JP6461085B2 - SDF-1 delivery for treating ischemic tissue - Google Patents

Description

(Cross-reference of related applications)
This application claims the benefit of US Provisional Application No. 61 / 794,742, filed Mar. 15, 2013, the entire contents of which are hereby incorporated by reference.

(Field of Invention)
The present application relates to SDF-1 delivery methods and compositions for treating cardiomyopathy and the use of SDF-1 delivery methods and compositions for treating ischemic cardiomyopathy. The present application further relates to SDF-1 delivery methods and compositions for treating severe ischemic limbs.

  Ischemia is a condition in which blood flow is completely blocked or significantly reduced in a localized part of the body, causing anoxia, reduced substance supply and accumulation of metabolites. The degree of ischemia varies depending on the acute degree of vascular occlusion, its duration, its sensitivity to tissue, and the degree of collateral blood vessel growth, but is usually dysfunctional in ischemic organs or tissues and is prolonged Ischemia causes atrophy, degeneration, apoptosis, and necrosis of the affected tissue.

  In ischemic cardiomyopathy (a disease in which coronary arteries are affected and cause myocardial ischemia), the extent of ischemic cardiomyocyte injury is from reversible cell damage as the time of coronary artery occlusion increases. Progresses to irreversible cell damage.

  Severe ischemic limb (CLI) represents the most advanced stage of atherolimb peripheral vascular disease (PVD) and is associated with a high rate of cardiovascular morbidity, mortality, and major amputation. The incidence of CLI is estimated to have 125,000-250,000 patients per year in the United States and is expected to increase as the population ages. The prevalence of PVD increases significantly with age, affecting approximately 20% of Americans over 65 years of age. Modern nursing standards for CLI individuals include open surgery, lower limb revascularization by either endovascular techniques, or lower limb amputation (ie, when revascularization has failed or is not feasible) . The one-year mortality rate for CLI patients is 25%, and can be as high as 45% for patients who have undergone amputation. Despite the evolution of vascular procedures and surgery, a significant percentage of CLI patients are not suitable for revascularization. Of these patients, only 20-30% of CLI patients are treated, 30% require major amputation, and 23% die within 3 months.

  Gene therapy strategies to open blocked blood vessels or stimulate angiogenesis have been intensively studied. In a trial of 6 CLI patients planned to undergo major amputation, the patient is a non-viral gene therapy that expresses fibroblast growth factor 1 (FGF1) 3-5 days prior to amputation Assigned to take NV1FGF. NV1FGF was administered at 8 intramuscular sites at a dosage of 0.4-4 mg. This trial shows FGF1 transgene expression up to 3 cm from the injection site at all dosages, indicating that plasmids that have spread to blood vessels were rapidly degraded. In a multicenter, phase II, double-blind, placebo-controlled trial conducted in the United States (TALISMAN 202), 71 patients with CLI were injected intramuscularly 8 times into the affected limb with placebo or 2-16 mg NV1FGF Were assigned to receive 5 treatment regimens to be administered. This trial showed that up to 16 mg of intramuscular NV1FGF was safe and well tolerated. Although the primary endpoint of TcPO2 increased above baseline in both patients treated with NV1FGF and those treated with placebo, ulcer healing did not improve in patients treated with NV1FGF. Similarly, in a randomized, double-blind, placebo-controlled trial of 125 NV1FGF patients, Nikol et al. Significantly reduced the risk of all amputations and limb amputations by a factor of two. , Showed a tendency to lower the risk of death. These clinical trials show the same degree of safety of the naked plasmid DNA treatment as the clinical trials proposed by the present inventors.

  The present application relates to a method of treating cardiomyopathy in a subject. Cardiomyopathy includes, for example, cardiomyopathy associated with pulmonary embolism, venous thrombosis, myocardial infarction, temporary ischemic stroke, peripheral vascular injury, atherosclerosis, and / or other myocardial damage or vascular disease be able to. This method involves testing at least one of the following parameters: left ventricular volume, left ventricular area, left ventricular dimensions, cardiac function, 6-minute walking test (6MWT), or New York Heart Association (NYHA) functional classification. Administration or local expression of an effective amount of SDF-1 to improve function in a weakened, ischemic and / or peri-infarct area of a person's myocardial tissue.

  In certain aspects of the present application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region is determined by: left ventricular end-systolic volume, left ventricular ejection fraction, wall motion score index Left ventricular end-diastolic length, left ventricular end-systolic length, left ventricular end-diastolic area, left ventricular end-systolic area, left ventricular end-systolic volume, 6 minute walking test (6 MWT), Alternatively, it is effective to improve the function in at least one of New York Heart Association (NYHA) function classifications. In another aspect of the present application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region is effective to improve the left ventricular end systolic volume. In a further aspect of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region is effective to improve left ventricular ejection fraction.

  In some aspects of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region is effective to increase the end-systolic volume of the left ventricle by at least about 10%. It is. In other aspects of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region is effective to increase the end-systolic volume of the left ventricle by at least about 15%. is there. In yet a further aspect of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region increases the left ventricular end systolic volume by at least about 10%, It is effective to improve ejection fraction by at least about 10%, improve wall motion score index by at least about 5%, improve walking distance for 6 minutes by at least about 30 meters, and improve NYHA classification by at least one classification. In a further aspect of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region is effective to improve left ventricular ejection fraction by at least about 10%. .

  In another aspect of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region is based on vascular density or myocardial perfusion imaging (eg, SPECT or PET) Substantially improves angiogenesis in at least about 20% of the weakened, ischemic, and / or peri-infarct area as measured by, and improves the resting score, loading score, And / or effective for a difference in deficit scores of at least about 10%. Injection of a solution comprising a plasmid expressing SDF-1 in the weakened, ischemic and / or peri-infarct region and expressing SDF-1 from the weakened, ischemic and / or peri-infarct region By doing so, SDF-1 can be administered. From the weakened, ischemic, and / or peri-infarct region, SDF-1 can be expressed in an amount effective to improve the left ventricular end systolic volume.

  In one aspect of the present application, an ischemic agent comprising an injection solution each containing about 0.33 mg / ml to about 5 mg / ml SDF-1 plasmid solution, the solution being injected multiple times, and the SDF-1 plasmid weakened. And / or may be administered to the area surrounding the infarct. In one example, the SDF-1 plasmid may be administered to ischemic and / or peri-infarct areas that have been attenuated by at least about 10 injections. Each injection administered to the weakened, ischemic, and / or peri-infarct area may have a volume of at least about 0.2 ml. SDF-1 may be expressed longer than about 3 days in weakened, ischemic, and / or peri-infarct areas.

  In an exemplary application, each injection of a solution containing a plasmid expressing SDF-1 has an injection volume of at least about 0.2 ml and an SDF-1 plasmid concentration per injection of about 0.33 mg / ml to 5 mg. / Ml. In another aspect of this application, the at least one functional parameter of the heart is such that the injection volume per site is at least about 0.2 ml, and at least about 10 injection sites have an SDF-1 plasmid concentration per injection of about Improvements can be made by injecting the SDF-1 plasmid at 0.33 mg / ml to about 5 mg / ml into the weakened, ischemic, and / or peri-infarct area of the heart.

  In a further example, the amount of SDF-1 plasmid administered to the weakened, ischemic, and / or peri-infarct region that can improve at least one functional parameter of the heart is greater than about 4 mg. The amount of SDF-1 plasmid administered to the weakened, ischemic, and / or peri-infarct area that can improve at least one functional parameter of the heart is at least about 10 ml.

  In another aspect of the present application, the subject to whom SDF-1 is administered may be a large mammal, such as a human or a pig. The SDF-1 plasmid may be administered to the subject by catheterization, such as intracoronary catheterization or intraventricular catheterization. Prior to administration of the SDF-1 plasmid, the subject's myocardial tissue may be imaged to determine areas of weakened, ischemic, and / or peri-infarct areas. May be administered to areas weakened, ischemic, and / or peri-infarcted. The imaging may include at least one of echocardiography, magnetic resonance imaging, coronary angiography, magnetic stereo mapping, or fluoroscopy.

  The present application relates to a method of treating large mammalian myocardial infarction by administering SDF-1 plasmid to a region surrounding the myocardial infarction of a mammal by catheterization, eg, intracoronary catheterization or intraventricular catheterization. Also related. SDF-1 administered by catheterization is at least one of the left ventricular volume, left ventricular area, left ventricular dimensions, cardiac function, 6-minute gait test (6MWT), or New York Heart Association (NYHA) functional class In an amount effective to improve function in one, it can be expressed from the area surrounding the infarct.

  In certain aspects of the present application, the amount of SDF-1 administered to the peri-infarct region is the left ventricular end systolic volume, left ventricular ejection fraction, wall motion score index, left ventricular end diastolic length, End-systolic length of ventricle, end-diastolic area of left ventricle, end-systolic area of left ventricle, end-systolic volume of left ventricle, 6-minute walk test (6MWT), or New York Heart Association (NYHA) functional classification It is effective to improve the function in at least one.

  In another aspect of the present application, the amount of SDF-1 administered to the area surrounding the infarct is effective to improve the left ventricular end systolic volume. In a further aspect of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region is effective to improve left ventricular ejection fraction.

  In some aspects of the present application, the amount of SDF-1 administered to the peri-infarct region is effective to improve the left ventricular end systolic volume by at least about 10%. In other aspects of the application, the amount of SDF-1 administered to the area surrounding the infarct is effective to increase the end-systolic volume of the left ventricle by at least about 15%. In yet a further aspect of the application, the amount of SDF-1 administered to the peri-infarct region increases the left ventricular end systolic volume by at least about 10% and increases the left ventricular ejection fraction by at least about 10%. It is effective to improve the wall motion score index by at least about 5%, improve the walking distance for 6 minutes by at least about 30 meters, and improve the NYHA classification by at least one classification. In a further aspect of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region is effective to improve left ventricular ejection fraction by at least about 10%. .

  In another aspect of the application, the amount of SDF-1 administered to the peri-infarct region is effective to substantially improve angiogenesis in the peri-infarct region by at least about 20% based on vascular density. It is.

  In one aspect of the present application, an ischemic agent comprising an injection solution each containing about 0.33 mg / ml to about 5 mg / ml SDF-1 plasmid solution, the solution being injected multiple times, and the SDF-1 plasmid weakened. And / or may be administered to the area surrounding the infarct. In one example, the SDF-1 plasmid may be administered to ischemic and / or peri-infarct areas that have been attenuated by at least about 10 injections. Each injection administered to the weakened, ischemic, and / or peri-infarct area may have a volume of at least about 0.2 ml. SDF-1 can be expressed in weakened, ischemic, and / or peri-infarct areas for more than about 3 days.

  In an exemplary application, each injection of a solution containing a plasmid expressing SDF-1 has an injection volume of at least about 0.2 ml and an SDF-1 plasmid concentration per injection of about 0.33 mg / ml to 5 mg. / Ml. In another aspect of this application, the at least one functional parameter of the heart is such that the injection volume per site is at least about 0.2 ml, and at least about 10 injection sites have an SDF-1 plasmid concentration per injection of about Improvements can be made by injecting the SDF-1 plasmid at 0.33 mg / ml to about 5 mg / ml into the weakened, ischemic, and / or peri-infarct area of the heart.

  In a further example, the amount of SDF-1 plasmid administered to the weakened, ischemic, and / or peri-infarct region that can improve at least one functional parameter of the heart is greater than about 4 mg. The volume of the solution of SDF-1 plasmid administered to the weakened, ischemic, and / or peri-infarct area that can improve at least one functional parameter of the heart is at least about 10 ml.

  The application further relates to a method of improving the end-systolic volume of the left ventricle of a large mammal after myocardial infarction. This method involves administering the SDF-1 plasmid to a region surrounding the infarct in a mammal by intraventricular catheterization. SDF-1 can be expressed from the area surrounding the infarct in an amount effective to improve the end-systolic volume of the left ventricle.

  In some aspects of the present application, the amount of SDF-1 administered to the peri-infarct region is effective to improve the left ventricular end systolic volume by at least about 10%. In other aspects of the application, the amount of SDF-1 administered to the area surrounding the infarct is effective to increase the end-systolic volume of the left ventricle by at least about 15%. In yet a further aspect of the application, the amount of SDF-1 administered to the peri-infarct region increases the left ventricular end systolic volume by at least about 10% and increases the left ventricular ejection fraction by at least about 10%. It is effective to improve the wall motion score index by at least about 5%, improve the walking distance for 6 minutes by at least about 30 meters, and improve the NYHA classification by at least one classification.

  In one aspect of the present application, an ischemic agent comprising an injection solution each containing about 0.33 mg / ml to about 5 mg / ml SDF-1 plasmid solution, the solution being injected multiple times, and the SDF-1 plasmid weakened. And / or may be administered to the area surrounding the infarct. In one example, the SDF-1 plasmid may be administered to ischemic and / or peri-infarct areas that have been attenuated by at least about 10 injections. Each injection administered to the weakened, ischemic, and / or peri-infarct area may have a volume of at least about 0.2 ml. SDF-1 can be expressed in weakened, ischemic, and / or peri-infarct areas for more than about 3 days.

  In an exemplary application, each injection of a solution containing a plasmid expressing SDF-1 has an injection volume of at least about 0.2 ml and an SDF-1 plasmid concentration per injection of about 0.33 mg / ml to 5 mg. / Ml. In another aspect of this application, the left ventricular end systolic volume has an injection volume of at least about 0.2 ml per site, and at least about 10 injection sites with a SDF-1 plasmid concentration per injection of about 0. From about 33 mg / ml to about 5 mg / ml can be improved by about 10% by injecting the SDF-1 plasmid into the weakened, ischemic, and / or peri-infarct area of the heart.

  In a further example, the amount of weakened, ischemic, and / or peri-infarct area that can improve the end-systolic volume of the left ventricle of the heart is greater than about 4 mg. The amount of weakened, ischemic and / or peri-infarcted SDF-1 plasmid that can improve the end-systolic volume of the left ventricle of the heart is at least about 10 ml.

  The application further relates to a method of treating severe ischemic limbs in an examiner. This method involves ACRX-100 (JVS), which is a sterile biological product (consisting of a plasmid having the nucleotide sequence of SEQ ID NO: 6, a naked DNA plasmid encoding human SDF-1 cDNA, and 5% dextrose). -100) (also known as -100) by direct injection into the ischemic limb. Preferably, the injection is made directly into the muscle tissue, eg, the upper limb (quadriceps) and / or the lower limb (mainly gastrocnemius) using multiple injection sites. The sequence of this plasmid is shown below.

Plasmid sequence used in ACRX-100 (aka JVS-100)
AGATCTCCTAGGGAGTCCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGCGGCCGGGAACGGTGCATTGGAACGCGGATTCCCCGTGCCAAGAGTGACGTAAGTACCGCCTATAGAGTCTATAGGCCCACCCCCTTGGCTTCTTATGCATGCTATACTGTTTTTGGCTTGGGGTCTATACACCCCCGCTTCCTCATGTTATAGGTGATGGTATAGCTTAGCCTATAGGTGTGGGTTATTGACCATTATTGACCACTCCCCTATTGGTGACGATACTTTCCATTACTAATCCATAACATGGCTCTTTGCCACAACTCTCTTTATTGGCTATATGCCAATACACTGTCCTTCAGAGACTGACACGGACTCTGTATTTTTACAGGATGGGGTCTCATTTATTATTTACAAATTCACATATACAACACCACCGTCCCCAGTG CCCGCAGTTTTTATTAAACATAACGTGGGATCTCCACGCGAATCTCGGGTACGTGTTCCGGACATGGGCTCTTCTCCGGTAGCGGCGGAGCTTCTACATCCGAGCCCTGCTCCCATGCCTCCAGCGACTCATGGTCGCTCGGCAGCTCCTTGCTCCTAACAGTGGAGGCCAGACTTAGGCACAGCACGATGCCCACCACCACCAGTGTGCCGCACAAGGCCGTGGCGGTAGGGTATGTGTCTGAAAATGAGCTCGGGGAGCGGGCTTGCACCGCTGACGCATTTGGAAGACTTAAGGCAGCGGCAGAAGAAGATGCAGGCAGCTGAGTTGTTGTGTTCTGATAAGAGTCAGAGGTAACTCCCGTTGCGGTGCTGTTAACGGTGGAGGGCAGTGTAGTCTGAGCAGTACTCGTTGCTGCCGCGCGCGCCACCAGACATAATAGCTGACAGACTAACAGACTGTTCCTTTCCATGGGTCTTTTCTGCAGTCACCGTCCTTGCCATCGGTGACCACTAGTGGCTCGCATCTCTCCTTCACGCGCCCGCCGCCCTACCTGAGGCCGCCATCCACGCCGGTTGAGTCGCGTTCTGCCGCCTCCCGCCTGTGGTGCCTCCTGAACTGCGTCCGCCGTCTAGGTAAGTTTAAAGCTCAGGTCGAGACCGGGCCTTTGTCCGGCGCTCCCTTGGAGCCTACCTAGACTCAGCCGGCTCTCCACGCTTTGCCTGACCCTGCTTGCTCAACTCTACGTCTTTGTTTCGTTTTCTGTTCTGCGCCGTTACAGATCGGTACCAAGCTTGCCACCACCATGAACGCCAAGGTCGTGGTCGTGCTGGTCCTCGTGCTGACCGCGCTCTGCCTCAGCGACGGGAAGCCCGTCAGCCTGAGCTACAGATGCCCATGCCGATTCTTCGAAAGCCATGTTGCCAGAGCCAACGTCAAGCATCTCAAAATTCTCAACACCCCAAACTGTGCCCTTCAGATTGTAGCCCGGCTGAAGAAC AACAACAGACAAGTGTGCATTGACCCGAAGCTAAAGTGGATTCAGGAGTACCTGGAGAAAGCCTTAAACAAGTAATCTAGAGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGGGCCGCGGTGGCCATCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGC CACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGAATTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGGAAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAGCCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAAGGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCTGACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATC (SEQ ID NO: 6)

The above and other features of the present application will become apparent to those of ordinary skill in the art to which the present application pertains upon reading the following description, while referring to the accompanying drawings.
FIG. 5 shows luciferase expression to change the amount and volume of DNA in a porcine model. It is a figure which shows the change rate% of the end-systolic volume of the left ventricle about various quantity of SDF-1 plasmids using the congestive heart failure pig model 30 days after SDF-1 injection. It is a figure which shows the change rate% of the ejection fraction of the left ventricle about various quantity of SDF-1 plasmids using the congestive heart failure pig model 30 days after SDF-1 injection. It is a figure which shows the change rate% of wall motion score index about various quantity of SDF-1 plasmids using the congestive heart failure pig model 30 days after SDF-1 injection. FIG. 7 is a graph showing% change in left ventricular end-systolic volume for various amounts of SDF-1 plasmid using a congestive heart failure pig model 90 days after SDF-1 injection. It is a figure which shows the change rate% of the blood vessel density about various amount of SDF-1 plasmids using the congestive heart failure pig model 30 days after SDF-1 injection. It is a schematic diagram of an SDF-1 plasmid vector. FIG. 2 is an image showing plasmid expression across a significant portion of a pig heart. FIG. 5 shows the left ventricular end systolic volume 30 days after baseline and the first injection. All groups show a similar increase in left ventricular end systolic volume on day 30. N = 3 for all data points. Data were expressed as mean ± SEM. FIG. 6 shows baseline and ventricular ejection fraction 30 days after the first injection. All groups show no improvement in left ventricular ejection fraction. N = 3 for all data points. Data were expressed as mean ± SEM. FIG. 3 is an image of luciferase expression in ischemic rat limbs 3 days after injection (A) and a time course of ACRX-100 vector expression in a rodent HLI model (B). It is a bioluminescence image of rabbit hind limb muscle 3 days after injection of ACL-01110L luciferase plasmid DNA. FIG. 6 is a diagram of ACL-01110L dosing parameters in the rabbit hind limb. FIG. 2 is an example of angiograms and scoring of ischemic rabbit hindlimbs 30 days (B and D) after baseline (A and C) and ACRX-100 injection. FIG. 4 is a graph of the rate of change of angiographic score 30 days and 60 days after injection of ACRX-100, normalized to control per group. It is a figure of ACRX-100 biodistribution after intracardiac injection. It is a figure of the relationship between SDF-1 expression after ischemic injury and CXCR4 expression. CXCR4 is the main receptor for SDF-1.

  Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Any patents, patent specifications, published specifications and publications, Genbank sequences, websites and other published materials mentioned throughout the disclosure of this specification are not indicated to have any other meaning. To the extent that the entire contents are incorporated herein by reference. In the event that there are a plurality of definitions for terms herein, those in this section prevail. Unless defined otherwise, all technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Commonly understood definitions of molecular biology terms include, for example, Rieger et al, Glossary of Genetics: Classical and Molecular, 5th edition, Springer Verlag: New York, 1991 and Lewin, Genes V, Oxford. New York, 1994.

  Methods including conventional molecular biology techniques are described herein. Such techniques are generally known in the art and can be found in methodology papers such as Molecular Cloning: A Laboratory Manual, 2nd edition, vol. 1-3, Sambrook et al. Edit, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.A. Y. 1989; and Current Protocols in Molecular Biology, Ausubel et al. Edit, Green Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods for chemically synthesizing nucleic acids are described, for example, in Beaucage and Caruthers, Tetra. Letts. 22: 1859-1862, 1981, and Matteucci et al, J. MoI. Am. Chern. Soc. 103: 3185, 1981, chemical synthesis of nucleic acids can be performed with a commercially available automated oligonucleotide synthesizer. Immunological methods (eg, preparation of antigen-specific antibodies, immunological and immunoblotting) are described, for example, in Current Protocols in Immunology, Coligan et al, John Wiley & Sons, New York, 1991; and Methods of Immunol. Analysis, edited by Masseyeff et al., John Wiley & Sons, New York, 1992. Conventional gene transfer and gene therapy methods can also be adapted for use in the present application. See, for example, Gene Therapy: Principles and Applications, T.A. Blackenstein, edited by Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), P.M. D. Edited by Robins, Humana Press, 1997; and Retro-vectors for Human Gene Therapy, C.I. P. See Hodgson Edit, Springer Verlag, 1996.

  When referring to URLs or other such identifiers or addresses, such identifiers can be changed and specific information on the Internet can be traversed, but by searching the Internet Equivalent information can be found. These references demonstrate the availability and distribution to the public of such information.

  As used herein, ACRX-100 is a sterile biological product composed of a plasmid having the nucleotide sequence of SEQ ID NO: 6, a naked DNA plasmid encoding human SDF-1 cDNA, and 5% dextrose. (ACRX-100 is sometimes referred to herein as JVS-100.)

  As used herein, “nucleic acid” refers to a polynucleotide containing at least two covalently linked nucleotides or nucleotide analog subunits. The nucleic acid may be deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or an analog of DNA or RNA. Nucleotide analogs are commercially available and methods for preparing polynucleotides containing such nucleotides are 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. For the purposes of the present invention, a nucleic acid is double stranded or is apparent from the contents unless otherwise stated.

  As used herein, “DNA” is meant to include DNA molecules of all types and sizes, including eDNA, plasmids and DNA, including modified nucleotides and nucleotide analogs.

  As used herein, “nucleotide” includes nucleoside monophosphates, diphosphates and triphosphates. Nucleotides also include modified nucleotides, which include, but are not limited to, phosphorothioate nucleotides, deazapurine nucleotides and other nucleotide analogs.

  As used herein, the term “subject” or “patient” refers to an animal into which large DNA molecules can be introduced. Higher organisms include mammals and birds, including humans, primates, rodents, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, cats, dogs, horses, chickens and others.

  As used herein, “large mammal” refers to a mammal having a typical adult body weight of at least 10 kg. Such large mammals can include, for example, humans, primates, dogs, pigs, cows, and exclude smaller mammals such as mice, rats, guinea pigs and other rodents. means.

  As used herein, “administer to a subject” means that one or more delivery agents and / or large nucleic acid molecules are introduced or applied to a subject, either together or separately. A procedure in which target cells present in the subject are finally contacted with the agent and / or the large nucleic acid molecule.

  As used herein, “delivery” is used interchangeably with “transduction” and refers to the process by which exogenous nucleic acid molecules migrate into a cell and are placed inside the cell. Nucleic acid delivery is a separate process from nucleic acid expression.

  As used herein, a “multicloning site (MCS)” is a nucleic acid region in a plasmid that contains multiple restriction enzyme sites, combining any of the restriction enzyme sites with standard recombination techniques, Can be digested. “Restriction enzyme digestion” refers to the catalytic cleavage of a nucleic acid molecule with an enzyme that functions only at specific locations in the nucleic acid molecule. Many of these restriction enzymes are commercially available. The use of such enzymes is widely understood by those skilled in the art. Frequently, the vector is linearized or fragmented with a restriction enzyme that can cut in MCS and ligate exogenous sequences to the vector.

  As used herein, an “origin of replication” (often referred to as “ori”) is a specific nucleic acid sequence from which replication is initiated. Alternatively, when the host cell is yeast, an autonomously replicating sequence (ARS) may be used.

  As used herein, a “selectable marker or screenable marker” provides a identifiable change to a cell that allows easy identification of the cell containing the expression vector. In general, a selectable marker is a marker that provides a selectable property. A selectable positive marker is a marker whose presence allows its selection, while a selectable negative marker is a marker whose presence interferes with its selection. An example of a selectable positive marker is a drug resistance marker.

  Usually, the inclusion of a drug selection marker assists in the cloning and identification of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. In addition to markers that provide a phenotype that can distinguish transformants based on the implementation of conditions, other types of markers that include screenable markers (eg, GFP) and are based on calorimetric analysis are also envisioned. The Alternatively, screenable enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. Those skilled in the art will also know how to use immunological markers, possibly in combination with FACS analysis. The marker used is considered unimportant if it can be expressed simultaneously with the nucleic acid encoding the gene product. Additional examples of selectable markers and screenable markers are well known to those skilled in the art.

  The term “transfection” is used to refer to the uptake of foreign DNA by a cell. A cell was “transfected” when exogenous DNA was introduced inside the cell. Many transfection techniques are generally known in the art. For example, Graham et al, Virology 52: 456 (1973); Sambrook et al, Molecular Cloning: A Laboratory Manual (1989); Davis et al, Basic Methods 6 198; 1981). Such techniques can be used to introduce one or more exogenous DNA molecules, such as nucleotide-integrated vectors and other nucleic acid molecules, into a suitable host cell. The term includes chemical procedures, electrical procedures and virus-mediated transfection procedures.

  As used herein, “expression” refers to the process by which a nucleic acid can be translated into a peptide or transcribed into RNA, eg, translated into a peptide, polypeptide, or protein. Where the nucleic acid is derived from genomic DNA, expression may include splicing of the mRNA when an appropriate eukaryotic host cell or organism is selected. In order for a heterologous nucleic acid to be expressed in a host cell, it must first be delivered to the cell and then remain in the nucleus upon entering the cell.

  As used herein, “gene therapy” includes the transfer of heterologous DNA into cells of a mammal (particularly human) having a disease or condition for which treatment or diagnosis is sought. The DNA is introduced into the selected target cells in such a way that the heterologous DNA is expressed, thereby producing the therapeutic product encoded by it. Alternatively, the heterologous DNA may mediate the expression of the DNA encoding the therapeutic product in several ways. The heterologous DNA may encode a product (eg, a peptide or RNA) that directly or indirectly mediates the expression of the therapeutic product in some manner. Gene therapy may also be used to deliver a nucleic acid encoding a gene product, replace the defective gene, or add a gene product produced by the introduced mammal or cell. Thus, the introduced nucleic acid is not normally produced in a mammalian host, or is not produced in a therapeutically effective amount or for a therapeutically useful time, eg, a growth factor or inhibitor thereof, or a tumor It may encode a necrosis factor or an inhibitor thereof, such as a receptor. To enhance or otherwise alter the product or its expression, the heterologous DNA encoding the therapeutic product may be modified prior to introduction into the affected host cell.

  As used herein, a “heterologous nucleic acid sequence” is typically DNA encoding RNA and proteins that are not normally produced in vivo by the cell in which expression occurs, or transcription, translation or other controllable. A mediator that alters the expression of endogenous DNA by undergoing a biochemical process, or a DNA that encodes this mediator. Heterologous nucleic acid sequences are sometimes referred to as foreign DNA. Any DNA that one skilled in the art recognizes or considers heterologous or exogenous to an internally expressed cell is encompassed by the heterologous DNA herein. Examples of heterologous DNA include, but are not limited to, DNA encoding traceable marker proteins (eg, proteins that confer drug resistance), DNA encoding therapeutically effective substances (eg, anticancer drugs, enzymes, and hormones), and the like And DNAs encoding these types of proteins (eg, antibodies). The antibody encoded by the heterologous DNA may 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 deterioration of the function of the myocardium (ie, the actual heart muscle) for any reason. Subjects with cardiomyopathy are often at risk of arrhythmia, sudden cardiac death, or hospitalization or death due to heart failure.

  As used herein, the term “ischemic cardiomyopathy” is a weakening of the heart muscle due to inadequate oxygen delivery to the myocardium, the most common cause of coronary artery disease. is there.

  As used herein, the term “ischemic heart disease” means that the heart muscle is damaged or does not work well due to lack of blood supply or relatively lack of Refers to any condition, most often caused by atherosclerosis, including angina, acute myocardial infarction, chronic ischemic heart disease and sudden death.

  As used herein, “myocardial infarction” refers to damage or death of a region of the heart muscle (myocardium) caused by blockage of blood flow to the region.

  As used herein, the term “6-minute walk test” or “6 MWT” refers to a test that measures the distance (6 MWD) that a patient can quickly walk on a flat, hard surface for 6 minutes. Assess the overall and integrated response of all systems involved during exercise, including the lung and cardiovascular system, systemic circulation, peripheral circulation, blood, neuromuscular units and muscle metabolism. This does not give specific information about the function of each of the different organs and systems involved in the mechanism of movement or movement limitation, as possible using the maximum cardiopulmonary exercise test. 6MWT at your own pace will evaluate the sub-maximum level of functional capacity. (See, for example, AM J Respir Crit Care Med, Vol. 166. Pp 111-117 (2002).)

  As used herein, “New York Heart Association (NYHA) functional classification” refers to a classification of the degree of heart failure. Based on how often you are constrained during physical activity, place the patient in one of four categories, which includes normal breathing and varying degrees of shortness of breath, and / or angina pain About.

  The present application relates to a method of treating cardiomyopathy in a subject that causes a decrease and / or failure of myocardial function. Cardiomyopathy to be treated by the compositions and methods of the present invention includes pulmonary embolism, venous thrombosis, myocardial infarction, temporary ischemic stroke, peripheral vascular injury, atherosclerosis, and / or other myocardial damage or blood vessels. Cardiomyopathy associated with other diseases. Methods for treating cardiomyopathy include weak myocardial tissue, ischemic myocardial tissue, and / or apoptotic myocardial tissue, for example, in the area surrounding the infarct of the heart after myocardial infarction, the volume of the left ventricle, the area of the left ventricle, An amount of stromal cell-derived factor-1 (SDF-1) effective to improve function in at least one of left ventricular dimensions, cardiac function, 6-minute walk test (6MWT), or New York Heart Association (NYHA) functional class ) May be administered locally (or locally delivered).

  Using a porcine heart failure model that mimics human heart failure, the enhancement of ischemic myocardial tissue depends on the amount, dosage and / or delivery of SDF-1 administered to the ischemic myocardial tissue, parameters of myocardial function, For example, optimizing the amount, dosage and / or delivery of SDF-1 to ischemic myocardial tissue such that left ventricular volume, left ventricular area, left ventricular dimensions, and cardiac function are substantially improved. I found out that As described below, in certain embodiments, the amount, concentration, and / or volume of SDF-1 administered to ischemic myocardial tissue reduces functional effects (eg, left ventricular volume, left ventricular volume, while reducing side effects). Area, left ventricular dimension, cardiac function, 6-minute walk test (6MWT) and / or New York Heart Association (NYHA) functional classification) can be controlled and / or optimized.

  In one example, SDF-I is an aggravation of cardiac functional parameters (eg, left ventricular volume, left ventricular area, left ventricular dimensions, or cardiac function as a result of ischemic cardiomyopathy, eg, myocardial infarction). Or it can be administered directly or locally to weakened, ischemic, and / or peri-infarcted areas of myocardial tissue in large mammals (eg, pigs or humans) where there is a catastrophe. Deterioration or aggravation of functional parameters may include, for example, increased left ventricular end-systolic volume as measured using echocardiography, decreased left ventricular ejection fraction, increased wall motion score index, left Increased ventricular end-diastolic length, increased left ventricular end-systolic length, increased left ventricular end-diastolic area (eg, mitral and papillary muscle insertion levels), left ventricular end-systolic An increase in area (eg, mitral valve level and papillary muscle insertion level) or an increase in the left ventricular end systolic volume may be mentioned.

  In certain aspects of the present application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct area of large mammalian myocardial tissue is measured using, for example, echocardiography. Decreased left ventricular end-systolic volume of myocardium, increased left ventricular ejection fraction, decreased wall motion score index, decreased left ventricular end-diastolic length, decreased left ventricular end-systolic length, Decreased left ventricular end-diastolic area (eg, mitral and papillary muscle insertion levels), reduced left ventricular end-systolic area (eg, mitral and papillary muscle insertion levels), or left ventricular Improve at least one functional parameter of the myocardium, such as reduction in end systolic volume and subject's 6-minute gait test (6MWT), or New York Heart Association (NYHA) functional classification It may be an amount effective to.

  In another aspect of the present application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region of large mammalian myocardial tissue with cardiomyopathy is It is effective to increase the left ventricular end systolic volume in mammals by at least about 10%, more specifically at least about 15%, 30 days after administration, as measured by examination. The rate of improvement is relative to each subject being treated and is based on respective parameters measured before or during therapeutic intervention or treatment.

  In a further aspect of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region of large mammalian myocardial tissue with cardiomyopathy is determined by echocardiography. 30 days after administration, the left ventricular end systolic volume is improved by at least about 10%, the left ventricular ejection fraction is improved by at least about 10%, and the wall motion score index is improved by at least about 5%. It is effective to make it.

  In yet a further aspect of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region of large mammalian myocardial tissue with cardiomyopathy is determined by SPECT imaging. Based on the measured increase in vascular density or cardiac perfusion, it is effective to improve angiogenesis in weakened, ischemic and / or peri-infarct areas by at least 20%. A 20% improvement in angiogenesis has been shown to be clinically significant (Losodo Circulation 2002; 105: 2012).

  In yet a further aspect of the application, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarct region of large mammalian myocardial tissue with cardiomyopathy is 6 minutes walking. It is effective to improve the distance by at least about 30 meters and to improve the NYHA classification by at least one classification.

  SDF-1 described herein weakens myocardial tissue after tissue injury (eg, myocardial infarction) for several hours, days, weeks or months after downregulation of SDF-1 occurs Can be administered to an area, ischemic area, and / or peri-infarct area. The period during which SDF-1 is administered to the cells includes from immediately after the occurrence of cardiomyopathy (eg, myocardial infarction) to days, weeks or months after the occurrence of ischemic injury or tissue injury. May be.

  The SDF-1 of the present application administered to the weakened, ischemic, and / or peri-infarct region of the peri-infarct region of myocardial tissue is substantially equivalent to the native mammalian SDF-I amino acid sequence. It may have a similar amino acid sequence. The amino acid sequences of many different mammalian SDF-1 proteins, including humans, mice and rats, are known. The human and rat SDF-1 amino acid sequences are at least 92% identical (eg, about 97% identical). SDF-1 may include two isoforms, SDF-1α and SDF-1β, both of which are referred to herein as SDF-1 unless specified otherwise.

  SDF-1 may have an amino acid sequence substantially identical to SEQ ID NO: 1. Overexpressed SDF-1 may have an amino acid sequence substantially similar to any of the mammalian SDF-1 proteins described above. For example, overexpressed SDF-1 may have an amino acid sequence substantially similar to SEQ ID NO: 2. SEQ ID NO: 2, substantially comprising SEQ ID NO: 1, is the amino acid sequence of human SDF-1 and is identified by Genbank accession number NP954637. Overexpressed SDF-1 may have an amino acid sequence substantially identical to SEQ ID NO: 3. SEQ ID NO: 3 comprises the amino acid sequence of rat SDF and is identified by Genbank accession number AAF01066.

  The SDF-1 of the present application may be a variant of a mammalian SDF-1, such as a fragment, analog and derivative of a mammalian SDF-1. Such variants include, for example, naturally-occurring allelic variants of the native SDF-1 gene (ie, naturally-occurring nucleic acids encoding naturally-occurring mammalian SDF-1 polypeptides). Encoded by a peptide, a polypeptide encoded by an alternating splicing form of the native SDF-1 gene, a polypeptide encoded by a homolog or ortholog of the native SDF-1 gene, and a non-naturally occurring variant of the native SDF-1 gene Polypeptide.

  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 characterized by the deletion, addition or substitution of one or more amino acids of the SDF-1 variant. The amino acid insertion is preferably an insertion of about 1 to 4 adjacent amino acids, and the deletion is preferably a deletion of about 1 to 10 adjacent amino acids. The variant SDF-1 polypeptide substantially maintains the functional activity of native SDF-1. Examples of SDF-1 polypeptide variants can be produced by expressing nucleic acid molecules characterized by silent or conservative changes. An example of an SDF-1 variant is listed in US Pat. No. 7,405,195, the entire contents of which are hereby incorporated by reference.

  SDF-1 polypeptide fragments that correspond to one or more particular motifs and / or domains or that correspond to any size are within the scope of this application. An isolated peptidyl moiety of SDF-1 can be obtained by screening peptides made recombinantly from corresponding fragments of nucleic acids encoding such peptides. For example, SDF-1 polypeptides may be arbitrarily divided into fragments of a desired length without fragment overlap, or preferably, may be divided into overlapping fragments of a desired length. This fragment may be produced recombinantly and tested to identify these peptidyl fragments, which can function as agonists of the native CXCR-4 polypeptide.

  Variants of SDF-1 polypeptide may also include recombinant forms of SDF-1 polypeptide. The recombinant polypeptide, in one embodiment, is encoded by a nucleic acid that may have at least 70% sequence identity within the nucleic acid sequence of the gene encoding mammalian SDF-1 in addition to the SDF-1 polypeptide. Is done.

  SDF-1 variants may include agonistic forms of proteins that constitutively express the functional activity of native SDF-1. Other SDF-1 variants may include those that are resistant to proteolytic cleavage, for example due to mutations that alter the target sequence of the protease. Whether a change in the amino acid sequence of the peptide yields a variant having one or more functional activities of native SDF-1 is easily determined by testing the variant for the functional activity of native SDF-1. Can do.

  The SDF-1 nucleic acid encoding the SDF-1 protein may be a native or non-native nucleic acid, and may be in the form of RNA or DNA (eg, eDNA, genomic DNA, and synthetic DNA). The DNA may be double-stranded or single-stranded, and if it is single-stranded, it may be a coding (sense) strand or a non-coding (antisense) strand. The nucleic acid coding sequence encoding SDF-1 may be substantially similar to the nucleotide sequence of the SDF-1 gene, eg, the nucleotide sequences shown in SEQ ID NO: 4 and SEQ ID NO: 5. SEQ ID NO: 4 and SEQ ID NO: 5 comprise the nucleic acid sequences of human SDF-1 and rat SDF-1, respectively, and are substantially similar to the nucleic acid sequences of Genbank accession number NM199168 and Genbank accession number AF189724.

  The nucleic acid coding sequence of SDF-1 may be a different coding sequence and encodes the same polypeptide as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 as a result of the redundancy or degeneracy of the genetic code.

  Other nucleic acid molecules that encode SDF-1 encode variants of native SDF-1, such as fragments, analogs and derivatives of native SDF-1. Such a variant may be, for example, a naturally occurring allelic variant of the native SDF-1 gene, a homolog or ortholog of the native SDF-1 gene, or a non-naturally occurring variant of the native SDF-1 gene. Good. These variants have a nucleotide sequence that differs from the native SDF-1 gene by one or more bases. For example, the nucleotide sequence of such a variant may be characterized by a deletion, addition or substitution of one or more nucleotides of the native SDF-1 gene. The nucleic acid insertion is preferably an insertion of about 1-10 contiguous nucleotides, and the deletion is preferably a deletion of about 1-10 contiguous nucleotides.

  In other applications, a variant SDF-1 that exhibits significant structural changes can be made by making nucleotide substitutions in the encoded polypeptide that do not result in conservative changes. Examples of such nucleotide substitutions are those that alter (a) the structure of the polypeptide backbone, (b) the charge or hydrophobicity of the polypeptide, or (c) the mass of amino acid side chains. Nucleotide substitutions that are expected to produce the greatest changes in protein properties generally result in non-conservative changes in codons. Examples of codon changes that would cause large changes in protein structure are: (a) from a hydrophobic residue (eg, serine or threonine) to a hydrophobic residue (eg, leucine, isoleucine, phenylalanine, valine or alanine). Substitution (or vice versa), (b) substitution of cysteine or proline to any other residue (or vice versa), (c) a residue with a positively charged side chain (eg lysine, arginine or Histidine) to a negatively charged residue (eg glutamine or aspartin) (or vice versa), or (d) a residue with a bulky side chain (eg phenylalanine) having a side chain This results in a substitution (or vice versa) for something that is not (eg glycine).

  A naturally occurring allelic variant of the native SDF-1 gene encodes a polypeptide having at least 70% sequence identity with the native SDF-1 gene and having structural similarity to the native SDF-1 polypeptide; A nucleic acid isolated from mammalian tissue. A homologue of a native SDF-1 gene is a nucleic acid isolated from other species that encodes a polypeptide having at least 70% sequence identity with the native gene and having structural similarity to the native SDF-1 polypeptide It is. Search public and / or intellectual property protected nucleic acid databases to identify other nucleic acid molecules with a high percentage (eg, 70% or more) of sequence identity to the native SDF-1 gene Can do.

  A non-naturally occurring SDF-1 gene variant has a sequence identity of at least 70% with the native SDF-1 gene and encodes a polypeptide having structural similarity to the native SDF-1 polypeptide. There are no nucleic acids (eg, made by human hands). Examples of non-naturally occurring SDF-1 gene variants are those that encode fragments of the native SDF-1 protein, or hybridize to the native SDF-1 gene or the complement of the native SDF-1 gene under stringent conditions. One that shares at least 65% sequence identity with the native SDF-1 gene, or the complement of the native SDF-1 gene.

  The nucleic acid encoding a fragment of the native SDF-1 gene is, in one embodiment, a nucleic acid encoding an amino acid residue of native SDF-1. Shorter oligonucleotides that encode or hybridize to nucleic acids encoding fragments of native SDF-1 can be used as probes, primers or antisense molecules. Longer oligonucleotides that encode or hybridize to nucleic acids encoding fragments of native SDF-1 can also be used in various aspects of the application. Nucleic acids encoding fragments of the native SDF-1 gene can be produced by enzymatic digestion (eg, using restriction enzymes) of the full length native SDF-1 gene or variants thereof, or by chemical denaturation.

  Nucleic acids that hybridize to any of the above nucleic acids under stringent conditions can also be used in the present invention. For example, such a nucleic acid may be a nucleic acid that hybridizes to any of the above nucleic acids under low stringency conditions, moderately stringent conditions, or highly stringent conditions.

  Nucleic acid molecules encoding SDF-1 fusion proteins may be used in certain embodiments. Such nucleic acids can be produced by preparing a construct (eg, an expression vector) that expresses an SDF-1 fusion protein when introduced into a suitable target cell. For example, such a construct encodes a first polynucleotide encoding an SDF-1 protein fused in frame with another protein such that expression of the construct in an appropriate expression system provides the fusion protein. Can be produced by ligating with a second polynucleotide.

  The nucleic acid encoding SDF-1 may be modified with a base moiety, sugar moiety or phosphate backbone, for example, to improve molecular stability, hybridization, and the like. The nucleic acids described herein can also be added to other added groups, such as peptides (eg, for targeting a target cell receptor in vivo), or passage to the cell membrane, triggering hybridization. It may contain an agent that facilitates cleavage. For this purpose, the nucleic acid may be conjugated to another molecule (eg, a peptide, a cross-linking agent that triggers hybridization, a transport agent, a cleavage agent that triggers hybridization, etc.).

  By administering SDF-1 protein to a weakened, ischemic and / or peri-infarct region, or to cells in a weak, ischemic and / or peri-infarct region of myocardial tissue, By introducing an agent that expresses 1 (ie, SDF-1 agent), increases expression, and / or upregulates SDF in the weakened, ischemic, and / or peri-infarct region of myocardial tissue -1 can be delivered. The SDF-1 protein expressed from the cell may be a genetically modified cell expression product.

  Agents that express, increase expression and / or upregulate SDF-1 can be incorporated into recombinant nucleic acid constructs (typically DNA constructs), introduced into cells of myocardial tissue, and intracellularly Natural or synthetic nucleic acids as described herein that can be replicated may be included. Such constructs may include replication systems and sequences that can transcribe and translate the polypeptide-encoding sequence into a given cell.

  One method for introducing drugs into target cells involves gene therapy. In certain embodiments of the present application, gene therapy may be used to express SDF-1 protein in vivo from cells in weakened, ischemic, and / or peri-infarct areas of myocardial tissue. it can.

  In one aspect of the present application, gene therapy can use a vector comprising a nucleotide encoding SDF-1 protein. “Vector” (sometimes referred to as gene delivery “vehicle” or gene transfer “vehicle”) refers to a macromolecule or molecular complex comprising a polynucleotide that is delivered to a target cell either in vitro or in vivo. . The polynucleotide to be delivered may include a coding sequence of interest in gene therapy. Vectors include, for example, viral vectors (eg, adenovirus (“Ad” retrovirus), adeno-associated virus (AAV) and retrovirus), non-viral vectors, liposomes, and other lipid-containing complexes, and to target cells. Other macromolecular complexes that can mediate delivery of the polynucleotide.

  The vector may also include other elements or functions that further regulate gene delivery and / or gene expression or otherwise provide beneficial properties to the targeted cells. Such other elements include, for example, elements that affect cell binding or targeting (including elements that mediate cell type or tissue specific binding), cells that affect the uptake of vector nucleic acid by the cell Elements, elements that affect the localization of the polynucleotide in the cell after uptake (eg, agents that mediate nuclear localization) and elements that affect the expression of the polynucleotide. Such elements may also include a marker, eg, a detectable marker and / or a selectable marker that can be used to detect or select cells that take up and express the nucleic acid delivered by the vector. Such elements may be provided as natural features of the vector (eg, the use of specific viral vectors containing elements or functions that mediate binding and uptake), or the vector may be rendered to provide such functions. It may be modified.

  The selectable marker may be positive, negative or bifunctional. Selectable positive markers can select cells carrying this marker, while selectable negative markers can selectively exclude cells carrying this marker. Various such marker genes have been described, including bifunctional (ie, positive / negative) markers (eg, Lupton, S., WO 92/08796 published May 29, 1992; and Lupton, S., see WO 94/28143 published December 8, 1994). Such marker genes can provide additional control results that can be advantageous in terms of gene therapy.

  Such a wide variety of vectors are known in the art and are generally available. Vectors for use in the present invention include viral vectors, liquid vectors, and others that can deliver nucleotides to cells in weakened, ischemic, and / or peri-infarcted areas of myocardial tissue Of non-viral vectors. The vector may be a targeting vector, particularly a targeting vector that preferentially binds to cells in the weakened, ischemic, and / or peri-infarct region of the myocardial tissue region. Viral vectors for use in the methods of the present invention exhibit low toxicity to cells in weakened, ischemic and / or peri-infarcted areas of myocardial tissue and are tissue specific. Mention may be made of those that induce the production of therapeutically useful amounts of SDF-1 protein in a manner.

  Examples of viral vectors are vectors derived from adenovirus (Ad) or adeno-associated virus (AAV). Human viral vectors and non-human viral vectors may be used, and the recombinant viral vector may be replication defective in humans. Where the vector is an adenovirus, the vector may comprise a polynucleotide having a promoter operably linked to the gene encoding SDF-1 protein and is replication defective in humans.

  Other viral vectors that can be used in the methods of the present application include herpes simplex virus (HSV) vectors. HSV vectors that are deficient in one or more immediate early genes (IE) are advantageous because they are generally non-toxic, maintain a state similar to latency in target cells, and provide effective transduction of target cells. . Recombinant HSV vectors can incorporate about 30 kb of heterologous nucleic acid.

  Retroviruses such as type C retroviruses and lentiviruses may be used in certain embodiments of the present application. For example, the retroviral vector may be derived from murine leukemia virus (MLV). 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 may contain up to 8 kb of heterologous (therapeutic) DNA instead of viral genes. The heterologous DNA may contain a tissue-specific promoter and SDF-1 nucleic acid. In the method of delivery to cells proximate the wound, the heterologous DNA may encode a ligand for a tissue specific receptor.

  Additional retroviral vectors that can be used are replication deficient lentiviral vectors, including human immunodeficiency (HIV) vectors. See, for example, Vigna and Naldini, J. et al. Gene Med. 5: 308-316, 2000 and Miyoshi et al, J. MoI. Viral. 72: 8150-8157, 1998. Lentiviral vectors are advantageous in that they can infect both actively dividing and non-dividing cells.

  Lentiviral vectors are also very effective when transducing human epithelial cells. Lentiviral vectors for use in the methods of the present invention may be derived from human and non-human (including SIV) lentiviruses. Examples of lentiviral vectors include nucleic acid sequences necessary for vector propagation and a tissue specific promoter operably connected to the SDF-1 gene. These formers may contain a viral LTR, primer binding site, polyprint lact, att site and encapsidation site.

  The lentiviral vector may optionally be wrapped in a suitable lentiviral capsid. Replacing one particle protein with another particle protein from a different virus is called “pseudotyping”. Vector capsids may include viral envelope proteins from other viruses including murine leukemia virus (MLV) or vesicular stomatitis virus (VSV). The use of VSV G protein results in a high titer vector and increases the stability of the vector virus particle.

  Alphavirus-based vectors such as those made from Semliki Forest virus (SFV) and Sindbis virus (SIN) may also be used herein. 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.

  Recombinant replication deficient alphavirus vectors are advantageous because they allow expression of high levels of heterologous (therapeutic) genes and can infect a wide range of target cells. Alphavirus replicons target specific cell types by exposing their virion surface to functional heterologous ligands or binding domains that can selectively bind to target cells that express homologous binding pairs. Also good. Alphavirus replicons may establish long-term heterologous nucleic acid expression in target cells by establishing latency. The replicon may also show transient heterologous nucleic acid expression in the target cell.

  For many viral vectors that are compatible with the methods of the present application, more than one type of promoter may be included in the vector so that more than one type of heterologous gene can be expressed by the vector. In addition, the vector may include a sequence encoding a signal peptide or other moiety that facilitates expression of the SDF-1 gene product from the target cell.

  To combine the advantageous properties of the two viral vector systems, hybrid viral vectors may be used to deliver SDF-1 nucleic acid to the target tissue. Standard techniques for constructing hybrid vectors are well known to those of skill in the art. Such techniques are described in, for example, Sambrook, et al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor, N.A. Y. Or it can be found in any number of laboratory manuals describing recombinant DNA technology. A double-stranded AAV genome in an adenoviral capsid containing a combination of AAV and adenoviral ITR may be used to transduce cells. In another variation, the AAV vector may be placed in a “gutless”, “helper dependent” or “high capacity” adenoviral vector. Adenovirus / AAV hybrid vectors are described in Lieber et al. J. et al. Viral. 73: 9314-9324, 1999. Retrovirus / adenovirus hybrid vectors are described in Zheng et al, Nature Biotechnol. 18: 176-186, 2000. The retroviral genome contained within the adenovirus may be integrated into the target cell genome for stable SDF-1 gene expression.

  Other nucleotide sequence elements that facilitate expression of the SDF-1 gene and vector cloning are further envisioned. For example, the presence of an enhancer upstream of the promoter or a terminator downstream of the coding region can facilitate expression, for example.

  According to another aspect of the present application, a tissue specific promoter may be fused to the SDF-1 gene. By fusing such a tissue-specific promoter within the adenovirus construct, transgene expression is limited to specific tissues. The efficacy and specificity of gene expression conferred by a tissue-specific promoter can be determined using the recombinant adenovirus system described herein.

  In addition to viral vector-based methods, non-viral methods may be used to introduce SDF-1 nucleic acid into target cells. A review of non-viral methods of gene delivery can be found in Nishikawa and Huang, Human Gene Ther. 12: 861-870, 2001. One example of a non-viral gene delivery method of the invention uses plasmid DNA to introduce SDF-1 nucleic acid into cells. Plasmid-based gene delivery methods are generally known in the art. In one example, the plasmid vector may have a structure as schematically shown in FIG. The plasmid vector of FIG. 7 contains a CMV enhancer and a CMV promoter upstream of the cDNA (RNA) sequence of SDF-1α.

  In some cases, the synthetic gene transfer molecule can be designed to produce multimolecular aggregates with the DNA of plasmid SDF-1. These aggregates can be designed to bind to cells in weakened, ischemic, and / or peri-infarct areas of myocardial tissue. Cationic amphiphilic compounds, including lipopolyamines and cationic lipids, may be used to perform receptor-independent SDF-1 nucleic acid transfer to target cells (eg, cardiomyocytes). In addition, previously prepared cationic liposomes or cationic lipids may be mixed with plasmid DNA to create complexes that transfect cells. Methods involving cationic lipid formulations are described in Feigner et al, Ann. N. Y. Acad. Sci. 772: 126-139, 1995 and Basic and Templeton, Adv. Drug Delivery Rev. 20: 221-266, 1996. For gene delivery, the DNA may be coupled to an amphiphilic cationic peptide (Fomaya et al, J. Gene Med. 2: 455-464, 2000).

  Methods involving viral and non-viral elements may be used in the present invention. For example, Epstein Barr virus (EBV) -based plasmids for therapeutic gene delivery are described in Cui et al, Gene Therapy 8: 1508-1513, 2001. In addition, methods involving DNA / ligand / polycationic adducts coupled to adenoviruses are described in Curiel, D. et al. T.A. Nat. Immun. 13: 141-164, 1994.

  Furthermore, SDF-1 nucleic acid can be introduced into target cells by transfecting the target cells using electroporation techniques. Electroporation techniques are well known and can be used to facilitate transfection of cells with plasmid DNA.

  A vector encoding the expression of SDF-1 can be delivered to target cells, if necessary, in the form of an injectable formulation containing a pharmaceutically acceptable carrier (eg, saline). Other pharmaceutical carriers, formulations and dosages can also be used in combination with the present invention.

  In one aspect of the invention, the vector may contain an SDF-1 plasmid (eg, as in FIG. 7). In a preferred embodiment, the SDF-1 plasmid comprises the nucleotide sequence of SEQ ID NO: 6. The SDF-1 plasmid can be transformed into at least one myocardial functional parameter (eg, left ventricular volume, left ventricular area, left ventricular dimension) or cardiac function, subject 6 minute walk test (6 MWT) or New York. By directly injecting an amount of SDF-1 plasmid vector effective to improve Heart Association (NYHA) functional class) into weakened, ischemic and / or peri-infarct areas of myocardial tissue, It can be delivered to cells in weakened, ischemic, and / or peri-infarct areas of myocardial tissue. Recombinant vectors more effectively target vector transfection by directly injecting the vector into or around weakened, ischemic, and / or peri-infarct areas of myocardial tissue Disappearance can be minimized. This type of injection allows local transfection of the desired number of cells, especially around weakened, ischemic and / or peri-infarct areas of myocardial tissue, thereby Maximize the therapeutic effect of gene transfer and minimize the possibility of an inflammatory response to viral proteins.

  In one aspect of the present application, an SDF-1 plasmid solution containing about 0.33 mg / ml to about 5 mg / ml of an SDF-1 plasmid solution is used, and an SDF-1 solution expressing plasmid DNA is injected multiple times. The plasmid may be administered to weakened, ischemic and / or peri-infarct areas. In one example, the SDF-1 plasmid is administered to the weakened, ischemic, and / or peri-infarct region by at least about 10 injections, at least about 15 injections, or at least about 20 injections. Can do. Area and / or number of weakened ischemic and / or peri-infarct areas to be treated by multiple injections of SDF-1 plasmid into the weakened, ischemic and / or peri-infarct areas Will increase.

  Each injection administered to the weakened, ischemic, and / or peri-infarct area may have a volume of at least 0.2 ml. The total volume of the solution containing an amount of SDF-1 plasmid capable of improving at least one functional parameter of the heart administered to the weakened, ischemic and / or peri-infarct area is at least about 10 ml is there.

  In one example, the SDF-1 plasmid can be administered to the weakened, ischemic, and / or peri-infarct region by at least about 10 injections. Each injection administered to the weakened, ischemic, and / or peri-infarct area may have a volume of at least about 0.2 ml. SDF-1 can be expressed in weakened, ischemic, and / or peri-infarct areas for more than about 3 days.

  For example, each injection of a solution containing a plasmid expressing SDF-1 has an injection volume of at least 0.2 ml, and the SDF-1 plasmid concentration per injection is from about 0.33 mg / ml to about 5 mg. / Ml. In another aspect of the application, the at least one functional parameter of the heart is such that the injection volume per site is at least about 0.2 ml, and at least about 10 injection sites have an SDF-1 plasmid concentration per injection of about Improvements can be made by injecting the SDF-1 plasmid at 0.33 mg / ml to about 5 mg / ml into the weakened, ischemic, and / or peri-infarct area of the heart.

  In a congestive heart failure pig model, the injection of SDF-1 plasmid solution has a concentration of less than about 0.33 mg / ml or greater than about 5 mg / ml for a pig model of heart failure and an injection volume per injection site of about 0.2 ml. Below, it was found that there was little, if any, functional improvement in the volume of the left ventricle of the heart to be treated, the area of the left ventricle, the dimensions of the left ventricle, or the function of the heart.

  In another aspect of the present application, the amount of SDF-1 administered to a weakened, ischemic, and / or peri-infarct region that can improve at least one functional parameter of the heart is the therapeutic intervention. Per, greater than about 4 mg and less than about 100 mg. The amount of SDF-1 plasmid administered by the therapeutic intervention herein is the amount of total SDF-1 plasmid administered to a subject during a treatment procedure designed to confer or induce a therapeutic effect. Point to. This amount includes the entire SDF-1 plasmid administered in a single injection for a particular therapeutic intervention, or the entire SDF-1 plasmid administered by multiple injections for a therapeutic intervention. Also good. Administration of 4 mg of SDF-1 plasmid DNA in the porcine model of congestive heart failure by direct injection of SDF-1 plasmid into the heart, the volume of the left ventricle, the area of the left ventricle, the size of the left ventricle or the heart It was found that there was no functional improvement of the function. Furthermore, when about 100 mg of SDF-1 plasmid DNA is administered to the heart by direct injection of SDF-1 plasmid, the volume of the left ventricle of the heart to be treated, the area of the left ventricle, the size of the left ventricle or the functional function of the heart There was no improvement.

  In certain aspects of the present application, in a region weakened, ischemic, and / or peri-infarcted for more than about 3 days after transfection with an SDF-1 plasmid vector, It can be expressed in a therapeutically effective amount or dosage. Expression of SDF-1 at a therapeutically effective dosage or amount for longer than about 3 days can have a therapeutic effect on weakened, ischemic and / or peri-infarct areas. Advantageously, SDF-1 is SDF-1 in the weakened, ischemic and / or peri-infarcted areas for less than about 90 days after transfection with the SDF-1 plasmid vector. -1 can be expressed in a therapeutically effective amount to mitigate potential chronic and / or cytotoxic effects that can inhibit the therapeutic efficacy of administering to a subject.

  The amount, volume, concentration, and / or dosage of SDF-1 plasmid administered to any animal or human is determined by subject size, body surface area, age, specific composition administered, sex, administration time It will be appreciated that and will depend on many factors, including the route of administration, overall health and other drugs administered together. Specific variations of the above-described amounts, volumes, concentrations and / or dosages of SDF-1 plasmid can be readily determined by one skilled in the art using the experimental methods described below.

  In another aspect of the application, the SDF-1 plasmid may be administered by direct injection by catheterization, eg, intracoronary or intraventricular catheterization. In one example, a deformable guide catheter device may be advanced retrogradely through the aortic valve into the left ventricle. Once the device is placed in the left ventricle, the SDF-1 plasmid may be injected into the area surrounding the infarct (both septal and lateral embodiments). Typically, 1.0 ml of SDF-1 plasmid solution may be injected over about 60 seconds. A subject to be treated may receive at least about 10 injections (eg, a total of about 15 to about 20 injections).

  Prior to administration of the SDF-1 plasmid, the myocardial tissue of the subject is imaged and the weakened, ischemic, and / or peri-infarct area is determined prior to administration of the SDF-1 plasmid. Good. Once the weakened, ischemic, and / or peri-infarct region is determined by imaging, more accurately intervene in the weakened, ischemic, and / or peri-infarct region and target the SDF-1 plasmid It becomes possible. Imaging techniques for determining weakened, ischemic, and / or peri-infarct areas of myocardial tissue may include any known cardiac imaging technique. Such imaging techniques may include, for example, at least one of sonocardiography, magnetic resonance imaging, coronary angiography, magnetic stereo mapping, or fluoroscopy. It will be appreciated that other imaging techniques that can determine weakened, ischemic, and / or peri-infarct areas can also be used.

  In some cases, other pharmaceutical agents other than SDF-1 nucleic acid (eg, SDF-1 plasmid) are introduced into weakened, ischemic and / or peri-infarct areas of myocardial tissue, resulting in weakened, ischemic. And / or promote expression of SDF-1 from cells in the peri-infarct region. For example, an agent that increases transcription of a gene encoding SDF-1 uses an agent that increases translation of mRNA encoding SDF-1 and / or decreases degradation of mRNA encoding SDF-1. Increase the level of SDF-1 protein. Increasing the rate of transcription from a gene in the cell can be achieved by introducing an exogenous promoter upstream of the gene encoding SDF-1. Enhancer elements that facilitate the expression of heterologous genes may also be used.

  Other agents include other proteins, chemokines and cytokines that can upregulate SDF-1 expression by weakened, ischemic, and / or peri-infarct areas of myocardial tissue when administered to target cells Can be mentioned. Such agents include, for example, insulin-like growth factor (IGF) -1 (Circ. Res.), Which has been shown to upregulate SDF-1 expression when administered to mesenchymal stem cells (MSC). 2008, Nov 21; 103 (11): 1300-98); Sonic hedgehog (Shh) (Nature Medicine, Volume) shown to upregulate SDF-1 expression when administered to adult fibroblasts. 11, Number 11, Nov. 23); transforming growth factor β (TGF-β), which was shown to upregulate the expression of SDF-1 when administered to human peritoneal mesothelial cells (HPMC); bone marrow stroma Expression of SDF-1 when administered to cells (BMSC), primary human osteoblasts (HOB) mixed with human osteoblast-like cell lines When administered to IL-1β, PDGF, VEGF, TNF-a and PTH (Bone, 2006, Apr; 38 (4): 497-508); bone marrow cells (BMC) that have been shown to regulate Thymosin beta (Curr. Pharm. Des. 2007; 13 (31): 3245-51); shown to upregulate expression; and upregulated SDF-1 expression when administered to bone marrow-derived progenitor cells Hypoxia-inducible factor 1α (HIF-1) (Cardiovasc. Res. 2008, E. Pub.) That has been shown to regulate. These agents are used to exist or be administered such cells that can treat certain cardiomyopathy and can upregulate the expression of SDF-1 for a particular cytokine.

  SDF-1 protein or SDF-1 agent that expresses, increases expression, and / or upregulates SDF-1, undiluted or in a pharmaceutical composition, attenuated myocardial tissue, ischemic And / or can be administered to the area surrounding the infarct. The pharmaceutical composition can locally release SDF-1 or the drug to cells in the weakened, ischemic, and / or peri-infarct area being treated. The pharmaceutical compositions of this application are generally mixed with an acceptable pharmaceutical diluent or excipient (eg, a sterile aqueous solution) to give a range of final concentrations, depending on its intended use. It will contain a fixed amount of SDF-1 or drug. Preparation techniques are generally well known in the art, as exemplified by Remington 'Pharmaceutical Sciences, 16th edition, Mack Publishing Company, 1980, which is incorporated herein by reference. In addition, for human administration, the formulation should meet sterility, pyrogenicity, overall safety and purity standards as required by FDA Office of Biological Standards.

  The pharmaceutical composition may be in unit dosage injectable 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 into sterile injectable solutions or dispersions. The carrier can be, for example, a solvent comprising water, ethanol, polyol (eg, glycol, propylene glycol, liquid polyethylene glycol, etc.), dextrose, saline, or phosphate buffered saline, suitable mixtures thereof and vegetable oils or It may be a dispersion medium. The 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. Non-aqueous vehicles (eg, cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil) and esters (eg, isopropyl myristate) can also be used as solvent systems for compound compositions. Good.

  In addition, various additives that enhance the stability, sterility and isotonicity of the composition may be added, including antimicrobial preservatives, antioxidants, chelating agents and buffers. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.

  In many cases, it will be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like. Prolonged absorption of injectable pharmaceutical forms can be brought about by the use of agents that delay absorption such as aluminum monostearate and gelatin. However, according to the methods described herein, any vehicle, diluent or additive used will have to be compatible with the compound.

  Sterile injectable solutions may be prepared, if desired, by incorporating the compounds utilized in carrying out the methods described herein in the required amount of a suitable solvent, including various amounts of other ingredients. Can do.

  Pharmaceutical “slow release” capsules or “sustained release” compositions or formulations may be used and are generally applicable. Slow release formulations are generally designed to provide a constant drug level over an extended period of time and may be used to deliver SDF-1 or drugs. Slow-acting formulations are typically implanted in the vicinity of weakened, ischemic and / or peri-infarct areas of myocardial tissue.

  Examples of sustained release formulations include a semi-permeable matrix of solid hydrophobic polymer containing SDF-1 or drug and are in the form of molded articles (eg, membranes or microcapsules). Examples of sustained release matrices include polyesters; hydrogels; eg, poly (2-hydroxyethyl-methacrylate) or poly (vinyl alcohol); polylactides, eg, US Pat. No. 3,773,919; L-glutamic acid and γ-ethyl A copolymer of L-glutamate; a non-degradable ethylene-vinyl acetate; a degradable lactic acid-glycolic acid copolymer, such as LUPRON DEPOT (injectable microspheres composed of a lactic acid-glycolic acid copolymer and leuprolide acetate); and poly- D-(-)-3-hydroxybutyric acid is mentioned.

  While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid can release molecules over 100 days, certain hydrogels release proteins for a shorter period of time. When encapsulated, SDF-1 or its drug may be maintained in the body for an extended period of time, resulting in denaturation or aggregation as a result of exposure to moisture at 37 ° C., thus reducing adult activity, and / or Or immunogenicity may change. Rational strategies are available based on the mechanism involved. For example, if the aggregation mechanism involves the generation of intramolecular S—S bonds by thio-disulfide exchange, the sulfhydryl residues are modified, lyophilized from acidic solution, the water content is controlled, and appropriate additives are added. Stabilization is achieved, for example, by developing specific polymer matrix compositions.

  In certain embodiments, liposomes and / or nanoparticles may be used with SDF-1 or a drug. The production and use of liposomes is generally known to those skilled in the art, as summarized below.

  Liposomes are made from phospholipids dispersed in an aqueous medium and spontaneously produce multilamellar concentric bilayer vesicles (referred to as multilamellar vesicles (MLV)). The MLV generally has a diameter of 25 nm to 4 μm. Sonication of the MLV produces small unilamellar vesicles (SUVs) with an aqueous solution in the core and a diameter in the range of 200-500 mm.

  Phospholipids may form various structures other than liposomes when dispersed in water, depending on the molar ratio of lipid to water. At low ratios, liposomes are the preferred structure. The physical characteristics of liposomes vary depending on pH, ionic strength and the presence of divalent cations. Liposomes may exhibit low permeability to ionic and polar substances, but at high temperatures undergo a phase transition and their permeability changes significantly. The phase transition involves a change from a tightly packed regular structure known as the gel state to a loosely packed irregular structure known as the fluid state. Phase transitions occur at characteristic phase transition temperatures and increase permeability to ions, sugars and drugs.

  Liposomes interact with cells by four different mechanisms. Endocytosis by phagocytic cells of the reticuloendothelial system (eg macrophages and neutrophils); either by non-specific weak hydrophobic forces or electrostatic forces, or by specific interactions with cell surface elements Adsorption to the cell surface; fusion with the plasma cell membrane by insertion of the lipid bilayer of the liposome into the plasma membrane with simultaneous release of the liposome content to the cytoplasm; and By movement of liposomal lipids into the intracellular membrane or vice versa. Changing the liposome formulation may change which mechanism works, but more than one mechanism may work simultaneously.

  Nanocapsules are generally capable of capturing compounds in a stable and reproducible manner. In order to avoid side effects due to excessive retention of the polymer in the cell, such ultrafine particles (particle size approximately 0.1 μm) are designed with a polymer that can be degraded in vivo. It should be. Biodegradable polyalkyl-cyanoacrylate nanoparticles that meet these requirements are envisioned for use in the present invention, and such particles are easily manufactured.

  For preparing pharmaceutical compositions from the compounds of the present application, pharmaceutically acceptable carriers can be in any form (eg, solid, liquid, gel, etc.). A solid carrier may be one or more substances and may function as a diluent, flavoring agent, binder, preservative and / or encapsulating material.

Severe ischemic limb The present application further relates to a method of treating a severe ischemic limb in an examiner. This method involves administering JVS-100 by intramuscular injection directly into the upper limb (quadriceps) and / or the lower limb (mainly gastrocnemius) using multiple injection sites. JVS-100 is a sterile biological product composed of a naked DNA plasmid encoding human SDF-1 cDNA (plasmid having the nucleotide sequence of SEQ ID NO: 6) and 5% dextrose.

  Severe ischemic limb (CLI) represents the most advanced stage of atherolimb peripheral vascular disease (PVD) and is associated with a high rate of cardiovascular morbidity, mortality, and major amputation. The incidence of CLI is estimated to have 125,000-250,000 patients per year in the United States and is expected to increase as the population ages. The prevalence of PVD increases significantly with age, affecting approximately 20% of Americans over 65 years of age. Modern nursing standards for CLI individuals include open surgery, lower limb revascularization by either endovascular techniques, or lower limb amputation (ie, when revascularization has failed or is not feasible) . The one-year mortality rate for CLI patients is 25%, and can be as high as 45% for patients who have undergone amputation. Despite the evolution of vascular procedures and surgery, a significant percentage of CLI patients are not suitable for revascularization. Of these patients, 30% require major amputation and 23% die within 3 months. Strategies for opening blocked blood vessels or stimulating angiogenesis are intensively studied.

  Therapeutic angiogenesis is first performed by Dr. Evaluated in 1994 in 71-year-old patients with severe PVD and limb gangrene by Jeffrey Isner, a strategy for treating CLI utilizing vascular-derived or vascular growth factors. Genes encoding these growth factors are injected into ischemic tissue and promote angiogenesis in an attempt to increase perfusion to the ischemic tissue by various mechanisms of action. In this study, the human plasmid phVEGF165 was applied to the distal popliteal artery by balloon angioplasty. Functional and angiographic parameters improve within 12 weeks and spider angiomas and edema grow unilaterally on the affected limb, indicating that this treatment has a local angiogenic effect It suggests. This pioneering experiment shows that experimental CLI therapy that attempts to increase the expression of angiogenic growth factors in ischemic tissue restores and preserves limb function for patients with poor surgical outcomes Suggested that it may be beneficial to give the potential for angiogenesis. Recent studies have shown that chemokines that stimulate angiogenesis can be an important component of therapy aimed at preserving and restoring limb function in patients with severe ischemic limbs.

  Non-viral gene delivery, i.e., applying naked plasmid DNA and expressing therapeutic proteins at specific sites, is a simple delivery method among methods that have been clinically tested in ischemic patients for 15 years. . The safety profile of non-viral gene delivery is also attractive because it does not produce a significant inflammatory response induced by viral vector delivery when compared to delivery by viral vector therapy. A significant amount of preclinical and clinical literature shows that non-viral delivery of therapeutic genes is safe and effective in disease models such as severe ischemic limbs, cardiomyopathy and wound healing. In particular, using plasmids encoding fibroblast growth factor (FGF), vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and injured by bad or damaged vasculature CLI patients are treated in an attempt to increase collateral circulation to Importantly, the clinical use of non-viral FGF and VEGF gene therapy has been shown to be safe with no significant safety issues reported to date. In some phase I and phase II trials, severe non-reconstructed PVD patients with resting pain or tissue necrosis are treated by intramuscular injection of non-viral FGF (NV1FGF). It was. The dose of NV1FGF was increased once (up to 16 mg) and repeatedly (up to 8 mg twice) and injected with naked plasmid DNA in the ischemic thigh and sural. NV1FGF is well tolerated, with a follow-up after 6 months in 38 patients with a marked decrease in pain scale and ischemic ulcer size and an increase in TcPO2 compared to baseline values It was done. A large phase III trial investigating the use of naked plasmid DNA to treat CLI (eg, a multicenter, double-blind, placebo comparison evaluating the efficacy and safety of NV1FGF in CLI patients with skin lesions Study, TAMARIS trial) will be ongoing and will include 490 patients assigned to placebo or NV1FGF intramuscular injection.

  These non-viral gene therapies have not shown safety issues to date, and NV1FGF appears to provide benefits to CLI subjects based on phase II clinical data. When combined with our previous preclinical study of ACRX-100 treatment of ischemic heart failure that shows improved angiogenesis and cardiac function without safety issues, ACRX-100 is safe It is hypothesized to improve angiogenesis and ultimately provide clinical benefit to patients with severe ischemic limbs.

  SDF-1 is up-regulated in multiple tissues after injury and has been determined to be expressed for 4-5 days. Several groups have shown the therapeutic potential of SDF-1 treatment in a wide range of diseases, including ischemic cardiomyopathy, peripheral vascular disease, wound healing, severe ischemic limbs and diabetes. SDF-1 is a potent chemoattractant of stem and progenitor cells, promoting tissue protection and blood vessel growth. Taken together, these reports point to a conserved pathway and mechanism of action by which SDF-1 can promote tissue injury repair and recovery functions. Accordingly, the present inventors have developed ACRX-100 (a non-viral naked DNA plasmid encoding SDF-1 in dextrose solution) for treating ischemic cardiovascular disease. In GLP safety and toxicity studies, ACRX-100 has shown functional benefits up to 30 mg and safety benefits up to 100 mg in a porcine model of heart failure. The present inventors are currently enrolled in a phase I, multicenter, open-label, dose-escalating clinical trial using ACRX-100 to treat patients with ischemic heart failure.

  Our studies have shown that ACRX-100 significantly increases angiogenesis in the heart, consistent with studies from several groups. These studies re-establish stem cell homing to chronically damaged tissues and suggest that from severe ischemic limbs, tissue repair can resume and powerfully improve blood flow. The group has intensively shown that direct intramuscular injection of ACRX-100 into rabbit ischemic hindlimbs promotes tissue revascularization compared to animals receiving control treatment. This suggests that ACRX-100 is a promising therapeutic candidate for treating patients with severe ischemic limbs (CLI). The incidence of CLI is estimated to have 125,000-250,000 patients per year in the United States and is expected to increase as the population ages. Modern nursing standards for patients with CLI include open leg surgery, lower limb revascularization by either intravascular techniques, or lower limb amputation (ie, when revascularization has failed or is not feasible) . The one-year mortality rate for CLI patients is 25%, and can be as high as 45% for patients who have undergone amputation. Despite the evolution of vascular procedures and surgery, a significant percentage of CLI patients are not suitable for revascularization.

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

  The plasmid having the nucleotide sequence of SEQ ID NO: 6 is a naked DNA plasmid designed to express human SDF-1 cDNA in mammalian tissue. The plasmid backbone consists of ColE1 and kanamycin resistance markers. SDF-1 transgene expression is driven by the CMV enhancer / promoter, CMV-intron A and the RU5 translation enhancer. Effective polyadenylation is ensured by the incorporation of the bovine growth hormone poly A sursayl sequence. This is the same plasmid and formulation used to treat heart failure patients. The inventors have developed ACRX-100 for the treatment of patients with severe ischemic limbs. ACRX-100 is formulated for direct intramuscular injection. This planned dosing regime consists of the administration of one or more doses to the upper limb (quadriceps) and / or the lower limb (mainly gastrocnemius) using multiple injection sites. The

  SDF-1 (also known as CXCL12) is a naturally occurring chemokine that is rapidly upregulated in response to tissue injury. Induction of SDF-1 stimulates many protective anti-inflammatory pathways, causes down-regulation of pro-inflammatory mediators (eg, MMP-9 and IL-8), and inhibits cascade-mediated activation of Akt By doing so, cells can be protected from apoptosis. In addition, SDF-1 is a potent chemoattractant of stem and progenitor cells derived from endogenous organ-specific bone marrow to the site of tissue injury, promoting tissue protection and blood vessel growth. Previous studies have shown that SDF-1 expression increases at the site of injury, but expression lasts less than a week, and thus the induced stem cell homing response disappears quickly. Although this short duration of SDF-1 expression reduces the potential for tissue repair, therapeutic intervention that prolongs or reintroduces the ability of SDF-1 to stimulate the homing process of stem cells may result in damaged tissue. It may be beneficial for patients with Based on this hypothesis, Dr. Penn's laboratory re-introduced SDF-1 by injecting cardiac fibroblasts overexpressing SDF-1 one month after MI, and was able to identify endogenous organs derived from bone marrow. Reestablishment of stem cell homing to the heart, growth of new blood vessels in damaged tissue, showed greater than 80% increase in cardiac function. When skeletal myoblasts overexpressing SDF-1 were injected 8 weeks after MI, cardiac function was significantly improved. In addition, SDF-1 improved cardiac function by increasing recruitment of circulating stem cells to the damaged tissue, creating new blood vessels, and increasing perfusion to the damaged area. These effects, along with significant protection of the myocardium, showed that when MSCs overexpressing SDF-1 were delivered to rats after acute MI, cardiac function increased by 240%. This biological response was conserved in many organ systems in response to ischemic injury.

  The benefits of SDF-1 treatment observed by the inventors have been demonstrated by recent studies in other independent laboratories. SDF-1 is delivered by nanofiber-encapsulated proteins in rats after acute MI, by recombinant proteins via fibrin patches in post-MI mice, or by direct intramyocardial injection of proteins in mice after acute MI If done, improves cardiac function in ischemic cardiomyopathy. Injection of plasmid encoding SDF-1 into the MI border region has been shown to attract circulating stem cells to the MI border region. Similarly, regenerative cell therapy using myoblasts or muscle stem cells grown from the patient's own muscle and genetically engineered to express SDF-1 has shown the full clinical efficacy of treating heart failure and ischemia Will be tested on patients in clinical trials to repair damaged tissue and increase function in REGEN trials. Together, these data published by multiple laboratories indicate that, independent of this delivery method, overexpression of SDF-1 provides a functional benefit in diseases with ischemic pathogenesis. Show.

  Restimulation of SDF-1 expression in ischemic muscles provides a high therapeutic potential for treatment of CLI by regenerating blood vessels damaged by poor blood flow. This provides an opportunity to repair and retain function in limb regeneration. Vascular regrowth has been shown to improve limb salvage in many clinical trials using VEGF cells or stem cells. Yamaguchi et al report that local delivery of SDF-1 protein enhances ischemic hindlimb angiogenesis after EPC administration, which enhances angiogenesis induced by EPC Suggests to do. Similarly, Hiasa et al have shown that SDF-1 gene transfer enhances ischemia-induced angiogenesis and angiogenesis in vivo by pathways associated with VEGF / eNOS.

  These observations were recently confirmed in an analysis of skeletal muscle obtained from patients undergoing chronic CLI peri-knee amputation. Expression of SDF-1 and its receptor CXCR4 increased in skeletal muscle fibers and microvessels, respectively. This suggests that the SDF-1 / CXCR4 repair axis was chronically upregulated in ischemic limb muscles in an attempt to stimulate microvascular growth for normal healing. A low level is not enough. By using ACRX-100 gene therapy to amplify the signal, we will be able to construct synergistically with the healing process that has already begun in damaged tissue. Thus, ACRX-100 represents a novel chemokine therapy for angiogenesis for the next generation of therapy.

Overview of Master Cell Bank and Bulk Plasmid Production The manufacture of drug substances used in phase I clinical trials for heart failure was accomplished on a 300 L scale using dedicated reagents and materials. For the Phase I / II CLI trial proposed by the inventors, the inventors have conducted a streamlined drug production using our authoritative master cell bank and an established quality testing service. The experience of the inventors of the present application obtained by using it can be utilized. All media were prepared using common animal-free ingredients and sterile USP grade water or higher quality. All buffers for bacterial lysates are marketed for production based on their respective certificate of analysis. Large scale dissolution is achieved with bubble elements without the use of RNase. All chromatographic buffers are marketed after USP grade or equivalent reagents are manufactured from Aldevron and meet internal specifications. All aseptic processing takes place in an environmentally controlled clean room. The clean room facilities consist of a preparation room (Class 10,000, ISO 7) for clothing with two doors and a wall window that leads to the main clean room (Class 10,000). ISO 6) room with Class-100 (ISO 5) biosafety food for final filling / finishing. Procedures for use and management, cleaning (after each batch) and environmental monitoring are performed in place. The bulk plasmid solution was sterilized by filtration, concentrated if necessary, and stored at −75 ° C. + 5 ° C. for final dispensing into drug product vials.

  The final step in this production is aseptic dilution of the sterile solution in a sterile bulk container. Thus, for early development, the drug substance was specified to be sterile.

Drug Product Manufacturing Overview Bulk plasmids are transferred to a Class ISO 6 clean room containing an ISO 5 biological safety cabinet (BSC). All contacting materials are pre-sterilized and pyrogen-free, and the disposable items marketed for use are based on the manufacturer's certificate of analysis. Processing is performed by BSC. First, the bulk plasmid solution was diluted with USP dextrose (5%) for injection to the final target concentration. After mixing, the sterile plasmid solution is then manually pipetted into a pre-sterilized final serum vial. The product is stored at -75 + 5 ° C. A representative list of media elements and downstream reagents will be given in IND along with their quality information.

Stability Stability studies have shown that the preclinical lot of ACRX-100 is stable at -20 ° C for up to 1 year after manufacture. Clinical grade ACRX-100 (Lot 24370D-F) is currently under stability (5 ± 3 ° C.) and standard (−20 ± 4 ° C.) stability studies (Table 1) and the results are: Expected to exceed preclinical stability findings. Results are reported in IND. The following tests are conducted as part of a stability program that includes appearance, identity (agarose gel electrophoresis), concentration (A260 / A280), homogeneity (density), effectiveness, and pH. The sterility test is conducted at the 6th month after launch, and once a year thereafter.

Ｘ＝すべての条件、保存無菌性Ｙ＝無菌性

X = all conditions, storage sterility Y = sterility

Non-clinical pharmacology and toxicology of ACRX-100 “Chemokine drugs” have received considerable attention in recent years due to their ability to stimulate stem cells and mobilize stem cells to the site of tissue injury. ACRX-100 is a gene therapy agent that delivers human chemokine SDF-1 by gene expression in human cells. The active protein SDF-1 produced by ACRX-100 enhances the function of the heart of the myocardium damaged by ischemia from a distance and temporarily recruits CXCR4 positive stem cells to recruit wounded epithelium. It has been shown to improve the healing rate. SDF-1 exhibits pro-angiogenic activity in patients with acute CLI and is down-regulated in patients with chronic CLI, indicating that treatment directed at new SDF-1 expression in chronic CLI is derived from bone marrow into adult blood vessels This suggests that angiogenesis can be enhanced by mobilizing these cells.

  A plasmid having the nucleotide sequence of SEQ ID NO: 6 and formulated into the drug product ACRX-100 was tested in animal models of ischemic cardiovascular disease and hindlimb ischemia. ACRX-100 was tested by intracardiac administration in a porcine model of heart failure for efficacy, safety and biodistribution, and a noxious dose (NOAEL) of 100 mg was established. Currently, the same formulation of ACRX-100 administered at a lower dosage than used in non-clinical and clinical trials for heart failure is evaluated to determine the potential therapeutic benefit provided to CLI. Single dose efficacy, toxicology and biodistribution studies with ACRX-100 were performed in hindlimb ischemic rabbits. This study shows that ACRX-100 has therapeutic potential for the treatment of severe ischemic limbs, and intramuscular injection of ACRX-100 into rabbit ischemic hind limbs is a sign of toxicity or histopathological changes. It was not. Furthermore, the plasmid having the nucleotide sequence of the plasmid of SEQ ID NO: 6 was essentially excreted from all organs, but not from the ischemic limb 60 days after treatment after a single dose. Repeated dose efficacy and safety (toxicology and biodistribution) studies are planned in a rabbit model of hindlimb ischemia to take up to 3 ACRX-100 doses in CLI patients.

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

Example 1:
Stromal cell derived factor-1, SDF-1, is a naturally occurring chemokine whose expression is rapidly upregulated in response to tissue injury. Induction of SDF-1 can stimulate many protective anti-inflammatory pathways, cause down-regulation of pro-inflammatory mediators (eg, MMP-9 and IL-8) and protect cells from apoptosis. In addition, SDF-1 is a potent chemoattractant of stem and progenitor cells derived from endogenous organ-specific bone marrow to the site of tissue injury, promoting tissue protection and blood vessel growth. Based on the observation that increased SDF-1 expression improves cardiac function in an ischemic animal model, we have developed a non-viral naked DNA SDF for the treatment of ischemic cardiovascular disease. The focus was on developing a plasmid encoding -1. During the series of developments, plasmids were optimized based on the results of the cell culture and small animal studies described below. The plasmid having the nucleotide sequence of SEQ ID NO: 6 was selected based on its ability to express the transgene in heart tissue and always improve cardiac function in preclinical animal models of ischemic cardiomyopathy. SDF-1 transgene expression into the plasmid having the nucleotide sequence of SEQ ID NO: 6 is driven by the CMV enhancer / promoter, CMV-intron A and the RU5 translation enhancer. The pharmaceutical product JVS-100 (formerly ACRX-100) is composed of a plasmid having the nucleotide sequence of SEQ ID NO: 6 in 5% dextrose.

  An initial study of a rat model of heart failure is that ACL-01110S (SDF-1 expression precursor to a plasmid having the nucleotide sequence of SEQ ID NO: 6) directly injected the plasmid into the infarct border region of the rat heart 4 weeks after MI. Later, it was shown that heart function was improved. The benefit lasted at least 8-10 weeks after injection and correlated with increased angiogenesis in animals treated with ACL-01110S. ACL-01110S was modified to optimize the expression profile.

Plasmid dose-dependent expression in the MI rat model To determine the plasmid dosage per injection that gives maximum expression in rat heart tissue, increasing the dosage of ACL-00011L luciferase plasmid (10, 50, 100, 500 μg) ) Was injected into the heart of infarcted rats. Lewis rats were incised midline sternum, the left anterior descending branch (LAD) was permanently ligated, and injected with ACL-00011L plasmid in 100 μl PBS at one location around the MI. Non-invasive bioluminescence imaging (Xenogen, Hopkinton, Mass.) Showed systemic luciferase expression at each dosage cohort (n = 3) at baseline, 1, 2, 3, 4, and 5 days after injection. It was measured. Peak expression increased to a dose of 100 11 g and was saturated at higher doses. Based on this dose-response curve, a dose of 100 μg was determined to be sufficient for maximal plasmid expression in the rat heart. ACL-00011L expressed the luciferase gene from a vector backbone equivalent to that used in the construction of ACL-00011S expressing SDF-1.

Comparison of Vector Expression in the Heart in a Rat Model of Ischemic Heart Failure Several SDF-1 plasmid candidate luciferase equivalents were examined for expression in heart tissue in a rat model of myocardial infarction (MI). Plasmid candidates differed in the presence of promoter elements and enhancer elements that drive expression. Lewis rats were median sternotomy and the left anterior descending branch (LAD) was permanently ligated and the chest closed. Four weeks later, the chest was reopened, and a plasmid expressing luciferase was directly injected into 4 sites around myocardial infarction (100 μl per injection, 100 μg). At 1 day, 2 days, 4 days, 6 days, 8 days, 10 days (and thereafter every 3-4 days) after injection, rats are anesthetized, injected with luciferin, and using a whole body Xenogen Luciferase imaging system. Imaged.

  Two CMV driven test plasmids, ACL-00011L and ACL-01110L, yielded detectable luciferase expression within 24 hours after injection, with the first peak of expression 2 days after injection.

  ACL-01110L peak expression was 7-fold greater than ACL-00011L, and expression was about 10 days longer (lasting 16 days after injection). In contrast, ACL-00002L (a plasmid driven by αMHC) did not show the first peak, but was expressed at a much lower level for 25 days after injection. These results can use a plasmid driven by CMV for localized transient protein expression in the heart, and the therapeutic protein expression timeframe can be adjusted by including an enhancer element. Supports previous work showing what can be done.

SDF-1 Plasmid Efficacy in a Rat Model of MI To determine whether the heart benefit for function can be achieved, plasmids encoding SDF-1 were tested in the MI rat model. Lewis rats were median sternotomy and LAD was permanently ligated in the immediate vicinity of the first bifurcation. After 4 weeks, the chest was reopened and one of three plasmids expressing SDF-1 (ACL-01110S, ACL-00011S or ACL-00021S) or saline was injected into 4 sites around the MI (injection). 100 μg per 100 μl of liquid).

  At baseline (before injection), 2 days, 4 days and 8 days after injection, the rats were anesthetized and imaged by M-mode echocardiography. LVEF, left ventricular diameter shortening and LV dimensions were measured by a trained sonographer who was randomized and blinded.

  The CMV driven plasmids, ACL-01110S and ACL-00011S, both were observed to have a strong tendency to improve cardiac function compared to saline controls. ACL-01110S induced a significant increase in left ventricular shortening rate at 4 weeks and persisted for up to 8 weeks after injection. In contrast, no functional difference was observed between plasmid ACL-00021S driven by αMHC and saline. Furthermore, compared to controls, animals treated with ACL-01110S and ACL-00011S have significantly increased macrovascular density (ACL-01110S: 21 ± 1.8 vessels / mm 2; ACL-00011S: 17 ± 1). .5 vessels / mm2; saline: 6 ± 0.7 vessels / mm2, versus saline, both p <0.001), reduced infarct size (ACL-01110S: 16.9 ±) 2.8%; ACL-00011S: 17.8 ± 2.6%; saline: 23.8 ± 4.5%). Importantly, treatment with ACL-01110S showed the greatest improvement in cardiac function and angiogenesis, indicating that the infarct size was the greatest decrease.

  In summary, in a rat model of ischemic heart failure, plasmids encoding SDF-1 driven by the CMV promoter both provide a cardiac benefit for function and angiogenesis compared to saline treatment. Increased and reduced infarct size. For all parameters tested, ACL-01110S gave the most significant benefits.

Transfection efficiency of the plasmid having the nucleotide sequence of SEQ ID NO: 6 and ACL-01010 Sk in H9C2 cells In vitro transfection of H9C2 cardiomyocytes without transfection reagent (ie naked plasmid DNA in cells in culture) In vivo transfection efficiencies yielding plasmid vectors, plasmids having the nucleotide sequence of SEQ ID NO: 6 and ACL-01010Sk were estimated from our GFP embodiment. H9C2 cells were cultured in vitro and various amounts of pDNA (0.5 μg, 2.0 μg, 4.0 μg, 5.0 μg) were added to 5% dextrose. A GFP vector was constructed from the backbone of plasmid (ACL-01110G) or ACL-01010Sk (ACL-01010G) having the nucleotide sequence of SEQ ID NO: 6. Three days after transfection, GFP fluorescence was assessed by FACS to estimate transfection efficiency. The transfection efficiency of ACL-01110G and ACL-01010G vectors in 5% dextrose ranged from 1.08 to 3.01%. For each amount of pDNAs tested, both vectors had similar transfection efficiencies in vitro. The inventors conclude that the 1-3% transfection efficiency observed in this study is consistent with trials from previous studies showing similar levels of transfection efficiency in vivo. Specifically, JVS-100, although limited, transfects a sufficient number of heart cells to produce a therapeutic amount of SDF-1.

Example 2:
Porcine Myocardial Plasmid Expression To test the efficacy and safety of ACRX-100, the left anterior descending branch (LAD) porcine occlusion / reperfusion MI model was selected as an appropriate large animal model. In this model, a 4-week recovery is given between MI and treatment to allow time for further cardiac remodeling and mimic chronic ischemic heart failure.

Surgical Procedure Yorkshire pigs were anesthetized, heparinized so that the activated clotting time (ACT) was 300 seconds or longer, and placed in the dorsal position. To determine the contour of the LV, left ventricular angiography was performed in an anterior-posterior, side view.

Delivery of Luciferase Plasmid to Porcine Myocardium A deformable guide catheter device is advanced through the aortic valve into the left ventricle, the guide wire is removed, the LV endocardial needle injection catheter is routed through the guide catheter, and the LV cavity. Put it in. The luciferase plasmid was injected at a given volume and concentration into 4 sites on either the septal or lateral side of the heart. A combination of five plasmid concentrations (0.5, 2, or 4 mg / ml) and site injection volume (0.2, 0.5, 1.0 ml) was tested. The plasmid was buffered at 0.5 mg / gl with USP dextrose and everything else was buffered with USP phosphate buffered saline. For each injection, a needle was inserted into the endocardium and the gene solution was injected at a rate of 0.8-1.5 ml / min. After injection, the needle was held in place for 15 seconds and then withdrawn. After the injection was completed, all instruments were removed, the incision was closed and the animal was allowed to recover.

Myocardial tissue collection Animals were dissected 3 days after injection. After euthanasia, the heart was removed, weighed, and perfused with lactated Ringer's solution until the blood was clear. The LV was opened and the injection site was identified. A 1 cm leg piece of tissue was collected around each injection site. Four cube pieces were collected as a negative control from the back wall away from the 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 5 ml glass tubes. Lysis buffer (0.5-1.0 ml) was added and the tissue was disrupted on ice using a Polytron homogenizer (PT1200 type). The tissue homogenate was centrifuged and the protein concentration of the supernatant was determined for each tissue sample using a Bio-rad Detergent-Compatible (DC) protein assay and a standard curve of known amounts of bovine serum albumin (BSA). Tissue sample homogeneity (1-10 μl) was assayed using a luciferase assay kit (Promega).

  The experimental results are shown in FIG. As shown in FIG. This data shows that the expression of the vector increases with increasing injection volume and increasing DNA concentration.

Example 3-Improvement of cardiac function by treatment with SDF-1 plasmid in a porcine model of ischemic cardiomyopathy Induction of myocardial infarction Heparin so that Yorkshire pigs are anesthetized and the activated clotting time (ACT) is 250 seconds or more. And put it in the dorsal position. The balloon catheter was introduced by advancing in the LAD direction through the guide catheter to the first major bifurcation of the LAD. The balloon was then inflated to a pressure sufficient to ensure complete occlusion of the artery and left inflated in the artery for 90-120 minutes. Using fluoroscopy, the balloon was completely inflated and deflated. The balloon was then removed, the incision was closed and the animal was allowed to recover.

Registration Criteria One month after MI, the function of each pig's heart was assessed by echocardiography. Pigs were enrolled in this study if the LVEF was less than 40% and the LVESV was greater than 56.7 ml.

Surgical Procedure Each registered pig was anesthetized, heparinized so that the activated clotting time (ACT) was 300 seconds or longer, and placed in the dorsal position. To determine the contour of the LV, left ventricular angiography was performed in an anterior-posterior, side view.

Delivery of the SDF-1 plasmid having the nucleotide sequence of SEQ ID NO: 6 to the myocardium Each pig was randomly divided into one of three types of death points at 3, 30, or 90 days after treatment and control ( Four (20 injections, buffer only), low (15 injections, 0.5 mg / ml), medium (15 injections, 2.0 mg / ml), or high (20 injections, 5.0 mg / ml) Randomly divided into one treatment group.

  All plasmids were buffered with USP dextrose. The injection procedure is described below. The deformable guide catheter device was advanced through the aortic valve into the left ventricle, the guide wire was removed, and the LV endocardial needle injection catheter was placed through the guide catheter into the LV cavity. Randomized doses of SDF-1 plasmid or buffer were placed in a 1 ml syringe and attached to a catheter. The volume of each injection was 1.0 ml. For each injection, a needle was inserted into the endocardium and this solution was injected over 60 seconds. After injection, the needle was held in place for 15 seconds and then withdrawn. After the injection was completed, all instruments were removed, the incision was closed and the animal was allowed to recover.

  At dissection, tissue samples from the heart and other organs were excised and snap frozen for PCR and histopathology analysis.

Assessment of cardiac function Each animal has cardiac function as assessed by standard two-dimensional echocardiography at 0, 30, 60, and 90 days (until dissection) after injection. It was. Measurements of left ventricular volume, area, and wall motion score were performed by an independent central laboratory. The measured efficacy parameters are shown in Table 1 below.

  The influence of the SDF-1 plasmid on function improvement is shown in FIGS. FIGS. 2-4 show that low dose and medium volume SDF-1 plasmid improves LVESV, LVEF and wall motion score index 30 days after injection compared to control, while showing no benefit at high dose It shows that. FIG. 5 shows that heart benefits at low and medium doses last for 90 days. Both show a significant attenuation of pathological remodeling, ie a small increase in LVESV compared to the control.

Evaluation of angiogenesis The animals sacrificed on day 30 were evaluated for left ventricular blood vessel density using 7-9 tissue samples collected from each formalin-fixed heart. Genomic DNA was extracted and effectively purified from formalin-fixed tissue samples using a mini-column purification procedure (Qiagen). Samples from SDF-1 treated animals and control animals were examined for the presence of plasmid DNA by quantitative PCR. Slides were prepared using 3-5 tissue samples (excluding control animals) found to contain at least 4 times higher copy of plasmid DNA than background for each animal and immunostained with isolectin. Cross sections were identified and blood vessels were measured in 20-40 randomly selected fields per tissue. Blood vessels per field of view were converted to blood vessels / mm 2 and averaged for each animal. For each dose, data is reported as mean vessel number / mm 2 from all animals that received that dose.

  FIG. 6 shows that both doses that benefited the function also significantly increased vascular density at day 30 compared to the control. In contrast, high doses that did not improve function did not substantially increase vascular density. This data provides a putative biological mechanism by which the SDF-1 plasmid improves ischemic cardiomyopathy heart function.

Biodistribution data JVS-100 distribution of cardiac and non-cardiac tissues was measured in key efficacy and toxicity studies in the MI pig model at 3, 30, and 90 days after injection. In heart tissue, the mean JVS-100 plasmid concentration increased with dose at each time point. At each dose, JVS-100 excretion was observed 3 days, 30 days, and 90 days after injection, with approximately 99.99999999% excreted from heart tissue on day 90. JVS-100 is distributed in non-cardiac organs with relatively high blood flow (eg heart, kidney, liver and lung), the highest concentration being shown 3 days after injection. JVS-100 was mainly present in the kidney, which was consistent with renal excretion of the plasmid. Low levels persisted on day 30, and JVS-100 was essentially undetectable in non-cardiac tissue on day 90.

Conclusion Treatment with JVS-100 significantly increased blood vessel production and improved cardiac function in pigs with ischemic heart failure after a single injection of 7.5 and 30 mg into the endocardial myocardium. The highest dose tested, JVS-100 (100 mg), showed a trend towards increased angiogenesis but no improvement in cardiac function. None of the doses of JVS-100 were associated with signs of toxicity, clinical pathological parameters or adverse effects on histology. JVS-100 was mainly distributed in the heart, and about 99.999999% was excreted from the heart tissue 90 days after treatment. JVS-100 is distributed in non-cardiac organs with relatively high blood flow (eg heart, kidney, liver and lung), with the highest concentration shown in the kidney 3 days after injection. JVS-100 was essentially undetectable in the body 90 days after injection, and only a negligible amount of the administered dose was found in non-cardiac tissue. Based on these findings, the MI pig model JVS-100 noxious amount (NOAEL) was 100 mg administered by endocardial myocardial injection.

Example 4-Pig Exploration Survey: LUC injection by hepatic artery injection in chronic MI pigs Method One pig with previous LAD occlusion / reperfusion MI and EF greater than 40% has the nucleotide sequence of SEQ ID NO: 6 The plasmid was injected using a hepatic artery catheter. Two injections into the LAD and two injections into the LCX were made with an injection volume of 2.5 ml for a total injection time of 125-130 seconds. An additional 3.0 ml injection of LCX was made with contrast mixed with the plasmid for a total injection time of 150 seconds.

Animal sacrifice and tissue collection Animals were euthanized 3 days after injection. After euthanasia, the heart was removed, blood drawn and placed on an ice-cold cutting board for further dissection by the investigator or pathologist. The right ventricle was opened and uninjected myocardium was obtained from the diaphragm. The right ventricle was cut from the heart and allowed to cool and protect the myocardium. A new surgical scalpel was used for each cut.

  Next, the entire left ventricle was cut by opening the left ventricle and dividing it into 6 parts from the apex to the bottom. The LV was uniformly divided into three pieces. After excision, each cut piece was laid flat. Each piece (three LV pieces, one RV piece, and one pectoral muscle) was placed in a labeled container separately, cooled with water ice to protect the myocardium, and analyzed for luciferase analysis. Moved for.

Luciferase imaging All collected tissues were soaked in luciferin and imaged with a Xenogen imaging system to determine plasmid expression.

Results A representative image of the heart is shown in FIG. A colored spot indicates a luciferase expression region. These spots had a relative light output (RLU) greater than 106 units and more than twice the background. This data indicated that the catheter delivered enough plasmid to produce significant plasmid expression in a significant portion of the heart.

Example 5-Clinical Trial Example JVS-100 was administered at increasing dosages to treat HF in subjects with ischemic cardiomyopathy. Safety is tracked by showing every adverse event (AE) at each dose, and the primary safety endpoint is the number of major heart AEs at day 30. In each cohort, the subject is given a single dose of JVS-100. In all cohorts, treatment efficacy includes standard ultrasound cardiography measurements, cardiac perfusion with single photon emission computed tomography (SPECT) imaging, New York Heart Association (NYHA) classification, 6-minute walking distance and quality of Assessed by measuring the effect of life on heart function.

  All subjects had a known medical history such as systolic dysfunction, previous MI and were confirmed to be currently not cancer by appropriate cancer screening to date. All subjects will be screened by an echocardiographic measurement with a physician. Further baseline testing is performed, eg SPECT perfusion imaging. Each subject received 15 1 ml injections of JVS-100 delivered by an endocardial needle catheter to a site within the infarct border region. Three cohorts (A, B, C) are examined. As shown in Table 2, the dosage is gradually increased by increasing the amount of DNA per injection site while keeping the number of injection sites constant at 15 and the injection volume at 1 ml. Subjects were monitored until approximately 18 hours after injection, and hospital visits were planned 3 and 7 days after injection to ensure no safety issues. The patient stays in the hospital until 18 hours after injection to ensure that all necessary blood (ie, amount of heart enzyme, plasma SDF-1 protein) is collected. All subjects were followed up to day 30 (1 month), day 120 (4 months), and day 360 (12 months) to assess safety and cardiac function. The primary safety endpoint is a major adverse cardiac event (MACE) within one month of treatment delivery. During the study, the AE is followed for each subject. Measure the following safety and efficacy endpoints.

Safety • Number of major adverse cardiac events (MACE) 30 days after injection • Adverse events throughout the 12-month follow-up period • Laboratory blood analysis (cardiac enzymes, CBC, ANA)
・ Plasma SDF-1 level ・ Physical assessment ・ Ultrasonography ・ AICD monitoring ・ ECG

efficacy:
Changes from baseline in LVESV, LVEDV, LVEF and wall motion score indices Changes from baseline in NYHA classification and quality of life Changes from baseline in perfusion determined by SPECT imaging Change from baseline in distance

  Based on preclinical data, delivery of JVS-100 is expected to elicit an improvement in cardiac function and symptoms at 4 months and persist until 12 months. 4 months after JVS-100 injection, 6 months walking distance improved by more than about 30 meters, quality of life improved by about 10%, and / or NYHA classification improved by about 1 class compared to baseline values Is expected to. Similarly, the inventors expect LVESV, LVEF, and / or WMSI to improve by about 10% relative to the baseline value.

Example 6-Evaluation of cardiac function by echocardiography in pigs with chronic heart failure after treatment with a plasmid having the nucleotide sequence of SEQ ID NO: 6 or ACL-01010 Sk Objectives The purpose of this study is in a porcine model of ischemic heart failure Comparing cardiac function against SDF-1 plasmid having the nucleotide sequence of SEQ ID NO: 6 and ACL-01010 Sk after endocardial myocardial delivery.

  This test compared the efficacy of ACL-01010 Sk with the SDF-1 plasmid having the nucleotide sequence of SEQ ID NO: 6 for improved function in a porcine ischemic heart failure model. In this study, the plasmid was delivered by an endocardial myocardial needle injection catheter. Efficacy was assessed by measuring the effect of treatment on cardiac remodeling (ie, left ventricular volume) and function (ie, left ventricular ejection fraction (LVEF)) by echocardiography.

Methods Briefly, male Yorkshire pigs were given myocardial infarction by LAD occlusion by 90 minutes of balloon angioplasty. Pigs with an ejection fraction of less than 40% were enrolled 30 days after infarction as measured by M-mode echocardiography. Pigs are injected in PBS with either phosphate buffered saline (PBS, control), a plasmid having the nucleotide sequence of SEQ ID NO: 6, or ACL-01010 Sk using an intraventricular needle injection catheter delivery system 3 Randomly divided into groups (Table 3).

  Ultrasound cardiograms were recorded before injection, 30 days and 60 days after injection. Table 4 below defines the variables as mentioned in this report.

Results Baseline ultrasound at the time of the first injection (30 days after MI) for all animals (n = 9) enrolled in this report, as reported in the central laboratory of the echocardiography. Cardiac examination characteristics are presented in Table 5 below.

  Table 5 shows LVESV, LVEF, LVEDV on days 0 and 30 after the first injection. Control PBS animals showed an increase in LVESV and LVEDV and no improvement in LVEF, consistent with this heart failure model. This treatment group did not decrease heart volume or increase LVEF compared to controls. Similar results were obtained 60 days after the first injection.

Example 7:
We observed a strategy to enhance stem cell homing to the peri-infarct region by transmyocardial delivery of SDF-1 catheter system in a porcine model of myocardial infarction to determine whether to improve left ventricular perfusion and function. Catheter techniques have been successfully used for cell transplantation and delivery of angiogenic growth factors in humans.

  Female German Landrace pigs (30 kg) were used. After fasting overnight, the animals were anesthetized and intubated.

  The animal was placed in a supine position and a 7 French sheath was placed on the femoral artery. The over-the-wire balloon was advanced to the distal LAD. The balloon was then inflated at 2 atm and agar beads were slowly injected over 1 minute into the distal LAD via a balloon catheter. After 1 minute, the balloon was deflated and distal LAD occlusion was shown by angiography. After induction of myocardial infarction, animals were monitored for 3-4 hours until heart rate and blood pressure were stable. The arterial sheath was removed, carprofen (4 mg / kg) was administered intramuscularly, and the animal was released from the ventilator. Two weeks after myocardial infarction, the animals were anesthetized. The animals were placed in a supine position and electromechanical mapping of the left ventricle was performed with an 8F femoral sheath. After a complete mapping of the left ventricle was obtained, human SDF-1 (Peprotec, Rocky-Hill, NJ) was injected with 18 injections (5 μg in 100 ml saline) by injection catheter and infarct area and peri-infarct. Delivered to the area. 5 μg per injection was used and adjusted to the reported efficiency of catheter injection. Only when the tip of the catheter is perpendicular to the wall of the left ventricle, the loop stability is less than 2 mm, and the protrusion of the needle into the myocardium causes ventricular ectopic beats, slowly over 20 seconds An injection was made. Control animals received the same procedure using sham injections. An echocardiography excluded post-intervention epicardial fluid.

  Twenty animals completed this test protocol. Eight are control animals and 12 are animals treated with SDF-1. Due to technical problems, only 6 control animals can be evaluated for myocardial perfusion imaging. The location of the infarct was an anterior septal infarction in all animals.

  The ratio of infarct size to the left ventricle as determined by tetrazolium staining was 8.9 ± 2.6% in the control group and 8.9 ± 1.2% in the SDF-1 group. Left ventricular muscle volume was similar in both groups (83 ± 14 ml vs. 95 ± 10 ml, p = ns). Immunofluorescence staining was found to be significantly more vWF-positive vessels in the peri-infarct area in animals treated with SDF-1 than in control animals (349 ± 17 / mm2 vs. 276 ± 21 / mm2). , P <0.05). When compared to control animals, a significant loss of collagen in the area surrounding the infarct was observed in animals treated with SDF-1 (32 ± 5% vs. 61 ± 6%, p <0.005). The number of inflammatory cells (neutrophils and macrophages) in the peri-infarct area was similar in both groups (332 ± 51 / mm2 vs. 303 ± 55 / mm2, p = ns). Overall myocardial perfusion did not change from baseline to follow up SPECT and was not different between groups. The final infarct size was similar in both groups and was well compared to the tetrazolium staining results. A segmental analysis of myocardial perfusion showed that tracer uptake at the apex and anterior septal regions was reduced and there was a significant difference between each part of the myocardium. However, baseline and follow-up tracer uptake was nearly identical in control animals and animals treated with SDF-1. There was no difference between groups in end-diastolic volume and end-systolic volume. However, stroke volume increased in control animals and slightly decreased in animals treated with SDF-1. The difference between both groups was significant.

  Similarly, ejection fraction increased in control animals and decreased in animals treated with SDF-1. The difference between groups showed a strong tendency (p = 0.05). Local shortening, another parameter of ventricular mechanical function, did not change in control animals. However, in animals treated with SDF-1, local shortening was significantly reduced, resulting in significant group differences. There was no significant difference in monopolar voltage within and between groups. A significant correlation was shown between baseline ejection fraction, stroke volume, and local shortening of baseline (EF and LS: r = 0.71, SV and LS: r = 0.59). Similar results were obtained for follow-up values (EF and LS: r = 0.49, SV and LS: r = 0.46). Changes in local shortening were significantly correlated with changes in ejection fraction (r = 0.52) and changes in stroke volume (r = 0.46). There was no correlation between local shortening and end-diastolic volume (baseline r = −0.03, follow-up r = 0.12), and there was no correlation between ejection fraction and end-diastolic volume ( Baseline r = −0.04, follow-up r = 0.05). Partial analysis of EEM data showed a decrease in monopolar voltage and local shortening in the anterior septal region, indicating a significant difference between myocardial portions at baseline. The distribution of monopolar voltage values in the myocardial region was similar at both baseline and follow-up. Partial local shortening 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 portions of the left ventricle. There was no significant interaction for assignments to SDF-1 and follow-up versus baseline.

  The above studies have shown that a single application of SDF-1 protein is not sufficient to provide a functional heart benefit.

Example 8-Expression of the ACRX-100 vector over time in a rat model of hindlimb ischemia Purpose The purpose of this study is to achieve ACRX-100 after direct intramuscular injection in a rodent model of hindlimb ischemia. It was to establish the duration of vector expression.

Method ACRX-100 (lot number 25637) was manufactured by Aldevron, LLC (Fargo, ND). Male Lewis rats were anesthetized and a longitudinal incision was made from the inguinal ligament to the knee joint to expose the femoral artery, which was ligated and removed. The animals were allowed to recover for 10 days and then anesthetized and 0.2 ml of 1.0, 2.0 or 4 mg / ml ACL-01110L (vector backbone containing luciferase cDNA) in 4 locations along the hind limb. The site was injected directly. Using a Xenogen Imaging Systems charge-coupled device camera, vector expression was routinely measured for luciferase expression on days 1, 2, 3, 8, 10, and 14. . The animals were anesthetized with 2% isofluorane and luciferin was injected intraperitoneally at a concentration of 125 mg / kg animal. After 10 minutes, real time images were obtained during 1 minute exposure to determine whole body chemiluminescence of luciferase expression. Data was measured as total luminous flux (pixels / second).

Results As in previous studies, the plasmid ACL-01110L driven by CMV had peak expression on day 3 (FIG. 11). Minimal expression was seen after 14 days.

CONCLUSION ACRX-100 expression in the ischemic rat hindlimb peaked on day 3 and was expressed until day 14, which is related to expression patterns measured in rat heart tissue and vector expression driven by the CMV promoter This is consistent with previously published studies. This data suggests that the 2-week dosing interval is reasonable for future studies evaluating the efficacy of repeated doses of ACRX-100. This dosing interval uses naked plasmid DNA to treat ischemic disease and has been reported in several trials using therapeutic gene expression driven by CMV-based vectors (FGF, HGF, VEGF, HIF1) There is also a correlation.

Example 9-In vivo characterization of ACRX-100 dosing in rabbit hind limb Objectives The purpose of this study was to determine the ACRX 3 days after injection volume, pDNA concentration, formulation was directly injected into rabbit hind limb muscles. It was to determine the effect on -100 pDNA expression.

Method A 3.0-4.0 kg male New Zealand white rabbit was anesthetized and injected with luciferase plasmid ACL-01110L. An incision was made from the inguinal ligament to the knee joint to expose the femoral artery. Four or eight injections of 0.5-1.0 ml plasmid ACL-01110L in 5% dextrose were added to the adductor muscles (2 injections), thin muscles (1 injection) of each rabbit limb. The half-tendon-like muscle (one injection) was injected at 0.1 ml / second. According to FIG. 13, the injections were divided into 6 groups. For future identification, each injury site was identified with a nylon suture and the wound was sewn and closed. Each hind limb was wrapped with a compression bandage for about 15 minutes. Three days after the injection, the animals were sacrificed, the hind limb muscles including the injection site were removed, immersed in luciferin (15 mg / ml) for 7 minutes, and bioluminescence imaging was performed using an IVIS Xenogen machine (FIG. 12). Total luminous flux (pixels per second) was evaluated after 1 minute exposure.

Results A macroscopic assessment of the injection site indicated no inflammation and the plasmid DNA was well tolerated at all doses in all animals. As shown in FIG. 13, expression is observed at all doses delivered and expression increases as a function of pDNA concentration. Expression appeared to peak at a concentration of 2 mg / ml and a total DNA dosage of 4 mg (groups 4-6). Higher volume or number of injection sites does not increase expression, suggesting that pDNA concentration is an important factor in adequate muscle cell transfection.

Conclusion This test shows that the luciferase aspect of the ACRX-100 vector can express the gene product in rabbit hind limb muscles in an amount sufficient to detect 3 days after injection. In addition, this study shows that naked plasmid DNA expression in the rabbit hind limb increases to 2 mg / ml with pDNA concentration. This study should use 0.5-1.0 ml per injection and should produce SDF-1 in the rabbit hind limb with 4-8 injection sites of 0.5-2.0 mg / ml pDNA. To suggest.

Example 10-Complete efficacy, safety and biodistribution study of ACRX-100 The efficacy, safety and biodistribution of ACRX-100 was determined after GLP after intracardiac injection directly via a catheter in a porcine heart failure model. Already determined in studies of efficacy, safety and biodistribution of pigs (summarized in Example 3). ACRX-100 exhibits an acceptable safety profile at dosages up to 100 mg after direct injection into ischemic porcine hearts. While this study will support the clinical trials planned at CLI, it does not mimic the specific clinical application being tested.

  The most reliable nonclinical assessment of the efficacy, safety and biodistribution of ACRX-100 in support of Phase 1 clinical trials in CLI patients is the rabbit model of hindlimb ischemia. This model provides an experimental setting that mimics a Phase 1 clinical trial in CLI patients. Safety and efficacy studies examining the toxicity and biodistribution of single doses of ACRX-100 with gradually increasing dosages were conducted in a rabbit HLI model and are summarized below. Based on these rabbits, we propose to evaluate the efficacy, toxicity and biodistribution of repeated doses of ACRX-100 in a rabbit model, as outlined in section 6.3 of the Pre-IND document. To do.

Safety and Efficacy of Single Dosing of ACRX-100 in Rabbit HLI Objectives The purpose of this study was to determine the efficacy, safety and biodistribution after a single dose of test article ACRX-100 in a rabbit model of hindlimb ischemia. It is to evaluate.

Methods New Zealand white rabbits (n = 5 / group; 2-3 males / female per group) received ligation of the femoral artery only on one side and 10 days after ligation, 4, 8 or 16 mg of ACRX- in the ischemic limb. Eight direct intramuscular 0.5 ml injections of 100 or 4 mg of control (luciferase) plasmid were made (Table 6).

  Safety endpoints were assessed 60 days after injection and histopathology and biodistribution from the hind limb (injection site), contralateral hind limb, heart, lung, liver, brain, spleen, lymph node, kidney and ovary. Was included. Global and microscopic examinations of paraffin sections stained with fixed hematoxylin and eosin were performed on specimens of the indicated tissues. Clinicopathology was assessed for all groups 60 days after injection. Efficacy was measured by% change in angiographic score compared to 30 and 60 days after treatment. The gastrocnemius muscle was excised and the difference in weight was evaluated 60 days after injection. The biodistribution of lung, liver, spleen, lymph node, kidney, brain, testis and ovary from animals of groups 1-4 was evaluated.

Results Angiographic analysis Angiograms were obtained on day 0 (before injection), 30 (± 2) days and 60 (± 2) days after injection and recorded in digital form (FIG. 14). Quantitative angiographic analysis of collateral blood vessel growth in ischemic limbs was performed using a superposition of a grid composed of 5 mm diameter squares in a row. The total number of grid intersections in the medial thigh region where the control turbid arteries intersect was measured in a single blind fashion. For each membrane, the angiographic score was calculated as the ratio of the intersection of the grids where the turbid arteries intersect the medial thigh divided by the total number of grid intersections.

Efficacy The angiographic data results were found to be similar for all animals on the day of dosing. In control animals, blood vessel density tended to decrease over 60 days after dosing. Animals treated with ACRX-100 improved at both time points at 1 mg / mL (group 2) and 2 mg / mL (group 3) compared to control animals injected with vectors that showed reduced blood flow Showed blood flow. Importantly, improvements at low and medium doses were observed on day 30, with a significant (p <0.05) benefit lasting 60 days in the medium dose group (FIG. 15). Improvement was also observed in group 4 (4 mg / ml) 60 days after injection. In the porcine heart failure test, a similar trend of increased angiogenesis was also observed in 5 mg / ml animals.

Pathology Mortality Except for one group of animals (505), all animals were alive until the time of planned dissection. Animal 505 died during 30 days of follow-up angiography. This animal was dissected. The cause of death is believed to be related to anesthesia and not the result of administration of the test article.

Animal observations Clinical findings were limited to observation of scabies and thinning hair in many animals. These findings are common observations in animals undergoing this procedure. In addition, anorexia, decreased activity, low or no feces were shown in 2 groups of animals (510) and hindlimb self-injury was shown in 4 groups of animals (519 and 520). . Observation of self-injury in animals 519 and 520 is likely the result of loss of sensation from injury in the hind limbs of these animals.

  Over a series of trials, animals initially lose weight during the first 3-4 weeks after injury. By the end of the study, the animals returned to their baseline weight. Weight loss is thought to be the result of multiple surgeries.

Macroscopic There were no macroscopic findings that were not related to the test article in any gender. Some macroscopic observations were accidental and considered unrelated to treatment.

Microscopic Several specimens of skeletal muscle were tested from the injection site in the normal area of the hind limb, the ischemic area of the hind limb, and the part of the opposite limb not injected. Tissue pieces were examined using hematoxylin and eosin, Masson trichrome staining. Tissue pieces were collected from ischemic areas that included various inconsistent ischemic areas. The ischemic area is mainly composed of minimal to moderate fibrosis, minimal to mild neovascularization (angiogenesis), a minor degree of subacute / chronic inflammation, hemorrhage, myofibril degeneration / necrosis And / or myofibril regeneration. The area of fibrosis and angiogenesis was mostly along the fascial surface of the muscle. Occasionally, foreign material (suture material) was observed and a minimal granulomatous inflammatory response often occurred around. Rare stones of myofibrils were also observed.

  There were no microscopic findings related to the test article in the remaining tissues microscopically examined (brain, heart, kidney, liver, lung, ovary, spleen). Some of the remaining microscopic observations were general background findings in rabbits or were accidental and were therefore considered unrelated to treatment.

Hematology and coagulation Sixty days after injection, there were no bio-related differences in hematology and coagulation parameters between treatment groups in either gender.

Clinical Chemistry Phosphorus was significantly reduced in groups 2, 3, and 4 of the ACRX-100 group versus the LUC plasmid control group in both males (6-36%) and females (7-30%). It was. There was also a slight increase in calcium in groups 3 and 4 of ACRX-100 in both males (4-7%) and females (7-9%) versus the LUC plasmid control group. For both calcium and phosphorus, all average and individual values always remained within the expected historical control range. None of these changes were considered harmful. In order to better evaluate the accuracy of these observations, we will evaluate these values in the repeated dose safety and efficacy studies described in section 6.3. Of the chemical parameters, no other alternative parameters were observed for any gender.

Biodistribution In this rabbit CLI test, biodistribution was assessed by quantitative PCR in Group 1 (1 mg / mL luciferase plasmid) and Group 4 (4 mg / mL ACRX-100) animals 60 days after injection. The results are shown in Table 7 below. As expected, no ACRX-100 vector was detected in tissues from the control group. In the high dose group, ACRX-100 was detected almost exclusively at the site of injection and only a trace amount of ACRX-100 was detected in the uninjected organ. The excretion rate of ACRX-100 plasmid DNA observed in the rabbit hind limb 60 days after injection (Table 7) is the ACRX from the injected heart site observed in the porcine heart failure safety study (Table 3, FIG. 16). It was consistent with -100 excretion. Furthermore, in the rabbit CLI test, the highest copy number (133, 360 copies / μg host DNA) detected 60 days after injection is the highest copy number (> 1 ×) detected in the 90 day cardiovascular test. The persistence was low because it was smaller than 106 copies / μg host DNA). This data suggests that ACRX-100 is excreted from the injected and non-injected organs of the rabbit, similar to that shown in the porcine heart failure test, and indeed the injections of the current rabbit test The duration at the selected site is less than that observed in the porcine heart failure model.

Conclusion All groups of animals injected with ACRX-100 showed an increase in ischemic hindlimb blood flow 60 days after injection. Animals injected once intramuscularly with ACRX-100 at a dosage of 1 mg / mL (low dose) or 2 mg / mL (medium dose) are compared to control animals that are infected with the vector and show decreased blood flow. The blood flow was improved 30 days and 60 days after the injection. Importantly, a benefit was observed on day 30 and a significant (p <0.05) benefit was maintained on day 60. These data are consistent with results from the porcine heart failure test where ACRX-100 injection (0.5-5.0 mg / mL) increases angiogenesis. Our data suggest that, independent of the total ACRX-100 delivered to the target tissue, the concentration of product delivered per injection significantly affects the angiogenic response. These results will help CLI patients benefit from a single dose of ACRX-100.

  Our data suggests that ACRX-100 is excreted from both injected and non-injected organs in rabbits, as demonstrated in the porcine heart failure test. Since the excretion of ACRX-100 is consistent with the cardiovascular test, the inventors consider that the remaining biodistribution findings of the porcine heart failure test hold the CLI test as well. That is, the expression of ACRX-100 at the end of the study is minimal, sub-therapeutic, and the ability to incorporate is lower than the natural displacement rate.

Example 11:
Test of safety and efficacy of the proposed repeated dose of ACRX-100 in a rabbit model of hindlimb ischemia The proposed new test is the ACRX-100 in the same rabbit model of hindlimb ischemia described in Example 10. We will evaluate the safety and efficacy of repeated dosing. As described below, three intramuscular doses of ACRX-100 are administered to support up to three treatments in LICLI patients.

Safety and Efficacy of Repeated Doses of ACRX-100 in Rabbit HLI Objective The purpose of this study is to determine the safety and efficacy of repeated doses of ACRX-100 in a rabbit model of hindlimb ischemia.

Rationale All non-viral gene therapies currently in clinical trials for severe ischemic limb deliver their non-viral therapies in repeated doses, including NV1FGF that has shown efficacy in phase II trials (HGF, PDGF). To date, our preclinical safety and efficacy studies have shown that ACRX-100 is 100 mg after a single dose to the heart (20 injection sites) or 16 mg hindlimb dosing (8 injection sites). Shows that it is safe. Therefore, we propose to deliver ACRX-100 in repeated doses at 0, 2, 4 weeks as part of our Phase 1 clinical trial for CLI patients. Repeated doses of ACRX-100 could provide additional benefits compared to single doses. CXCR4, a receptor for SDF-1, is upregulated indefinitely after injury, while SDF-1 is transiently after an acute ischemic event (FIG. 17) or in response to an injection procedure. Only up-regulated. Delivery of SDF-1 multiple times after injury takes advantage of increased local expression of CXCR4 expression in the damaged tissue and increases stem cell homing to the SDF-1 expression site. In CLI, repeated injections have the potential to synergistically increase angiogenesis, collateral vessel growth, and wound healing in the ischemic limb. The safety profile observed after injecting 100 mg into the pig heart suggests that smaller dose regimens share similar safety results.

  In order to evaluate the preclinical safety and efficacy of ACRX-100 repeated dosing in the same rabbit hindlimb ischemia model used in the above study, we propose a protocol as detailed below. The repeated dose regimen will be the same as the proposed repeated dose cohort of the Phase I / II trial in all aspects, except for the volume of injection (described in Example 12). In rabbits, 0.5 mL is used instead of 1 mL for humans due to the large area of human thigh / calf skeletal muscle compared to rabbit hind limbs.

Methods New Zealand white rabbits (n = 5 / male and female group) receive the same unilateral femoral artery ligation as done in Example 10 above (day -10). Ten days after ligation (Day 0), each animal received 3 doses (over 15 days) of 1 mg / mL delivered 16 times directly into each 0.5 mL ischemic limb. Of control luciferase plasmids (groups 1 and 2) or ACRX-100 (groups 3 and 4) (Table 8). Safety endpoints include histopathology (total and normal) and biodistribution from the hind limb (injection site), contralateral hind limb, heart, lung, liver, brain, spleen, lymph nodes, kidney and ovary, Assessed on day 3 (day 33) and day 30 (day 60) from the last injection. Global and microscopic examinations of paraffin sections stained with fixed hematoxylin and eosin were performed on specimens of the indicated tissues. Before hindlimb ischemia (day -10), before each dose (day 0, 15, 30), 3 days after dose (day 3, day 18, day 33), last dose 7 days after (day 37), 30 days after the last dose (day 60) or until sacrifice, clinical chemistry and pathology are assessed for all groups. Observations next to Ori are performed twice a day and detailed clinical experiments are performed once a week. Efficacy is measured by% change in angiogenic score from baseline compared to controls on day 30 (all cohorts) and day 60 (cohorts 2 and 4) from the first injection Let's go.

  Samples for biodistribution are collected from lung, liver, spleen, lymph node, kidney, brain, testis, ovary, 3 days after last dose (day 33) and 30 days after last dose (day 60) Are assessed by qPCR at two death points.

The duration of 60 days from the initial treatment of ACRX-100 in this study was chosen to align the time points of efficacy using the single treatment study reported in Example 10. Since this time point is 30 days after the last dose, the biodistribution results are based on the findings from the porcine heart failure safety and efficacy studies reported in Example 8 and the ACRX present at the injection site. It is expected to have −100 copies (103-105 copies / μg host DNA) and a limited number (<103 copies / μg host DNA). Report the results of this proposed test to IND. Given that the safety results of this study are similar to those found in the porcine heart failure safety and efficacy studies, this study should justify repeated dosing of ACRX-100 in CLI patients.

Example 12:
Proposed Clinical Trials The inventors have completed trials showing that ACRX-100 is safe and effective in preclinical models of heart failure and CLI. The results of these completed pre-clinical trials and the proposed pre-clinical trials support the Phase I / II clinical trial proposed by the inventor to evaluate the safety and efficacy of ACRX-100 treatment in CLI patients.

  66 patients will be combined with the proposed Phase I / II study to observe the safety and initial efficacy of using ACRX-100 to treat Rutherford Class 5 CLI patients. In each group, safety is monitored by showing all adverse events (AEs) and measuring standard blood laboratory values (eg, whole blood count, plasma SDF-1 level) The primary sex endpoint is the number of major AEs at 30 days (groups 1 and 2) or 60 days (3 groups, 4 groups, 5 groups) after enrollment.

  In the Phase I portion of the study (Groups 1 to 4), patients were either single doses of ACRX-100 (Groups 1 and 2) or repeated dosing sessions over 4 weeks (Groups 3 and 4) The upper and lower limbs will receive 8 or 16 injections (Table 9). Dose escalation consists of doubling the number of injections from 8 (group 1) to 16 (group 2), moving from single dosing (group 2) to 3 repeated dosing sessions (group 3) Doubling the number of injections per dose from 8 (group 3) to 16 (group 4). Under the assumption that repeated dosing is more effective than single dosing, groups 3 and 4 are randomly divided into 2: 1 receiving treatment or placebo (5% dextrose injection). The group is tested for preliminary efficacy of taking only active drug. In all groups, efficacy is assessed over 12 months after the first dose by the following endpoints: (1) major amputation, (2) generation of a fully wounded closure, (3) change in survival from baseline, (4) rate of change of ulcer healing index, (5) transcutaneous oxygen ( TcPO2) and (6) Pain at rest.

  In the phase II part of the trial (group 5), 30 patients will receive either ACRX-100 (a more effective dosing regimen from groups 3 or 4) or vehicle (5% dextrose solution) (Table 9). Safety and efficacy are measured by the same endpoint as Phase I, and the primary endpoint of efficacy is survival without major amputation. All dose escalations in Phase I of this study and the start of the Phase II portion will be done only in accordance with the DSMC review as described below.

If Phase I / II trial results show that ACRX-100 is effective in improving CLI using DSMC recommendations, patients who were initially randomly divided into control groups were: Under certain conditions, the patient may be given ACRX-100 treatment for free.
1. The patient completes the study follow-up plan.
2. Leading researchers will determine if patients are benefiting from this treatment.

  When treated, the patient follows a plan consisting of (minimum) desirable safety endpoints.

  All enrolled patients will receive normal nursing standards for CLI, including pharmacology (analgesics) and wound treatment (debridement, infection control with antibiotics) if necessary. However, per selection / exclusion criteria, patients must be registered as bad candidates for revascularization and must not have undergone revascularization within 6 weeks of enrollment. Not treated by surgery or interventional techniques. The reason for being a bad candidate for revascularization is that blocking multiple arteries causes ischemia and cannot all generate resumption or bypass. This can create new blood vessels that are derived from the blood vessels of the entire ischemic limb and restore blood flow over a large area, thereby improving symptoms, limb function, and outcomes. Angiogenic therapy (eg, ACRX-100) would be attractive.

  Without treatment, these patients have a poor prognosis. CLI patients who are poor candidates for revascularization (also referred to as “non-reconstructable” disease) may have 40% amputation within 6 months. Furthermore, the one-year mortality rate for CLI patients is 25%, which is 3 to 5 times higher risk of cardiovascular mortality than patients without CLI.

  A summary of the proposed Phase I / II trial is given in Table 11. 66 patients with ulcers that do not heal (Rutherford Class 5). Registered continuously at up to 15 medical centers. Each patient is directly injected intramuscularly with ACRX-100 and left for 12 hours after the initial dosing. ACRX-100 may be delivered using a 27 gauge needle to the injection site extending above the knee and in the lower limb. Safety and efficacy endpoints are collected as outlined in Table 11. In the open-label part (Groups 1 and 2), descriptive statistics are used to compare consecutive efficacy variables across the dosing groups. Where appropriate, descriptive statistics may collect safety parameters, evaluate them qualitatively, or quantify them. In the randomized portion of this study (Groups 3-5), ACRX-100 or vehicle control was administered at 8 or 16 injection sites by the same technique as Phase I at 0, 2, 4 weeks from enrollment. Delivered later. Each treatment group is statistically compared to the control group for all efficacy variables with statistical significance defined as p-values less than 0.05.

  The logic of this test is as follows. Prior to enrollment, all patients must be given written informed consent to participate in this study. Prior to planning the first injection of ACRX-100, patients will be screened within 2 weeks using tests to determine study eligibility including ABI and Rutherfords (Table 11). Additional safety trials (CMP, PT / PTT and baseline values are established during the screening period for efficacy endpoints (TcPO2, quality of life, ulcer size). In the open-label part (Groups 1 and 2), consecutive patients are enrolled and all receive ACRX-100 treatment. (Groups 3-5), each patient is randomly divided and before the injection procedure, the medical center will notify the randomization, all patients will evaluate safety and efficacy, Shake follow-up of 1 week, 2 weeks, 4 weeks, 5 weeks, 3 months, 6 months, 12 months from the first injection (Table 11) Enrollment of the first 3 patients in each group 1 to 4 Is small It is divided into at least 7 days After the last dose of the last patient in groups 1 to 4, all safety data is collected for 7 days, after which each subject takes ACRX-100 The DSMC is responsible for monitoring safety, determining adverse events, and summarizing subject data that meet the suspension rules.The committee has a medical monitor (no voting) and at least three people The DSMC must recommend escalation to the next dose in the first phase of this study and recommend the initiation of a phase II study.

  Prior to submission of CLI IND, national investigators and medical monitors will develop a list of withdrawal rules and will require DSGS data summaries to delay and temporarily suspend study registration if: .

Reasons for the proposed clinical dosage The proposed clinical dosage is based on the results of a non-clinical single-dose safety and efficacy study (see Example 11). As shown in Table 10 below, the proposed human dosing start is 1 mg / mL DNA at 1 mL per injection site (8 mg total). This starting dose was based on the results of a single dose rabbit safety and efficacy CLI study. The starting human dosage is an effective dosage at the same concentration and the same number of injections as the single-dose rabbit test (1 mg / mL, 0.5 mL, 8 sites, 4 mg total). In addition, the starting human dosage is half of the largest total DNA tested in the nostalgic rabbit test (4 mg / mL, 0.5 mL, 8 sites, 16 mg total). The higher volume / site (1.0 mL) used in the Phase I study is that of the 0.5 mL volume of the non-clinical study because the human lower limb has a much higher muscle weight compared to the rabbit hind limb. 2 times.

  The proposed CLI Phase 1 is also supported by data from heart failure tests of GLP safety and efficacy from the porcine preclinical described in Example 3 above. The total DNA dosage at the start of the proposed Phase 1 CLI is 8 mg, which is more than 10 times more safe for the 100 mg NOAEL found in the pig heart failure safety study. The maximum amount of ACRX-100 proposed in the Phase 1 CLI study (16 mg x 3 dose) is the amount of total DNA defined as NOAEL for nostalgic dosing in the efficacy and heart failure of the porcine heart failure test (100 g ) Less than half.

  Finally, the volume per injection (1 ml) used in the Phase 1 CLI test is used for the porcine heart failure test and several published CLI clinic studies.

  Following a successful Phase I / II study, follow-up in a Phase II study to demonstrate the safety and efficacy of ACRX-100 at one or more dosages in CLI Rutherford Class 5 patients , Or an important third machine test is designed.

  From the above description of this specification, those skilled in the art will perceive improvements, changes and modifications. Such improvements, changes and modifications within the skill of the art are intended to be covered by the appended claims. All patents, patent specifications, and publications cited herein are hereby incorporated by reference in their entirety.

  Selected sequences disclosed:

SEQ ID NO: 1
KPVSLLYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

SEQ ID NO: 2
MNAKVVVVLVLVLTALCLSDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKNNNRQVCIDPKLKWIQEYLEKALNK

SEQ ID NO: 3
MDAKVVAVLALVLAALCISDGKPVSLSYRCPCRFFESHVARANVKHLKILNTPNCALQIVARLKSNNRQVCIDPKLKWIQEYLDKALNK

SEQ ID NO: 4
gccgcactttcactctccgtcagccgcattgcccgctcggcgtccggcccccgacccgcgctcgtccgcccgcccgcccgcccgcccgcgccatgaacgccaaggtcgtggtcgtgctggtcctcgtgctgaccgcgctctgcctcagcgacgggaagcccgtcagcctgagctacagatgcccatgccgattcttcgaaagccatgttgccagagccaacgtcaagcatctcaaaattctcaacactccaaactgtgcccttcagattgtagcccggctgaagaacaacaacagacaagtgtgcattgacccgaagctaaagtggattcaggagtacctggagaaagctttaaacaagtaagcacaacagccaaaaaggactttccgctagacccactcgaggaaaactaaaaccttgtgagagatgaaagggcaaagacgtgggggagggggccttaaccatgaggaccaggtgtgtgtgtggggtgggcacattgatctgggatcgggcctgaggtttgccagcatttagaccctgcatttatagcatacggtatgatattgcagcttatattcatccatgccctgtacctgtgcacgttggaacttttattactggggtttttctaagaaagaaattgtattatcaacagcattttcaagcagttagttccttcatgatcatcacaatcatcatcattctcattctcattttttaaatcaacgagtacttcaagatctgaatttggcttgtttggagcatctcctctgctcccctggggagtctgggcacagtcaggtggtggcttaacagggagctggaaaaagtgtcctttcttcagacactgaggctcccgcagcagcgcccctcccaagaggaaggcctctgtggcactcagataccgactggggctgggcgccgccactgccttcacctcctctttcaacctcagtgattggctctgtgggctccatgtagaagccactattactgggact gtgctcagagacccctctcccagctattcctactctctccccgactccgagagcatgcttaatcttgcttctgcttctcatttctgtagcctgatcagcgccgcaccagccgggaagagggtgattgctggggctcgtgccctgcatccctctcctcccagggcctgccccacagctcgggccctctgtgagatccgtctttggcctcctccagaatggagctggccctctcctggggatgtgtaatggtccccctgcttacccgcaaaagacaagtctttacagaatcaaatgcaattttaaatctgagagctcgctttgagtgactgggttttgtgattgcctctgaagcctatgtatgccatggaggcactaacaaactctgaggtttccgaaatcagaagcgaaaaaatcagtgaataaaccatcatcttgccactaccccctcctgaagccacagcagggtttcaggttccaatcagaactgttggcaaggtgacatttccatgcataaatgcgatccacagaaggtcctggtggtatttgtaactttttgcaaggcatttttttatatatatttttgtgcacatttttttttacgtttctttagaaaacaaatgtatttcaaaatatatttatagtcgaacaattcatatatttgaagtggagccatatgaatgtcagtagtttatacttctctattatctcaaactactggcaatttgtaaagaaatatatatgatatataaatgtgattgcagcttttcaatgttagccacagtgtattttttcacttgtactaaaattgtatcaaatgtgacattatatgcactagcaataaaatgctaattgtttcatggtataaacgtcctactgtatgtgggaatttatttacctgaaataaaattcattagttgttagtgatggagcttaaaaaaaa

SEQ ID NO: 5
ccatggacgccaaggtcgtcgctgtgctggccctggtgctggccgcgctctgcatcagtgacggtaagccagtcagcctgagctacagatgcccctgccgattctttgagagccatgtcgccagagccaacgtcaaacatctgaaaatcctcaacactccaaactgtgcccttcagattgttgcaaggctgaaaagcaacaacagacaagtgtgcattgacccgaaattaaagtggatccaagagtacctggacaaagccttaaacaagtaagcacaacagcccaaaggactt

Claims (21)

  A SDF-1 plasmid comprising a polynucleotide having the sequence of SEQ ID NO: 6.   A formulation comprising the SDF-1 plasmid of claim 1 and a pharmaceutically acceptable carrier.   The formulation of claim 2, wherein the formulation is injectable.   4. An injectable formulation according to claim 3, wherein the pharmaceutically acceptable carrier is 5% dextrose. 0 . 5. An injectable formulation according to claim 4, comprising 33 mg / ml to 5 mg / ml of the SDF-1 plasmid. 6. An injectable formulation according to claim 5, comprising 2 mg / ml of the SDF-1 plasmid. 6. An injectable formulation according to claim 5, comprising 1 mg / ml of the SDF-1 plasmid. For the treatment of ischemic conditions of the patient, the formulation according to any one of claims 2 to 7. The preparation according to claim 8, wherein the ischemic state is ischemic cardiomyopathy. Said treatment weakened the patient's heart, comprising administering a pre-SL manufactured agent ischemic and / or peri-infarct region A formulation according to claim 9. 11. The formulation of claim 10, wherein the amount of SDF-1 plasmid administered to the weakened, ischemic and / or peri-infarct area of the patient's heart is greater than 4 mg. Weakened patient's heart by direct injections, are administered in ischemic and / or peri-infarct region A formulation according to claim 10 or 11. Wherein the formulation is administered at least 10 injections, also to the volume of each injection is small 0. 13. The formulation of claim 12, which is 2 ml. The total amount of the formulation administered is 1 0 ml also less formulation according to claim 13. Is administered via the catheters, the formulation according to any one of claims 9 14. The formulation according to claim 15, wherein the catheter is an intravascular catheter or an intramyocardial catheter. The preparation according to claim 8, wherein the ischemic state is a severe ischemic limb. It is administered directly to specific intramuscular limb afflicted patient by injection, formulation of claim 17. Wherein the formulation is administered 5 to 2 0 injections, 1 also to the volume of each injection is small. 19. A formulation according to claim 18 which is 0 ml. 20. The formulation of claim 19, wherein the amount of SDF-1 plasmid administered is between 5 and 20 mg. 21. A formulation according to any one of claims 17 to 20, wherein the administration is repeated up to 3 times, each administration being separated by a period of 2 weeks.

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