who

- 9 031308 Paradise led to a decrease and / or impaired functional abilities of the myocardium. Cardiomyopathy to be treated with the compositions and methods provided herein may include cardiomyopathies associated with pulmonary embolism, venous thrombosis, myocardial infarction, transient ischemic attack, peripheral vascular disease, atherosclerosis, ischemic heart disease, and / or other myocardial disease. . A method for treating cardiomyopathy may include local administration (or local delivery) to a weakened myocardial tissue, ischemic myocardial tissue and / or apoptotic myocardial tissue, such as a peri-infarction region of the heart, resulting from a myocardial infarction, a certain amount of stromal growth factor-1 (SDF-1) which is effective for obtaining a functional improvement of at least one of the following parameters: the volume of the left ventricle, the area of the left ventricle, the size of the left ventricle, the function of the heart, the result of the test Ia with the six-minute walking (6MWT) or evaluation for functional classification of New York Heart Association (NYHA).

Using a model of heart failure in pigs that mimics human heart failure, it was found that the functional improvement of ischemic myocardial tissue depends on the amount, dose, and / or delivery of SDF-1 introduced into the ischemic myocardial tissue, and that the amount, dose, and / or the delivery of SDF-1, introduced into the ischemic myocardial tissue, can be optimized so that the parameters of the functional ability of the myocardium, such as the volume of the left ventricle, the area of the left ventricle, size left ventricular or heart function, essentially improved. As discussed below, in some aspects, the amount, concentration, and volume of SDF-1 injected into ischemic myocardial tissue can be adjusted and / or optimized to significantly improve functional parameters (for example, left ventricular volume, left ventricular area, left ventricular size, heart function , a six-minute walk test result (6MWT) or an assessment by the New York Heart Association's (NYHA) functional classification while reducing unwanted side effects.

In one example, SDF-1 can be injected directly or locally into a weakened area, ischemic area and / or peri-infarction area of myocardial tissue of a large mammal (for example, a pig or a human) in which a deterioration or distortion of the functional function of the heart, such as the volume of the left ventricle, is observed , the area of the left ventricle, the size of the left ventricle or the function of the heart, which is a consequence of ischemic cardiomyopathy, such as myocardial infarction. Deterioration or distortion of the functional parameter may include, for example, an increase in the left ventricular end-systolic volume, a decrease in the left ventricular ejection fraction, a decrease in the index of local myocardial contractility, an increase in the end-diastolic length of the left ventricle, an increase in the end-systolic length of the left ventricle, an increase in the end-diastolic area left ventricle (for example, at the level of the mitral valve and the level of attachment of the papillary muscle), an increase in the end-systolic area left ventricle (for example, at the level of the mitral valve and the level of attachment of the papillary muscle) or an increase in the end-systolic volume of the left ventricle, measured, for example, using echocardiography.

In an aspect of this application, the amount of SDF-1 introduced into a weakened, ischemic and / or peri-infarction portion of the myocardial tissue of a large mammal may be an amount effective to improve at least one of the functional parameters of the myocardium, such as reducing the left ventricular end-systolic volume , an increase in the left ventricular ejection fraction, a decrease in the index of impaired local myocardial contractility, a decrease in the end-diastolic length of the left ventricle, a decrease in the end-systolic left ventricular length, decreased left ventricular end-diastolic area (for example, at the level of the mitral valve and papillary muscle attachment level), reduced left-ventricular end-systolic area (for example, at the level of the mitral valve and the level of papillary muscle attachment) or reduced systolic volume of the left ventricle, measured, for example, with the help of echocardiography, as well as patient's improvement of the test result with a six-minute walk (6MWT) or evaluation by functional classification H w York Heart Association (NYHA).

In another aspect of the present application, the amount of SDF-1 introduced into a weakened, ischemic and / or peri-infarction portion of the myocardial tissue of a large mammal with cardiomyopathy is effective for improving the left-ventricular left-ventricular volume in a mammal at least about 10%, more specifically less at least about 15%, 30 days after administration, as determined using echocardiography. The percentage improvement is relative for each patient who has undergone treatment, and is based on the corresponding parameter, the value of which was measured before or during the treatment intervention or treatment.

In an additional aspect of the present application, the amount of SDF-1 introduced into a weakened, ischemic and / or peri-infarction portion of the myocardial tissue of a large mammal with cardiomyopathy is effective in improving the left ventricular end-systolic volume by at least about 10%, improving the left ventricular ejection fraction in at least at

- 10,031,308 at about 10% and an improvement in the index of impaired local myocardial contractility by about 5% 30 days after administration, as determined using echocardiography.

In yet another additional aspect of the present application, the amount of SDF-1 administered in a weakened, ischemic and / or peri-infarctional portion of the myocardial tissue of a large mammal with cardiomyopathy is effective for improving vasculogenesis in a weakened, ischemic, and / or peri-infarcting region of at least 20% determined according to vascular density or an increase in cardiac perfusion, established using single-photon emission computed tomography. A 20% improvement in vasculogenesis has been shown to be clinically significant (Losordo Circulation 2002; 105: 2012).

In yet another additional aspect of the present application, the amount of SDF-1 introduced into a weakened, ischemic and / or peri-infarctional portion of the myocardial tissue of a large mammal with cardiomyopathy is effective for improving the distance when tested with six minutes walking at least 30 m or improving the NYHA class at least measure 1 class.

SDF-1 described in this document can be inserted into a weakened, ischemic and / or peri-infarction region of myocardial tissue, starting from the moment of tissue damage (for example, due to myocardial infarction), and continue for hours, days, weeks, or months after the start of a decrease in SDF-expression one. The period of introduction of SDF-1 into cells can begin almost immediately after the onset of cardiomyopathy (for example, myocardial infarction) and continue for days, weeks, or months after the onset of ischemic disorder or tissue damage.

In accordance with the present application, SDF-1, introduced into a weakened ischemic and / or peri-infarction region of myocardial tissue, may have an amino acid sequence that is substantially similar to mammalian SDF-1 own amino acid sequence. The amino acid sequence of the SDF-1 protein found in many different mammals, including humans, mice and rats, is known. Amino acid sequences of human SDF-1 and rat are at least approximately 92% identical (for example, approximately 97% identical). SDF-1 may have two isoforms, SDF-1a and SDF-1P, which are referred to herein as SDF-1, unless otherwise indicated.

SDF-1 may have an amino acid sequence substantially identical to SEQ ID NO: 1. SDF-1, overexpressed, also has an amino acid sequence substantially identical to one of the mammalian SDF-1 proteins mentioned above. For example, SDF-1, over-expressed, may have an amino acid sequence substantially identical to SEQ ID NO: 2. SEQ ID NO: 2, which essentially contains SEQ ID NO: 1, is the amino acid sequence of human SDF-1 and has an access number in the GenBank database NP954637. SDF-1, subject to overexpression, may have an amino acid sequence that is substantially identical to SEQ ID NO: 3. SEQ ID NO: 3 contains the rat SDF amino acid sequences and has an access number in the GenBank database AAF01066.

In accordance with the present application, the SDF-1 may also be a variant of the mammal SDF-1, such as a fragment, analogue and derivative of the mammal SDF-1. Such variants include, for example, a polypeptide encoded in a naturally occurring allelic variant of the native gene SDF-1 (i.e., in a naturally occurring nucleic acid that encodes the naturally occurring mammalian SDF-1 polypeptide), encoded in the form of a native the SDF-1 gene subjected to alternative splicing, a polypeptide encoded in the homologue or ortholog of the native gene SDF-1, and a polypeptide encoded in the version of the native gene SDF-1, which is not found in nature.

SDF-1 variants have a peptide sequence that differs from the native SDF-1 polypeptide by one or more amino acids. The peptide sequence of such variants may be subject to deletion, addition or replacement of one or more amino acids in comparison with the SDF-1 variant. Amino acid inserts are preferably composed of approximately 1-4 consecutive amino acids, and the deletion preferably affects approximately 1 to 10 consecutive amino acids. SDF-1 variant polypeptides essentially retain the functional activity of native SDF-1. Examples of polypeptide variants of SDF-1 can be obtained by expressing nucleic acid molecules that allow for changes in conservative and silent regions. One example of an SDF-1 variant is listed in US Pat. No. 7,405,195, which is incorporated herein by reference in its entirety.

Fragments of an SDF-1 polypeptide corresponding to one or more specific motifs and / or domains, or of arbitrary size, are included in the scope of the invention as the subject of this application. Isolated peptidyl parts of SDF-1 can be obtained by screening peptides made recombinantly from the corresponding nucleic acid fragment encoding such peptides. For example, an SDF-1 polypeptide can be arbitrarily divided into fragments of the required length without overlapping the fragments or preferably divided into overlapping fragments of the required length. Fragments can be obtained recombinantly and investigated to determine the identity of these peptidyl fragments, which can

- 11 031308 to enhance the function of agonists of native CXCR-4 polypeptides.

Versions of the SDF-1 polypeptides may also include recombinant forms of the SDF1 polypeptides. In some embodiments, the implementation of the recombinant polypeptides in addition to the SDF-1 polypeptides are encoded in a nucleic acid, the sequence of which is at least 70% identical to the nucleic acid sequence of the gene encoding a mammal SDF-1.

SDF-1 variants may include agonistic forms of a protein that constantly possesses the functional activity of native SDF-1. Other variants of SDF-1 may include substances that are resistant to proteolytic cleavage, for example, in connection with mutations that alter target sequences for proteases. Since a change in the amino acid sequence of a peptide results in a variant that has one or more forms of the functional activity of native SDF-1, such a change can be easily detected by examining the variant for the presence of functional activity characteristic of native SDF-1.

The SDF-1 nucleic acid, which encodes the SDF-1 protein, can be a native or non-native nucleic acid, and also be in the form of RNA or DNA (for example, eDNA, genomic DNA and synthetic DNA). DNA can be double-stranded or single-stranded, and single-stranded DNA can be represented by a coding (sense) or non-coding (antisense) chain. A given nucleic acid coding sequence in which SDF-1 is encoded may be essentially the same as the nucleotide sequence of the SDF-1 gene, such as the nucleotide sequence shown in SEQ ID NO: 4 and SEQ ID NO: 5. SEQ ID NO: 4 and SEQ ID NO: 5 contain, respectively, the nucleic acid sequences of human SDF-1 and SDF-1 rats, which are essentially the same as the nucleotide sequences with the access number in the GenBank NM199168 database and the access number in the GenBank AF189724 database .

The SDF-1 nucleotide acid coding sequence may also be another coding sequence that, due to the redundancy or degeneracy of the genetic code, encodes the same polypeptide as SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.

Other nucleic acid molecules that encode SDF-1 are variants of native SDF-1, which encode fragments, analogs, and derivatives of native SDF-1. Such variants can be, for example, a naturally occurring allelic variant of the native SDF-1 gene, a homologue or ortholog of the native SDF-1 gene, and also a non-naturally occurring allelic variant of the native SDF-1 gene. Such variants of SDF-1 have a nucleotide sequence that differs from the native SDF-1 gene by one or more bases. For example, the nucleotide sequence of such variants may be subject to deletion, addition or replacement of one or more nucleotides in comparison with the native SDF-1 gene. Nucleic acid inserts are preferably composed of about 1-10 consecutive nucleotides, and the deletion preferably affects about 1 to 10 consecutive nucleotides.

In other applications in the SDF-1 variant, significant changes in the structure are observed, which can be obtained by nucleotide substitutions, causing less conservative changes in the encoded polypeptide. Examples of such nucleotide substitutions can serve as substitutions that cause changes (a) to the structure of the polypeptide backbone; (b) the charge or hydrophobicity of the polypeptide; or (c) the size of the amino acid side chain. Nucleotide substitutions, in which large changes in protein properties are usually expected, are associated with non-conservative changes in codons.

Examples of changes in codons that are likely to cause large changes in the structure of a protein are changes causing (a) a hydrophilic residue (for example, serine or threonine) to be replaced by a hydrophobic residue (for example, leucine, isoleucine, phenylalanine, valine or alanine ); (b) cysteine or proline to another residue; (c) a residue having a positive side charge chain (eg, lysine, arginine or histidine), a negative charge residue (eg glutamine or asparagine), or (d) a residue having a bulk side chain (eg phenylalanine), for a residue that does not have a side chain (for example, glycine).

Naturally occurring allelic variants of the native SDF-1 gene are nucleic acids isolated from mammalian tissue and have a sequence that is at least 70% identical to the native SDF-1 gene, and encode polypeptides that have structural similarity to the native SDF-1 polypeptide. Homologues of the native SDF-1 gene are nucleic acids isolated from other species and have a sequence that is at least 70% identical to the native gene, and encode polypeptides that have structural similarity to the native SDF-1 polypeptide. In public and / or private databases of nucleic acids, a search can be conducted to determine other nucleic acid molecules whose sequence has a large percentage of similarity (for example, 70% or more identical) with the native SDF-1 gene.

Non-naturally occurring variants of the SDF-1 gene are nucleic acids that are not found in nature (for example, can be made by humans), have a sequence that is identical to the SDF-1 gene by at least 70%, and encode polypeptides with structural similarity with native SDF-1 polypeptide. Examples of non-naturally occurring variants.

- 12 031308 of the SDF-1 gene serve as variants encoding a fragment of the native SDF-1 protein, which hybridizes with the native SDF-1 gene or the complementary chain to the SDF-1 gene under harsh conditions, and the sequence is identical to at least 65% of the native gene SDF-1 or complementary chain to the native gene SDF-1.

Nucleic acids encoding fragments of the native SDF-1 gene, in some embodiments, encode the amino acid residues of the native SDF-1. Shorter oligonucleotides that encode fragments of native SDF-1 or hybridize with nucleic acids that encode fragments of native SDF-1 can be used as probes, primers, or antisense molecules. Longer polynucleotides that encode fragments of native SDF-1 or hybridize with nucleic acids that encode fragments of native SDF-1 can also be used in various aspects of this application. Nucleic acids encoding fragments of native SDF-1 can be obtained by enzymatic cleavage (for example, using a restriction enzyme) or by chemical decomposition of the full-size native SDF-1 gene or its variants.

Nucleic acids that hybridize under stringent conditions with one of the nucleic acids mentioned above can also be used in this document. For example, such nucleic acids can hybridize with one of the above-mentioned nucleic acids under conditions of low, medium and high stringency.

Nucleic acid molecules encoding a hybrid protein SDF-1 can also be used in some embodiments. Such nucleic acids can be obtained by preparing a construct (for example, an expression vector) that expresses the SDF-1 fusion protein when introduced into a suitable target cell. For example, a similar construct can be made by binding a first polynucleotide encoding an SDF-1 protein hybridized relative to a reading frame with a second polynucleotide encoding another protein, so that the expression of the construct in a suitable expression system allows for the production of a hybrid protein.

Nucleic acids encoding SDF-1 can be modified at the level of a nitrogenous base, carbohydrate residue or phosphate backbone, for example, to improve the stability of the molecule, its hybridization, etc. The nucleic acids described herein may additionally contain other additional groups, such as peptides (for example, for receptors specific for target cells in vivo) or agents that facilitate movement across the cell membrane and hybridization-activated cleavage of molecules. For this purpose, nucleic acids can be conjugated to another molecule (for example, a peptide), activated by hybridization with a cross-linking agent, carrier, activated by hybridization with a cleaving agent, etc.

SDF-1 can be delivered to a weakened, ischemic and / or peri-infarction region of myocardial tissue by injecting SDF-1 protein into a weakened, ischemic and / or peri-infarction region, or introducing a weakened, ischemic and / or peri-infarction portion of myocardial tissue of the factor that causes , increases and / or activates the expression of SDF-1 (i.e. SDF-1 factor).

Cell-expressed SDF-1 protein can be the product of expression of a genetically modified cell.

The factor that causes, enhances and / or activates the expression of SDF-1 may contain the natural or synthetic nucleic acids described in this document, which can be incorporated into recombinant nucleic acid constructs, usually DNA constructs, intended for introduction and replication in cells myocardial tissue. Such a construct may contain a replication system and sequences intended for transcription and translation of the sequence encoding the polypeptide in the specified cell.

One way of introducing a factor into a target cell involves the use of gene therapy. In some embodiments of this application, gene therapy can be used to express SDF-1 protein in a weakened, ischemic, and / or peri-infarction portion of myocardial tissue in vivo.

In an aspect of this application, a vector comprising a nucleotide encoding an SDF-1 protein can be used as part of gene therapy. A vector (sometimes referred to as a gene delivery vehicle or gene transfer vehicle) refers to a macromolecule or complex of molecules containing a polynucleotide that must be delivered to a target cell in vitro or in vivo. A polynucleotide to be delivered may contain the coding sequence necessary for gene therapy. Vectors include, for example, viral vectors (such as adenoviruses (Ad), adeno-associated viruses (AAV) and retroviruses), non-viral vectors, liposomes and other lipid-containing complexes, as well as other macromolecular complexes designed to ensure the delivery of the polynucleotide into the target cell.

Vectors may also contain other components or functional groups that additionally modulate gene delivery and / or gene expression or otherwise impart useful

- 13 031308 properties to target cells. Other such components include, for example, components that affect binding or specificity to the cell (including components that modulate specific binding to a cell of a particular type or tissue); components that affect cell uptake of a nucleic acid from a vector; components that influence the location of the polynucleotide in the cell after absorption (such as factors that modulate penetration into the nucleus), and components that influence the expression of the polynucleotide. Such components may also include markers, such as detectable and / or selectable markers, which can be used to detect or select cells that must absorb and express the nucleic acid delivered by the vector. Such components may be a natural feature of the vector (such as using some viral vectors that contain components or functional groups that modulate binding and uptake), or vectors must be modified to provide such functionality.

Selectable markers can be positive, negative or bifunctional. Positive selectable markers allow for the selection of cells carrying markers, while negative selectable markers provide for selective removal of cells carrying markers. Many such marker genes have been described, including bifunctional (i.e., positive / negative) markers (see, for example, Lupton, S., international patent application WO 92/08796, published May 29, 1992; and Lupton S., international patent application WO 94/28143, published December 8, 1994). Such marker genes may be an additional method of control, which may be preferred as part of gene therapy. A large number of similar vectors are known and widely available in this area.

Vectors for use for the purposes described in this document include viral vectors, lipid-based vectors, and other non-viral vectors designed to deliver the nucleotide into cells of a weakened, ischemic, and / or peri-infarction region of myocardial tissue. The vector may be a specific vector, in particular a specific vector, which preferably binds to the cells of the weakened, ischemic and / or peri-infarction portion of the myocardial tissue. Viral vectors for use in the methods provided for in this document may include vectors with low toxicity for cells of the weakened, ischemic, and / or peri-infarction portion of myocardial tissue and causing the production of SDF-1 protein, which has therapeutic value, the tissue to which they are specific.

Examples of viral vectors include vectors derived from adenovirus (Ad) or adeno-associated virus (AAV). Human and non-human viral vectors can be used, the recombinant viral vector may not be able to replicate in humans. If the vector is represented by an adenovirus, the vector may contain a polynucleotide that has a promoter functionally linked to the gene encoding the SDF-1 protein, and may not have the ability to replicate in humans.

Other viral vectors that can be used in accordance with the method presented in this application include herpes simplex virus (HSV) vectors. HSV-based vectors from which one or more of the early gene (IE) genes have been removed are preferred because they are generally not cytotoxic, persist in a state similar to the latent one in the target cell, and provide efficient transduction in the target cell. Recombinant HSV-based vectors can contain approximately 30 thousand nucleotides of a heterologous nucleic acid.

Retroviruses, such as C-type retroviruses and lentiviruses, can also be used in some embodiments of this application. For example, retroviral vectors can be based on the mouse leukemia virus (MLV) (see, for example, Hu and Pathak, Pharmacal. Rev. 52: 493-511, 2000 and Pong et al., Crit. Rev. Ther. Drug Carrier Syst 17: 1-60, 2000). MLV-based vectors can contain up to 8,000 heterologous (therapeutic) DNA nucleotides instead of viral genes. This heterologous DNA may contain a tissue-specific promoter and SDF-1 nucleic acid. In methods of delivery to cells located in the immediate vicinity of the affected area, it can also encode a ligand for a tissue-specific receptor.

Additional retroviral vectors that can be used are lentivirus-based vectors that are not replicable, including vectors based on the human immunodeficiency virus (HIV) (see, for example, Vigna and Naldini, J. Gene Med. 5: 308- 316, 2000 and Miyoshi et al., J. Viral. 72: 8150-8157, 1998). Lentivirus-based vectors are preferred because they are capable of infecting cells that are actively dividing and not dividing. They are also highly effective for transduction in human epithelial cells.

Lentiviral vectors used in the methods presented in this document can be derived from human and other organ lentiviruses (including monkey immunodeficiency virus). Examples of lentivirus-based vectors contain nucleic acid sequences necessary for virus propagation and a tissue-specific promoter that is functionally linked to the SDF-1 gene. The former may include viral long terminal repeats, prime binding site

- 14,031,308 pa, polypurine site, site of attachment and site of capsidation.

A lentivirus-based vector can be packaged in any suitable lentivirus capsid. This replacement of one capsomer with a protein from another virus is called pseudotyping. The vector capsid may contain proteins from the membranes of other viruses, including the mouse leukemia virus (MLV) or vesicular stomatitis virus (VSV). Using the VSV G-protein allows you to get a high titer of the vector and achieve greater stability of the virus particles of the vector.

Alphavirus-based vectors derived from the Semliki forest virus (SFV) and Sindbis virus (SIN) can also be used in this document. The use of alphaviruses is described in Lundstrom K., Intervirology 43: 247-257, 2000 and Perri et al., Journal of Virology 74: 9802-9807, 2000.

He replicable recombinant alphavirus-based vectors are preferred because they provide increased expression of the heterologous (therapeutic) gene and infect a wide range of target cells. The alphavirus replicons can be specific to certain cell types by presenting on the surface of the virion a functional heterologous ligand or binding domain that can provide selective binding to target cells expressing a recognized binding partner. The alphavirus replicons can be in a latent state, which results in long-term expression of the heterologous nucleic acid in the target cell. Replicons can also induce short-term expression of a heterologous nucleic acid in a target cell.

In many viral vectors compatible with the methods of this application, more than one promoter may be included in the vector to allow the expression of more than one heterologous gene using a vector. In addition, the vector may contain a sequence that encodes a signal peptide or other compound that facilitates the expression of the SDF-1 gene product in a target cell.

In order to combine the beneficial properties of the two systems of viral vectors, hybrid viral vectors can be used to deliver SDF-1 nucleic acid to target tissue. Standard techniques for constructing hybrid vectors are well known to those skilled in the art. Similar techniques can be found, for example, in Sambrook, et al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor, NY, or in a variety of laboratory instructions that discuss techniques for recombinant DNA production. The double-stranded AAV genomes in adenovirus capsids containing a combination of inverted terminal AAV repeats and adenoviruses can also be used for cell transduction. In another embodiment, an AAV-based vector may be placed in a gutted, helper-dependent or high-capacity adenovirus-based vector. Adenovirus / AAV hybrid vectors are discussed in Lieber et al., J. Viral. 73: 9314-9324, 1999. Retrovirus / adenovirus hybrid vectors are discussed in Zheng et al., Nature Biotechnol. 18: 176-186, 2000. Retroviral genomes contained within adenovirus can be inserted into the genome of the target cell and stably affect the expression of the SDF-1 gene.

Additionally, other nucleotide sequence elements that facilitate the expression of the SDF-1 gene and cloning a vector are considered. For example, the presence of enhancers against the course of transcription relative to the promoter or terminators along the transcription relative to the coding region, for example, may facilitate expression.

In accordance with another aspect of the present application, the tissue-specific promoter can be hybridized with the SDF-1 gene. Due to the hybridization of a tissue-specific promoter with an adenovirus-based construct, transgene expression is limited to a specific tissue. The efficiency of gene expression and the degree of specificity provided by the tissue-specific promoters can be determined using the recombinant adenovirus system described in this document.

In addition to viral-based methods, non-viral methods can also be used to introduce an SDF-1 nucleic acid into a target cell. An overview of non-viral gene delivery methods is provided in Nishikawa and Huang, Human Gene Ther. 12: 861-870, 2001. In accordance with the present invention, in the example of a non-viral gene delivery method, plasmid DNA is used to introduce the SDF-1 nucleic acid into the cell. Plasmid-based gene delivery methods are well known in the art. In one example, the plasmid vector may have a structure that is shown schematically in FIG. 7. The plasmid vector shown in FIG. 7, contains a cytomegalovirus (CMV) enhancer and the CMV promoter against transcription relative to the SDF-1a cDNA sequence (RNA). Optionally, synthetic molecules can be developed for gene transfer to form multimolecular aggregates with SDF-1 plasmid DNA. These aggregates can be designed to bind to cells of the weakened, ischemic, and / or peri-infarction portion of myocardial tissue. Cationic amphiphiles, including lipopolyamines and cationic lipids, can be used to provide receptor-independent transfer of SDF-1 nucleic acid to target cells (for example, cardiomyocytes). In addition, pre-prepared cationic liposomes or cationic lipids can be mixed with plasmid DNA to create complexes for transfecting cells. Methods involving cationic lipid formulations are discussed in Feigner et al., Ann. NY Acad. Sci. 772: 126-139, 1995 and basic and Templeton, Adv. Drug de

- 15 031308 livery Rev. 20: 221-266, 1996. For gene delivery, DNA can also be attached to an amphipathic cationic peptide (Fominaya et al., J. Gene Med. 2: 455-464, 2000).

Techniques involving the simultaneous use of viral and non-viral components can also be used in this document. For example, a plasmid based on Epstein-Barr virus (EBV) for delivery of a therapeutic gene is described in Cui et al., Gene Therapy 8: 1508-1513, 2001. In addition, the method includes the use of adenovirus-linked DNA / ligand / polycation auxiliary, described in Curiel Dl ·., Nat. Immun. 13: 141-164, 1994.

In addition, the SDF-1 nucleic acid can be introduced into a target cell by transfecting target cells using electro-impulse cell-opening techniques. Electropulse cell pore opening techniques are well known and can be used to facilitate the transfection of cells using plasmid DNA.

If necessary, the vectors encoding the expression of SDF-1 can be delivered to the target cell as an injectable preparation containing a pharmaceutically acceptable carrier, such as saline. Other pharmaceutical carriers, formulations and dosages may also be used in accordance with the present invention.

In one aspect of the present invention, a vector may contain a plasmid SDF-1, as shown, for example, in FIG. 7. In preferred embodiments, the implementation of the plasmid SDF-1 contains the nucleotide sequence of SEQ ID NO: 6. Plasmid SDF-1 can be delivered to the cells of the weakened, ischemic and / or peri-infarction region of the myocardial tissue by directly injecting the plasmid vector with SDF-1 into the weakened, ischemic and / or peri-infarct portion of the myocardial tissue in an amount effective to improve at least one myocardial function, such as the volume of the left ventricle, the area of the left ventricle, the size of the left ventricle or the work of the heart, as well as the patient’s improvement in the test Minute walking (6MWT) or assessments by the functional classification of the New York Heart Association (NYHA). By directly injecting the vector into the weakened, ischemic and / or peri-infarction portion of myocardial tissue or along its periphery, it is possible to carry out an even more specific transfection using a vector with minimal loss of recombinant vectors. This type of injection administration provides local transfection of the required number of cells, especially if they are close to the weakened, ischemic and / or peri-infarction region of myocardial tissue, thereby maximizing the therapeutic efficacy of gene transfer and minimizing the possibility of an inflammatory response to viral proteins.

In an aspect of this application, a plasmid SDF-1 can be introduced into a weakened, ischemic and / or peri-infarction region by repeated injections of an SDF-1 solution expressing plasmid DNA, each injection containing from about 0.33 mg / ml to about 5 mg / ml plasmid SDF-1. In one example, the plasmid SDF-1 may be injected into a weakened, ischemic, and / or peri-infarct region during at least about 10 injections, at least about 15 injections, or at least about 20 injections. Repeated injections of the plasmid SDF-1 into the weakened, ischemic and / or peri-infarction region allow therapeutic effects on a large area and / or number of cells of the weakened, ischemic and / or peri-infarct region.

Each injection injected into a weakened, ischemic and / or peri-infarct area may have a volume of at least about 0.2 ml. The total volume of the solution, which includes the amount of plasmid SDF-1 introduced into the weakened, ischemic and / or peri-infarct region, which can improve at least one functional parameter of the heart, is at least about 10 ml.

In one example, the plasmid SDF-1 may be injected into a weakened, ischemic, and / or peri-infarct region during at least about 10 injections. Each injection carried out in a weakened, ischemic and / or peri-infarct area may have a volume of at least about 0.2 ml. SDF-1 can be expressed in a weakened, ischemic, and / or peri-infarct region for more than three days.

For example, each injection of a solution containing plasmids expressing SDF-1 can be carried out in a volume of at least about 0.2 ml and at a concentration of plasmid SDF-1 from about 0.33 mg / ml to about 5 mg / ml per injection. In another aspect of the present application, at least one functional parameter of the heart can be improved by injecting plasmid SDF-1 into a weakened, ischemic and / or peri-infarction region of the heart with an injection volume of at least one injection site of at least about 0.2 ml , at least about 10 injection sites with a plasmid SDF-1 concentration of about 0.33 mg / ml to about 5 mg / ml per injection.

Using a model of congestive heart failure in pigs, it was found that injections of a solution of plasmid SDF-1, having a concentration of less than about 0.33 mg / ml or more than about 5 mg / ml, with an injection volume per injection site of at least approximately

- 16 031308

0.2 ml, within the model of congestive heart failure in pigs, resulted in a small, if any, functional improvement in the volume of the left ventricle, the area of the left ventricle, the size of the left ventricle, or the function of the heart undergoing treatment.

In another aspect of this application, the amount of plasmid SDF-1 introduced into a weakened, ischemic and / or peri-infarct region that can improve at least one of the functional parameters of the heart should be more than about 4 mg and less than about 100 mg per therapeutic intervention . The amount of plasmid SDF-1 introduced during therapeutic intervention in this document corresponds to the total amount of plasmid SDF-1 administered to the patient during the therapeutic procedure and calculated to provide or obtain a therapeutic effect. It may include the total amount of plasmid SDF-1 administered per injection for a specific therapeutic intervention, or the total amount of plasmid SDF-1 administered for multiple injections as part of a therapeutic intervention. Using a model of congestive heart failure in pigs, it was found that the introduction of approximately 4 mg of plasmid SDF-1 DNA through direct injection of plasmid SDF-1 into the heart resulted in no functional improvement in left ventricular volume, left ventricular area, left ventricular size or heart function, subjected to treatment. Moreover, the introduction of approximately 100 mg of SDF-1 plasmid DNA through direct injection of plasmid SDF-1 into the heart resulted in no functional improvement in left ventricular volume, left ventricular area, left ventricular size, or treated heart function.

In some aspects of the present application, SDF-1 may be expressed in a therapeutically effective amount or dose in a weakened, ischemic, and / or peri-infarct region after transfection with a plasmid vector with SDF-1 for more than three days. Expression of SDF-1 in a therapeutically effective dose or amount for more than three days may provide a therapeutic effect in a weakened, ischemic, and / or peri-infarct region. Preferably, SDF-1 can be expressed in a weakened, ischemic, and / or peri-infarct region after transfection with a plasmid vector with SDF-1 in a therapeutically effective amount for less than 90 days to reduce possible chronic and / or cytotoxic effects, which can reduce therapeutic efficacy. SDF-1 administration to the patient.

It should be borne in mind that the amount, volume, concentration and / or dosage of plasmid SDF-1 administered to any animal or person depends on various factors, including patient size, body surface area, age, specific composition to be entered, gender , time and mode of administration, general health and other drugs, administered simultaneously. Specific variants of the above quantities, volumes, concentrations and / or dosages of the plasmid SDF-1 can be easily determined by a person skilled in the art using experimental methods, which are described below.

In another aspect of the present application, plasmid SDF-1 may be administered via direct injection using catheterization, such as intraventricular catheterization or intramyocardial catheterization. In one example, a device with a deflectable guide catheter may be advanced into the left ventricle in the opposite direction along the aortic valve. After installing the device in the left ventricle, the plasmid SDF-1 can be injected into the peri-infarction region (from the septal and lateral side) of the left ventricle. Typically, 1.0 ml of the plasmid SDF-1 solution can be injected for approximately 60 seconds. A patient undergoing treatment may be given at least about 10 injections (for example, a total of about 15 to about 20 injections).

Patient's myocardial tissue can be visualized prior to the introduction of the plasmid SDF-1 to determine the area of the weakened, ischemic and / or peri-infarct region prior to the introduction of the plasmid SDF-1. The definition of a weakened, ischemic and / or peri-infarction region by visualization provides greater accuracy of intervention and the specificity of the effect of the plasmid SDF-1 on the weakened, ischemic and / or peri-infarction region. The imaging technique used to determine the weakened, ischemic, and / or peri-infarct portion of the myocardial tissue may include any known heart imaging technique. Such imaging techniques may include, for example, at least one of echocardiography, magnetic resonance imaging, coronary artery angiography, electroanatomical mapping, or fluoroscopy. It should be borne in mind that other imaging techniques can also be used with which the weakened, ischemic and / or peri-infarction myocardial region can be determined.

Optionally, other substances other than SDF-1 nucleic acids (for example, the SDF-1 plasmid) can be introduced into a weakened, ischemic, and / or peri-infarct region of myocardial tissue to allow the expression of SDF-1 by weakened, ischemic, and / or peri-infarction cells plot. For example, substances that enhance transcription of a gene encoding SDF-1, enhance translation of mRNA encoding SDF-1, and / or reduce the cleavage of mRNA encoding SDF-1 can be used to increase the content of SDF-1 protein. Speed increase

- 17 031308 transcription of a gene in a cell can be achieved by introducing an exogenous promoter against transcription relative to the gene encoding SDF-1. Enhancer elements that contribute to the expression of a heterologous gene can also be used.

Other factors may include other proteins, chemokines and cytokines, which, when injected into target cells, activate the expression of SDF-1 in a weakened, ischemic, and / or peri-infarct region of myocardial tissue. Such factors may include, for example, insulin-like growth factor (IGF) -1, for which the ability to activate the expression of SDF-1 when introduced into mesenchymal stem cells (MSC) was shown (Circ. Res. 2008, Nov 21; 103 (11) : 1300-98); protein sonic hedgehog (Shh), for which the ability to activate the expression of SDF-1 when introduced into mature fibroblasts was shown (Nature Medicine, volume 11, number 11 of November 23); transforming growth factor β (TGF-β), for which the ability to activate the expression of SDF-1 when introduced into human mesothelial cells of the human peritoneum (HPMC) was shown to be activated; interleukin-shch (IL-1 β), platelet growth factor (PDGF), vascular endothelial growth factor (VEGF), tumor necrosis factor-alpha (TNF-a) and parathyroid hormone (PTH), for which the ability to express has been shown SDF-1 when injected into human primary osteoblasts (NOV) mixed with bone marrow stromal cells (BMSC) and human osteoblast-like cell lines (Bone, 2006, Apr; 38 (4): 497-508); Thymosin - ['> 4. for which it has been shown to activate expression when introduced into bone marrow cells (IUDs) (Curr. Pharm. Des. 2007; 13 (31): 3245-51); and a hypoxia-inducible factor, 1a (HIF-1), for which the ability to activate SDF-1 expression was shown when introduced into progenitor cells isolated from bone marrow (Cardiovasc. Res. 2008, E. Pub.). Such factors can be used to treat specific cardiomyopathies, since such cells are methods for activating the expression of SDF-1 in response to the presence or introduction of a specific cytokine.

The SDF-1 protein or factor that causes the enhancement and / or activation of the expression of SDF-1 can be introduced into the attenuated, ischemic and / or peri-infarction region of myocardial tissue in pure form or as part of a pharmaceutical composition. The pharmaceutical composition may provide for the local release of SDF-1 or factor into the cells of the weakened, ischemic and / or peri-infarct region that is being treated. In accordance with the present application, pharmaceutical compositions typically contain some amount of SDF-1 or factor mixed with a pharmaceutically acceptable diluent or adjuvant, such as a sterile aqueous solution, to obtain a wide range of final concentrations depending on the intended use. Methods of obtaining well-known in this field, their examples can be found in Pharmaceutical Sciences, 16th edition, Mack Publishing Company, 1980, which is incorporated into this document by reference. Moreover, for human administration, the drugs must comply with the standards of sterility, pyrogenicity, general safety and purity set by the Department of Biological Standards of the Food and Drug Administration (FDA).

The pharmaceutical composition may be in the form of a single dose injectable dosage form (eg, solution, suspension, and / or emulsion). Examples of pharmaceutical formulations that can be used for injection include sterile aqueous solutions or dispersions and sterile powders for reconstitution in sterile injectable solutions or dispersions. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a polyhydric alcohol (for example, glycerin, propylene glycol, liquid polyethylene glycol, etc.), dextrose, saline or phosphate-buffered saline, their suitable mixtures and vegetable oils.

Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Anhydrous carriers such as cottonseed oil, sesame oil, soybean oil, corn oil, sunflower oil, peanut oil, and esters such as isopropyl maleate can also be used as a solvent system for compounds of the composition.

In addition, various additives may be added that maintain the stability, sterility, and isotonicity of the compositions, including antimicrobial preservatives, antioxidants, chelating agents, and buffers. Prevention of the influence of microorganisms can be achieved by using various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, etc. In many cases it may be necessary to include isotonic agents, for example, sugars, sodium chloride, etc. Prolonged absorption of the pharmaceutical injectable form can be achieved by the use of agents that delay absorption, for example, aluminum monostearate and gelatin. In accordance with the methods described in this document, any carriers, diluents or additives used must be compatible with the compounds.

If necessary, sterile solutions for injection can be obtained by adding the required number of compounds used in the methods described in this document, in the appropriate solvent, containing various amounts of other ingredients.

- 18 031308

Slow-release pharmaceutical capsules or formulations and sustained-release preparations can also be used in connection with general availability. Slow release formulations are generally designed to provide a constant concentration of the drug over a long period of time and can be used to deliver SDF-1 or factor. Slow release formulations are usually implanted in the vicinity of a weakened, ischemic, and / or peri-infarction region of myocardial tissue.

Examples of drugs for long-term absorption include semipermeable matrices of solid hydrophobic polymers containing SDF-1 or factor, and these matrices are in the form of shaped articles, such as films or microcapsules. Examples of matrices of continuous absorption include polyesters, hydrogels, for example poly-2-hydroxyethyl methacrylate or polyvinyl alcohol, polylactides, for example, from US Pat. No. 3,773,919; copolymers of L-glutamic acid and y-ethyl-b-glutamate; non-biodegradable ethylene vinyl acetate; degradable copolymers of lactic acid and glycolic acid, such as LUPRON DEPOT (injection microspheres consisting of a copolymer of lactic acid and glycolic acid, as well as leuprolide acetate); and poly-e - (-) - 3-hydroxybutyric acid.

While polymers such as ethylene vinyl acetate and copolymers of lactic acid and glycolic acid, provide the release of molecules for more than 100 days, some hydrogels provide the release of proteins for shorter periods of time. In encapsulated form, SDF-1 or a factor can remain in the body for a long time, their denaturation or aggregation occurs when exposed to moisture and a temperature of 37 ° C, thereby reducing the biological activity and / or changes in immunogenicity. There are appropriate stabilization strategies that depend on the mechanism used. For example, if the aggregation mechanism involves the formation of intramolecular SS bonds during the thiodisulfide exchange reaction, stabilization is achieved by modifying the sulfhydryl residues, freeze-drying acidic solutions, regulating the water content, using appropriate additives, developing special polymer matrix compositions, etc.

In some embodiments, the implementation of liposomes and / or nanoparticles can also be used with SDF-1 or factor. The formation and use of liposomes is generally known to those skilled in the art and is summarized below.

Liposomes are formed from phospholipids that are dispersed in an aqueous medium and spontaneously form multi-layer concentric bilayer vesicles (also called multi-layer vesicles. CEMs. CEMs usually have a diameter of 25 nm to 4 microns. The destruction of SLE by ultrasound leads to the formation of small single-layer vesicles (MOB). with a diameter in the range from 200 to 500 A, in the middle of which there is an aqueous solution.

Phospholipids can form many structures that differ from liposomes when dispersed in water, depending on the molar ratio of lipids to water. At low ratios, liposome is the preferred structure. The physical characteristics of liposomes depend on pH, ionic strength, and the presence of divalent cations. Liposomes may have low permeability to ionic and polar substances, but at elevated temperatures a phase transition occurs, which significantly changes their permeability. The phase transition involves the transformation of a tightly packed ordered structure, known as a gel state, into a loosely packed, less ordered structure known as a liquid state. This occurs at a certain phase transition temperature and leads to an increase in permeability to ions, sugars, and drugs.

Liposomes interact with cells through four different mechanisms: endocytosis by phagocytic cells of the reticuloendothelial system, such as macrophages and neutrophils; adsorption on the surface of cells, provided by weak non-specific hydrophobic or electrostatic forces, as well as specific interactions with components of the cell surface; cell membrane fusion by inserting a liposome lipid bilayer into the plasma membrane with simultaneous release of the liposome contents into the cytoplasm and transfer of the liposomal lipids into the cell and subcellular membranes, or vice versa, without any interaction with the liposome contents. Changes in the composition of liposomes can influence which mechanism is present, although more than one mechanism can act simultaneously.

Nanocapsules can generally hold up compounds in a stable and reproducible manner. In order to avoid the side effects associated with intracellular overloading with polymers, such ultra-fine particles (with a size of about 0.1 μm) can be developed using polymers capable of degradation in vivo. Biodegradable polyalkyl cyanoacrylate particles that meet similar requirements are proposed for use in these methods, such particles can be easily obtained.

In order to obtain pharmaceutical compositions of the compounds described in this application, pharmaceutically acceptable carriers can have any form (for example, solids, liquids, gels, etc.). A solid carrier may contain one or more substances, which may also serve as diluents, flavoring agents, binders, preservatives, and / or materials.

- 19 031308 to encapsulate.

Critical limb ischemia.

This application further relates to a method for treating critical limb ischemia in a patient. The method comprises administering JVS-100 by direct intramuscular injection into the upper leg (quadriceps femoris) and / or the lower leg (preferably gastrocnemius muscle), using multiple injection sites. JVS-100 is a sterile biological product consisting of free plasmid DNA encoding human SDF-1 cDNA (the plasmid contains the nucleotide sequence of SEQ ID NO: 6) and a 5% dextrose solution.

Critical limb ischemia (CIC) is the latest stage of peripheral vascular disease (BPS) of the lower limbs of atherosclerotic nature and is associated with high morbidity and mortality from both cardiovascular diseases and large amputation. The estimated frequency of CICs in the United States ranges from 125,000 to 250,000 patients per year, and it is assumed that it increases as patients age. The prevalence of BPS increases dramatically with age, the disease affects about 20% of Americans aged 65 years and older. The current standard of care for people with CIC includes revascularization of the lower extremities either through open and endovascular surgical interventions on peripheral vessels, or by amputation of the extremity (if revascularization was unsuccessful or impossible). One-year mortality of patients with CIC is 25% and can reach 45% among those who underwent amputation. Despite the advanced techniques of vascular and surgical interventions, a significant proportion of patients with CIC are not subject to revascularization. Among these patients, 30% would require a large amputation, and 23% would die within 3 months. The strategies for opening closed vessels or stimulating angiogenesis are being actively studied.

Therapeutic angiogenesis, first evaluated by Dr. Jeffrey Eisner in a 71-year-old patient with severe BPS and gangrene of the toe in 1994, is a CIC treatment strategy that uses angiogenic and vasculogenic growth factors. The genes encoding these growth factors are injected into ischemic tissue to ensure neovascularization in order to increase the blood supply to the ischemic tissues through various mechanisms of action. In this study, plasmid human phVEGF165 was used with balloon angioplasty in the distal popliteal artery. Functional and angiographic parameters improved within 12 weeks, and the stellate hemangioma and edema were unilateral in the affected limb, suggesting that the treatment had a local angiogenic effect. This preliminary study confirmed that experimental CIC treatment methods suggesting an increase in the expression of angiogenic growth factors in ischemic tissue may be useful in increasing the ability to angiogenesis of patients with unfavorable surgical outcomes in order to restore function and preserve limb. Recent studies have shown that chemokines that stimulate angiogenesis can be critical components of treatment methods aimed at preserving and restoring the function of limbs in patients suffering from critical limb ischemia.

Non-viral gene delivery or the use of free plasmid DNA for the expression of a therapeutic protein in a specific area is a simple delivery method that has been clinically tested for more than 15 years in patients suffering from ischemia. The safety profile of non-viral gene delivery is also attractive compared to the delivery of genes based on viral vectors, since it does not develop a significant inflammatory response to the delivery of the viral vector. A considerable amount of literature, both preclinical and clinical, has shown that non-viral delivery of therapeutic genes is safe and effective in models of diseases such as critical limb ischemia, cardiomyopathy, and wound healing. In particular, plasmid DNA encodes fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF) and is used to treat patients with CIC with the aim of increasing collateral circulation in areas weakened due to poor or damaged vascular network . It is important that it was shown that the clinical use of non-viral gene therapy of FGF and VEGF was safe, at the moment there is no information about significant safety issues. In some studies of phase I and II, patients suffering from severe non-reconstructive intervention BPS severe, accompanied by pain at rest or necrosis of the tissue, were prescribed treatment with intramuscular injections of non-viral FGF (NV1FGF). Elevated single-dose (up to 16 mg) and repeated (from 2 to 8 mg) doses of NV1FGF were injected into the ischemic hip and shin using free plasmid DNA. NV1FGF was well tolerated, with subsequent follow-up after 6 months in 38 patients there was a significant decrease in the assessment of pain and the size of the ischemic ulcer, and an increase in the transdermal oxygen partial pressure in comparison with the initial values. Phase III large-scale clinical trials are being conducted on the use of free plasmid DNA to treat CICs, such as a multicenter, double-blind, placebo-controlled TAMARIS clinical trial to evaluate the efficacy and safety of NV1FGF in patients with CIC and skin lesions, including 490 patients who have been prescribed

- 20 031308 placebo or intramuscular injection of NV1FGF.

It was shown that today it is not known about any problems with the safety of the use of such non-viral methods of gene therapy; Based on data from a Phase II clinical trial, it can be assumed that the administration of NVIFGF has a beneficial effect on patients with CIC. Thus, in combination with previous clinical work devoted to treating ischemic heart failure with the help of ACRX-100 and showing an improvement in vasculogenesis and heart function without safety problems, it can be assumed that the use of ACRX-100 will be safe, will improve vasculogenesis and, ultimately , will have a beneficial clinical impact on patients with critical limb ischemia.

It was determined that the production of SDF-1 is activated in many tissues after injury, this gene is expressed for 4-5 days. A study in several groups showed that the use of SDF1 is promising for the treatment of a wide range of diseases, including ischemic myopathy, peripheral vascular disease, wound healing, critical limb ischemia and diabetes. SDF-1 is a strong chemoattractant for stem cells and progenitor cells that preserve tissue and develop blood vessels. Taken together, such messages indicate a stored reaction cascade and mechanism of action by which SDF-1 can restore and restore tissue function after it has been damaged. This has led applicants to develop ACRX-100 (non-viral free plasmid DNA encoding SDF-1, in dextrose solution) for the treatment of ischemic diseases of the cardiovascular system. In a study of safety and toxicity, conducted according to the rules of good laboratory practice, within the framework of a model of heart failure in pigs, it was shown that the drug ACRX-100 has a favorable functional effect in a dose of up to 30 mg and is safe in a dose of up to 100 mg. Currently, applicants are recruiting test subjects in a multicenter, open phase I clinical trial with dose escalation in which ACRX-100 is used to treat patients with ischemic heart failure.

Applicants' studies showed that ACRX-100 significantly increased heart vasculogenesis, which was consistent with studies in several groups. Similar studies confirm that stem cells that tend to return to chronically damaged tissue during critical limb ischemia can initiate tissue repair and probably improve blood flow. The company showed that direct intramuscular injection of ACRX-100 into the rabbit ischemic hind limb provided tissue revascularization compared to control animals, which confirms that ACRX-100 is a promising drug candidate for treating patients with critical limb ischemia (CIC). The estimated frequency of CICs in the United States ranges from 125,000 to 250,000 patients per year, and it is assumed that it increases as patients age. The current standard of care for patients with CIC includes revascularization of the lower extremities either through open and endovascular surgical procedures on peripheral vessels, or by amputation of the limb (if revascularization was not successful or impossible). One-year mortality of patients with CIC is 25% and can reach 45% among those who underwent amputation. Despite the advanced techniques of vascular and surgical interventions, a significant proportion of patients with CIC are not subject to revascularization.

The plasmid having the nucleotide sequence of SEQ ID NO: 6 contains free plasmid DNA encoding human SDF-1 cDNA. ACRX-100 is a sterile biological product consisting of a plasmid having the nucleotide sequence of SEQ ID NO: 6 and a 5% dextrose solution.

A plasmid having the nucleotide sequence of SEQ ID NO: 6 is a free plasmid DNA developed for the expression of human SDF-1 in mammalian tissue. The plasmid backbone is derived from the plasmid ColE1 and contains a kanamycin resistance marker. The expression of the SDF1 transgene is activated by the enhancer / promoter from CMV, intron A from CMV and the translational enhancer RU5. Effective polyadenylation is achieved by incorporating a bovine growth hormone signaling polyadenylic sequence. The same plasmid and composition is used to treat patients with heart failure. Applicants have developed ACRX-100 for the treatment of patients with critical limb ischemia. ACRX-100 has a composition suitable for direct intramuscular injection. The planned application contains one or more doses in the upper leg (the quadriceps muscle of the thigh) and the lower leg (preferably the gastrocnemius muscle), using multiple injection sites.

SDF-1 (also known as CXCL12) is a naturally occurring chemokine that is easily activated in response to tissue damage. Induction of SDF-1 stimulates a variety of protective anti-inflammatory reactions causing a decrease in the production of inflammatory mediators (such as MMP-9 (matrix metalloproteinase-9) and IL-8), and protects cells from apoptosis by suppressing caspase-modulated activation of protein kinase B. Moreover, SDF-1 is a strong chemoattractant for stem cells and progenitor cells specific for internal organs and bone marrow, promoting their movement into the damaged tissue site, which ensures the preservation of

- 21 031308 tissue and development of blood vessels. Previous studies have shown that SDF1 expression is elevated at the site of the lesion, however, expression lasts less than a week, so the induced response of stem cells quickly stops. The short duration of the expression of SDF-1 reduces the possibility of tissue repair, but confirms that therapeutic interventions, prolonging or re-inducing SDF-1 to stimulate the process of stem cell movement, may be useful for patients with damaged tissues. Based on this assumption, in the laboratory of Dr. Penn, it was shown that repeated administration of SDF-1 by injection of cardiac fibroblasts with increased expression of SDF-1 a month after myocardial infarction (MI) resulted in the resumption of stem cell-specific aspiration stem cells brain, moving to the heart, the growth of new blood vessels in the affected tissue and the improvement of heart function by more than 80%. Injection of skeletal myoblasts with increased expression of SDF-1 eight weeks after myocardial infarction also significantly improved heart function. Moreover, SDF-1 improved the function of the heart by increasing the attraction of circulating stem cells to the damaged tissue and the formation of new blood vessels to enhance the blood supply to the affected area. These effects, along with significant preservation of the myocardium, were shown when MSC was delivered to rats with increased SDF-1 expression after acute MI, which resulted in a 240% improvement in heart function. The biological response to ischemic damage was stable in a variety of organ systems.

The benefits of SDF-1 treatment observed by the applicants have been confirmed by the latest work carried out by independent laboratories. SDF-1 improved cardiac function in ischemic cardiomyopathy when it was delivered as a protein embedded in nanofibers, to rats who had acute MI, recombinant protein in a fibrin film for mice that had MI, or to direct intramyocardial protein injection to mice that had MI. It was shown that when the plasmid encoding SDF-1 was injected, circulating stem cells were attracted into the marginal zone of MI. Similarly, in the case of regenerative cell therapy, in which myoblasts are used, or muscle stem cells that were grown from human muscle and genetically modified for increased expression of SDF-1, the preclinical efficacy of heart failure treatment was shown and clinical studies were conducted with the participation of patients to assess the recovery of ischemic tissue and improve functioning in a REGEN clinical study. In aggregate, similar published data obtained from many laboratories showed that, regardless of the delivery method, increased expression of SDF-1 provides a positive functional effect on diseases of the ischemic nature.

Repeated stimulation of the expression of SDF-1 in ischemic muscle has a great therapeutic potential for treating CICs by regenerating the vasculature damaged by insufficient blood flow. This makes it possible to restore and preserve the function of the affected limbs. In a variety of clinical studies that used stem cells or VEGF, it has been shown that the re-growth of the blood vessel network increases the frequency of cases of limb preservation. Yamaguchi et al. reported that local delivery of SDF-1 protein resulted in increased neovascularization in the ischemic hind limb after the introduction of embryonic stem cells (EPC), which confirms that SDF-1 enhances EPC-induced vasculogenesis. Similarly, Hiasa et al. showed that SDF-1 gene transfer enhanced ischemic-induced vasculogenesis and in vivo angiogenesis through a cascade of reactions associated with VEGF / eNOS (endothelial NO-synthase).

Similar observations were recently confirmed during the analysis of skeletal muscle obtained from patients who underwent amputation in the area of the knee due to chronic CIC. The expression of SDF-1 and its receptor, CXCR4, was increased in skeletal muscle fibers and capillaries, respectively. This confirms that the cascade of reductive reactions associated with SDF-1 / CXCR4 was chronically activated in the ischemic muscles of the legs to naturally stimulate the growth of the microvasculature, but it was not sufficient with such a low content of these substances. With the help of gene therapy ACRX-100 to enhance the signal can synergistically stimulate the healing process, which has already begun in the damaged tissue. Thus, ACRX-100 is a fundamentally new chemokine treatment for therapeutic next-generation neovascularization.

Brief information about the main cell bank and the production of bulk plasmid solution.

The production of the active ingredient used to treat heart failure in a Phase I clinical trial was carried out in a volume of 300 liters with the help of dedicated reagents and materials. In our proposed clinical trials for the treatment of CICs of phase I / II, it was possible to apply the experience gained by applicants using the prepared main cell bank and improving the drug production process using pre-established quality tests. All media were prepared from the usual ingredients that do not contain components of animal origin, and sterile water according to the requirements of F. USA or even higher quality. All bacteria lysis buffers have been released for production at

- 22 031308 on the basis of relevant certificates of analysis. Lysis on a large scale was carried out using an ultrasound device without the use of ribonuclease. All chromatographic buffers were manufactured by Aldevron using reagents. meet the requirements of F. USA. or their equivalents. and released after checking for compliance with internal specifications. All aseptic processing was carried out in clean rooms with a controlled environment. A clean room structure consists of an auxiliary room (cleanliness class 10,000. ISO 7) for changing clothes with two doors and a transmission window. overlooking the main clean room (cleanliness class 10,000); inside there is a room with a cleanliness class of 1000 (ISO 6) with a microbiological safety cap for the final filling / treatment with a cleanliness class of 100 (ISO 5). Established procedures. used for maintenance. cleaning (after each series) and environmental control. All unpackaged plasmid solution is sterilized using a filter. if necessary, its concentration is adjusted. after which the solution is stored at a temperature of 75 + 5 ° C for final distribution in vials for the drug.

The final stage of production is aseptic dilution of sterile solutions in sterile containers for bulk materials. In this way. for use in the early stages of development, the active ingredient must be sterile.

Brief information about the production of medicines.

The unpackaged plasmid solution is transferred to a clean room of ISO 6 class. In which is located the box of biological safety (BSC) of ISO 5. Purity class. All materials. with whom contact is possible. are disposable items. which are pre-sterilized. do not contain pyrogens and are issued for use on the basis of the manufacturer's certificate of analysis. Processing takes place in the BBB. The unpackaged plasmid solution is first diluted to the final target concentration with a 5% dextrose solution (F. USA) for injection. After mixing, the sterile plasmid solution is manually pipetted and transferred to the final penicillin vials. which were sterilized beforehand. The product is stored frozen at -75 + 5 ° C. Full list of media components and auxiliary reagents. as well as quality information. given in the application for conducting clinical trials of a new drug.

Stability.

Stability studies have shown. that the preclinical batches of the ACRX-100 are stable at -20 ° C for a year after production. ACRX-100 for clinical use (batches 24370D-F) is currently included in stability studies under accelerated degradation (5 ± 3 ° C) and standard conditions (-20 ± 4 ° C) (Table 1). expected. that the results will exceed the data. obtained during preclinical stability studies. The results are shown in the application for conducting clinical trials of a new drug. As part of the stability study program, the following tests should be carried out: appearance. authenticity (agarose gel electrophoresis). concentration (A260 / A280). homogeneity (measurement of optical density). activity and pH. Sterility testing should be carried out at release. six months later and thereafter annually.

Chemokine-based drugs have recently attracted considerable attention due to their ability to stimulate and attract stem cells to damaged tissue. ACRX-100 is a gene therapy agent. which delivers the human chemokine SDF-1 by expressing the gene by human cells. It is shown. that the active protein is SDF-1. produced using the ACRX-100. improves heart function in the myocardium. ischemic for some time. and increases the frequency of preservation of the affected epithelium by attracting CXCR4-positive stem cells. It is shown. that SDF-1 has pro-angiogenic activity in patients with acute CIC. moreover, its production is suppressed in patients with chronic CIC. which is evidence of that. what are the treatments. aimed at the resumption of the expression of SDF-1 in chronic CIC. may enhance vasculogenesis due to the transformation of cells. from bone marrow. into a mature vascular network.

Plasmid. having the nucleotide sequence of SEQ ID NO: 6 and part of the drug ACRX-100. was investigated on models of cardiovascular disease and ischemia

- 23,031,308 hind limbs on animals. Within the framework of the model of heart failure in pigs, the ACRX-100 was investigated after intracardiac administration in terms of efficacy, safety and biodistribution, and a maximum non-toxic dose (MND) of 100 mg was established. The identical composition ACRX-100, administered in smaller doses than the doses used in preclinical and clinical studies of heart failure, is currently being evaluated to determine the possible therapeutic effect in CIC. A study was conducted of the effectiveness, toxicity and biodistribution of a single dose of ACRX-100 administered to rabbits with ischemic hind limb. This study showed that ACRX-100 may have a therapeutic effect in critical limb ischemia, and that intramuscular injection of ACRX-100 into the ischemic rabbit hind limbs did not lead to any signs of toxicity or histopathological changes. In addition, a plasmid having the nucleotide sequence of SEQ ID NO: 6 is a plasmid that was substantially removed from all organs, except the ischemic limb, 60 days after the therapeutic administration of a single dose. A study was planned on the efficacy and safety (toxicology and biodistribution) of repeated doses in the model of rabbit hind limb ischemia to confirm that up to 3 doses of ACRX-100 can be used in patients with CIC.

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the attached claims.

Example 1

Stromal growth factor-1, or SDF-1, is a naturally occurring chemokine whose expression is rapidly activated in response to tissue damage. Induction of SDF-1 stimulates a variety of protective anti-inflammatory reactions that cause a decrease in the production of inflammatory mediators (such as MMP-9 and IL-8), and protects cells from apoptosis. Moreover, SDF-1 is a strong chemoattractant for stem cells and progenitor cells that are specific for organs and bone marrow, facilitating their transfer to the damaged tissue site, which ensures the preservation of tissue and the development of blood vessels. Based on the observation that increased expression of SDF-1 leads to improved heart function in animal models of ischemia, the applicants focused on the development of a non-viral free plasmid with DNA encoding SDF-1 for the treatment of ischemic diseases of the cardiovascular system. During development, the plasmid was optimized based on the results of cell culture studies and small animal studies below. The plasmid having the nucleotide sequence of SEQ ID NO: 6 was selected on the basis of its ability to express transgene in heart tissue and, as a result, improve heart function in preclinical models of ischemic cardiomyopathy in animals. The expression of the SDF-1 transgene in a plasmid with the nucleotide sequence of SEQ ID NO: 6 is activated by an enhancer / promoter from CMV, an intron A from CMV and a translational enhancer RU5. Drug JVS-100 (previously known as ACRX-100) consists of a plasmid having the nucleotide sequence of SEQ ID NO: 6, and a 5% dextrose solution.

Preliminary studies in a rat model of heart failure showed that ACL-01110S (a precursor of a plasmid having the nucleotide sequence SEQ ID NO: 6 expressing SDF-1) improved heart function after injection of the plasmid directly into the marginal infarction zone in the hearts of rats after four weeks after MI. The therapeutic effect persisted for at least 8-10 weeks after injection and corresponded to increased vasculogenesis in animals treated with ACL-01110S. ACL-01110S has been modified to optimize the expression profile.

Dose-dependent plasmid expression in the IM model in rats.

To determine the plasmid dose per injection, which can maximize expression in rat cardiac tissue, increasing doses (10, 50, 100, 500 μg) of the plasmid with ACL-00011L and luciferase were introduced into the hearts of rats after a heart attack. Lewis rats were subjected to median sternotomy, a permanent ligature was applied to the anterior descending branch of the left coronary artery (PNVLKA), after which 100 μl of the plasmid with ACL-00011L in phosphate buffer (FBI) was injected in one area near the source of MI. Luciferase expression throughout the body was measured in each of the dosing groups (n = 3) by non-invasive bioluminescent imaging (Xenogen, Hopkinton, Mass.) At the initial time and 1, 2, 3, 4 and 5 days after injection. Peak expression increased to a dose of 100 mg and reached a limit at high doses. Based on the dose-dependency curve, it was determined that a dose of 100 mg is sufficient to obtain maximum plasmid expression in rat hearts. In ACL-00011L, the luciferase gene was expressed from the backbone of the vector equivalent to the ACL-00011S construct, which expresses SDF-1.

Comparison of vector expression in the heart within the model of ischemic heart failure in rats.

Several candidate plasmids with SDF-1, equivalent to a plasmid expressing luciferase, were evaluated from the point of view of expression in the heart tissue within the framework of the myocardial infarction (MI) model in rats. Candidate plasmids differed in expression promoters and in the presence of an enhancer.

- 24 031308 sulfur elements. Lewis rats were subjected to median sternotomy, a permanent ligature was applied to the anterior descending branch of the left coronary artery (FNLA), after which the thorax was sutured. Four weeks later, the thorax was re-opened, after which a plasmid expressing luciferase was injected directly into four sites in the vicinity of the myocardial infarction center (100 μg in 100 μl per injection). At 1, 2, 4, 6, 8, and 10 days after injection (and then every 3-4 days), rats were anesthetized, luciferin was injected into them, and the whole body was visualized using the Xenogen Luciferase system.

In a study of two plasmids based on CMV, ACL-00011L and ACL-01110L, it was found that detectable luciferase expression was reached within 24 hours after injection with an initial expression peak 2 days after injection.

The peak expression of ACL-01110L was 7 times greater than that of ACL-00011L, and the expression lasted about 10 days longer (lasted up to 16 days after injection). In contrast, for ACL00021L (plasmid with the AMHS promoter), there was no initial peak, however, expression continued at a low level for 25 days after injection. These results confirm data from previous studies that showed that CMV-based plasmids can be used for local transient expression of the protein in the heart and that the duration of expression of the therapeutic protein can vary with the introduction of enhancer elements.

The effectiveness of plasmids with SDF-1 in the model of MI in rats.

Plasmids encoding SDF-1 were studied in the framework of the MI model in rats to determine whether a therapeutic effect on the heart could be obtained. Lewis rats were subjected to a median sternotomy, a permanent ligature distal to the first bifurcation was superimposed on the CNTLA rat. Four weeks later, the chest was re-opened, and one of three plasmids expressing SDF-1 (ACL-01110S, ACL-00011S or ACL-00021S) or saline (100 μg per 100 μl per injection) was injected into four tissue sections near focus of IM. At baseline (pre-injection) and 2, 4, and 8 weeks after injection, rats were anesthetized and imaged with M-mode echocardiography. The left ventricular ejection fraction (LVEF), the shortening fraction and the size of the left ventricle were determined by an experienced ultrasound ultrasound specialist, who was not aware of the randomization results.

A steady trend towards an improvement in heart function was observed in the case of two plasmids based on CMV, ACL-01110S and ACL-00011S, in comparison with the control administration of saline. The introduction of ACL-01110S caused a statistically significant increase in the shortening fraction four weeks later, which persisted for 8 weeks after injection. On the contrary, no difference was found in the functioning of the heart with the introduction of the plasmid ACL-00021S with the aMHC promoter and saline. Moreover, compared to control, animals treated with ACL01110S and ACL-00011S experienced a significant increase in the density of large vessels (ACL-01110S: 21 ± 1.8 vessels / mm ² ; ACL-00011S: 17 ± 1.5 vessels / mm ² ; saline: 6 ± 0.7 vessels / mm ² , p <0.001 for both cases compared with saline) and a reduction in the size of the infarction focus (ACL-01110S: 16.9 ± 2.8%; ACL-00011S : 17.8 ± 2.6%; saline: 23.8 ± 4.5%). It is important that the treatment of ACL-01110S showed the greatest improvement in heart function and vasculogenesis, it also caused the greatest decrease in the size of the hearth lesion.

As a result, within the rat model of ischemic heart failure, the use of both plasmids encoding SDF-1 and having a CMV promoter provided an improvement in heart function, increased vasculogenesis, and a reduced infarct size in comparison with the introduction of saline. For all parameters studied, the introduction of ACL-01110S provided the most reliable therapeutic effect.

The transfection efficiency of a plasmid having the nucleotide sequence of SEQ ID NO: 6 and ACL-01010Sk in H9C2 cells.

Transfection in H9C2 myocardial cells in vitro without transfection reagents (i.e., adding free plasmid DNA to cell culture) was used to determine the transfection efficiency in vivo versions of the basic invented plasmid vectors with green fluorescent protein (RFP); SEQ ID NO: 6 and ACL-01010Sk. H9C2 cells were cultured in vitro, various amounts of pDNA (0.5, 2.0, 4.0, and 5.0 μg) were added to a 5% dextrose solution. Vectors with GFP were constructed from the cores of a plasmid having the nucleotide sequence of SEQ ID NO: 6 (ACL-01110G) or ACL-01010Sk (ACL-01010G). On the third day after transfection, the fluorescence of GFP was evaluated by sorting cells with activated fluorescence to determine the efficiency of the transfection. The transfection efficiency for the vectors ACL-01110G and ACL-01010G in a 5% dextrose solution was in the range of 1.08–3.01%. In the case of each amount of pDNA tested, both vectors had similar in vitro transfection efficiency values. The applicants concluded that the transfection efficacy observed in this study, which was 1-3%, corresponded to data from previous studies, in which a similar level of transfection efficiency was obtained in vivo. More specific,

- 25 031308

JVS-100 transfects a limited, but sufficient number of heart cells to produce therapeutically effective amounts of SDF-1.

Example 2. Plasmid expression in pig myocardium.

The MI model in pigs with occlusion / reperfusion of the anterior descending branch of the left coronary artery (ANVL) was selected as a suitable large animal model for evaluating the effectiveness and safety of the ACRX-100. In this model, between the MI and treatment provided 4 weeks of recovery, during which additional restructuring of the cardiac activity and imitation of chronic ischemic heart failure should occur.

Surgical intervention.

Yorkshire breed pigs were given anesthesia and heparin until an activated clotting time (ABC)> 300 s was obtained, after which they were placed in a position on the back with their legs bent at the knees. To determine the LV contour, left ventriculography was performed in the anteroposterior and lateral projections.

Delivery of the plasmid with luciferase in the myocardium of the pig.

A device with a deflectable guide catheter was advanced into the left ventricle (LV) in the opposite direction along the aortic valve, then the guide wire of the catheter was removed, and a needle catheter for endocardial injection was inserted through the guide catheter into the LV cavity. A plasmid with luciferase was injected in a given volume and concentration in 4 sites on the septal or side wall of the heart. Five combinations of plasmid concentrations (0.5, 2 or 4 mg / ml) and volumes for injection (0.2, 0.5, 1.0 ml) were investigated. The plasmid at a concentration of 0.5 mg / ml was buffered in a dextrose solution (F. USA), all other concentrations were buffered in phosphate-buffered saline (PBS, F. USA). For each injection, the needle was inserted into the endocardium, where the gene solution was introduced at a rate of 0.8-1.5 ml / min. After injection, the needle was left in place for 15 seconds and then removed. After the completion of the injections, all the tools were removed, the incision was sutured, and the animals were allowed to recover.

Myocardial tissue intake.

On the third day after the injection, the animals were autopsied. After euthanasia, the heart was extracted, weighed and washed with Ringer's lactate solution until it was free of blood. The LV was opened, after which the injection sites were established. A cube tissue sample with a side of 1 cm ² was separated from each injection site. As a negative control, four (4) cubic samples were taken from the back wall away from any injection site. Tissue samples were frozen in liquid nitrogen and stored at -20 to -70 ° C.

Evaluation of luciferase expression.

Tissue samples were thawed and placed in a 5 ml glass tube. A lysis buffer (0.5-1.0 ml) was added, the tissue was destroyed with a Polytron homogenizer (model PT1200) on ice. The tissue homogenate was centrifuged, the protein concentration in the supernatant was determined for each tissue sample by quantitative analysis of protein compatible with surfactant, Bio-rad and a standard curve based on known quantities of bovine serum albumin (BSA). A tissue sample homogenate (1-10 μl) was quantified using a luciferase assay kit (Promega).

The results of the study are presented in FIG. 1. Data indicates that vector expression increases with increasing injection volume and increasing DNA concentration.

Example 3. Improvement of heart function in the treatment of plasmid SDF-1 in the framework of a model of ischemic cardiomyopathy in pigs.

Induction of myocardial infarction.

Yorkshire breed pigs were given anesthesia and heparin until an activated clotting time (ABC)> 250 s was obtained, after which they were placed in a supine position with their legs bent at the knees. A balloon catheter was inserted by advancing along the guide catheter into the FNVLKA below the first main bifurcation of FNVLKA. Then the balloon was inflated to obtain pressure sufficient to ensure complete arterial occlusion, and left in the artery in a swollen state for 90-120 minutes. The completeness of inflating and deflating the balloon was checked by fluoroscopy. Then the balloon was removed, the incision was sutured, and the animal was allowed to recover.

Inclusion criteria

One month after MI, the functioning of the heart was checked by echocardiography. If LVEF was less than 40%, and CSOLV was more than 56.7 ml, the pig was included in the study.

Surgical intervention.

Each included pig was given anesthesia and heparin until an activated clotting time (ABC)> 300 s was obtained, after which it was placed in a position on the back with the legs bent at the knees. To determine the LV contour, left ventriculography was performed in anterior-posterior and lateral projections.

- 26 031308

Delivery to the myocardium of a plasmid with SDF-1 having the nucleotide sequence of SEQ ID NO: 6

With the help of randomization for each pig, one of three dates of killing was determined: 3 days, 30 days or 90 days after treatment, they were also identified in one of four treatment groups: control (20 injections, only buffer), with low concentration ( 15 injections, 0.5 mg / ml), with an average concentration (15 injections, 2.0 mg / ml) or with a high concentration (20 injections, 5.0 mg / ml). All plasmids were buffered in a dextrose solution (F. USA). The injection procedure is described below.

A device with a deflectable guide catheter was advanced into the left ventricle (LV) in the opposite direction along the aortic valve, then the guide wire of the catheter was removed, and a needle catheter for endocardial injection was inserted through the guide catheter into the LV cavity. Plasmid solution with SDF-1 or buffer were recruited in an amount determined by randomization into 1 ml syringes that were connected to a catheter. The volume of each injection was 1.0 ml. For each injection, the needle was inserted into the endocardium, where the solution was injected for 60 seconds. After injection, the needle was left in place for 15 seconds and then removed. After the completion of the injections, all the tools were removed, the incision was sutured, and the animal was allowed to recover.

At killing, tissue samples from the heart and other major organs were excised and immediately frozen for polymerase chain reaction (PCR) and histopathological analysis.

Evaluation of heart function.

The functioning of the heart of each animal was assessed using a standard two-dimensional echocardiograph at 0, 30, 60 and 90 after injection (or before killing). Measurements of the volume, area and index of violation of local contractility of the left ventricle were carried out by an independent central laboratory. The measured efficiency parameters are presented below in table. one.

Table 1

Echocardiographic parameters

Variable name Value KSOLV The end-systolic volume measured in the parasternal position of the long axis KDOLV End-diastolic volume, measured in the parasternal position of the long axis LVEF (KDOLV-KSOLV) / KDOLV * SO% INLSM The average value of all read indices of violation of local myocardial contractility is based on the 17-segment model of the American Society of Echocardiography and the rating system from 0 to 5

The effect of plasmid SDF-1 on functional improvement is shown in FIG. 2-5. FIG. 2-4 show how low and medium doses of plasmid SDF-1 improve CSOLV, LVEF and the index of impaired local myocardial contractility 30 days after injection compared to control, while the administration of a high dose did not lead to a therapeutic effect. FIG. 5 shows that the therapeutic effect on the heart of low and medium doses lasted up to 90 days, in both cases a noticeable slowdown of the pathological restructuring was observed, consisting in a smaller increase in CSOLV, in comparison with the control.

Evaluation of vasculogenesis.

Animals that were sacrificed after 30 days were used to estimate vascular density in the left ventricle using 7–9 tissue samples taken from each heart, fixed with formalin. Genomic DNA was extracted and efficiently purified from a formalin-fixed tissue sample using a minicolumn cleaning procedure (Qiagen). Samples obtained from animals treated with SDF-1 and in the control group were examined for the presence of plasmid DNA using quantitative PCR. Three to five tissue samples of each animal containing copies of plasmid DNA in an amount greater than the background one at least 4 times (except for animals from the control group) were used to prepare sections that were immunostained with isolectin. The cross sections were determined, after which the vessels were counted in 20-40 random fields per one tissue sample. This number of vessels in one field was converted to vessels / mm ² , and then averaged for each animal. For each dose, data were provided on the average number of vessels / mm ² for each animal that received such a dose.

FIG. 6 shows that both doses, which showed an improvement in function, also significantly increased the density of the vessels within 30 days compared to the control. In contrast, high

- 27 031308 dose, which showed no improvement in function, showed no increase in vascular density. This data is provided by the putative biological mechanism by which the plasmid SDF-1 improves heart function in ischemic cardiomyopathy.

Biodistribution of data.

The distribution of JVS-100 in cardiac and non-cardiac tissues was measured at 3, 30 and 90 days after injection in a study of key efficacy and toxicology in the porcine MI model. In cardiac tissue, at each time point, the average plasmid concentration of JVS-100 increases with dose. In each dose, excretion of JVS-100 was observed at 3, 30 and 90 days after administration, with approximately 99.999999% removal from cardiac tissue on day 90. JVS-100 spread to non-cardiac organs with relatively high blood flow (for example, heart, kidney, liver and lungs) with the highest marked concentrations on day 3 after injection. JVS-100 was present primarily in the kidney according to the renal clearance of the plasmid. Low levels of persistence were observed on day 30, and JVS-100 was essentially undetectable in non-cardiac tissue after 90 days.

Conclusions

Treatment with JVS-100 resulted in a significant increase in blood vessel formation and improved heart function in pigs with ischemic heart failure after a single endomyocardial injection of 7.5 and 30 mg. The highest dose tested JVS-100 (100 mg) showed a tendency to increase the formation of blood vessels, but showed no improvement in cardiac function. None of the doses of JVS-100 was not associated with manifestations of toxicity, adverse effects on the parameters of clinical pathology or histopathology. JVS-100 primarily spread to the heart, with approximately 99.999999% elimination from cardiac tissue 90 days after treatment. JVS-100 has spread to non-cardiac organs with relatively high blood flow (for example, heart, kidney, liver and lungs), with the highest concentration in the kidney on day 3 after injection. JVS-100 was essentially undetectable in non-cardiac tissue 90 days after administration, with a minor amount of dose administered in non-cardiac tissues.

Based on these data, it was concluded that the highest non-toxic dose (NOAEL) for JVS-100 on the porcine myocardial infarction model is 100 mg when administered endomyocardially.

Example 4. A preliminary study on the pig model.

LUC injections by transarterial administration to pigs with chronic myocardial infarction.

Ways.

One pig with previous myocardial infarction with concomitant occlusion / reperfusion of the HMWH and FV> 40% received an injection of a plasmid having the nucleotide sequence of SEQ ID NO: 6 using a transarterial catheter. Two injections in the HMWH and 2 in the OV were performed with an injection volume of 2.5 ml and a total injection time of 125-130 s. One additional introduction of the contrast mixed with the plasmid into the RH 3.0 ml with a total administration time of 150 s was also carried out.

Sleep and collect tissue.

Three days after the injection, the animal is euthanized. After euthanasia, the heart was removed, drained of blood, placed on an ice cutting board and then opened by an autopsy specialist or a pathologist. The myocardium that did not receive the injection was obtained from the septum through the opening in the right ventricle. The right ventricle was separated from the heart and placed in cold cardioplegia. New scalpel blades were used for each of the cuts.

Next, the left ventricle was opened, and the entire left ventricle was extracted by cutting into 6 sections, cuts were made from the top to the bottom. LV was evenly divided into 3 slices. After excision, each section could be placed flat on the surface. Each slice (3 LV slices, 1 slice of the pancreas, and 1 slice of the pectoral muscle) was placed in separate labeled containers with cold cardioplegia on wet ice and transported for analysis using luciferase.

Luciferase images.

All harvested tissues were immersed in luciferin, and their images were obtained using the Xenogen imaging system to determine plasmid expression.

Results.

A representative image of the heart is shown in FIG. 8. Colored spots indicate areas of luciferase expression. The value of the relative light units (RLUS) of these spots is more than 10 ⁶ units, which is more than 2 orders of magnitude higher than the background. These data indicate that plasmid delivery through the catheter is sufficient to generate significant plasmid expression for a significant portion of the heart.

Example 5. An example of a clinical study.

Increasing doses of JVS-100 are administered to treat heart failure in patients with ischemic cardiomyopathy. Safety is monitored for each dose by documenting all adverse events (AEs) with a series of large heart AEs occurring for 30 days as the primary endpoint of safety. In each group, patients received a single dose of JVS-100. In all groups, the effectiveness of treatment is assessed by measuring the effects on cardiac function using standard echocardiography, cardiac perfusion using single-photon emission

- 28 031308 computed tomography (SPECT) images, classification of the New York Heart Association (NYHA), walking for six minutes and quality of life.

All patients have a history of known systolic dysfunction, preliminary MI and the absence of cancer, confirmed prior to the examination for the detection of cancer, age-appropriate. All patients were examined with a visit to the doctor and cardiac echocardiography. Conducted further basic research, such as SPECT perfusion. Each patient was made 15 injections of 1 ml of JVS-100, delivered through the endocardial stylet catheter to the zone lying within the border of the infarction. The study will undergo three groups: (A, B, C). As shown in the table. 2, doses will be increased by increasing the amount of DNA per injection, while maintaining the same number of injection sites - 15 and the volume of injections - 1 ml. Patients were monitored for approximately 18 hours after administration and visits were scheduled for 3 and 7 days after administration to confirm that there were no safety hazards. The patient remains in the clinic for 18 hours after the injection to collect all necessary blood samples (for example, for cardioferment analysis, plasma SDF-1 protein level). All patients were scheduled for repeated examinations after 30 days (1 month), 120 days (4 months) and 360 days (12 months) for the study of AE and cardiac function. The primary endpoint of safety is the manifestation of serious adverse cardiovascular events (MACE) within 1 month after treatment. For each patient during the entire study will be monitored AE. The following efficacy and safety endpoints will be measured:

Security.

The number of serious adverse cardiovascular events (MACE) within 30 days after administration.

Adverse events in the next 12 months.

Laboratory blood tests (cardiofermental, general, antinuclear factor).

SDF-1 plasma levels.

Evaluation of the physical condition of the patient.

Echocardiography.

Monitoring the work of AICD.

ECG.

Efficiency.

Changes in LVESV, LVEDV, LVEF and index of violation of local myocardial contractility relative to baseline values.

Changes regarding baseline NYHA classification values and changes in quality of life.

Change of perfusion relative to baseline values according to SPECT images.

Changes relative to baseline results of a six-minute walk test.

table 2

Clinical dosage schedule quotes Group Patient number DNA number / place The amount of injected drug / place No. of injection sites Total dose of DNA Group A four 0.33 mg 1, 0 ml 15 5 mg Group B 6 1.0 mg 1.0 ml 15 15 mg Group C 6 2.0 mg 1.0 ml 15 3 0 mg

Based on preclinical studies, JVS-100 delivery is expected to cause an improvement in cardiac function and symptoms for 4 months, and the effect will last for 12 months. Four months after the introduction of the JVS-100, there were improvements regarding the baseline values of the six-minute walk test results over 30 m and improvements in the quality of life estimate by about 10% and / or a possible improvement by about grade 1 in the NYHA classification. Also, applicants expect a relative improvement in LVESV, LVEF and / or WMSI by approximately 10% relative to baseline values.

Example 6. Evaluation of heart function according to echocardiography in pigs with chronic heart failure after treatment with a plasmid having the nucleotide sequence of SEQ ID NO: 6 or ACL-01010Sk.

Purpose.

The purpose of this study is to compare the functional cardiovascular response to SDF-1 plasmids having the nucleotide sequence of SEQ ID NO: 6 with ACL-01010Sk after delivery through an endomyocardial catheter in a porcine model of ischemic heart failure.

This study compares the effectiveness of a plasmid having the nucleotide sequence of SEQ ID NO: 6 and ACL-01010Sk to improve function in a swine ischemic model of heart failure. In this study, the plasmids were delivered via an endoventricular 29 031308 stylet catheter.

Efficacy was assessed by measuring the effect of therapy on cardiac remodeling (i.e., left ventricular volume) and function (i.e., left ventricular ejection fraction (LVEF)) measured by echocardiography.

Ways.

Briefly, in male Yorkshire pigs, myocardial infarction was caused by provoking the occlusion of the LAD in balloon balloon angioplasty for 90 minutes. Pigs that showed an ejection fraction of <40%, determined by M-mode echocardiography within 30 days after a heart attack, were allowed to participate in the study. Pigs were randomly assigned to one of 3 groups for the introduction of phosphate buffer solution (FBI, control), a plasmid having the nucleotide sequence of SEQ ID NO: 6 or ACL-01010Sk in the FBI, through the delivery system of an endoventricular stylet catheter (Table 3 ).

Table 3

The initial design of the clinical study. SDF-1 chronic therapy. heart failure in pigs

Group Candidate Plasmids No pig The amount of injected drug / place DNA number / place No. of injection sites Total DNA one Carrier 3 200 μl N / 3 ten N / 3 2 ACL-01010Sk 3 200 μl 400 μl ten 4 mg 3 plasmid having the nucleotide sequence of SEQ ID N0: 6 3 200 μl 400 μl ten 4 mg

Echocardiograms were removed before administration and 30 and 60 days after administration. In tab. 4 below are the variables referenced in this report.

Table 4

Variable definition

Variable name Value KSOLV The end-systolic volume measured in the parasternal position of the long axis KDOLV End-diastolic volume, measured in the parasternal position of the long axis LVEF (KDOLV-KSOLV) / KDOLV * SO%

Results.

The main echocardiographic indicators of time from the initial injection (day 30 after MI) of all animals included in the study (n = 9) are presented in Table 1 of the leading echocardiographic laboratory. 5 below.

Table 5

Main characteristics

Parameter Basic group score 1 Baseline Grade Group 2 Baseline score group 3 KSOLV 78 ± 18 ml 67 ± 2 ml 86 ± 31 ml KDOLV 132 + 30 ml 114 + 11 ml 130 + 36 ml LVEF 41 + 1% 41 + 5% 34 + 10%

In tab. 5 shows the LVESV, LVEF and LVEDV values at 0 and 30 days after the initial injection of SDF-1. The control group of animals treated with the FBI showed an increase in LVESV and LVEDV and the absence of improvements in LVEF corresponding to this model of heart failure. The treated group did not show a decrease in cardiac volume or an improvement in LVEF compared with the control. Similar results were obtained on day 60 after the initial injection.

Example 7

The strategy of increasing the homing of stem cells in the near-infarction region with the help of transendocardial delivery of SDF-1 through a catheter in a porcine myocardial infarction model was examined to determine the possibility of increasing perfusion and left ventricular function. Introduction through a catheter has been used successfully for cell transplantation and the delivery of blood vessel growth factors in humans.

Were used female pigs breed German landrace (30 kg). After fasting overnight, the animals were anesthetized and intubated.

French guides were placed in the femoral artery of the animals in a supine position. The cylinders delivered by conductor were placed at the distal end of the LAD. Cylinders were

- 30 031308 were inflated to 2 atmospheres, and the agarose granules were slowly introduced over 1 minute using a balloon catheter at the distal end of the LAD. After 1 min, the balloon was deflated, and the occlusion at the distal end of the LAD was determined using angiography. After the induced heart attack, the animals were monitored for 3–4 hours until the heart rate and blood pressure stabilized. Arterial vehicles were removed, carprofen (4 mg / kg) was administered intramuscularly to the animals, and animals were disconnected from the respirator. Two weeks after the infarction, the animals were anesthetized. An electromechanical mapping of the left ventricle was carried out through section 8F of the femoral sheath in lying animals. After completion of the left ventricular mapping, human SDF-1 (Peprotec, Rocky-Hill, NJ) was injected via 18 injections (5 μg in 100 ml of saline) into the infarction region and near infarction region using a catheter for administration. 5 μg per injection were used to adjust previously determined efficacy when administered after SDF-1 injection. The insertion was carried out slowly over 20 seconds, and only when the tip of the catheter was installed perpendicular to the left ventricular wall and when the loop stability was less than 2 mm and when the outgoing part of the needle in the myocardium provoked ectopic additional ventricular contractions. The control group of animals underwent the same procedure with the introduction of the dummy. Echocardiography ruled out postoperative pericarditis.

animals completed the study protocol: 8 control animals and 12 animals treated with SDF-1. Only 6 animals could be assessed by visualizing myocardial perfusion due to technical problems. All animals had anterior septal infarction.

The infarct size in percent relative to the left ventricle, determined by tetrazol staining, was 8.9 ± 2.6% in the control group and 8.9 ± 1.2% in the group receiving SDF-1. The muscle volume of the left ventricle was similar in both groups (83 ± 14 ml relative to 95 ± 10 ml, p = ns). Immunofluorescent staining showed a significantly greater number of vessels positive for von Willebrand factor in the near-infarction region in animals treated with SDF-1 than in the control group of animals (349 ± 17 / mm ² relative to 276 ± 21 / mm ² , p <0.05) . A significant loss of collagen in the near-infarction region was observed in animals treated with SDF-1 compared with the control group of animals (32 ± 5% versus 61 ± 6%, p <0.005). The data of inflammatory cells (neutrophils and macrophages) in the near-infarction region were similar in both groups (332 ± 51 / mm ² relative to 303 ± 55 / mm ² , p = ns). Global myocardial perfusion did not change from baseline values to subsequent SPECT, and there was no difference between the groups. The final infarct size was the same in both groups and was comparable to the results of tetrazole staining. Segment analysis of myocardial perfusion showed a decrease in label uptake in the apical and antero-posterior regions with significant differences between the myocardial sections. However, the label uptake in the initial and subsequent measurements was almost identical in animals treated with SDF-1 and in the control group. There were no differences in the final diastolic and final systolic volumes between the groups. However, the systolic volume of the heart increased in the control group of animals and slightly decreased in animals treated with SDF-1. The difference between the two groups was significant.

Similarly, the ejection fraction increased in the control group and decreased in the group treated with SDF-1. The difference between the groups showed a significant trend (p = 0.05). Local shortening, another parameter of the mechanical functions of the ventricle, has not changed in the control group of animals. However, local shortening decreased significantly in the group of animals treated with SDF-1, which resulted in a significant difference between the groups. There was no significant difference in unipolar voltage within groups and between groups. Significant correlations between the baseline ejection fraction, the contraction volume, and the base local shortening (PV and MU: r = 0.71, OS and MU: r = 0.59) were also observed. Similar results were obtained for the following values (PV and MU: r = 0.49, OS and MU: r = 0.46). Changes in local shortening significantly correlated with changes in the ejection fraction (r = 0.52) and volume of reduction (r = 0.46). There was no correlation between local shortening and the final diastolic volume (base r = -0.03, subsequent r = 0.12), or between the ejection fraction and the final diastolic volume (base r = -0.04, subsequent r = 0, 05). A segmental analysis of the data obtained by the electronegativity equalization method showed a decrease in unipolar voltage and local shortening in the anteroluminal divisions with a significant difference between the segments of the myocardium initially. The distribution of unipolar voltage values in myocardial segments was similar in both groups initially and with subsequent observation. Local shortening of the segments did not change in the control group. However, it decreased in the SDF-1 group, mainly due to a decrease in the lateral and posterior segments of the left ventricle. There was a significant relationship between the assignment of SDF-1 and the subsequent relationship of the baseline and subsequent levels.

The study described above shows that a single application of the SDF-1 protein is not enough for the manifestation of significant improvements in cardiac function.

Example 8. Expression of the vector ACRX-100 depending on time on the model of the posterior ischemia

- 31 031308 impairment of the rat.

Purpose.

The purpose of this study is to establish the duration of expression of the ACRX-100 vector after direct intramuscular injection into the rat hind limb ischemia model.

Ways.

ACRX-100, batch No. 25637, was manufactured by Aldevron, LLC (Fargo, ND). Male Lewis rats were anesthetized and a longitudinal incision was made in the mid-thigh from the inguinal ligament to the knee joint, the open femoral artery was ligated and removed. Animals were left for recovery for 10 days, then anesthetized and directly injected 1.0, 2.0 or 4 mg / ml ACL-01110L (vector skeleton with luciferase cDNA) in an amount of 0.2 ml in 4 areas along the hind limb. The expression vector was measured by standard methods for determining luciferase expression on days 1, 2, 3, 8, 10, 14 using a cooled CCD camera, Xenogen Imaging Systems. The animals were anesthetized with 2% isofluran, and luciferin was administered intraperitoneally at a concentration of 125 mg / kg animal weight. After 10 minutes, real-time images were obtained during the 1-minute exposure to determine the total chemiluminescence of luciferase expression. Data was measured as a total stream (pixels / second).

Results.

Similar to previous studies, a plasmid based on the CMV ACL-01110L showed an expression peak at day 3 (Fig. 11). Minimal expression was measured on day 14.

Conclusions

ACRX-100 expression in rats with ischemia on the hind limbs showed a peak value on day 3 and expressed up to day 14 according to the expression patterns measured in rat cardiac tissue and previously published vector expression studies driven by the CMV promoter. These data suggest that for future studies evaluating the effectiveness of repeated doses of ACRX-100, it is wise to choose the interval between dosing 2 weeks. This dosing interval also correlates with dosing regimens presented in several clinical trials using CMV-based vectors that induce therapeutic gene expression (FGF, HGF, VEGF, HIF1) using pure plasmid DNA to treat ischemic diseases.

Example 9. In vivo characterization of the dosage of ACRX-100 in rabbit hind limbs.

Purpose.

The purpose of this study was to determine the effect of the injected volume, the concentration of pDNA and the technology for producing the dosage form on the expression of ACRX-100 pDNA 3 days after direct injection into the muscles of the rabbit's hind limb.

Ways.

Male New Zealand white rabbits weighing 3.0-4.0 kg were anesthetized and received an injection of plasmid ACL-01110L with luciferase. An incision was made in the medial part of the thigh from the inguinal ligament of the knee to open the femoral artery. Four to eight injections of 0.5-1.0 ml of plasmid ACL-01110L in 5% dextrose were introduced at a rate of 0.1 ml per second into adductor (2 injections), thin (1 injection) and semi-corrosive (1 injection) muscles on each rabbit hind paw. Injections were divided into 6 groups according to FIG. 13. Each injection site was marked with a nylon thread for further identification, and the wound was closed with a suture. Each limb was wrapped in a compression bandage for approximately 15 minutes. Three days after the injection, the animals were euthanized, and the muscles of the hind limb, including the injection sites, were removed, soaked in luciferin (15 mg / ml) for 7 minutes and bioluminescently visualized using an IVIS Xenogen instrument (Fig. 12). The total flow (pixels per second) was estimated after exposure for 1 min.

Results.

Macroscopic assessment of the injection sites showed no inflammatory processes, and plasmid DNA was well tolerated by all animals and in all doses. As shown in FIG. 13, expression was observed at all doses administered with an increase in expression as a function of pDNA concentration. Plateau expression was observed at a concentration of 2 mg / ml and a total dose of 4 mg of DNA (groups 4-6). More volume or number of injection sites does not increase expression, suggesting that pDNA concentration is an important factor in proper transfection of muscle cells.

Conclusions

This study demonstrated that the addition of luciferase to the ACRX-100 vector is capable of expressing the gene product in the muscles of the hind limbs of the rabbit in quantities sufficient for detection on day 3 after administration. Further studies have shown that the expression of pure plasmid DNA in the rabbit hind limbs is increased in direct proportion to the concentration of pDNA up to 2 mg / ml. This study suggests that 4–8 injection sites using volumes of 0.5–1.0 ml per injection with a pDNA concentration of 0.5–2.0 mg / ml should lead to the production of SDF-1 in the rabbit's hind limbs.

Example 10. The study of final efficacy, safety and biodistribution of ACRX- 32 031308

100.

The efficacy, safety and biodistribution of ACRX-100 were previously determined in a study of efficacy, toxicity and biodistribution in swine models in accordance with the principles of NLP after a direct catheter-mediated cardiac injection in swine heart failure models (briefly described in Example 3). The ACRX-100 demonstrated an acceptable safety profile in doses up to 100 mg after direct administration to ischemic pig hearts. This study is generally considered to support the planned clinical studies in the CLI, but does not coincide with the specific clinical criteria being studied.

The final preclinical evaluation of the effectiveness, safety and biodistribution of ACRX100, supporting phase 1 clinical trials in patients with CLI, is an estimate based on a rabbit model of hind limb ischemia. This model provides experimental conditions that model the proposed clinical trials of phase 1 in patients with CLI. Safety and efficacy studies evaluating the toxicology and biodistribution of increasing single doses of ACRX-100 were performed on a rabbit model of hind limb ischemia, and its results are shown below. Based on these results, applicants propose to evaluate the efficacy, toxicology and biodistribution of ACRX-100 on the rabbit models described in section 6.3 of the presented preliminary study on the use of a new drug.

Safety and efficacy of a single dose of ACRX-100 in rabbits with hind limb ischemia.

Purpose.

The purpose of this study is to evaluate the efficacy, safety and biodistribution after a single dose of the test product, ACRX-100, in a rabbit model of hind limb ischemia.

Ways.

New Zealand white rabbits (n = 5 / group; 2-3 males / females per group) underwent unilateral ligation of the femoral artery and 10 days after ligation received 4, 8 or 16 mg of the plasmid ACRX-100 or 4 mg of the control plasmid (luciferase) 8 direct intramuscular injections of 0.5 ml and a limb with ischemia (Table 6).

Table 6

Clinical study design of the safety and efficacy of a single dose of ACRX-100 in a rabbit model of hind limb ischemia

Group Number of animals Number of doses pdnc / dose Injection Number Place Volume / place Cone (mg / ml) pdnc / place Repeated dose Efficiency Safety assessment (day 60) one five one Control (4 mg person) eight 0.5 ml one 0.5 mg Not 0, 30, 60 biodistribution / histopathology 2 five one 4 mg eight 0.5 ml one 0.5 mg Not 0, 30, 60 histopathology 3 five one 8 mg eight 0.5 ml 2 1, 0 mg Not 0, 30, 60 histopathology four five one 16 mg eight 0.5 ml four 2.0 mg Not 0, 30, 60 bio-distribution / histopathology

Safety endpoints were evaluated at 60 days after injection and included histopathology and biodistribution from the hind limbs (injection sites) to the opposite hind limb, heart, lungs, liver, brain, spleen, lymph nodes, kidneys and ovaries. A macro- and microscopic examination of paraffin-fixed and hematoxylin and eosin stained sections of the corresponding tissues was carried out. Clinical pathology was assessed in all groups on day 60 after injection. Efficiency was measured by the percentage change in the angiographic assessment compared to control at 30 and 60 days after treatment. The calf muscles were excised and evaluated for weight differences 60 days after injection. Biodistribution was assessed in the lungs, liver, spleen, lymph nodes, kidneys, brain, testes and ovaries of animals of groups 1 and 4.

Results.

Angiographic analysis.

Angiograms were taken on day 0 (before administration), 30 (± 2) and 60 (± 2) days after administration and recorded in digital format (Fig. 14). A quantitative angiographic analysis of the development of collateral vessels in the ischemic limb was performed with an overlay of a grid consisting of squares with a diameter of 5 mm arranged in a row. The total number of intersections of the mesh in the middle region of the thighs, as well as the total number of intersections intersected by the contrast opaque artery, were calculated by one-sided blind method.

Angiographic assessment was calculated for each film as the ratio of the intersections of the grid,

- 33 031308 intersected by an opaque artery divided by the total number of grid intersections in the middle part of the thigh.

Efficiency.

The results obtained by the method of angiography showed that on the day of dosing all animals were similar. In the control group of animals, there was a tendency to a decrease in vascular density after a 60-day period after the dose. Animals treated with ACRX-100 showed improved blood flow at both time points, in groups receiving doses of 1 mg / ml (group 2) and 2 mg / ml (group 3), compared with the control group of animals receiving a vector in which worsened blood flow. It is important to note that improvement in the groups of low and medium doses was observed on day 30 with a significant (p <0.05) advantage of the group of average dose on day 60 (Fig. 15). Improvement was also observed in animals in group 4 (4 mg / ml) 60 days after injection. A similar tendency to an increase in vasculogenesis was observed in the porcine model of heart failure at a dose of 5 mg / ml.

Pathology.

Mortality.

All animals survived to the planned hibernation, with the exception of one animal from group 1 (505). The animal 505 died within 30 days after the angiogram. The animal was opened. The cause of death was considered to be related to anesthesia, and not the consequence of the administration of the investigational drug.

Observation of animals.

Clinical observations were limited to observing scab, and sparse coat in most animals. Such observations are standard for animals that have undergone a similar procedure. In addition, loss of appetite, decreased activity and a small amount or absence of feces was observed in one animal of group 2 (510), and self-damage to the hind limb was observed in two animals of group 4 (519 and 520). Observed self-harm in animals 519 and 520 most likely resulted from the loss of sensation of damage to the hind limbs of these animals.

During the study, the animals initially lost weight during the first 3-4 weeks after the injury. By the end of the study, the animals returned to their original body weight. Weight loss is considered the result of numerous surgical procedures.

Macroscopy.

A macroscopic examination did not reveal any research-related phenomena in animals of both sexes. Few macroscopic observations are considered random and not related to the study.

Microscopy.

Were examined several sections of skeletal muscles from the places of injections on the hind limb, from the intact areas of the hind limb, from the ischemic area of the hind limb and intact areas of the opposite limb. Tissue sections were examined using hematoxylin, eosin staining and Masson's trichrome staining. The tissue sections of the ischemic region contained areas of ischemia, which are alternately and inconsistently located. Ischemia sites were initially characterized by minimal to moderate fibrosis, and minimal to medium capillary formation, and to a lesser extent subacute / chronic inflammation, hemorrhage and muscle fiber degradation / necrosis and / or muscle fiber regeneration. Often, areas of fibrosis and neovascularization are followed by fascial muscle surfaces. Sometimes extraneous material (suture material) was observed, often surrounded by minimal granulomatous inflammatory response. Mineralization of muscle fibers has also been observed occasionally.

There were no microscopic test-related findings in the remaining tissues examined with a microscope (brain, heart, kidney, liver, lung, ovaries, and spleen). The few remaining microscopic observations were either common background findings in rabbits or random and thus were considered irrelevant to treatment.

Hematology and coagulation.

No biologically relevant differences were found in the hematological and coagulation parameters between any treated groups of either sex after 60 days after the injection.

Clinical chemistry.

Phosphorus was gradually reduced in the ACRX-100 2, 3, and 4 groups relative to the LUC control group of plasmid 1 in both male (from 6 to 36%) and female (from 7 to 30%) individuals. There were also average increases in calcium in the ACRX-100 3 and 4 groups relative to the LUC control group of plasmid 1 in both male (4–7%) and female (7–9%) individuals. All averages and individual values for calcium and phosphorus always remained within the expected historical control ranges. None of these changes were deemed unfavorable. For a better assessment of the reliability of these observations, applicants evaluate these values in a re-examination of the safety and efficacy of the dosage described in section 6.3. No other changes in chemical parameters were observed in any gender.

- 34 031308

Biodistribution

In this study, biodistribution in rabbits with a CIC was evaluated by quantitative PCR 60 days after injection in the group of animals 1 (1 mg / ml of luciferase plasmid) and 4 (4 mg / ml of ACRX-100). The results are shown in Table. 7 below. As expected, the ACRX-100 vector was not detected in the tissues of any of the control groups. In the high dose group, ACRX-100 was found almost exclusively at the injection site, and only trace amounts of ACRX-100 were found in non-injected organs. The rate of elimination of the plasmid's ACRX-100 DNA, which was observed in the rabbit's hind limb 60 days after injection (Table 7), is consistent with the elimination of ACRX-100 from cardiac injections observed in the study of safety measures in pig heart failure (Table 3, Fig. 16). Additionally, resistance levels were lower in the CIC study in rabbits, since the largest number of copies found in this study 60 days after injection (133360 copies / μg of the host organism DNA) was lower than the maximum number of copies found during a cardiovascular study days (> 1x10 ⁶ copies / µg of host DNA). These data suggest that the ACRX-100 was derived from both injected and non-injected rabbit organs in a manner similar to that shown in a study of heart failure in pigs, in fact, the resistance of the injected area in the current study of rabbits is less than that observed in the model of heart failure in pigs.

Table 7 The biodistribution of ACRX-100 in the IZK tissues of the rabbit 60 days after injection

LLOD - below detection limit.

Conclusions

All groups of animals injected with ACRX-100 showed an increase in blood flow in the ischemic hind limb 60 days after injection. Animals injected with a single intramuscular dose of 1 mg / ml (low dose) or 2 mg / ml (medium dose) of ACRX-100 showed improved blood flow at 30 and 60 days after injection compared to the control animals that received vector injection, which showed reduced blood flow . It is important that the benefits observed after 30 days are largely (p <0.05) preserved even after 60 days. These data are consistent with the results of a study of heart failure in pigs, where injections of ACRX-100 (0.5-5.0 mg / ml) led to an increase in vasculogenesis. The data obtained suggest that, regardless of the total amount of ACRX-100 administered to the target tissue, the concentration of the product administered by injection significantly affects the vasculogenesis response. These results indicate that CIC patients can benefit from a single dose of ACRX-100.

The findings suggest that ACRX-100 is derived from both injected and non-injected rabbit organs in a manner similar to that demonstrated in a study of heart failure in pigs. Since the elimination of ACRX-100 was consistent with cardiovascular

- 35 031308 research, the applicants suggest that other observations regarding biodistribution in the study of heart failure in pigs are also valid for the CIC study. In a literal sense, any expression of ACRX-100 at the end of the study is minimal, subtherapeutic, and the potential for integration is less than the level of spontaneous mutation.

Example 11. The proposed study of the safety of repeated doses and the effectiveness of ACRX-100 in a model for rabbit with hind limb ischemia.

The proposed new study will evaluate the safety and efficacy of repeated doses of ACRX-100 in the same rabbit model with hind limb ischemia, which is described in Example 10. As described below, 3 intramuscular doses of ACRX-100 will be administered to support up to 3 treatments for patients with CIC.

The safety and effectiveness of ACRX-100 re-dosing in rabbits with ICL.

Purpose.

The purpose of this study is to determine the safety and efficacy of repeated doses of the ACRX-100 in a rabbit model with hind limb ischemia.

Justification.

All types of non-viral therapy that are currently being clinically examined for critical limb ischemia, are administered non-viral drugs in repeated doses (HGF, PDGF), including NV1FGF, which has been shown to be effective in a phase II study. Currently, the preclinical safety and efficacy studies conducted by the applicants have shown that ACRX-100 is safe up to 100 mg after a single injection into the heart (20 injection points) or 16 mg when administered to the hind limb (8 injection points). Thus, applicants propose to administer ACRX-100 in repeated doses at 0, 2, and 4 weeks as part of our phase I trial in patients with CIC. Repeated administration of ACRX-100 may provide additional benefits over a single dose. CXCR4, the SDF-1 receptor, is indefinitely regulated to increase after injury; while SDF-1 is regulated with an increase in transient after an acute ischemic event (Fig. 17) or in response to an injection procedure. Delivery of SDF-1 to numerous points at a later time immediately after injury is capitalized on increased localized expression of CXCR4 expression in damaged tissue and increases the homing of stem cells at the site of SDF-1 expression. With CIC, repeated injections have the potential for a synergistic increase in vasculogenesis, collateral vascular growth and healing of a wound in the ischemic limb. The safety profile, which was observed after injection of 100 ml into the pig heart, suggests that adjusting the dosage of lower volumes will give similar safety results.

To assess preclinical safety and the effectiveness of re-dosing the ACRX-100 in the same rabbit hind-limb ischemia model that was used in the study above, the applicants suggest a protocol described in detail below. The re-dosing schedule will be identical to the re-dosing categories that were proposed during the phase I / II trial, in all aspects, except for the injection volume (described in Example 12). On a rabbit, 0.5 ml will be used instead of 1 ml assigned to a person, due to the larger skeletal muscle area in the human thigh / calf compared to the rabbit's hind limb.

Ways.

The New Zealand white rabbits U = 5 / group of males and females) will undergo the procedure of unilateral ligation of the femoral artery (day -10), identical to that performed in example 10 above. Ten days after banding (day 0), each animal will receive 3 doses (15 days apart) of 1 mg / ml of the control luciferase plasmid (groups 1 and 2) or ACRX-100 (groups 3 and 4), introduced by 16 direct intramuscular injections into the ischemic limb, 0.5 ml each (Table 8). Safety endpoints will be evaluated on day 3 (day 33) and day 30 (day 60) of post-final injection, including histopathology (gross and normal) and bio-distribution from the hind limb (injection site) opposing the hind limb, heart, lung, liver, brain, spleen, lymph nodes, kidneys and ovaries. A coarse and microscopic study of fixed hematoxylin and a section of eosin-colored paraffin will be performed on tissue sections. Clinical chemistry and pathology will be assessed for all groups before hind limb ischemia (day -10), before each dose (day 0, 15, 30), 3 days after a dose (day 3, 18, 33), 7 days after a post-final dose (day 37) and 30 days after the post-final dose (day 60) or before killing. Cell monitoring will be carried out twice a day, and detailed clinical examinations will be conducted weekly. Efficacy will be measured by the percentage of changes in the angiographic deviation from the main line compared to control by 30 (all categories) and 60 (categories 2 and 4) days of post-final injection.

Samples for studying biodistribution will be taken from the lung, liver, spleen, lymph node, kidney, brain, testicles and ovaries and evaluated using quantitative PCR at two killing points: 3 days post-final dosage (day 33) and 30 days post-final dosage (day 60 ).

The 60-day duration after the first injection of ACRX-100 in the study was chosen to reconcile the time points of efficacy with the one-time treatment study described in Example 10. Due to the fact that the time point is 30 days post-final dosage, expect

- 36 031308 that the biodistribution results will have copies of ACRX-100 present at the injection sites (on the order of 10 ³ -10 ⁵ copies / μg of host DNA) and limited amounts (less than 10 ³ copies / μg of host DNA) based on the results of a study of the safety and efficacy of heart failure in pigs described in Example 8. The results of this proposed study will be presented in a new drug study (IND). Based on the fact that the safety results in this study are similar to those obtained in the study of safety and efficacy in heart failure in pigs, the above study should justify repeated dosing of ACRX-100 in patients with CIC.

Table 8

Suggested dosage regimen

Category Number of animals Number of doses pdnc / dose Number of places / dose Volume / place Concentration pdnc / place Time of processing Killing point Efficiency 1 Control 4 (2 males, 2 females) Day 33 Angiography on day 0, day 30 2 Control 4 (2 males, 2 females) 3 8 mg sixteen 0.5 ml 1 mg / ml 0.5 mg Day 0, 15, 30 Day 60 Angiography on day 0, day 30, day 60 3 ACRX-100 5 (2 males, 3 females) Day 33 Angiography on day 0, day 30 4 ACRX-100 5 (3 males, 2 females) Day 60 Angiography on day 0, day 30, day 60

Safety assessment in ALL categories.

Before death: clinical observations, pathology, chemistry.

After death: histopathology and biodistribution.

Example 12. The proposed clinical study.

The applicants have performed studies indicating that ARCX-100 is both safe and effective in preclinical models of heart failure and CIC. The results of these completed and proposed studies support the clinical trial phase I / II proposed by the applicants, assessing the safety and efficacy of treating patients with CIC with ARCX100.

The proposed combined study in 66 patients, phase II, will examine the safety and initial efficacy of using ACRX-100 for treating patients with CIC grade 5 according to Rutherford. Safety will be observed in each group by documenting all adverse events (AE) and measuring standard laboratory values for blood (for example, total blood count, plasma SDF-1 levels), with an initial safety end point of most AE set at 30 days (group 1 and 2) or 60 days (groups 3, 4 and 5) after listing.

During Phase I of the study (groups 1-4), patients will receive 8 or 16 injections in the upper and lower legs of either the single dose (groups 1 and 2) or repeated dose sessions for 4 weeks (groups 3 and 4) of the ACRX-100 ( Table 9). Increasing the dose includes doubling the number of injections from 8 injections (group 1) to 16 injections (group 2), moving from a single dose (group 2) to 3 repeated dosage sessions (group 3) and doubling the number of injections per dose from 8 (group 3) up to 16 (group 4). Assuming that repeated doses will be more effective than single doses, groups 3 and 4 will be randomly mixed 2: 1 to receive either medication or placebo (5% dextrose injection) to test preliminary efficacy; Groups 1 and 2 will receive only the active drug. In all groups, the effectiveness will be assessed within 12 months after the first dose by evaluating the following endpoints: 1) significant amputations, 2) the case of complete wound closure, 3) survival, the difference from the main line, 4) the degree of change in the ulcer healing index, 5 ) transcutaneous oxygen (TsRO2) and 6) pain at rest.

In Phase II trials (group 5), 30 patients will be randomly mixed 2: 1 to obtain either ACRX-100 (more effective than groups 3 or 4), or vehicle (5% dextrose solution) (Table 9 ). Safety and efficacy will be measured at the same end points as in phase I, the initial end point of effectiveness is survival without major amputation. All dosage escalations in phase I and the start of phase II research will only occur in accordance with the DSMC review, as described below.

- 37 031308

If the results of phase I / II indicate that ACRX-100 is effective in improving CIC with the recommendation of DSMC patients initially mixed with control groups, treatment with ACRX-100 may be offered for free to the patient in the following condition:

1. The patient follows the schedule after the test.

2. The local head of research determines that the patient can still benefit from treatment.

If treatment is carried out, the patient will have to follow a schedule consisting of (at least) the described safety endpoints.

Table 9

The proposed mode of clinical dosing phase I / II

Group Number of patients Number of doses pdnc / dose Number of injection sites Volume / place pDNA / concentration pdnc / place Time of processing Open or random one 3 one 8 mg eight 1 ml 1 mg / ml 1 mg Day 0 Open 2 3 one 16 mg sixteen Day 0 Open 3 15 3 8 mg eight Day 0, 14, 28 Random 2: 1 (10 processed, 5 control) four 15 3 16 mg sixteen Day 0, 14, 28 Random 2: 1 (10 processed, 5 control) five thirty The best dose of groups 3 and 4 Random 2: 1 (20 processed, 10 control)

All enrolled patients receive normal, standardized care for CICs, including pharmacological (painkillers) and wound care (cleaning, infection control with antibiotics, etc.) as needed. However, according to the inclusion / exclusion criteria, the patient will not receive surgical treatment or treatment with an internal intervention technique, as listed patients should be weak candidates for revascularization and should not receive revascularization procedures for 6 weeks before listing. One of the reasons that they are weak candidates for revascularization is that ischemia is the result of blockage in numerous arteries that cannot be unblocked or bypassed. This makes pro-angiogenic therapy, such as ACRX-100, attractive because it has the potential to create new blood vessels originating from the vessels of the ischemic leg, which can restore blood flow in a large area and thus improve symptoms, limb function and overall results. .

In the absence of therapy, these patients have a poor prognosis. For patients with CIC who are weak candidates for revascularization (also called patients with non-recoverable disease), there is a 40% probability of amputation over 6 months. Moreover, they have a mortality rate of 25% over the course of a year, including a 3-5-fold increase in the risk of cardiovascular death compared with those who are not sick with a CFC.

The synopsis of the proposed phase I / II study is presented in table. 11. 66 patients with non-healing ulcers (class 5 according to Rutherford) will be sequentially listed in no more than 15 clinical centers. Each patient will receive direct intramuscular injections of ACRX-100 and will be observed for 12 months after the initial injection. The ACRX-100 will be inserted using a 27 gauge needle with injections located on the thigh above the knee and lower leg. Safety and efficiency endpoints will be collected as shown in Table. 11. In the open part (groups 1 and 2), descriptive statistics will be used to compare variations of the long-lasting effect by groups receiving the drug. Safety parameters will be collected and evaluated qualitatively or summarized quantitatively using descriptive statistics, where appropriate. In the mixed part of the study (groups 3-5), either the ACRX-100 or the filling control will be introduced using the same techniques as in phase I, at 8 or 16 injection sites at 0, 2, and 4 weeks after listing. A statistical comparison of each treatment group with a control group will be performed for all efficacy variables with static importance, defined as p-values less than 0.05.

Logistics research is as follows. Before listing, all patients must provide written confirmation of familiarization and consent for participation in the study. Patients will be monitored for 2 weeks before the first scheduled injection of ACRX-100 with

- 38 031308 testing to determine the acceptability of the study. including the ABI and Rutherford class (Table 11). Further safety checks (CMP. PT / PTT) and the establishment of the baseline values for the end points of effectiveness (TCP2. Quality of life. Ulcer size) will be performed during the observation period. The subject will be considered listed. when he or she comes to the clinic to prepare for the injection procedure. In the open part (groups 1 and 2), patients will be sequentially listed. and everyone will get ACRX-100 treatment. In a randomly mixed part (groups 3-5), each patient will be randomly selected. and the clinical center will be given a random selection before the injection procedure. All patients will follow the schedule 1. 2. 4. 5 weeks. 3. 6 and 12 months after the first injection to assess safety and efficacy (Table 11). For groups 1-4, each of the first 3 patients from the list will be separated from the rest by at least 7 days. After the final dose of the last patient in groups 1-4 all safety data. collected over 7 days. following administration of a dose of ACRX-100 to each subject. will be reviewed by an independent DSMC. DSMC will be responsible for reviewing security data. deciding on adverse events and reviewing any data about the subject. that comply with the rules of termination. The committee will consist of a medical observer (without the right to vote) and at least 3 other members. DSMC should recommend an increase in the next dose in phase I of the study and the start of phase II of the study.

Before submitting to CLI IND, the national research head and the medical observer will develop a list of discontinuation rules. which if any of them is executed. will require a temporary suspension of the list of treatment in accordance with the data viewed by the DSMC.

The validity of the proposed clinical dosing.

The proposed clinical doses are based on the results of a non-clinical study of the safety and efficacy of the dosage (see Example 11). As shown in the table. 10 below. The proposed starting dose of a human is 1 mg / ml of DNA per injection site per 1 ml (8 mg in total). This starting dose was based on the results of a single dose safety and efficacy study for a rabbit with a CFC. The starting dose of a person has the same concentration and number of injections. as an effective dose in the study of a single dose for a rabbit (1 mg / ml in 0.5 ml at 8 injection sites. 4 mg in total). Further. The starting human dose is half the maximum total DNA dose. tested in a single dose rabbit study (4 mg / ml in 0.5 ml at 8 injection sites. 16 mg in total). A higher volume / location in phase I study. 1.0 ml is a doubling of a volume of 0.5 ml from a non-clinical study. since the human lower limb is significantly larger in muscular weight. than the rabbit's hind limb.

Table 10

Doses of ACRX-100 for humans and animals

Parameter Starting dose of a person Maximum human dose Effective single dose in rabbit hind limb ischemia The highest single dose tested on a rabbit model The highest multiple dose tested on a rabbit model NOAEL in the Pig Heart Failure Model (IND # 14203) Concentration (mg / ml) one one one four one five Volume of dose (ml) one one 0.5 0.5 0.5 one Number of injection sites eight sixteen eight eight sixteen 20 Full dose of DNA (mg) eight sixteen four sixteen eight 100

The proposed dose of CIC phase I is also supported by data from a preclinical safety and efficacy study in heart failure in the pig. described in example 3 above. The proposed starting dose of phase I CIC 8 mg total DNA provides a more than 10-fold level of safety compared with NOAEL 100 mg. found in the study of safety in pig heart failure. The maximum amount of ACRX-100. the proposed CIC phase I study (16 mgx3 doses) is smaller. than half the volume of total DNA (100 mg). defined as NOAEL for a single dose in a study of the efficacy and safety of heart failure in a pig.

Last thing. volume per injection. which should be used in the test phase I CIC (1 ml). consistent with volume. used in the study of heart failure in pigs. and several published CIC clinical studies.

Upon successful completion of the phase I / II study, either a subsequent phase II study or a phase III phase study will be developed to demonstrate the safety and efficacy of ACRX-100 in single or multiple doses for the target population of class 5 according to Rutherford with a CIC.

Table 11

Synopsis of the I / II study phase

Protocol name: A phase I / II study to evaluate the safety and preliminary efficacy of the ACRX-100, administered by intramuscular injection to adult categories with critical limb ischemia. Research Phase: I / ii Objectives of the study: Primary: Examine the safety and tolerability of single and repeated doses of ACRX-100 administered by direct intramuscular injection to subjects with CIC. Background: Examine the preliminary efficacy of single and repeated doses of ACRX-100, administered by direct intramuscular injection to subjects with CIC. Sample size: 66 (n = 36 phase 1, n = 30 phase 2) Study population: The main inclusion criteria are: - Men and women aged 4–5 years or older - Non-healing ulcers (category 5 according to Rutherford) with no infection in the wound - ABI 0.4 or less - Systolic pressure in the ankle 9.3 kPa (70 mmHg). Art.) or less, or systolic pressure in the toe of 6.7 kPa (50 mmHg) or less - Weak predictions for surgery, angioplasty or bypass surgery - Subjects with diabetes who take optimal diabetic drugs with HbA1c < 8% Key exclusion criteria: - Life expectancy of less than a year - Pre Procession major amputation of the leg, in need of treatment or planned major amputation during the first month following the introduction of the list - Symptoms of osteomyelitis - Revascularization with angiographic signs of improved blood flow in the leg, which is in need of treatment, for 6 weeks before making the list

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- NYHA class IV heart failure - Uncontrolled blood pressure, defined as SBP> 24 kPa (> 180 mmHg) or DBP> 15 kPa (> 110 mmHg), despite adequate antihypertensive treatment during follow-up or listing - Burger's disease is known - Significant hepatitis disease (defined as more than 3-fold increase in ALT / AST), carriers of hepatitis B or C - Active proliferative retinopathy - Immunodeficiency states (for example, HIV-positive or patient is a recipient transplanted organ) or a subject receiving immunosuppressive drugs - History of a malignant neoplasm (with the exception of treatable skin malignant neoplasms not related to melanoma) - Pregnant or breastfeeding women, or patients of childbearing age who are not protected by an effective birth control method Investigator’s point of view may affect any aspect of the test - Cardiac angioplasty with or without a shunt or CABG for 3 months before listing - Zn Severe negative cardiovascular event for 3 months before listing - Previous treatment with angiogenic growth factor or with stem cell therapy for 1 year Study design The study will be conducted in five consecutive groups with dosing and random mixing characteristics, as described in Error! Reference source not found In groups 1 and 2 (n = 3 in each) each suitable subject will be sequentially written into an open category. Within each category, all patient records will be separated by at least 7 days. After the final dose of the last patient in each category, all available safety data collected during the 7 days following the administration of the dose of ACRX-100 to each subject will be reviewed by an independent data safety monitoring committee (DSMC). DSMC should recommend increasing the next dose. In groups 3 and 4 (n = 15 each), patients will be randomly mixed 2: 1 to obtain either ACRX-100 or control vehicle. In each group, the records of the first three patients will be separated by at least 7 days. After the final dose of the last patient in each category, all safety data collected during the 7 days following the administration of the dose of ACRX-100 to each subject will be reviewed by an independent data safety monitoring committee (DSMC). The DSMC should recommend increasing the next dose (following group 3) or starting phase II of the study (following group 4).

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Studies in the group: 1 for receiving repeated doses of either ACRX for dextrose supplement;

listing.

Methods

Methods

Each patient will research on safety and efficacy.

ACRX-100 or Placebo Equivalent

Product research

ACRX-100 filler using needle 2 / gauge dosage and syringe procedure;

intravenous lower leg

Parameters major safety and adverse days after injection

Secondary endpoints:

full list of familiarization table

Groups 1 and

Descriptive parametric statistics standard deviation nonparametric; the average statistics of the interv values of the artillery range will be used for a long comparison of variables

The parameters will be evaluated summarized in descriptive quantitative using the estimated each temporal raw variable the main difference of each patient.

Groups statistics, standard deviation, nonparametric statistics, the values of the quartile range will be applied to the long-term control of each effectiveness of each time variable, the main difference between each patient will be.

be considered

Parameters evaluated summarized descriptive quantitatively using

Duration of observation throughout the study days) after the initial dose of ACRX-100

Centers of research conducted by more than 15 clinical centers of primary efficacy studies:

survival efficiency will be a statistic parameter where appropriate.

randomly mixed patients will be dosed in groups.

efficacy between dosage groups.

efficiency will be a parameter

Every patient where appropriate. The data of each statistic

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Table 12 Subsequent schedules for phase I / II CIC clinical trial

Evaluation Observation Injection procedure Subsequent visits Visits out of schedule X Time point of the study Written confirmation Inclusion / exclusion Medical history HIV / HC complementary drugs Epatitis B / C (taken 3 ml of blood) Pregnancy test (taken 3 ml of blood) Total metabolic panel (CMR) (taken 3 ml of blood From day 14 to day -1 x x x x x x x Day -1 (within 24 hours from the injection procedure) X X X X Day 0 (up to 4 hours after injection) X Day 7 (7 ± 1 days) X Day 14 (14 ± 1 days) X Day 28 (28 ± 1 days) X Day 35 (35 ± 2 days) X 3 months (90 ± 7 days) X 6 months (180 ± 14 days) X 12 months (360 ± 14 days) X Tests for clotting of PT / PTT and INR (3 ml of blood taken) X X Analysis of urine X X X (post procedure) X X X X X X X X Security Physical examination X X X X X X X X X X Vital statistics X X 2 measurements *: before injection, before administration X X X X X X X X Adverse events X X X X X X X X X 12 leading ECGs SDF-1 plasma levels (4 ml of blood taken) Full blood count (3 ml of blood taken) Additional blood sampling for potential anti-ACRX-100 assessment (3 ml) Injection procedure Efficacy Dose of painkillers Quality of life X x x x x X x Before administration 4 measurements *: before injection, 15 minutes, 3 hours, before administration Before administration Before administration X X x x x X x x x x x x X x x x x x x X x x x x x X x x x x x X x x x x X x x x x X x Visual analogue of pain intensity X X X X X X X Ulcer healing X X X X X X X

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Stirring Index (ABI) X X X X X X X Finger Brachial Index (TBI) X X X X X X X TsRO ₂ X X X X X X X Rescue limbs X X X X X X X X X Survival X X X X X X X X X The total amount of blood taken (ml) 15 (18 if female) 9 22 ten ten ten ten ten 7 7 3

From the above description of the application, specialists in this field will perceive information about improvements, changes and modifications. Such improvements, changes and modifications within the knowledge in this area should be covered by the attached claims. All patents, patent applications and publications cited in this document are hereby incorporated by reference in their entirety.

Selected sequences are disclosed:

SEQ ID NO: 1

KPVSLLYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLE

KALNK

SEQ ID NO: 2

MNAKWWLVLVLTALCLSGGPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARL

KNNNRQVCIDPKLKWIQEYLEKALNK

SEQ ID NO: 3

MDAKWAVLALVLAALCISDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARL

KSNNRQVCIDPKLKWIQEYLDKALNK

SEQ ID NO: 4 gccgcactttcactctccgtcagccgcattgcccgctcggcgtccggcccccgacccgcgctc gtccgcccgcccgcccgcccgcccgcgccatgaacgccaaggtcgtggtcgtgctggtcctcg tgctgaccgcgctctgcctcagcgacgggaagcccgtcagcctgagctacagatgcccatgcc gattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaa actgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccga agetaaagtggattcaggagtacctggagaaagctttaaacaagtaagcacaacagccaaaaa ggactttccgctagacccactcgaggaaaactaaaaccttgtgagagatgaaagggcaaagac gtgggggagggggccttaaccatgaggaccaggtgtgtgtgtggggtgggcacattgatctgg gatcgggcctgaggtttgccagcatttagaccctgcatttatagcatacggtatgatattgca gcttatattcatccatgccctgtacctgtgcacgttggaacttttattactggggtttttcta agaaagaaattgtattatcaacagcattttcaagcagttagttccttcatgatcatcacaatc atcatcattctcattctcattttttaaatcaacgagtacttcaagatctgaatttggcttgtt tggagcatctcctctgctcccctggggagtctgggcacagtcaggtggtggcttaacagggag ctggaaaaagtgtcctttcttcagacactgaggctcccgcagcagcgcccctcccaagaggaa ggcctctgtggcactsadataccgactggggctgggcgccgccactgccttcacctcctettt caacctcagtgattggctctgtgg gctccatgtagaagccactattactgggactgtgctcag agacccctctcccagctattcctactctctccccgactccgagagcatgcttaatcttgcttc tgcttctcatttctgtagcctgatcagcgccgcaccagccgggaagagggtgattgctggggc tcgtgccctgcatccctctcctcccagggcctgccccacagctcgggccctctgtgagatccg tctttggcctcctccagaatggagctggccctctcctggggatgtgtaatggtccccctgctt acccgcaaaagacaagtctttacagaatcaaatgcaattttaaatctgagagctcgctttgag tgactgggttttgtgattgcctctgaagcctatgtatgccatggaggcactaacaaactctga ggtttccgaaatcagaagcgaaaaaatcagtgaataaaccatcatcttgccactaccccctcc tgaagccacagcagggtttcaggttccaatcagaactgttggcaaggtgacatttccatgcat aaatgcgatccacagaaggtcctggtggtatttgtaactttttgcaaggcatttttttatata tatttttgtgcacatttttttttacgtttctttagaaaacaaatgtatttcaaaatatattta tagtcgaacaattcatatatttgaagtggagccatatgaatgtcagtagtttatacttctcta ttatctcaaactactggcaatttgtaaagaaatatatatgatatataaatgtgattgcagctt ttcaatgttagccacagtgtattttttcacttgtactaaaattgtatcaaatgtgacattata tgcactagcaataaaatgctaattgtttcatggtataaacgtcctactgtatgtgggaattta tttacctgaaataaaattcattagttgttagtgatggagcttaaaaaaaa

SEQ ID NO: 5 ccatggacgccaaggtcgtcgctgtgctggccctggtgctggccgcgctctgcatcagtgacg gtaagccagtcagcctgagctacagatgcccctgccgattctttgagagccatgtcgccagag ccaacgtcaaacatctgaaaatcctcaacactccaaactgtgcccttsadattgttgcaaggc tgaaaagcaacaacagacaagtgtgcattgacccgaaattaaagtggatccaagagtacctgg acaaagccttaaacaagtaagcacaacagcccaaaggactt

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SEQUENCE LIST <110> THE CLEVELAND CLINIC FOUNDATION

JUVENTAS THERAPEUTICS, INC.

<120> DELIVERY SDF-1 FOR THE TREATMENT OF CARDED TISSUE <130> 101521.000091 <140>

<141>

<150> 61/794742 <151> 2013-03-15 <160> 6 <170> PatentIn version 3.5 <210> 1 <211> 68 <212> Protein <213> Artificial sequence <220>

<223> Description of artificial sequence: synthetic polypeptide <400> 1

Lys 1 Pro Val Ser Leu 5 Leu Tyr Arg Cys Pro 10 Cys Arg Phe Phe Glu 15 Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys Ile Leu Asn Thr Pro 20 25 thirty Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys Asn Asn Asn Arg Gln 35 40 45 Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln Glu Tyr Leu Glu Lys

55 60

Ala Leu Asn Lys <210> 2 <211> 89 <212> Protein <213> Homo sapiens <400> 2

Met Asn ala lys val val val val leu val leu one five ten 15

Cys Leu Ser Asp Gly Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys 20 25 thirty

- 45 031308

Arg Phe Phe 35 Glu Ser His Val Ala 40 Arg Ala Asn Val Lys 45 His Leu Lys Ile Leu Asn Thr Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys 50 55 60 Asn Asn Asn Arg Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln 65 70 75 80 Glu Tyr Leu Glu Lys Ala Leu Asn Lys

<210> 3 <211> 89 <212> Protein <213> Rattus norvegicus

<400> 3 Lys Val 5 Val Ala val Leu Ala 10 Leu Val Leu ala Ala 15 Leu Met 1 Asp Ala Cys Ile Ser Asp Gly Lys Pro Val Ser Leu Ser Tyr Arg Cys Pro Cys 20 25 thirty Arg Phe Phe Glu Ser His Val Ala Arg Ala Asn Val Lys His Leu Lys 35 40 45 Ile Leu Asn Thr Pro Asn Cys Ala Leu Gln Ile Val Ala Arg Leu Lys 50 55 60 Ser Asn Asn Arg Gln Val Cys Ile Asp Pro Lys Leu Lys Trp Ile Gln 65 70 75 80

Glu Tyr Leu Asp Lys Ala Leu Asn Lys <210> 4 <211> 1940 <212> DNA <213> Homo sapiens <400> 4

gccgcacttt cactctccgt cagccgcatt gcccgctcgg cgtccggccc ccgacccgcg 60 ctcgtccgcc cgcccgcccg cccgcccgcg ccatgaacgc caaggtcgtg gtcgtgctgg 120 tcctcgtgct gaccgcgctc tgcctcagcg acgggaagcc cgtcagcctg agctacagat 180 gcccatgccg attcttcgaa agccatgttg ccagagccaa cgtcaagcat ctcaaaattc 240 tcaacactcc aaactgtgcc cttcagattg tagcccggct gaagaacaac aacagacaag 300 tgtgcattga cccgaagcta aagtggattc aggagtacct ggagaaagct ttaaacaagt 360

- 46 031308

aagcacaaca gccaaaagg actttccgct agacccactc gaggaaaact aaaaccttgt 420 gagagatgaa agggcaaaga cgtgggggag ggggccttaa ccatgaggac caggtgtgtg 480 tgtggggtgg gcacattgat ctgggatcgg gcctgaggtt tgccagcatt tagaccctgc 540 atttatagca tacggtatga tattgcagct tatattcatc catgccctgt acctgtgcac 600 gttggaactt ttattactgg ggtttttcta agaaagaaat tgtattatca acagcatttt 660 caagcagtta gttccttcat gatcatcaca atcatcatca ttctcattct cattttttaa 720 atcaacgagt acttcaagat ctgaatttgg cttgtttgga gcatctcctc tgctcccctg 780 gggagtctgg gcacagtcag gtggtggctt aacagggagc tggaaaaagt gtcctttctt 840 cagacactga ggctcccgca gcagcgcccc tcccaagagg aaggcctctg tggcactcag 900 ataccgactg gggctgggcg ccgccactgc cttcacctcc tctttcaacc tcagtgattg 960 gctctgtggg ctccatgtag aagccactat tactgggact gtgctcagag acccctctcc 1020 cagctattcc tactctctcc ccgactccga gagcatgctt aatcttgctt ctgcttctca 1080 tttctgtagc ctgatcagcg ccgcaccagc cgggaagagg gtgattgctg gggctcgtgc 1140 cctgcatccc tctcctccca gggcctgccc cacagctcgg gccctctgtg agatccgtct 1200 ttggcctcct ccagaatgga gctggccctc tcctggggat gtgtaatggt ccccctgctt 1260 acccgcaaaa gacaagtctt tacagaatca aatgcaattt taaatctgag agctcgcttt 1320 gagtgactgg gttttgtgat tgcctctgaa gcctatgtat gccatggagg cactaacaaa 1380 ctctgaggtt tccgaaatca gaagcgaaaa aatcagtgaa taaaccatca tcttgccact 1440 accccctcct gaagccacag cagggtttca ggttccaatc agaactgttg gcaaggtgac 1500 atttccatgc ataaatgcga tccacagaag gtcctggtgg tatttgtaac tttttgcaag 1560 gcattttttt atatatattt ttgtgcacat ttttttttac gtttctttag aaaacaaatg 1620 tatttcaaaa tatatttata gtcgaacaat tcatatattt gaagtggagc catatgaatg 1680 tcagtagttt atacttctct attatctcaa actactggca atttgtaaag aaatatatat 1740 gatatataaa tgtgattgca gcttttcaat gttagccaca gtgtattttt tcacttgtac 1800 taaaattgta tcaaatgtga cattatatgc actagcaata aaatgctaat tgtttcatgg 1860 tataaacgtc ctactgtatg tgggaattta tttacctgaa ataaaattca ttagttgtta 1920 gtgatggagc ttaaaaaaaa 1940

<210> 5 <211> 293 <212> DNA <213> Rattus norvegicus <400> 5 ccatggacgc caaggtcgtc gctgtgctgg ccctggtgct ggccgcgctc tgcatcagtg 60

- 47 031308

acggtaagcc agtcagcctg agctacagat gcccctgccg attctttgag agccatgtcg 120 ccagagccaa cgtcaaacat ctgaaaatcc tcaacactcc aaactgtgcc cttcagattg 180 ttgcaaggct gaaaagcaac aacagacaag tgtgcattga cccgaaatta aagtggatcc 240 aagagtacct ggacaaagcc ttaaacaagt aagcacaaca gcccaaagga ctt 293

<210> 6 <211> 3948 <212> DNA <213> Artificial sequence <220>

<223> Description of artificial sequence: synthetic polynucleotide

<400> 6 agatctccta gggagtccgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 60 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg 120 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 180 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 240 tggcattatg cccagtacat gaccttatgg gactttccta cttggctata catctacgta 300 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 360 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 420 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 480 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 540 cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga 600 tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac 660 gtaagtaccg cctatagagt ctataggccc acccccttgg cttcttatgc atgctatact 720 gtttttggct tggggtctat acacccccgc ttcctcatgt tataggtgat ggtatagctt 780 agcctatagg tgtgggttat tgaccattat tgaccactcc cctattggtg acgatacttt 840 ccattactaa tccataacat ggctctttgc cacaactctc tttattggct atatgccaat 900 acactgtcct tcagagactg acacggactc tgtattttta caggatgggg tctcatttat 960 tatttacaaa ttcacatata caacaccacc gtccccagtg cccgcagttt ttattaaaca 1020 taacgtggga tctccacgcg aatctcgggt acgtgttccg gacatgggct cttctccggt 1080 agcggcggag cttctacatc cgagccctgc tcccatgcct ccagcgactc atggtcgctc 1140 ggcagctcct tgctcctaac agtggaggcc agacttaggc acagcacgat gcccaccacc 1200 accagtgtgc cgcacaaggc cgtggcggta gggtatgtgt ctgaaaatga gctcggggag 1260 cgggcttgca ccgctgacgc atttggaaga cttaaggcag cggcagaaga agatgcaggc 1320

- 48 031308

agctgagttg ttgtgttctg ataagagtca gaggtaactc ccgttgcggt gctgttaacg 1380 gtggagggca gtgtagtctg agcagtactc gttgctgccg cgcgcgccac cagacataat 1440 agctgacaga ctaacagact gttcctttcc atgggtcttt tctgcagtca ccgtccttgc 1500 catcggtgac cactagtggc tcgcatctct ccttcacgcg cccgccgccc tacctgaggc 1560 cgccatccac gccggttgag tcgcgttctg ccgcctcccg cctgtggtgc ctcctgaact 1620 gcgtccgccg tctaggtaag tttaaagctc aggtcgagac cgggcctttg tccggcgctc 1680 ccttggagcc tacctagact cagccggctc tccacgcttt gcctgaccct gcttgctcaa 1740 ctctacgtct ttgtttcgtt ttctgttctg cgccgttaca gatcggtacc aagcttgcca 1800 ccaccatgaa cgccaaggtc gtggtcgtgc tggtcctcgt gctgaccgcg ctctgcctca 1860 gcgacgggaa gcccgtcagc ctgagctaca gatgcccatg ccgattcttc gaaagccatg 1920 ttgccagagc caacgtcaag catctcaaaa ttctcaacac cccaaactgt gcccttcaga 1980 ttgtagcccg gctgaagaac aacaacagac aagtgtgcat tgacccgaag ctaaagtgga 2040 ttcaggagta cctggagaaa gccttaaaca agtaatctag agggccctat tctatagtgt 2100 cacctaaatg ctagagctcg ctgatcagcc tcgactgtgc cttctagttg ccagccatct 2160 gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc cactgtcctt 2220 tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc tattctgggg 2280 ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag gcatgctggg 2340 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gggccgcggt ggccatcatg 2400 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc 2460 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa 2520 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag 2580 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta 2640 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta 2700 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag 2760 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg 2820 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg 2880 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag 2940 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc 3000 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa 3060 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg 3120 aattcagaag aactcgtcaa gaaggcgata gaaggcgatg cgctgcgaat cgggagcggc 3180

- 49 031308

gataccgtaa agcacgagga agcggtcagc ccattcgccg ccaagctctt cagcaatatc 3240 acgggtagcc aacgctatgt cctgatagcg gtccgccaca cccagccggc cacagtcgat 3300 gaatccagaa aagcggccat tttccaccat gatattcggc aagcaggcat cgccatgggt 3360 cacgacgaga tcctcgccgt cgggcatgct cgccttgagc ctggcgaaca gttcggctgg 3420 cgcgagcccc tgatgctctt cgtccagatc atcctgatcg acaagaccgg cttccatccg 3480 agtacgtgct cgctcgatgc gatgtttcgc ttggtggtcg aatgggcagg tagccggatc 3540 aagcgtatgc agccgccgca ttgcatcagc catgatggat actttctcgg caggagcaag 3600 gtgagatgac aggagatcct gccccggcac ttcgcccaat agcagccagt cccttcccgc 3660 ttcagtgaca acgtcgagca cagctgcgca aggaacgccc gtcgtggcca gccacgatag 3720 ccgcgctgcc tcgtcttgca gttcattcag ggcaccggac aggtcggtct tgacaaaaag 3780 aaccgggcgc ccctgcgctg acagccggaa cacggcggca tcagagcagc cgattgtctg 3840 ttgtgcccag tcatagccga atagcctctc cacccaagcg gccggagaac ctgcgtgcaa 3900 tccatcttgt tcaatcatgc gaaacgatcc tcatcctgtc tcttgatc 3948

Claims (1)

CLAIM 1. Plasmid for expression of stromal growth factor-1 (SDF-1), containing a polynucleotide encoding SDF-1, with the sequence SEQ ID NO: 6. 2. The drug for the treatment of the ischemic condition of the patient, containing from about 0.33 mg / ml to about 5 mg / ml plasmid SDF-1 according to claim 1 and a pharmaceutically acceptable carrier. 3. The drug according to claim 2, where the specified drug is injected. 4. The preparation according to claim 3, where the specified pharmaceutically acceptable carrier is 5% dextrose. 5. The preparation according to claim 3, containing approximately 2 mg / ml of the indicated plasmid SDF-1. 6. The preparation according to claim 3, containing approximately 1 mg / ml of the indicated plasmid SDF-1. 7. The use of the drug plasmid SDF-1 according to any one of claims 2-6 for the treatment of the ischemic condition of the patient. 8. The use according to claim 7, wherein said ischemic condition is ischemic cardiomyopathy. 9. The use according to claim 7, wherein said ischemic condition is a critical limb ischemia. 10. A method of treating an ischemic condition of a patient, comprising administering to a patient a preparation according to any one of claims 2-6. 11. The method according to claim 10, wherein said ischemic condition is ischemic cardiomyopathy. 12. The method according to claim 11, wherein said drug is administered to the weakened, ischemic and / or peri-infarction region of the patient's heart. 13. The method according to claim 12, wherein the amount of SDF-1 plasmid introduced into the weakened, ischemic and / or peri-infarction region of the patient’s heart is more than about 4 mg. 14. The method according to item 12 or 13, in which the drug is injected into the weakened, ischemic and / or peri-infarction region of the patient's heart by direct injection. 15. The method of claim 14, wherein the preparation is administered at least 10 injections, and each injection has a volume of at least about 0.2 ml. 16. The method of claim 15, wherein the total amount of injected drug is at least about 10 ml. 17. A method according to any one of claims 10-16, wherein said preparation is administered through a catheter. 18. The method according to claim 17, wherein said catheter is an endoventricular catheter or an intra myocardial catheter. 19. The method according to claim 10, in which the specified ischemic condition is critical ischemia - 50,031,308 limbs. 20. The method according to claim 19, in which the drug is injected into the injured limb of the patient by direct intramuscular injection. 21. The method of claim 20, wherein the preparation is administered by from about 5 to about 20 injections, and each injection has a volume of at least about 1.0 ml. 22. The method according to claim 21, wherein the amount of the inserted SDF-1 plasmid is from about 5 to about 20 mg. 23. A method according to any one of claims 19-22, wherein the administration is repeated up to three times and each administration is separated from the others by a period of about 2 weeks. 100,000 DNA / cell (mg) Amount / place (ml) Concentration (mg / ml) Formula FIG. one > 30 _Ί cl sh 20 > —1 f ten S Z 0 f X F -ten Σ <* uh S -20 'h® about ⁴ -30 ^j > 40 ω w thirty > i 20 f s ten z f 0 z f Σ -ten ω s -20 h © -thirty FIG. 2 FIG. 3 Control (n = 7) Low (n = 6) Medium (n = 8) High (n = 6) FIG. four - 51 031308 45.0 y Control (n = 3) Low (n = 4) Medium (n = 2) High (n = 3) FIG. 5 _; Low (n = 2) Medium (n = 3) High (n = 1) FIG. 6 FIG. 7 - 52 031308 120 100 s; FIG. 8> with w> - Phosphate salt buffer - ACL-01110Sk ΑACL-01010Sk thirty Days after injection FIG. 9 t salt buffer ................................................ .................................................. ...... j-in-ACL-01110Sk Τ '- · * - ACL-01010Sk; ........................................ .......... thirty Days after injection FIG. 10 {Image i Min. = -6.0393e + 05i ί Max. = 2.4485e + 06 p-ce </ cm ^L 2 / zg 1.0E + 08 8,0Е + 07 6,0Е + 07 4.0 E + 07 2.0E + 07 0.0E + 00 jbkg sub ί plateau space 8 10 Day after injection FIG. eleven - 53 031308 Click # AV00090327114300 Series: 73 rabbit right limb FIG. 12 j 1.0E + 08; F :: 1,0Е + 07 F S f 1,0Е + 06 о δ = 1.0Е + 05 S § 1.0E + 04 Group one 2 3 four five 6 Places eight eight eight four eight four Volume (ml) 0.5 0.5 0.5 0.5 0.5 one Concentration (mg / ml) 0.05 0.1 0.5 2 2 2 DNA / place (mg) 0.025 0.05 0.25 one one 2 Total amount DNA / limb 0.2 mg 0.4 mg 2.0 mg 4.0 mg 8.0 mg 8.0 mg FIG. 13 FIG. 14 - 54 031308 Group 2 (4 mg) Group 3 (8 mg) FIG. 15 ACRX-100 Biodiversity • Quad right ventricle. Basis a Quad. And the middle ▼ Quad. And the top ♦ Quad. D basis About Quad. D middle D Quad. D top