stromal-derived factor 1 plasmid (sdf-1) and injectable preparation comprising said plasmid

专利摘要:
SDF-1 PLASMIDE, INJECTABLE PREPARATION AND USE OF THAT PREPARATION. One method of treating cardiomyopathy in an individual includes administering directly or expressing locally in a region of peri-infarction and / or weakened, ischemic, of the individual's myocardial tissue, an amount of SDF-1 effective to cause functional improvement in at least one of the following parameters: left ventricular volume, left ventricular area, left ventricular dimension, cardiac function, 6-minute walk test, or New York Heart Association (NYHA) functional classification.
公开号:BR112012004395B1
申请号:R112012004395-1
申请日:2010-08-30
公开日:2021-01-12
发明作者:Marc S. Penn；Rahul Aras；Joseph Pastore；Timothy R. Miller
申请人:The Cleveland Clinic Foundation；
IPC主号:

专利说明:

[0001] This application relates to SDF-I delivery methods and compositions to treat cardiomyopathy and to the use of SDF-I delivery methods and compositions to treat ischemic cardiomyopathy. Fundamentals of the Invention
[0002] Ischemia is a condition in which blood flow is completely obstructed and considerably reduced in localized parts of the body, resulting in anoxia, reduced supply of substrates and accumulation of metabolites. Although the extent of ischemia depends on the severity of the vascular obstruction, its duration, tissue sensitivity to it, and extension of the development of collateral vessels, dysfunction in ischemic organs or tissues usually occurs, and prolonged ischemia results in atrophy, denaturation, apoptosis and necrosis of the affected tissues.
[0003] In ischemic cardiomyopathy, which are diseases that affect the coronary artery and cause myocardial ischemia, the extent of ischemic myocardial cell damage proceeds from reversible cell damage to irreversible cell damage with increased coronary artery obstruction. Summary of the Invention
[0004] This application refers to a method for treating cardiomyopathy in an individual. Cardiomyopathy may include, for example, cardiomyopathies associated with pulmonary embolism, venous thrombosis, myocardial infarction, transient ischemic attack, a peripheral vascular disorder, atherosclerosis, and / or other myocardial injury or vascular disease. The method includes directly administering or expressing locally in a weakened, ischemic and / or peri-infarction region of the individual's myocardial tissue an amount of SDF-I effective to cause functional improvement in at least one of the following parameters: left ventricular volume , left ventricular area, left ventricular dimension, cardiac function, 6-minute walk test (6MWT), or New York Cardiac Association (NYHA) functional classification.
[0005] In one aspect of the order, the amount of SDF-I administered to the weakened, ischemic and / or peri-infarction region is effective in causing functional improvement in at least one of the left and systolic ventricular volume, left ventricular ejection fraction, classification of wall movement, diastolic length of left ventricular extremity, systolic length of left ventricular extremity, diastolic area of left ventricular extremity, systolic area of left ventricular extremity, diastolic volume of left ventricular extremity, 6-minute walk test (6MWT) , or New York Cardiac Association (NYHA) functional classification. In another aspect of the order, the amount of SDF-I administered to the weakened, ischemic, and / or peri-infarction region is effective in improving the left ventricular and stroke volume. In another aspect of the order, the amount of SDF-I administered to the weakened, ischemic, and / or peri-infarction region is effective in improving left ventricular ejection fraction.
[0006] In some aspects of the application, the amount of SDF-I administered to the weakened, ischemic, and / or peri-infarction region is effective in improving left ventricular stroke systolic volume by at least about 10%. In other aspects of the application, the amount of SDF-I administered to the weakened, ischemic and / or peri-infarction region is effective in improving the stroke volume of the left ventricular extremity by at least about 15%. In other aspects of the order, the amount of SDF-I administered to the weakened, ischemic and / or peri-infarction region is effective in improving the stroke volume of the left ventricular end by at least about 10%, improving the ventricular ejection fraction left by at least about 10%, improve wall movement rating index by at least about 5%, improve 6-minute walking distance by at least about 30 meters, and improve NYHA class by at least 1 class. In another aspect of the application, the amount of SDF-I administered to the weakened, ischemic, and / or peri-infarction region is effective in improving the left ventricular ejection fraction by at least about 10%.
[0007] In another aspect of the application, the amount of SDF-1 administered to the weakened, ischemic and / or peri-infarcted region is effective in substantially improving vasculogenesis of the weakened, ischemic, and / or peri-infarcted region by at least about 20 % based on vessel density or measured by myocardial perfusion imaging (eg, SPECT or PET) with an improvement in added rest score, added stress score and / or added difference score by at least about 10%. SDF-I can be administered by injecting a solution comprising SDF-I that expresses plasmid in the weakened, ischemic, and / or peri-infarction region and which expresses SDF-I from the weakened, ischemic, and / or peri region -infarction. SDF-I can be expressed from the weakened, ischemic, and / or peri-infarction region in an amount effective to improve the stroke volume of the left ventricular extremity.
[0008] In one aspect of the application, the SDF-1 plasmid can be administered to the weakened, ischemic and / or peri-infarction region in multiple injections of the solution with each injection comprising about 0.33 mg / ml to about 5 mg / ml of SDF-1 plasmid solution. In one example, plasmid SDF-1 can be administered to the weakened, ischemic and / or peri-infarction region in at least about 10 injections. Each injection given to the weakened, ischemic and / or peri-infarction region can have a volume of at least about 0.2 ml. SDF-1 can be expressed in the weakened, ischemic, and / or peri-infarction region for more than about three days.
[0009] In an order example, each injection of solution comprising SDF-1 that expresses plasmid can have an injection volume of about 0.2 ml and an SDF-1 plasmid concentration per injection of about 0.33 mg / ml at about 5 mg / ml. In another aspect of the order, at least one functional parameter of the heart can be improved by injecting plasmid SDF-1 into the weakened, ischemic and / or peri-infarcted region of the heart in an injection volume per site of at least about 0.2 ml, at least about 10 injection sites, and at a concentration of plasmid SDF-1 per injection of about 0.33 mg / ml to about 5 mg / ml.
[0010] In another example, the amount of plasmid SDF-1 administered to the weakened, ischemic and / or peri-infarction region that can improve at least one functional parameter of the heart is greater than about 4 mg. The volume of the SDF-1 plasmid solution administered to the weakened, ischemic and / or peri-infarction region that can improve at least one functional parameter of the heart is at least about 10 ml.
[0011] In another aspect of the application, the individual to whom SDF-1 is administered may be a large mammal, such as a human or pig. The SDF-1 plasmid can be administered to the individual by catheterization, such as coronary catheterization or endo-ventricular catheterization. The individual's myocardial tissue can be subjected to imaging to define an area of the weakened, ischemic and / or peri-infarcted region before administration of plasmid SDF-1, and plasmid SDF-1 can be administered to the weakened, ischemic region. and / or peri-infarction defined by imaging. Imaging can include at least one of echocardiography, magnetic resonance imaging, coronary angiogram, electroanatomical mapping or fluoroscopy.
[0012] The application also relates to a method for treating a myocardial infarction in a large mammal by administering plasmid SDF-1 to the mammalian myocardial peri-infarction region by catheterization, such as intra-coronary catheterization or endo-ventricular catheterization. SDF-1 administered by catheterization can be expressed from the peri-infarction region in an amount effective to cause functional improvement in at least one of the following parameters: left ventricular volume, left ventricular area, left ventricular dimension, cardiac function, 6-minute walk (6MWT), or New York Heart Association (NYHA) functional classification.
[0013] In one aspect of the application, the amount of SDF-1 administered to the peri-infarction region is effective to cause functional improvement in at least one of the left ventricular end systolic volume, left ventricular ejection fraction, wall movement 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 diastolic volume, 6-minute walk test (6MWT), or functional classification of New York Heart Association (NYHA). In another aspect of the application, the amount of SDF-1 administered to the peri-infarction region is effective in improving the stroke volume of the left ventricular end. In another aspect of the order, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarction region is effective in improving the left ventricular ejection fraction.
[0014] In some aspects of the application, the amount of SDF-1 administered to the peri-infarction region is effective in improving the stroke volume of the left ventricular end by at least about 10%. In other aspects of the application, the amount of SDF-1 administered to the peri-infarction region is effective in improving the stroke volume of the left ventricular end by at least about 15%. In still other aspects of the application, the amount of SDF-1 administered to the peri-infarction region is effective in improving the stroke volume of the left ventricular end by at least about 10%, improving the left ventricular ejection fraction by at least about 10%, improve the wall movement score index by about 5%, improve the six-minute walk distance by at least 30 meters, or improve NYHA class by at least 1 class. In another aspect of the order, the amount of SDF-1 administered to the weakened, ischemic, and / or peri-infarction region is effective in improving the left ventricular ejection fraction by at least about 10%.
[0015] In another aspect of the application, the amount of SDF-1 administered to the peri-infarction region is effective in substantially improving vasculogenesis of the peri-infarction region by at least about 20% based on the density of the containers.
[0016] In one aspect of the application, plasmid SDF-1 can be administered to the weakened, ischemic, and / or peri-infarction region in multiple injections of the solution with each injection comprising from about 0.33 mg / ml to about 5 mg / ml of SDF-1 plasmid / solution. In one example, plasmid SDF-1 can be administered to the weakened, ischemic, and / or peri-infarction region in at least about 10 injections. Each injection given to the weakened, ischemic, and / or peri-infarction region can have a volume of at least about 0.2 ml. SDF-1 can be expressed in the weakened, ischemic, and / or peri-infarction region for more than about three days.
[0017] In an application example, each injection of the solution comprising SDF-1 which plasmid expression can have 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 about 5 mg / ml. In another aspect of the application, at least one functional parameter of the heart can be improved by injecting the SDF-1 plasmid into the weakened, ischemic and / or peri-infarcted region of the heart in an injection volume per site of at least about 0 , 2 ml, at least about 10 injection sites, and at an SDF-1 plasmid concentration per injection of about 0.33 mg / ml to about 5 mg / ml.
[0018] In another example, the amount of SDF-1 plasmid administered to the weakened, ischemic, and / or peri-infarcted region that can improve at least one functional parameter of the heart is greater than about 4 mg. The volume of SDF-1 plasmid solution administered to the weakened, ischemic and / or peri-infarction region that can improve at least one functional parameter of the heart is at least about 10 ml.
[0019] The application also refers to a method to improve the stroke volume of the left ventricular extremity in a large mammal after myocardial infarction. The method includes administering the SDF-1 plasmid to the mammalian peri-infarction region by endo-ventricular catheterization. SDF-1 can be expressed from the peri-infarction region in an amount effective to cause functional improvement in the stroke volume of the left ventricular extremity.
[0020] In some aspects of the application, the amount of SDF-1 administered to the peri-infarction region is effective in improving the stroke volume of the left ventricular end by at least about 10%. In other aspects of the application, the amount of SDF-1 administered to the peri-infarction region is effective in improving the stroke volume of the left ventricular end by at least about 15%. In still other aspects of the application, the amount of SDF-1 administered to the peri-infarction region is effective in improving the stroke volume of the left ventricular end by at least about 10%, improving the left ventricular ejection fraction by at least about 10%, improve the wall movement score index by at least about 5%, improve the six-minute walking distance by at least about 30 meters, or improve NYHA class by at least 1 class.
[0021] In one aspect of the application, the SDF-1 plasmid can be administered to the weakened, ischemic, and / or peri-infarction region in multiple injections of the solution with each injection comprising from about 0.33 mg / ml to about 5 mg / ml SDF-1 plasmid / solution. In one example, the SDF-1 plasmid can be administered to the weakened, ischemic and / or peri-infarction region in at least about 10 injections. Each injection given to the weakened, ischemic and / or peri-infarction region can have a volume of at least about 0.2 ml. SDF-1 can be expressed in the weakened, ischemic, and / or peri-infarction region for more than about three days.
[0022] In an order example, each injection of solution comprising SDF-1 that expresses plasmid can have an injection volume of at least about 0.2 ml and a concentration of SDF-1 plasmid can injection of about 0.33 mg / ml to about 5 mg / ml. In another aspect of the order, the stroke volume of the left ventricular end of the heart can be improved by about 10% by injecting the SDF-1 plasmid into the weakened, ischemic, and / or peri-infarcted region of the heart in one injection volume per site of at least about 0.2 ml, at least about 10 injection sites, and at an SDF-1 plasmid concentration per injection of about 0.33mg / ml to about 5mg / ml.
[0023] In another example, the amount of SDF-1 plasmid delivered to the weakened, ischemic and / or peri-infarction region that can improve the left ventricular stroke volume is greater than about 4 mg. The volume of the SDF-1 plasmid solution administered to the weakened, ischemic and / or peri-infarction region that can improve the systolic volume of the left ventricular end of the heart is at least about 10 ml. Brief Description of Drawings
[0024] The previous characteristics and other characteristics of the application will become evident to those versed in the technique to which the application refers after reading the following description in relation to the attached drawings. Fig. 1 is a table illustrating expression of luciferase for varying amounts and volumes of DNA in a porcine model; Fig. 2 is a table illustrating% change in stroke volume from the left ventricular end to various amounts of SDF-1 plasmid using a porcine model of congestive heart failure 30 days after injection of SDF-1; Fig. 3 is a table illustrating% change in left ventricular ejection fraction for various amounts of SDF-1 plasmid using a porcine model of congestive heart failure 30 days after SDF-1 injection; Fig. 4 is a table illustrating% change in the wall movement score index for various amounts of SDF-1 plasmid using a porcine model of congestive heart failure 30 days after injection of SDF-1; Fig. 5 is a table illustrating% change in stroke volume from the left ventricular end to various amounts of SDF-1 plasmid using a porcine model of congestive heart failure 90 days after injection of SDF-1; and Fig. 6 is a table illustrating% change in container density for various amounts of SDF-1 plasmid using a porcine model of congestive heart failure 30 days after injection of SDF-1. Fig. 7 is a schematic diagram of a plasmid vector in accordance with an aspect of the application. Fig. 8 is an image showing plasmid expression over a substantial portion of a swine heart. Fig. 9 is a table illustrating the stroke volume of the left ventricular end at baseline and 30 days after initial injection. All groups show similar increases in stroke volume of the left ventricular end in 30 days. N = 3 for all data points. Data presented as ± SEM average. Fig. 10 is a table showing left ventricular ejection fraction at baseline and 30 days after initial injection. All groups showed a lack of improvement in left ventricular ejection fraction. N = 3 for all data points. Data presented as ± SEM average. Detailed Description
[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as generally understood by those versed in the technique to which the orders belong. All patents, patent applications, published orders and publications, Banco Genético sequences, websites and other published materials relating to all disclosure here, unless otherwise noted, are incorporated by reference in their entirety. In the event that there are a plurality of definitions for terms here, those in this section will prevail. Unless otherwise defined, all the thermal terms used here have the same meaning as generally understood by those skilled in the art to which this application belongs. Generally understood definitions of molecular biology terms can be found in, for example, Rieger et al., Glossary of Genetics: Classical and Molecular, 5th edition, Springer-Verlag: New York, 1991; and Lewin, Genes V, Oxford University Press: New York, 1994.
[0026] Methods that involve conventional molecular biology techniques are described here. Such techniques are generally known in the art and described in detail in methodology treatises, such as Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; and Current Protocols in Molecular Biology, ed. Ausubel et al., Greene Publishing and Wiley-Interscience, New York, 1992 (with periodic updates). Methods for chemical synthesis of nucleic acids are discussed, for example, in Beaucage and Carruthers, Tetra. Letts. 22: 1859-1862, 1981, and Matteucci et al., J. Am. Chem. Soc. 103: 3185, 1981. Chemical synthesis of nucleic acids can be carried out, for example, in automatic oligonucleotide synthesizers. Immunological methods (e.g., preparation of antigen specific antibodies, immunoprecipitation and immunoblotting) are described, e.g., in Current Protocols in Immunology, ed. Coligan et al., John Wiley & Sons, New York, 1991; and Methods of Immunological Analysis, ed. Masseyeff et al., John Wiley & Sons, New York, 1992. Conventional methods of gene transfer and gene therapy can also be adapted for use in the application. See, e.g., Gene Therapy: Principles and Applications, ed. T. Blackenstein, Springer Verlag, 1999; Gene Therapy Protocols (Methods in Molecular Medicine), ed. P. D. Robbins, Humana Press, 1997; and Retro-vectors for Human Gene Therapy, ed. C. P. Hodgson, Springer Verlag, 1996.
[0027] When reference is made to a URL or other identifier or address, it is understood that such identifiers can change and specific information on the internal can come and go, but equivalent information can be found by searching the internet. References to them prove the availability and public dissemination of such information.
[0028] As used herein, "nucleic acid" refers to a polynucleotide containing at least two covalently linked nucleotide or nucleotide analog subunits. A nucleic acid can be a deoxyribonucleic acid (DNA), a ribonucleic acid (RNA), or a DNA or RNA analog. Nucleotide analogs are commercially available and methods for preparing polynucleotides containing such nucleotide analogs 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, or a mixture thereof. For the purposes of this document, unless otherwise specified, nucleic acid is double-stranded, or apparent from context.
[0029] As used herein, "DNA" must include all types and sizes of DNA molecules including cDNA, plasmids and DNA including modified nucleotides and nucleotide analogs.
[0030] As used herein, "nucleotides" include nucleoside mono-, di-, and triphosphates. Nucleotides also include modified nucleotides, such as, but are not limited to, phosphorothioate nucleotides and deazapurine nucleotides and other nucleotide analogs.
[0031] As used here, the term "individual" or "patient" refers to animals that large DNA molecules can be introduced in. Larger organisms, such as mammals and birds, including humans, primates, rodents, cattle, pigs, rabbits, goats, sheep, mice, rats, guinea pigs, cats, dogs, horses and chicken and others.
[0032] As used here, "large mammal" refers to mammals having a typical adult weight of at least 10 kg. Such large mammals may include, for example, humans, primates, dogs, pigs, cattle and should exclude smaller mammals, such as mice, rats, guinea pigs and other rodents.
[0033] As used here, "administer to an individual" is a procedure whereby one or more agents and / or large nucleic acid molecules are introduced into or applied to an individual so that the target cells that are present in the individual are eventually contacted with the agent and / or the nucleic acid molecules.
[0034] As used here, "distribution", which is used interchangeably with "transduction", refers to the process by which exogenous nucleic acid molecules are transferred in a cell so that they are located within the cell. The distribution of nucleic acids is a different process from the expression of nucleic acids.
[0035] As used here, a "multiple cloning site" (MCS) "is a region of nucleic acid in a plasmid that contains multiple restriction enzyme sites, any of which can be used in conjunction with standard recombinant technology to digest the vector." Digestion restriction enzyme "refers to the catalytic cleavage of a nucleic acid molecule with an enzyme that works only at specific locations on a 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. Often, a vector is linearized or fragmented using a reaction enzyme that cuts into the MCS so that exogenous sequences are linked to the vector.
[0036] As used here, "origin of replication" (often called "ori"), is a specific nucleic acid sequence that replication is initiated in. Alternatively, an autonomous replication sequence (ARS) can be employed if the host cell is yeast.
[0037] As used here, "selectable or triple markers" confer an identifiable change to a cell that allows easy identification of cells containing an expression vector. Generally, a selectable marker is one that confers a property that allows selection. A positive selectable marker is a where the presence of the marker allows selection, while a selectable marker is one in which its presence prevents selection. An example of a positive selectable marker is a drug resistance marker.
[0038] Generally, the inclusion of a drug selection marker helps 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 confer a phenotype that allows discrimination of transformants based on the implementation of conditions, other types of markers that include triple markers such as GFP, whose basis is calorimetric analysis, are also contemplated. Alternatively, triple enzymes such as simple virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) can be used. Those skilled in the art would also know how to employ immunological markers, possibly in conjunction with FACS analysis. The negative marker is not believed to be important, as long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Other examples of selectable and triple markers are well known to those skilled in the art.
[0039] The term "transfection" is used to refer to the absorption of foreign DNA by a cell. A cell was "transfected" when exogenous DNA was introduced into the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al., Virology 52: 456 (1973); Sambrook et al., Molecular Cloning: A Laboratory Manual (1989); Davis et al., Basic Methods in Molecular Biology (1986); Chu et al., Gene 13: 197 (1981). Such techniques can be used to introduce one or more portions of exogenous DNA, such as a vector for integrating nucleotides and other nucleic acid molecules, into host cells. The term covers chemical, electrical and virus-mediated transfection procedures.
[0040] As used here, "expression" refers to the process by which the nucleic acid is translated into peptides or is transcribed into RNA, which, for example, can be translated into peptides, polypeptides or proteins. If the nucleic acid is derived from genomic DNA, the expression may, if an appropriate eukaryotic cell or organism is selected, include splicing of the mRNA. For heterologous nucleic acid to be expressed in a host cell, it must initially be distributed in the cell and then, being in the cell, reside in the nucleus.
[0041] As used here, "gene therapy" involves the transfer of heterologous DNA to cells of a mammal, specifically a human, with a disorder or conditions for which therapy or diagnosis is requested. DNA is introduced into selected target cells so that heterologous DNA is expressed and a therapeutic product encoded therefrom is produced. Alternatively, heterologous DNA can somehow mediate expression of DNA encoding the therapeutic product; it can encode a product, such as a peptide or RNA that somehow directly or indirectly mediates the expression of a therapeutic product. Gene therapy can also be used to deliver nucleic acid that encodes a gene product to replace a defective gene or to supplement a gene product produced by the mammal or the cell in which it is introduced. The nucleic acid introduced can encode a therapeutic compound, such as a growth factor inhibitor, or a necrosis tumor factor or inhibitor thereof, such as a receptor for it, which is not normally produced in the mammalian host or which does not it is produced in therapeutically effective amounts or in a therapeutically useful time. The heterologous DNA encoding the therapeutic product can be modified prior to introduction into the cells of the affected host to increase or otherwise alter the product or its expression.
[0042] As used here, “heterologous nucleic acid sequence” is generally DNA that encodes RNA and proteins that are not normally produced in vivo by the cell in which it is expressed, or that mediates or encodes mediators that alter expression of endogenous DNA affecting transcription, translation or other adjustable biochemical processes. A heterologous nucleic acid sequence can also be referred to as foreign DNA. Any DNA that those skilled in the art would recognize or consider to be heterologous or foreign to the cell in which it is expressed is here understood by heterologous DNA. Examples of heterologous DNA include, but are not limited to, DNA encoding traceable marker proteins, such as a protein that confers drug resistance, DNA encoding therapeutically effective substances, such as anti-cancer agents, enzymes and hormones, and DNA encoding other types of proteins, such as antibodies. Antibodies that are encoded by heterologous DNA can be secreted or expressed on the cell surface where the heterologous DNA was introduced.
[0043] As used here, the term "cardiomyopathy" refers to the deterioration of myocardial function (i.e., the actual cardiac muscle) for any reason. Individuals with cardiomyopathy are often at risk for arrhythmia, sudden cardiac death, or hospitalization or death due to heart failure.
[0044] As used here, the term "ischemic cardiomyopathy" is a weakness in the heart muscle due to inadequate delivery of oxygen to the myocardium with coronary artery disease being the most common cause.
[0045] As used here, the term "ischemic heart disease" refers to any condition in which the heart muscle is damaged or malfunctioned due to the relative absence or deficiency of its blood supply; most often caused by atherosclerosis, including angina pectoris, acute myocardial infarction, chronic ischemic heart disease, and sudden death.
[0046] As used here, the term “myocardial infarction” refers to the damage or death of an area of the heart muscle (myocardium) resulting from a blocked blood supply to that area.
[0047] As used here, the term "6-minute walk test" or "6MWT" refers to a test that measures the distance a patient can quickly walk on a flat, rigid surface for a period of 6 minutes (the 6MWD). It assesses the global and integrated responses of all systems involved during exercise, including the pulmonary and vascular systems, systemic circulation, peripheral circulation, blood, neuromuscular units, and muscle metabolism. It does not provide specific information about the function of each of the different organs and systems involved in exercise or the exercise limitation mechanism, as is possible with maximum cardiopulmonary exercise testing. Self-paced 6MWT assesses the sub-maximum level of functional capacity. (See, for example, AM J Respir Crit Care Med, Vol. 166. Pp 111-117 (2002))
[0048] As used here, "New York Heart Association (NYHA) Functional Classification" refers to a classification for the proportion of heart failure. This classifies patients into one of four categories based on how limited they are during physical activity; limitations / symptoms refer to normal breathing and varying degrees of shortness of breath / or pain in angina:
[0049] This application refers to compositions and methods for treating cardiomyopathy in an individual that results in reduced and / or compromised myocardial function. Cardiomyopathy treated by the compositions and methods here may include cardiomyopathy associated with a pulmonary embolus, a venous thrombosis, a myocardial infarction, a transient ischemic attack, a peripheral vascular disorder, atherosclerosis, ischemic heart disease and / or other myocardial injury or disease vascular. The method for treating cardiomyopathy may include administering locally (or distributing locally) to weakened myocardial tissue, ischemic myocardial tissue and / or apoptotic myocardial tissue, as a peri-infarction region of a heart after myocardial infarction, an amount of stroma cell-derived factor 1 (SDF-1) that is effective in causing functional improvement in at least one of the following parameters: left ventricular volume, left ventricular area, left ventricular dimension, cardiac function, 6-minute walk test (6MWT ), or New York Cardiac Association (NYHA) functional classification.
[0050] It was discovered using a porcine model of heart failure that mimics heart failure in a human in which the functional improvement of ischemic myocardial tissue is dependent on the amount, dose and / or distribution of SDF-1 administered to ischemic myocardial tissue and in which The amount, dose and / or distribution of SDF-1 to the ischemic myocardial tissue can be optimized so that the functional parameters of the myocardium, such as left ventricular volume, left ventricular area, left ventricular dimension, or cardiac function are substantially improved. As discussed below, in some respects, the amount, concentration, and volume of SDF-1 delivered to ischemic myocardial tissue can be controlled and / or optimized to substantially improve functional parameters (eg, left ventricular volume, left ventricular area, size left ventricular, cardiac function, 6-minute walk test (6MWT), and / or New York Cardiac Association (NYHA) functional classification to mitigate adverse side effects.
[0051] In one example, SDF-1 can be administered directly or locally to a weakened region, ischemic region and / or myocardial tissue peri-infarction region of a large mammal (eg, pig or human) in which there is deterioration or worsening of a functional parameter of the heart, such as left ventricular volume, left ventricular area, left ventricular dimension, left ventricular dimension, or cardiac function as a result of ischemic cardiomyopathy, such as a myocardial infarction. Deterioration or worsening of the functional parameter may include, for example, an increase in stroke volume of the left ventricular extremity, a decrease in the left ventricular ejection fraction, an increase in the movement index of the wall, an increase in the diastolic length of the left ventricular end, increase in left ventricular end systolic length, increase in left ventricular end diastolic area (eg, mitral valve level and level of papillary muscle insertion), increase in left ventricular end systolic area (eg, mitral valve level and level insertion of the papillary muscle), or increase in diastolic volume of the left ventricular extremity as measured using, for example, echocardiography.
[0052] In one aspect of the application, the amount of SDF-1 administered to the region, weakened, ischemic region, and / or peri-infarction region of large mammalian myocardial tissue may be an effective amount to improve at least one functional parameter of the myocardium, such as a decrease in left ventricular end systolic volume, increase in left ventricular ejection fraction, decrease in wall movement score index, decrease in left ventricular end diastolic length, decrease in left ventricular end systolic length, decrease in diastolic area of the left ventricular extremity (eg, mitral valve level and level of insertion of the papillary muscle), decrease in systolic area of the left ventricular extremity (eg, mitral valve level and level of muscle insertion), or decrease in volume diastolic diameter of the left ventricular extremity measured using, for example, echocardiography as well as r the 6-minute walk test (6MWT) or functional classification of the New York Heart Association (NYHA) of the individual.
[0053] In another aspect of the application, the amount of SDF-1 administered to the weakened region, ischemic region and / or peri-infarction region of myocardial tissue of the large mammal with a cardiomyopathy is effective in improving the stroke volume of the left ventricular end in the mammal at least about 10%, and more specifically at least about 15%, after 30 days following administration as measured by echocardiography. The percentage of improvement is relative to each individual treated and is based on the respective parameter measured before or at the time of intervention or therapeutic treatment.
[0054] In another aspect of the application, the amount of SDF-1 administered to the weakened region, ischemic region, and / or myocardial tissue peri-infarction of the large mammal with a cardiomyopathy is effective in improving the stroke volume of the left ventricular end in at least minus about 10%, improve the left ventricular ejection fraction by at least about 10%, and improve the index of wall movement scores by about 5%, after 30 days following administration as measured by echocardiography.
[0055] In yet another aspect of the application, the amount of SDF-1 administered to the weakened region, ischemic region, and / or myocardial tissue peri-infarction region of the large mammal with a cardiomyopathy is effective in improving vasculogenesis of the weakened region, ischemic region , and / or peri-infarction region by at least about 20% based on vessel density or an increase in cardiac perfusion measured by SPECT imaging. A 20% improvement in vasculogenesis proved to be clinically significant (Losordo Circulation 2002; 105: 2012).
[0056] In yet another aspect of the application, the amount of SDF-1 administered to the weakened region, ischemic region, and / or myocardial tissue peri-infarction region of the large mammal with a cardiomyopathy is effective in improving the six-minute walk distance at least 30 meters or improve NYHA class by at least 1 class.
[0057] SDF-1 described here can be administered to the weakened region, the ischemic region and / or myocardial tissue peri-infarction region after tissue injury (eg, myocardial infarction) about hours, days, weeks or months after the down regulation start of SDF-1. The length of time that SDF-1 is administered to cells can range from about immediately after the onset of cardiomyopathy (eg, myocardial infarction) to about days, weeks, or months after the onset of the ischemic disorder or tissue injury .
[0058] [0002] SDF-1 in accordance with the request, which is administered to the weakened, ischemic and / or peri-infarction region of the peri-infarction region of myocardial tissue may have an amino acid sequence that is substantially similar to a sequence of native mammalian SDF-1 amino acids. The amino acid sequence of a series of SDF-1 proteins from different mammals is known including human, mouse and rat. The amino acid sequences of human and rat SDF-1 are at least about 92% identical (e.g., about 97% identical). SDF-1 can comprise two isoforms, SDF-1 alpha and SDF-1 beta, both of which are referred to here as SDF-1 unless otherwise identified.
[0059] SDF-1 can have an amino acid sequence substantially identical to SEQ ID No. 1. SDF-1 that is overexpressed can have an amino acid sequence substantially similar to that of the SDF-1 proteins of the previous mammal. For example, SDF-1 that is overexpressed may have an amino acid sequence substantially similar to SEQ ID No. 2. SEQ ID No. 2, which substantially comprises SEQ ID No. 1, is the amino acid sequence for SDF- 1 human and is identified by Access to Genetic Bank N ° NP954637. SDF-1 that is overexpressed can have an amino acid sequence that is substantially identical to SEQ ID No. 3. SEQ ID No. 3 includes the amino acid sequences for rat SDF and is identified by Access to Genetic Bank No. AAF01066.
[0060] SDF-1 in accordance with the application may have a mammalian SDF-1 variant, as a fragment, analog and derivative of mammalian SDF-1. Such variants include, for example, a polypeptide encoded by a naturally occurring allelic variant of the native SDF-1 gene (ie, a naturally occurring nucleic acid encoding a naturally occurring mammal SDF-1 polypeptide), a polypeptide encoded by an alternative form of division of a native SDF-1 gene, a polypeptide encoded by a homolog or ortholog of a native SDF-1 gene, and a polypeptide encoded by a naturally occurring variant of a native SDF-1 gene.
[0061] SDF-1 variants have a peptide sequence that differs from a native SDF-1 polypeptide in one or more amino acids. The peptide sequence of such variants may characterize a deletion, addition or substitution of one or more amino acids from an SDF-1 variant. Amino acid insertions are preferably about 1 to 4 contiguous amino acids, and deletions are preferably about 1 to 10 contiguous amino acids. Variant SDF-1 polypeptides substantially maintain a functional activity of native SDF-1. Examples of SDF-1 polypeptide variants can be made by expressing nucleic acid molecules that characterize silent or conservative changes. An example of a variant of SDF-1 is listed in U.S. Patent No. 7,405,195, which is incorporated herein by reference in its entirety.
[0062] SDF-1 polypeptide fragments corresponding to one or more specific motifs and / or domains or to arbitrary sizes are included in the scope of this application. Isolated peptidyl portions of SDF-1 can be obtained by screening recombinantly produced peptides from the corresponding fragment of the nucleic acid encoding such peptides. For example, an SDF-1 polypeptide can be arbitrarily divided into fragments of desired length with no fragment overlap, or preferably divided into overlapping fragments of desired length. The fragments can be produced recombinantly and tested to identify those peptidyl fragments, which can function as agonists of native CXCR-4 polypeptides.
[0063] Variants of SDF-1 polypeptides may also include recombinant forms of SDF-1 polypeptides. Recombinant polypeptides in some embodiments, in addition to SDF-1 polypeptides, are encoded by a nucleic acid that can have at least 70% sequence identity with the nucleic acid sequence of a gene encoding a mammalian SDF-1.
[0064] SDF-1 variants can include agonistic forms of the protein that constitutively expresses the functional activities of native SDF-1. Other variants of SDF-1 may include those that are resistant to proteolytic degradation, such as, for example, due to mutations, that alter target protease sequences. Whether a change in the amino acid sequence of a peptide results in a variant having one or more functional activities of a native SDF-1 can be immediately determined by testing the variant for a native SDF-1 functional activity.
[0065] The SDF-1 nucleic acid encoding the SDF-1 protein can be a native or non-native nucleic acid and in the form of RNA or in the form of DNA (e.g., cDNA, genomic DNA, and synthetic DNA). DNA can be single-stranded or double-stranded, and if single-stranded it can be the encoding (sense) or non-encoding (antisense) filament. The nucleic acid coding sequence encoding SDF-1 can be substantially similar to a nucleotide sequence of the SDF-1 gene, such as the nucleotide sequence shown in SEQ ID No. 4 and SEQ ID No. 5. SEQ ID No. 4 and SEQ ID No. 5 comprise, respectively, the nucleic acid sequences for human SDF-1 and rat SDF-1 and are substantially similar to the nucleic acid sequences of Access to Genetic Bank No. NM199168 and Access to Genetic Bank N ° AF 189724. The nucleic acid coding sequence for SDF-1 can also be a different coding sequence which, as a result of redundancy or degeneration of the genetic code, encodes the same polypeptide as SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID No. 3.
[0066] Other nucleic acid molecules that encode SDF-1 are variants of a native SDF-1, such as those that encode fragments, analogs and derivatives of synthetic SDF-1. Such variants can be, for example, a naturally occurring allelic variant of a native SDF-1 gene, a homolog or orthologue of a native SDF-1 gene, or a naturally occurring variant of a native SDF-1 gene. These variants have a nucleotide sequence that differs from an SDF-1 gene in one or more bases. For example, the nucleotide sequence of such variants may characterize a deletion, addition or substitution of one or more nucleotides from a native SDF-1 gene. Nucleic acid inserts are preferably about 1 to 10 contiguous nucleotides, and deletions are preferably about 1 to 10 contiguous nucleotides.
[0067] In other applications, SDF-1 variant exhibiting substantial changes in structure can be generated by making nucleotide substitutions that cause less conservative changes in the encoded polypeptide. Examples of such nucleotide substitutions are those that cause changes (a) in the structure of the polypeptide backbone; (b) the charge or hydrophobicity of the polypeptide; or (c) the volume of a side chain of amino acids. The nucleotide substitutions that are generally expected to produce the biggest changes in protein properties are those that cause non-conservative changes in codons. Examples of changes in codons that are likely to cause major changes in protein structure are those that cause replacement of (a) a hydrophilic residue (eg, serine or threonine), to (or through) a hydrophobic residue (eg, leucine, isoleucine, phenylalanine, valine or alanine); (b) a cysteine or proline for (or through) any other residue; (c) a residue having an electropositive side chain (e.g., lysine, arginine, or histidine), to (or through) an electronegative residue (e.g., glutamine or aspartate); or (d) a residue having a bulky side chain (e.g., phenylalanine), for (or through) one not having a side chain, (e.g., glycine).
[0068] Naturally occurring allelic variants of a native SDF-1 gene are nucleic acids isolated from mammalian tissue that have at least about 70% sequence identity with a native SDF-1 gene, and encode polypeptides having structural similarity to a polypeptide Native SDF-1. Homologues of an SDF-1 gene are nucleic acids isolated from other species that have at least 70% sequence identity with the native gene, and encode polypeptides having structural similarity to a native SDF-1 polypeptide. Public and / or private nucleic acid databases can be searched to identify other nucleic acid molecules having a high percentage sequence identity (e.g., 70% or more) for a native SDF-1 gene.
[0069] Non-naturally occurring SDF-1 gene variants are non-naturally occurring nucleic acids (eg, they are man-made), have at least 70% sequence identity with a native SDF-1 gene, and encode polypeptides having structural similarity to a native SDF-1 polypeptide. Examples of non-naturally occurring gene variants are those that encode a fragment of a native SDF-1 protein, those that hybridize to an SDF-1 gene or a complement to, a native SDF-1 gene under strict conditions, and those that share at least 65% sequence identity with a native SDF-1 gene or a complement to a native SDF-1 gene.
[0070] Nucleic acids that encode fragments of a native SDF-1 gene in some embodiments are those that encode amino acid residues of native SDF-1. Shorter oligonucleotides that encode or hybridize to nucleic acids that encode fragments of native SDF-1 can be used as probes, primers or antisense molecules. Longer polynucleotides that encode or hybridize to nucleic acids that encode fragments of a native SDF-1 can also be used in several aspects of the application. Nucleic acids that encode fragments of a native SDF-1 can be made by enzymatic digestion (e.g., using a restriction enzyme) or chemical degradation of the full-length native SDF-1 gene or its variants.
[0071] Nucleic acids that hybridize under strict conditions to one of the previous nucleic acids can also be used here. For example, such nucleic acids can be those that hybridize to one of the foregoing nucleic acids under low rigidity conditions, moderate rigidity conditions or high rigidity conditions.
[0072] Nucleic acid molecules that encode an SDF-1 fusion protein can also be used in some embodiments. Such nucleic acids can be made by preparing a construct (e.g., an expression vector) that expresses an SDF-1 fusion protein when introduced into a suitable target cell. For example, such a construct can be made by linking a first polynucleotide that encodes a SDF-1 protein fused in structure with a second polynucleotide that encodes another protein so that expression of the construct in a suitable expression system provides a fusion protein.
[0073] Nucleic acids encoding SDF-1 can be modified in the base portion, sugar portion, or phosphate backbone, for example, to improve molecule stability, hybridization, etc. The nucleic acids described herein can additionally include other appended groups such as peptides (e.g., to target target cell receptors in vivo), or agents that facilitate cell membrane transport, hybridization-driven divination. For this, the nucleic acids can be conjugated to another molecule, (e.g., a peptide), cross-linking agent activated by hybridization, transport agent, dividing agent activated by hybridization etc.
[0074] SDF-1 can be delivered to the weakened, ischemic, and / or peri-infarcted region of myocardial tissue by administering an SDF-1 protein to the weakened, ischemic, and / or peri-infarcted region, or by introducing an agent into the cells of the region weakened, ischemic and / or peri-infarction of myocardial tissue that causes, increases and / or upward regulates the expression of SDF-1 (ie, SDF-1 agent). The SDF-1 protein expressed from cells can be an expression product of a genetically modified cell.
[0075] The agent that causes, increases and / or upward regulates SDF-1 expression can comprise natural or synthetic nucleic acids as described here that are incorporated into recombinant nucleic acid constructs, usually DNA constructs, capable of introduction and replication in cells myocardial tissue. Such a construct can include a replication system and sequences that are capable of transcribing and translating a sequence that encodes polypeptide in a given cell.
[0076] One method of introducing the agent into a target cell involves using gene therapy. Gene therapy in some modalities of the application can be used to express SDF-1 protein from a cell in the weakened, ischemic and / or myocardial tissue peri-infarction region in vivo.
[0077] In one aspect of the application, gene therapy may use a vector that includes a nucleotide that encodes an SDF-1 protein. A "vector" (sometimes called a gene distribution or gene transfer "vehicle") refers to a macromolecule or complex of molecules comprising a polynucleotide to be distributed to a target cell, either in vitro or in vivo. The polynucleotide to be delivered may comprise a coding sequence of interest in gene therapy. Vectors include, for example, viral vectors (such as adenovirus ('Ad'), adeno-associated viruses (AAV), and retrovirus), nonviral vectors, liposomes and other complexes containing lipids, and other macromolecular complexes capable of mediating distribution from a polynucleotide to a target cell.
[0078] Vectors can also comprise other components or features that still modulate gene distribution and / or gene expression, or that otherwise provide beneficial properties to targeted cells. Such other components include, for example, components that influence binding or targeting cells (including components that mediate binding of specific cell type or tissue); components that influence the absorption of the vector's nucleic acid by the cell; components that influence the location of the polynucleotide within the cell after absorption (such as agents that measure nuclear localization); and components that influence polynucleotide expression. Such components can also include markers, such as detectable and / or selectable markers that can be used to detect or select cells that have been absorbed and are expressing nucleic acid distributed by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors that have components or functionality that mediate binding and absorption), or the vectors can be modified to provide such functionality.
[0079] Selectable markers can be positive, negative or bi-functional. Positive selectable markers allow selection of cells that carry the marker, while negative selectable markers allow cells that carry the marker to be selectively deleted. A variety of such marker genes have been described, including bi-functional (ie, positive / negative) markers (see, eg, Lupton, S., WO 92/08796, published on May 29, 1992; and Lupton, S., WO 94/28143, published December 8, 1994). Such marker genes can provide an added measure of control that can be advantageous in gene therapy settings. A wide variety of such vectors are known in the art and are generally available.
[0080] Vectors for use here include viral vectors, lipid-based vectors and other non-viral vectors that are capable of delivering a nucleotide to cells in the weakened region, ischemic region, and / or myocardial tissue peri-infarction. The vector can be a targeted vector, especially a targeted vector that preferentially binds to cells in the weakened, ischemic and / or myocardial tissue peri-infarction region. Viral vectors for use in the methods here may include those that require low toxicity to cells in the weakened, ischemic, and / or myocardial tissue peri-infarction region and induce production of therapeutically effective amounts of tissue-shaped SDF-1 protein specific.
[0081] Examples of viral vectors are those derived from adenovirus (Ad) or adeno-associated virus (AAV). Both human and non-human viral vectors can be used and the recombinant viral vector may have replication deficiency in humans. When the vector is an adenovirus, the vector may comprise a polynucleotide having a promoter operationally linked to a gene that encodes the SDF-1 protein and has replication deficiency in humans.
[0082] Other viral vectors that can be used in accordance with the fart method include vectors based on herpes virus (HSV). HSV vectors deleted from one or more immediate initial genes (IE) are advantageous because they are generally non-cytotoxic, persist in a latency-like state in the target cell, and provide effective target cell transduction. Recombinant HSV vectors can incorporate approximately 30 kb of heterologous nucleic acid.
[0083] Retroviruses, such as type C retroviruses and lentiviruses, can also be used in some forms of order. For example, retroviral vectors can be based on murine leukemia virus (MLV). See, e.g., Hu and Pathak, Pharmacol. Rev. 52: 493-511, 2000 and Fong et al., Crit. Rev. Ther. Drug Carrier Syst. 17: 1-60, 2000. MLV-based vectors can contain up to 8 kb of heterologous (therapeutic) DNA instead of viral genes. The heterologous DNA can include a specific tissue promoter and an SDF-1 nucleic acid. In distribution methods for cells close to the fold, a ligand can also be encoded to a specific tissue receptor.
[0084] Additional retroviral vectors that can be used are vectors based on replication-deficient lentiviruses, including vectors based on human immunodeficiency (HIV). See, e.g., Vigna and Naldini, J. Gene Med. 5: 308-316, 2000 and Miyoshi et al., J. Virol. 72: 8150-8157, 1998. Lentiviral vectors are advantageous in that they are able to infect both active dividing cells and non-dividing cells. They are also highly effective in transducing human skin cells.
[0085] Lentiviral vectors for use in the methods here can be derived from human and non-human lentiviruses (including SIV). Examples of lentiviral vectors include nucleic acid sequences required for vector propagation as well as a specific tissue promoor operably linked to an SDF-1 gene. These first may include viral LTRs, a primer binding site, a polipurin tract, att sites and an encapsidation site.
[0086] A lentiviral vector can be packaged in any lentiviral capsid. Replacing one particle protein with another from a different virus is called "pseudotyping". The vector capsid may contain viral envelope proteins and other viruses, including murine leukemia virus (MLV) or vesicular stomatitis virus (VSV). The use of VSV-G protein provides a high vector grade and results in greater stability of the vector virus particles.
[0087] Alfavirus-based vectors, such as those made from semliki forest viruses (SFV) and syndbis viruses (SIN) can also be used here. The use of alphavirus is described in Lundstrom, K., Intervirology 43: 247-257, 2000 and Perri et al., Journal of Virology 74: 9802-9807, 2000.
[0088] Recombinant alpha-virus vectors with replication deficiency are advantageous because they are capable of high-level heterologous (therapeutic) gene expression, and can infect a wide variety of target cells. Replicas of alphaviruses can be targeted to specific cell types by displaying a functional heterologous ligand or binding domain on their virion surface that would allow selective binding to target cells that express a cognate binding partner. Alphavirus replicates can establish latency, and therefore, long-term heterologous nucleic acid expression in a target cell. The replicates may also exhibit heterologous transient nucleic acid expression in the target cell.
[0089] In many of the viral vectors compatible with the methods of the application, more than one promoter can be included in the vector to allow more than one heterologous gene to be expressed by the vector. In addition, the vector may comprise a sequence that encodes a signal peptide or other portion that facilitates the expression of an SDF-1 gene product from the target cell.
[0090] To combine advantageous properties of two viral vector systems, hybrid viral vectors can be used to deliver an SDF-1 nucleic acid to a target tissue. Standard techniques for the construction of hybrid vectors are well known to those skilled in the art. Such techniques can be found, for example, in Sambrook, et al., In Molecular Cloning: A laboratory manual. Cold Spring Harbor, N.Y. or any number of laboratory manuals that discuss recombinant DNA technology. Double-stranded AAV genomes in adenoviral capsids containing a combination of AAV and adenoviral ITRs can be used to transduce cells. In another variation, an AAV vector can be placed in a "cowardly" adenoviral vector "that depends on help" or "high capacity". Hybrid adenovirus / AAV vectors are discussed in Lieber et al., J. Virol. 73: 9314-9324, 1999. Hybrid retrovirus / adenovirus vectors are discussed in Zheng et al., Nature Biotechnol. 18: 176-186, 2000. Retroviral genomes contained in an adenovirus can integrate into the target cell's genome and perform stable SDF-1 gene expression.
[0091] Other elements of nucleotide sequence that facilitate the expression of the SDF-1 gene and cloning of the vector are still contemplated. For example, the presence of enhancers upstream of the promoter or terminators downstream of the coding region, for example, can facilitate expression.
[0092] In accordance with another aspect of the application, a specific tissue promoter, can be fused to an SDF-1 gene. By fusing such a specific tissue promoter within the adenoviral construct, transgene expression is limited to a specific tissue. The efficacy of gene expression and the degree of specificity provided by specific tissue promoters can be determined using the recombinant adenoviral system described herein.
[0093] In addition to viral vector-based methods, non-viral methods can also be used to introduce SDF-1 nucleic acid into a target cell. A review of non-viral methods of gene distribution is provided in Nishikawa and Huang, Human Gene Ther. 12: 861-870, 2001. An example of a non-viral gene delivery method according to the invention employs plasmid DNA to introduce an SDF-1 nucleic acid into a cell. Plasmid-based gene delivery methods are generally known in the art. In one example, the plasmid vector may have a structure as shown schematically in Fig. 7. The plasmid vector in Fig. 7 includes a CMV enhancer and CMV promoter upstream of an SDF-1α cDNA (RNA) sequence .
[0094] Optionally, synthetic gene transfer molecules can be designed to form multimolecular aggregates with SDF-1 plasmid DNA. These aggregates can be designed to bind to cells in the weakened, ischemic and / or myocardial tissue peri-infarction region. Cationic amphiphiles, including lipopolyamines and cationic lipids, can be used to provide transfer of independent receptor SDF-1 in target cells (e.g., cardiomyocytes). In addition, cationic liposomes or preformed cationic lipids can be mixed with plasmid DNA to generate cell transfection complexes. Methods involving cationic lipid formulations are reviewed in Feigner et al., Ann. N.Y. Acad. Sci. 772: 126-139, 1995 and Lasic and Templeton, Adv. Drug Delivery Rev. 20: 221-266, 1996. For gene distribution, DNA can also be coupled to an amphipathic cationic peptide (Fominaya et al., J Gene Med. 2: 455-464, 2000).
[0095] Methods that involve both viral and non-viral based components can be used here. For example, an Epstein Barr virus (EBV), based on a plasmid for the delivery of the therapeutic gene is described in Cui et al., Gene Therapy 8: 1508-1513, 2001. In addition, a method involving a DNA / ligand / polycationic adjuvant coupled to an adenovirus is described in Curiel, DT, Nat. Immun. 13: 141-164, 1994.
[0096] In addition, SDF-1 nucleic acid can be introduced into the target cell by transfecting the target cells using electroporation techniques. Electroporation techniques are well known and can be used to facilitate transfection of cells using plasmid DNA.
[0097] Vectors encoding SDF-1 expression can be delivered to the target cell in the form of an injectable preparation containing a pharmaceutically acceptable carrier, such as saline, as needed. Other pharmaceutical vehicles, formulations and dosages can also be used in accordance with the present invention.
[0098] In one aspect of the invention, the vector can comprise an SDF-1 plasmid, such as for example in fig. 7. SDF-1 plasmid can be delivered to cells in the weakened region, ischemic region, and / or myocardial tissue peri-infarction region by direct injection of the SDF-1 plasmid vector into the weakened region, ischemic region and / or region of peri-infarction of myocardial tissue in an amount effective to improve at least one functional myocardial parameter, such as left ventricular volume, left ventricular area, left ventricular size, or cardiac function, as well as improving the walking test 6 minutes (6MWT) of the individual or New York Heart Association (NYHA) functional classification. By injecting the vector directly into or on the periphery of the weakened region, ischemic region and / or myocardial tissue peri-infarction region, it is possible to achieve vector transfection quite effectively, and minimize the loss of recombinant vectors. This type of injection allows local transfection of a desired number of cells, especially over the weakened region, ischemic region and / or myocardial tissue peri-infarction region, thus maximizing the therapeutic effectiveness of gene transfer, and minimizing the possibility of an inflammatory response to viral proteins.
[0099] In one aspect of the application, the SDF-1 plasmid can be administered to the weakened, ischemic and / or peri-infarction region in multiple injections of an SDF-1 solution expressing plasmid DNA at each injection comprising about 0.33 mg / ml to about 5 mg / ml of SDF-1 plasmid / solution. In one example, the SDF-1 plasmid can be administered to the weakened, ischemic and / or peri-infarcted region in at least about 10 injections, at least about 15 injections, or at least about 20 injections. Multiple injections of the SDF-1 plasmid into the weakened, ischemic and / or peri-infarcted region allows a larger area and / or number of cells in the weakened, ischemic and / or peri-infarcted region to be treated.
[0100] Each injection given to the weakened, ischemic and / or peri-infarction region can have a volume of at least about 0.2 ml. The total volume of solution that includes the amount of SDF-1 plasmid administered to the weakened, ischemic and / or peri-infarction region that can improve by at least one functional parameter of the heart is at least 10 ml.
[0101] In one example, the SDF-1 plasmid can be administered to the weakened, ischemic and / or peri-infarcted region in at least about 10 injections. Each injection given to the weakened, ischemic and / or peri-infarction region can have a volume of at least about 0.2 ml. SDF-1 can be expressed in the weakened, ischemic and / or peri-infarcted region for more than about three days.
[0102] For example, each injection of solution including SDF-1 expressing plasmid can have an injection volume of at least about 0.2 ml and a concentration of SDF-1 plasmid per injection of about 0.33 mg / ml at about 5 mg / ml. In another aspect of the application, at least one functional parameter of the heart can be improved by injecting SDF-1 plasmid into the weakened, ischemic and / or peri-infarcted heart region in an injection volume per site of at least about 0.2 ml, at least about 10 injection sites, and at a concentration of SDF-1 plasmid per injection of about 0.33 mg / ml to about 5 mg / ml.
[0103] In a porcine model of congestive heart failure, injections of a solution of SDF-1 plasmid with a concentration of less than about 0.33 mg / ml or greater than about 5 mg / ml and an injection volume per site injection of less than about 0.2 ml to a porcine model of heart failure resulted in little or no functional improvement in left ventricular volume, left ventricular area, left ventricular size, or cardiac function of the treated heart.
[0104] In another aspect of the application, the amount of SDF-1 plasmid administered to the weakened, ischemic and / or peri-infarcted region that can improve by at least one functional parameter of the heart is greater than about 4 mg and less to about 100 mg per therapeutic intervention. The amount of SDF-1 plasmid administered by therapeutic intervention here refers to the total SDF-1 plasmid administered to the subject during a therapeutic procedure designed to affect or obtain a therapeutic effect. This can include the total SDF-1 plasmid administered in a single injection for a particular therapeutic intervention or the total SDF-1 plasmid which is administered by multiple injections for a therapeutic intervention. In a porcine model of congestive heart failure, administration of about 4 mg of plasmid SDF-1 DNA by direct injection of the plasmid SDF-1 to the heart did not result in any functional improvement in left ventricular volume, area left ventricle, left ventricular size, or cardiac function of the treated heart. In addition, administration of about 100 mg of SDF-1 plasmid DNA by direct injection of the SDF-1 plasmid into the heart did not result in any functional improvement in left ventricular volume, left ventricular area, left ventricular size, or cardiac function of the treated heart.
[0105] In some aspects of the application, SDF-1 can be expressed in an effective therapeutic amount or dose in the weakened, ischemic and / or peri-infarcted region after transfection with the plasmid SDF-1 vector for more than about three days . Expression of SDF-1 at an effective therapeutic dose or amount greater than three days can provide a therapeutic effect for the weakened, ischemic and / or peri-infarcted region. Advantageously, SDF-1 can be expressed in the weakened, ischemic and / or peri-infarction region after transfection with the plasmid SDF-1 vector in an effective therapeutic amount for less than about 90 days to mitigate potentially chronic and / or cytotoxic agents that may inhibit the therapeutic efficacy of the administration of SDF-1 to the subject.
[0106] It will be understood that the amount, volume, concentration and / or dosage of SDF-1 plasmid that is administered to any one animal or human depends on many factors, including the size of the individual, body area or age, particular composition being administered , sex, time and route of administration, general health, and other drugs being administered concurrently. Specific variations in the amounts, volumes, concentrations and / or dosages of SDF-1 plasmid mentioned above can be readily determined by one skilled in the art using the experimental methods described below.
[0107] In another aspect of the application, plasmid SDF-1 can be administered by direct injection using catheterization, such as endo-ventricular catheterization or intra-myocardial catheterization. In one example, a deflectable guide catheter device can be advanced into a retrograde left ventricle through the aortic valve. Once the device is positioned inside the left ventricle, SDF-1 plasmid can be injected into the peri-infarction region (both septal and lateral aspect) of the left ventricular area. Typically, 1.0 ml of SDF-1 plasmid solution can be injected over a period of about 60 seconds. The subject to be treated can receive at least about 10 injections (for example, about 15 to about 20 injections in total).
[0108] The subject's myocardial tissue can be visualized, before the administration of the SDF-1 plasmid to define the area of weakened, ischemic and / or peri-infarcted region, before the administration of the SDF-1 plasmid. The definition of the weakened, ischemic and / or peri-infarcted region by image allows a more precise intervention and targeting of the SDF-1 plasmid to the weakened, ischemic and / or peri-infarcted region. The imaging technique used to define the weakened, ischemic and / or peri-infarcted region of myocardial tissue can include any known cardio-imaging technique. Such imaging techniques may include, for example, at least one echocardiography, MRI, coronary angiogram, electroanatomical mapping, or fluoroscopy. It will be appreciated that other imaging techniques that can define the weakened, ischemic and / or peri-infarcted region can also be used.
[0109] Optionally, agents other than SDF-1 nucleic acids (for example, SDF-1 plasmids) can be introduced into the weakened, ischemic and / or peri-infarcted region of myocardial tissue to promote SDF-1 expression from cells of the weakened, ischemic and / or peri-infarction region. For example, agents that increase the transcription of a gene that encodes SDF-1 increase the translation of an mRNA that encodes SDF-1, and / or those that decrease the degradation of an mRNA that encodes SDF-1 could be used to increase levels of SDF-1 protein. The increase in the rate of transcription of a gene within a cell can be accomplished by introducing an exogenous promoter upstream of the gene encoding SDF-1. Enhancing elements, which facilitate the expression of a heterologous gene, can also be used.
[0110] Other agents may include other proteins, chemokines and cytokines, which, when administered to target cells, can upregulate SDF-1 expression that forms the weakened, ischemic and / or peri-infarcted region of myocardial tissue. Such agents may include, for example: insulin-like growth factor (IGF) -1, which has been shown to upregulate SDF-1 expression when administered to mesenchymal stem cells (MSCs) (Circ. Res. 2008, November 21; 103 (11): 1300-98); the Sonic hedgehog gene (Shh), which has been shown to upregulate SDF-1 expression when administered to adult fibroblasts (Nature Medicine, Volume 11, Number 11, Nov. 23); transformation growth factor β (TGF-β); which has been shown to upregulate SDF-1 expression when administered to human peritoneal mesothelial cells (HPMCs); IL-1β, PDGF, VEGF, TNF-α, and PTH, which has been shown to upregulate SDF-1 expression when administered to primary human osteoblasts (hOBs) mixed with stromal marrow cells (BMSCs), and lines human osteoblast-like cells (Bone, 2006, Apr; 38 (4): 497-508); thymosin β4, which has been shown to upregulate expression when administered to bone marrow cells (BMC) (Curr. Pharm Des 2007; 13 (31): 3245-51, and inducible hypoxia factor 1a (HIF-1) , which has been shown to positively regulate SDF-1 expression, when administered to bone marrow-derived progenitor cells (Cardiovasc. Res. 2008, Pub E ..). These agents can be used to treat specific cardiomyopathies where such cells are capable of positive regulation of SDF-1 expression with respect to the specific cytokine are present or administered.
[0111] The expression SDF-1 protein or agent, which causes it to increase, and / or upregulate SDF-1, can be administered to the weakened, ischemic and / or peri-infarcted region of pure myocardial tissue or in a pharmaceutical composition. The pharmaceutical composition can provide localized release of SDF-1 or agent to cells in the weakened, ischemic and / or peri-infarcted region to be treated. The pharmaceutical compositions according to the application will generally include an amount of SDF-1 or the agent mixed with an acceptable pharmaceutical diluent or excipient, such as a sterile aqueous solution, to provide a range of final concentrations, depending on the intended use. Preparation techniques are generally well known in the art, as exemplified by Remington's Pharmaceutical Sciences, 16th ed. Mack Publishing Company, 1980, incorporated herein by reference. In addition, for administration to humans, preparations must meet sterility, pyrogenicity, general safety and purity standards, as required by the FDA's Office of Biological Standards.
[0112] The pharmaceutical composition can be in an injectable unit dosage form (for example, 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 vehicle can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), dextrose, saline, or phosphate buffered saline, its suitable mixtures and vegetable oils.
[0113] Adequate fluidity can be maintained, for example, by using a coating, such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Non-aqueous vehicles such as cottonseed oil, sesame oil, olive oil, soy oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate, can also be used as systems of solvents for compositions of compounds.
[0114] In addition, various additives, which improve the stability, sterility, and isotonicity of the compositions, including anti-microbial preservatives, chelating agents, antioxidants, and buffers, can be added. The 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. The prolonged absorption of the injectable pharmaceutical form can be caused by the use of agents that delay absorption, for example, aluminum monostearate and gelatin. According to the methods described herein, however, any vehicle, diluent or additive used would have to be compatible with the compounds.
[0115] Sterile injectable solutions can be prepared by incorporating the compounds used in the practice of the methods described herein in the required amount of the appropriate solvent with various amounts of other ingredients, as desired.
[0116] "Slow-release" pharmaceutical capsules or "sustained-release" compositions or preparations can be used and are generally applicable. Slow release formulations are generally designed to give a constant drug level over an extended period and can be used to deliver SDF-1 or an agent. Slow-release formulations are typically implanted in the vicinity of the region, weakened, ischemic and / or peri-infarction of myocardial tissue.
[0117] Examples of sustained release preparations include semi-permeable matrices of solid hydrophobic polymers containing the SDF-1 or agent, which are matrices in the form of shaped articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters; hydrogels, for example, poly (2-hydroxyethyl methacrylate) or poly (vinyl alcohol); polylactides, for example, U.S. Patent No. 3,773,919; copolymers of L-glutamic acid and γ ethyl-L-glutamate; non-degradable ethylene-vinyl acetate; degradable lactic acid-glycolic acid copolymers, such as Lupron Depot (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D - (-) -3-hydroxybutyric.
[0118] Although polymers such as ethylene vinyl acetate and lactic acid-glycolic acid allow molecules to be released for more than 100 days, certain hydrogels release proteins for shorter periods of time. When encapsulated, SDF-1 or the agent may remain in the body for a long time, and may denature or aggregate as a result of exposure to moisture at 37 0 C, thereby reducing biological activity and / or changing immunogenicity. Rational strategies are available for stabilization depending on the mechanism involved. For example, if the aggregation mechanism involves the formation of intermolecular SS bonds through transfer thiodisulfide, stabilization is achieved by modifying sulfhydryl residues, freeze-drying from acidic solutions, controlling moisture content, using appropriate additives, developing polymer-specific matrix compositions, and the like.
[0119] In certain embodiments, liposomes and / or nanoparticles can also be used with SDF 1 or an agent. The formation and use of liposomes is generally known to those skilled in the art, as summarized below.
[0120] Liposomes are formed from phospholipids which are dispersed in an aqueous medium and bilayered concentric vesicles in a spontaneously multilamellar form (also called multilamellar vesicles (MLV). MLVs generally have diameters from 25 nm to 4 µm. Sonication of MLV results in the formation of small unilamellar vesicles (SUVs), with diameters in the range of 200 to 500 Á, containing an aqueous solution in the nucleus.
[0121] Phospholipids can form a wide variety of other liposome structures when dispersed in water, depending on the molar ratio of lipid to water. In low proportions, the liposome is the preferred structure. The physical characteristics of liposomes depend on the pH, ionic strength and the presence of divalent cations. Liposomes may show low permeability to ionic and polar substances, but at high temperatures they undergo a phase transition, which markedly alters their permeability. The phase transition involves a change from a compact, orderly structure, known as the gel state, to a loosely packaged structure, the less orderly structure, known as the fluid state. This occurs at a characteristic phase transition temperature and results in an increased permeability to ions, sugars and drugs.
[0122] Liposomes interact with cells through four different mechanisms: endocytosis by phagocytic cells in the reticuloendothelial system, such as macrophages and neutrophils; adsorption to the cell surface, either by weak non-specific hydrophobic or electrostatic forces, or by specific interactions with cell surface components; fusion with the plasma cell membrane by inserting the lipid bilayer of the liposome into the plasma membrane, with the simultaneous release of liposome content into the cytoplasm and by transferring liposomal lipids to cell or subcellular membranes, or vice versa, without any association of liposome content. Varying the liposome formulation can change which mechanism is operative, although more than one can operate at the same time.
[0123] Nanocapsules can generally trap compounds in a stable and reproducible manner. To avoid side effects due to polymeric intracellular overload, such ultrafine particles (size about 0.1 µm) must be designed using polymers capable of being degraded in vivo. Biodegradable polyalkyl-cyanoacrylate nanoparticles that satisfy these requirements are contemplated for use in the methods, and such particles can be easily prepared.
[0124] To prepare pharmaceutical compositions from the application compounds, pharmaceutically acceptable carriers can be in any form (for example, solids, liquids, gels, etc.). A solid carrier can be one or more substances, which can also act as diluents, flavoring agents, binders, preservatives, and / or an encapsulating material.
[0125] The following examples are for illustrative purposes only and are not intended to limit the scope of the claims, which are attached. Examples Example 1
[0126] Factor 1 derived from the stromal cell or SDF-1 is a naturally occurring chemokine whose expression is rapidly up-regulated in response to tissue damage. SDF-1 stimulates induction in a number of anti-inflammatory protection pathways, causes down-regulation of pro-inflammatory mediators (such as MMP-9 and IL-8), and can protect cells from apoptosis. In addition, SDF-1 is a strong chemo attractant for specific organs, bone marrow and stem cells derived and progenitor cells for the site of tissue damage, which promotes tissue preservation and blood vessel development. Based on the observations that increased SDF-1 expression led to improved cardiac function in ischemic animal models, we focused on the development of naked-DNA, non-viral SDF-1-plasmid coding for the treatment of ischemic cardiovascular disease . During the course of development, the plasmid was optimized based on cell culture and the results of the small animal study are described below. Plasmid ACL-01110Sk was selected based on its ability to express transgenes in cardiac tissue and to consistently improve cardiac function in pre-clinical animal models of ischemic cardiomyopathy. SDF-1 expression of the transgene in ACL-01110Sk is driven by the CMV enhancer / promoter, CMV-intron A, and the translational enhancer RU5. The drug product, JVS-100 (formerly ACRX-100), is composed of plasmid ACL-01110Sk in 5% dextrose.
[0127] The initial study in a mouse model with heart failure demonstrated that ACL-01110S (a precursor SDF-1 with ACL-01110Sk) improved cardiac function after the injection of the plasmid directly into the infarct frontier zone of rat hearts four weeks after a myocardial infarction. Benefits were maintained for at least 8-10 weeks after injection and correlated with increased vasculogenesis in the treated ACL-01110S animals. ACL_ 01110S has been modified to optimize your expression profile. Dose-Dependent Plasmid Expression in a Ml Mouse Model
[0128] To determine the dose of plasmid by injection that would provide maximum expression in cardiac rat tissue, increasing doses (10, 50, 100, 500 µg) of the plasmid ACL-00011L luciferase were injected into the hearts of infarcted rats. Lewis rats underwent a median sternotomy and the left anterior descending artery (LAD) was permanently ligated, and injected peri-MI into a site with 100 µl ACL-00011L plasmid in PBS. Luciferase expression throughout the body was measured at each dose (n = 3) by non-invasive bioluminescent imaging (Xenogen, Hopkinton, MA) at baseline and at 1, 2, 3, 4 and 5 days after injection. Peak expression increased to a dose of 100 µg and saturated with higher doses. Based on this dose-response curve, a dose of 100 µg was determined to be sufficient for maximum expression plasmid in rat hearts. ACL-00011L expresses the luciferase gene from a vector backbone equivalent to that used in the construction of ACL-00011S, which expresses SDF-1. Comparison of Cardiac Vector Expression in a Mouse Model of Ischemic Heart Failure
[0129] Luciferase equivalents expressing various candidates for SDF-1 plasmid were tested for expression in cardiac tissue in a rat model of myocardial infarction (M1). Plasmid candidates differed in the promoters of conduction expression and the presence of enhancer elements. Lewis rats underwent a median sternotomy and the left anterior descending artery (LAD) was permanently attached and the chest was closed. Four weeks later, the chest was reopened, and plasmids expressing luciferase were injected directly (100 µg in 100 µl per injection) into 4 peri-infarction sites of the myocardium. After 1, 2, 4, 6, 8 and 10 days after the injection (and every 3-4 days thereafter), the rats were anesthetized, injected with lucifenna and photographed with a full body image of the Xenogen Luciferase system.
[0130] The two CMV oriented plasmids tested, ACL-00011L and ACL-01110L, produced detectable luciferase expression within 24 hours of injection with an initial peak of expression minus 2 days after injection.
[0131] ACL-01110L peak expression was 7 times greater than ACL-00011L and expression was approximately 10 days longer (lasting up to 16 days after injection). In contrast, ACL-00021L (αMHC driven plasmid) showed no initial peak, but expressed at a low level until day 25 post-injection. These results support previous studies demonstrating that the targeted CMV plasmids can be used for the expression of the localized, transient protein in the heart and that the term of expression of the therapeutic protein can be modulated through the inclusion of enhancer elements. Efficiency of SDF-1 Plasmids in a Ml Mouse Model
[0132] The SDF-1-encoding plasmids were tested in a mouse model of M1 to determine whether functional cardiac benefit can be achieved. Lewis rats underwent a median sternotomy and the LAD was permanently attached immediately distal to the first bifurcation. Four weeks later, the chest was reopened, and one of the three SDF-1 expressing plasmids (ACL-01110S, ACL-00011S, or ACL-00021S) or saline was injected (100 µg per 100 µl injection) in 4 peri sites -MI: At baseline (pre-injection), and 2, 4 and 8 weeks after injection, the rats were anesthetized and photographed with M echocardiography mode. LVEF dimensions, shortening fraction, and LV were measured by a trained ultrasonographer who was unaware of randomization.
[0133] A strong trend in improving cardiac function was observed with both CMV plasmids, ACL-01110S and 00011S-ACL, compared to saline controls. ACL-01110S caused a statistically significant increase in shortening at four weeks that was sustained 8 weeks after injection. In contrast, no difference in function was observed between aMFIC-oriented plasmid ACL-00021S and saline. In addition, compared to the control, the ACL-01110S and the ACL-00011S-treated animals showed significant increases in the density of large vessels (ACL-01110S: 21 ± 1.8 vessels / mm2; ACL-00011S: 17 ± 1.5 vessels / mm2; saline: 6 ± 0.7 vessels / mm2, p <0.001 for both saline vessels) and 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 demonstrated the greatest improvement in cardiac function and vasculogenesis, and caused the greatest reduction in the size of the infarction.
[0134] In summary, in a mouse model of ischemic heart failure both SDF-1 encoding plasmids driven by a CMV promoter, provided cardiac functional benefit, increased vasculogenesis, and reduced infarct size compared to saline treatment . In all the parameters tested, the ACL-01110S provided the most significant benefit. Transfection efficiency of ACL-01110Sk and ACI-01010Sk in H9C2 cells
[0135] In in vitro transfection of H9C2 myocardial cells without transfection reagents (that is, naked plasmid DNA was added to cells in culture) were used to estimate in vivo transfection efficiencies of GFP versions of Juventas carrying plasmid vectors, ACL- 01110Sk and ACL-01010Sk. 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 in 5% dextrose. The GFP vectors were constructed from the ACL-01110Sk (ACL-01110G) or ACL-01010Sk (ACL-01010G) backbone. On Day 3 post-transfection, GFP fluorescence was assessed by FACS to estimate transfection efficiency. The transfection efficiencies for vectors ACL-01110G and ACL-01010G in 5% dextrose ranged from 1.08 to 3.01%. For each amount of pDNA tested, both vectors had similar in-vitro transfection efficiency. We conclude that the transfection efficiency 1-3% observed in the present study is in line with the results of previous studies that demonstrate a similar level of transfection efficiency in vivo. Specifically, JVS-100 will transfect a limited but sufficient number of cardiac cells to produce therapeutic amounts of SDF-1. Example2 Plasmid Expression in Porcine Myocardium
[0136] A porcine Ml occlusion / reperfusion model of the left anterior descending artery (LAD) was selected as an appropriate large animal model to test the efficacy and safety of ACRX-100. In this model, 4 weeks of recovery is given between Ml and treatment to allow time for additional cardiac remodeling and to simulate chronic ischemic heart failure. Surgical procedure
[0137] Yorkshire pigs were anesthetized and heparinized for an activated clotting time (ACT) of 300 seconds or more, and placed in the supine position. To determine the LV contour, left ventriculography was performed in both the anterior - posterior and lateral views. Delivery of the Luciferase Plasmid to Porcine Myocardium
[0138] A deflectable guide catheter device was advanced into the left ventricular retrograde through the aortic valve, the guide wire was removed, and an injection needle from the endocardial LV catheter was inserted through the guide catheter into the LV cavity. . Plasmid luciferase was injected into 4 sites of a given volume and concentration were made into either the lateral septal or heart wall. Five combinations of plasmid concentration (0.5, 2 or 4 mg / ml) and injection volumes (0.2, 0.5, 1.0 ml) were tested. The 0.5 mg / ml plasmid was buffered in USP Dextrose, all the others were buffered with USP buffered saline phosphate. For each injection, the needle was inserted into the endocardium, and the gene solution was injected at a rate of 0.8-1.5 ml / minute. After the injection, the needle was held in place for 15 seconds and then removed. After the injections were completed, all instrumentation was removed, the incision was closed, and the animal was left to recover. Myocardial tissue collection
[0139] On Day 3 after the injection, the animals underwent necropsy. After euthanasia, the heart was removed, weighed, and perfused with lactated Ringer's solution until it was clear of blood. The LV was opened and the injection sites identified. A 1-centimeter square cube of tissue was taken around each injection site. Four (4) cubes harvested from the remote back wall from any injection sites served as negative controls. The tissue samples were frozen in liquid nitrogen and stored at -20 to -70 ° C. Evaluation of luciferase expression
[0140] The tissue samples were thawed and placed in a 5 ml glass tube. Lysis buffer (0.5 - 1.0 ml) was added and the tissue was interrupted using Politron homogenization (model PT1200) on ice. Tissue homogenate was centrifuged and the surfactant protein concentration was determined for each tissue sample using the Bio-Rad Detergent-Compatible Protein Assay (DC) and a standard curve of known amounts of bovine serum albumin (BSA). Homogenate tissue sample (1-10 µl) was assayed using the luciferase assay kit (Promega).
[0141] The results of the experiment are shown in fig. 1. The data shows that the expression of the vector increases with an increasing injections volume and increasing DNA concentration. Example 3 Improvement of cardiac function by SDF-1 plasmid treatment in a porcine model of ischemic cardiomyopathy Myocardial Infarction Induction
[0142] Yorkshire pigs were anesthetized and heparinized at an activated clotting time (ACT) of 250 seconds or more, and placed in the supine position. A balloon catheter was introduced by advancing through one of the guide catheter into the LAD below the first major fork in 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 minutes -120. Insufflation of the complete balloon and deflation was verified with fluoroscopy. The balloon was then removed, the incision was closed, and the animal was left to recover. Inclusion criteria
[0143] One month after MI, cardiac function in each pig was assessed by echocardiogram. If the LVEF was less than 40% and the LVESV was greater than 56.7 ml, the pig was included in the study. Surgical procedure
[0144] Each enrolled pig was anesthetized and heparinized at an activated clotting time (ACT) of 300 seconds or more, and placed in the supine position. To determine the LV contour, left ventriculography was performed in both the anterior - posterior and lateral views. Delivery of SDF-1 plasmid (ACL-01110Sk) in Myocardium
[0145] Each pig was chosen at random for one of the 3 sacrifice points: 3 days, 30 days, or 90 days post-treatment, and one of the four treatment groups: control (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). All plasmids were buffered in USP Dextrose. The injection procedure is described below.
[0146] A deflectable guide catheter device was advanced to retrograde the left ventricle through the aortic valve, the guidewire was removed, and an endocardial LV catheter injected the needle and was inserted through the guide catheter into the LV cavity. SDF-1 plasmid or buffer, at random dose gall loaded in 1 ml syringes that were attached to the catheter: Each injection volume was 1.0 ml. For each injection, the needle was inserted into the endocardium, and the solution was injected for 60 seconds. After the injection, the needle was held in place for 15 seconds and then removed. After the injections were completed, all instrumentation was removed, the incision was closed, and the animal was left to recover.
[0147] At the time of sacrifice, tissue samples from the heart and other major organs were excised and flash frozen by PCR and histopathological analysis. Assessment of cardiac function
[0148] Each animal had cardiac function assessed by the 2-dimensional echocardiography standard on day 0, 30, 60 and 90 after injection (or until sacrifice). The measurements of left ventricular volume, area and wall score were performed by an independent central laboratory. The measured effectiveness parameters are shown below in Table 1.
[0149] The impact of the SDF-1 plasmid on functional improvement is shown in Figs. 2-5. Figs. 2-4 show that low and medium doses of SDF-1 plasmid improve LVESV, LVEF, and movement index on the wall 30 days after injection compared to control and that the higher dose has no benefit. Fig. 5 demonstrates that the cardiac benefit in the low and medium dose is sustained for 90 days, as much as they show a significant attenuation in pathological remodeling, that is, a lower increase in LVESV, compared to the control. Assessment of vasculogenesis
[0150] Animals that were sacrificed on day 30 were evaluated for vessel density in the left ventricle, with 7 to 9 tissue samples taken from each heart fixed in formalin. Genomic DNA was efficiently extracted and purified from a fixed formalin tissue sample using a mini-column purification procedure (Qiagen). Samples of treated SDF-1 and control animals were tested for the presence of plasmid DNA by quantitative PCR. Three to five tissue samples were found to contain copies of plasmid DNA at least four times above the bottom (except in control animals) for each animal were used to prepare the slides and immunostained with isolectin. The cuts were identified and vessels counted in random fields by 20-40 tissue. The vessels per field were converted to vessels / mm2 and averaged for each animal. For each dose, data are reported as the vessel / mm2 mean of all animals that received the dose.
[0151] Fig. 6 shows that both doses that provided functional benefit also significantly increase vessel density in 30 days compared to control. In contrast, the higher dose, which does not improve function, does not substantially increase the density of the vessel. These data provide a biological mechanism by which putative SDF-1 plasmid is improving cardiac function in ischemic cardiomyopathy. Biodistribution Data
[0152] Distribution of JVS-100 in cardiac and non-cardiac tissues was measured 3, 30 and 90 days after pivot injection on the efficacy and toxicology study in the porcine model of myocardial infarction. In cardiac tissue, at each time point, the concentration of JVS-100 medium plasmid increased with dose. Art each dose, JVS-100 clearance was observed at 3, 30 and 90 days after injection with approximately 99.999999% clean from cardiac tissue On day 90. JVS-100 was distributed to non-cardiac organs with blood flow relatively high (eg, heart, kidney, liver, and lung) with the highest concentrations seen 3 days after injection. JVS-100 was present mainly in the kidney, according to the renal clearance of the plasmid. There were low levels of persistence at 30 days and JVS-100 was essentially undetectable in non-cardiac tissues at 90 days. Conclusions
[0153] Treatment with JVS-100 resulted in the formation of a significant increase in blood vessel and improved heart function in pigs with ischemic heart failure after a single endomyocardial injection of 7.5 and 30 mg. The highest dose of JVS-100 tested (100 mg) showed a tendency to increase blood vessel formation, but did not show improved cardiac function. None of the doses of JVS-100 were associated with signs of toxicity, adverse effects on clinical pathology parameters or histopathology. JVS-100 was distributed mainly to the heart with approximately 99.999999% clean from cardiac tissue, 90 days after treatment. JVS-100 was distributed to non-cardiac organs with relatively high blood flow (eg, heart, kidney, liver, and lung) with the highest concentrations in the kidneys 3 days after injection. JVS-100 was essentially undetectable in the body 90 days after injection with only insignificant amounts of the administered dose found in non-cardiac tissues. Based on these results, the level of unobserved adverse effect (NOAEL) for JVS-100 in the Ml pig model was 100 mg administered by endomyocardial injection Example 4 Porcine Exploratory Study: LUC Injections by Transarterial Injection in Chronic IM Pigs Methods
[0154] A pig with an anterior LAD occlusion / reperfusion Ml and an EF> 40% was injected with ACL-01110Sk with a transarterial catheter. Two injections in the LAD and 2 in the LCX were performed with an injection volume of 2.5 ml and a total injection time of 125-130 sec. An additional 3.0 ml LCX injection with a total injection time of 150 sec was performed with contrast mixed with the plasmid. Sacrifice and tissue collection
[0155] Three days after the injections, the animal was sacrificed. After euthanasia, the heart was removed, drained of blood, placed on an icy cutting board and dissected by the autopsy technician or pathologist. The myocardium not injected from the septum was obtained through the opening of the right ventricle. The right ventricle was cut from the heart and placed on cold cardioplegia. New scalpel blades were used for each of the sections.
[0156] Then, the left ventricle was opened and the entire left ventricle was excised by cutting into 6 cutting sections from the apex to the base. The LV was divided into 3 slices. After excision, each section was able to remain flat. Each section (3 LV sections, 1 RV section, and a pectoral muscle) was placed in separate containers marked with cold cardioplegia on wet ice, and transported for luciferase analysis. Luciferase image
[0157] All collected tissues were immersed in luciferin and photographed with a Xenogen imaging system to determine plasmid expression. Results
[0158] A representative image of the heart is shown in fig. 8. Colored dots denote areas of expression of luciferase. These spots showed relative light units (RLUs) greater than 106 units, more than 2 orders of magnitude above the bottom. These data demonstrated that the catheter delivered sufficiently to generate the plasmid plasmid of substantial expression over a significant portion of the heart. Example 5 Example of Clinical Study
[0159] Rising doses of JVS-100 are administered to treat HF in patients with ischemic cardiomyopathy. Safety is monitored at each dose, documenting all adverse events (AEs), with the primary safety objective being the large number of adverse cardiac events in 30 days. At each cut, individuals will receive a single dose of JVS-100. In all groups, therapeutic efficacy is assessed by measuring the impact on cardiac function using standard echocardiography, cardiac perfusion via Single Photon Emission Computed Tomography (SPECT) imaging, New York Heart Association (NYHA) class, six minutes of distance, and quality of life.
[0160] • Número de eventos cardíacos adversos maiores (MACE), 30 dias após a injeção • Eventos adversos ao longo dos 12 meses de seguimento • Laboratório de Análise de Sangue (enzimas cardíacas, CBC, ANA) • Os SDF-1 Níveis Plasmáticos • Avaliação Física • Ecocardiografia • Monitoramento AICD • ECG Eficiência: • Mudança da linha de base no VSF, VDF, FE e índice de escore de movimento de parede • Mudança da linha de base na classificação NYHA e qualidade de vida • Alteração dos valores basais na perfusão como determinado por SPECT • Mudança da linha de base na distância do Teste de Seis Minutos de Caminhada
[0161] Based on preclinical data, delivery of JVS-100 is expected to elicit cardiac function and improve symptoms in 4 months, reaching 12 months. At 4 months after the JVS-100 injections, compared to baseline, an improvement in distance in six minutes of walking of over 30 meters, an improvement in the quality of life score of about 10% and / or an improvement of about 1 NYHA class are anticipated. Likewise, a relative improvement in VSF, LVEF, and / or LVMI of approximately 10% is expected in relation to baseline values. Comparative Example 1 Evaluation of cardiac function by echocardiogram in pigs with chronic heart failure after treatment with ACL-01110Sk or ACL-01010Sk objective
[0162] The aim of this study is to compare the functional cardiac response to SDF-1 plasmids ACL-01110Sk or ACL-01010Sk after delivery of the endomyocardial catheter in a porcine model of ischemic heart failure
[0163] This study compared the effectiveness of ACL-01110Sk and ACL-01010Sk in improving function in a porcine model of ischemic heart failure. In this study, the plasmids were delivered by an injection of an endoventricular catheter needle. Efficacy was assessed by measuring the impact of therapy on cardiac remodeling (ie, left ventricular volumes) and function (ie, left ventricular ejection fraction (LVEF)) using echocardiography. Methods
[0164] Briefly, myocardial infarctions due to LAD occlusion were caused in male Yorkshire pigs via balloon angioplasty for 90 minutes. Pigs with an ejection fraction <40%, measured by M-mode echocardiogram 30 days after infarction were included. The pigs were chosen at random for one of the 3 groups to be injected either with saline phosphate buffer (PBS, control), ACL-01110Sk in PBS, or ACL-01010Sk in PBS using a catheter injection needle delivery system endoventricular (Table 3). Table 3. Initial Study Design: SDF-1 Therapy for Chronic Heart Failure in Pigs
[0165] Echocardiograms were recorded before the injection and after 30 and 60 days after the injection. Table 8 below defines the variables as they are referred to in this report. Table 4. Definition of variables
[0166] The baseline echocardiographic characteristics at the time of the initial injection (30 days post-MI) for all animals enrolled in this report (n = 9) as reported by the central echocardiography laboratory, are provided in Table 5 below.
[0167] Table 5 shows LVESV, LVEF and LVEDV at 0 and 30 days after the initial injection. The PBS control animals demonstrated an increase in LVESV and LVEDV and no improvement in LVEF consistent with this model of heart failure. Treatment groups did not reduce cardiac volumes or increase LVEF over control. Similar results were obtained at 60 days after the initial injections. Comparative Example 2
[0168] A strategy to increase stem cells homing to the peri-infarction region by delivering SDF-1 based on a transendocardial catheter in a porcine myocardial infarction model was investigated to determine whether it would improve perfusion and left ventricular function. The catheter-based approach has been used successfully for cell transplantation and delivery of angiogenic growth factors in humans.
[0169] German female Landrace pigs (30 kg) were used. After a night of fasting, the animals were anesthetized and intubated.
[0170] A French 7 sheath was placed in the femoral artery with the animal in a supine position. A wire over the balloon was advanced to the distal LAD. The balloon was inflated with 2 atm beads and agarose was injected slowly over 1 min, through the balloon catheter in the distal anterior descending artery. After 1 minute, the balloon was deflated and the distal LAD occlusion was documented by angiography. After the induction of myocardial infarction, the animals were monitored for 3-4 hours until the rhythm and blood pressure were stable. The arterial sheath was removed, carprofen (4 mg / kg) was administered intramuscularly and the animals were removed from the respirator. Two weeks after myocardial infarction, the animals were anesthetized. Electromechanical mapping of the left ventricle was performed through an 8F femoral sheath with the animal in the supine position. After a complete map of the left ventricle had been obtained, human SDF-1 (Peprotec, Rocky-Hill, NJ) was delivered by 18 injections (5 µg in 100 µml of saline) into the infarction and peri-infarction region via one of the injection catheter. 5 µg per injections was used to adjust the reported efficiency of catheter injections. The injections were performed slowly over 20 s, and only when the tip of the catheter was perpendicular) to the left ventricular wall, when the loop stability was <2 mm and when the protruding needle in the myocardium caused extra ventricular ectopic beats. Control animals were subjected to an identical process with sham injections. Echocardiography excluded post-interventional pericardial effusion.
[0171] Twenty (20) animals completed the study protocol: 8 control animals and 12 SDF-1 treated animals. For myocardial perfusion images, only 6 animals in the control group could be evaluated due to technical problems. Infarction location was anteroseptal in all animals.
[0172] The infarct size as a percentage of 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 group-1. Left ventricular muscle volume was similar in both groups (83 ± 14 ml versus 95 ± 10 ml, p = ns). Immunofluorescence technique showed significantly more vWF-positive vessels in the peri-infarction area in SDF-1 treated animals than in control animals (349 ± 17 / mm2 vs. 276 ± 21 / mm2, p <0.05). The profound loss of collagen in the peri-infarction area was observed in animals treated with SDF-1 compared to control animals (32 ± 5% vs 61 ± 6%, p <0.005). The number of inflammatory cells (neutrophils and macrophages) within the peri-infarction area was similar in both groups (332 ± 51 / mm2 vs. 303 ± 55 / mm2, p = ns). Global myocardial perfusion did not change from baseline to SPECT follow-up and there was no difference between groups. The size of the final infarct was similar in both groups and compared well with the tetrazolium staining results. Segmental analysis of myocardial perfusion revealed decreased uptake of the tracer in the apical and anteroid segments with significant differences between the myocardial segments. However, uptake of the marker at the beginning of the study and follow-up were almost identical in the control and SDF-1 treated animals. There were no differences in final diastolic and end systolic volume between groups. However, stroke volume increased in control animals and decreased slightly in SDF-1 treated animals. The difference between the two groups was significant.
[0173] Likewise, the ejection fraction increased in the control animals, with a reduction in SDF-1 treated animals. The difference between the groups showed a strong trend (p = 0.05). Local shortening, another parameter of mechanical ventricular function, did not change in control animals. However, local shortening decreased significantly in SDF-1 treated animals, resulting in a significant difference between groups. There were no significant differences in unipolar tension within and between groups. Significant correlations between baseline ejection fraction and local baseline ejection volume and shortening (EF and LS: r = 0.71, SV and LS: r = 0.59) were observed. Similar results were obtained for monitoring values (EF and LS: r = 0.49, SV and LS: r = 0.46). The change in local shortening was significantly correlated with the change in ejection fraction (r = 0.52) and ejection volume (r = 0.46). There was no correlation between the reduction in local and final diastolic volume (baseline r = -0.03, r = 0.12) or between the ejection fraction and final diastolic volume (baseline r = -0.04, r = 0.05). Segmental analysis of EME data showed decreased unipolar shortening of tension and locations in the anteroid segments with significant differences between myocardial segments at baseline. The distribution of unipolar tension values in myocardial segments was similar in both groups at baseline and at follow-up. Local segmental 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 segment of the left ventricle. There was a significant interaction between the assignment of SDF-1 and basal vessel monitoring.
[0174] The study described above demonstrated that a single application of SDF-1 protein was insufficient to produce functional cardiac benefit.
[0175] From the description above the application, those skilled in the art will notice improvements, changes and modifications. Such improvements, changes and modifications within the skill of the technique are intended to be covered by the attached claims. All patents, patent applications and publications cited herein are incorporated by reference in their entirety.

权利要求:
Claims (15)
[0001]
Stromal cell-derived factor 1 (SDF-1) plasmid characterized by the fact that it comprises an SDF-1α cDNA sequence whose expression is driven by a cytomegalovirus (CMV) enhancer and CMV-intron A and translational enhancer RU5 .
[0002]
SDF-1 plasmid according to claim 1, characterized in that said plasmid comprises, in an order of 5 'to 3', nucleotide sequences for a CMV enhancer, a CMV promoter, a CMV-intron A , RU5, SDF-1α, BGH poliA, ColE1origin and Kanamycin reversed oriented.
[0003]
Injectable preparation, characterized in that it comprises an SDF-1 plasmid defined in claim 1 and a pharmaceutically acceptable carrier.
[0004]
Injectable preparation according to claim 3, characterized in that said pharmaceutically acceptable carrier is dextrose.
[0005]
Injectable preparation according to claim 3, characterized in that it comprises from about 0.33 mg / ml to about 5 mg / ml of said plasmid SDF-1.
[0006]
Injectable preparation according to any one of claims 3 to 5, characterized in that it is for use in a method of treating a cardiomyopathy.
[0007]
Injectable preparation according to claim 6, characterized by the fact that the method involves administering the preparation to the weakened, ischemic and / or peri-infarcted region of myocardial tissue.
[0008]
Injectable preparation according to claim 7, characterized in that the total amount of plasmid SDF-1 administered is greater than 4mg.
[0009]
Injectable preparation according to claim 7 or 8, characterized by the fact that the method involves administration of the preparation by direct injection to the weakened, ischemic, and / or myocardial tissue peri-infarction region.
[0010]
Injectable preparation according to any one of claims 7 to 9, characterized in that the method involves administering the preparation to the weakened, ischemic, and / or peri-infarction region in at least 10 injections with each injection with a volume of hair minus 0.2 ml.
[0011]
Injectable preparation according to claim 10, characterized by the fact that the total amount of preparation administered is at least about 10 ml.
[0012]
Injectable preparation according to any of claims 7 to 11, characterized in that the preparation is administered to the weakened, ischemic, and / or peri-infarction region in at least 15 injections with each injection comprising 0.33 mg / ml at 5 mg / ml of plasmid SDF-1.
[0013]
Injectable preparation according to any of claims 6 to 12, characterized in that the method involves administration by direct injection using catheterization.
[0014]
Injectable preparation according to claim 13, characterized by the fact that the catheterization is selected from endoventricular catheterization or intra-myocardial catheterization.
[0015]
Plasmid expressing SDF-1 according to claim 1 or 2, characterized by the fact that it is for use in a method of improving the vasculogenesis of a weakened, ischemic and / or peri-infarcted region by at least 20%, based on vessel density or measured by myocardial perfusion imaging with an improvement in added rest score, added stress score and / or added difference score of at least 10%, the method comprising administering, by injection, a solution comprising the plasmid expressing SDF-1 to the weakened, ischemic and / or peri-infarction region.

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112012004395B1|2021-01-12|stromal-derived factor 1 plasmid | and injectable preparation comprising said plasmid

---

JP2006509043A|2006-03-16|Cell-based VEGF delivery

---

AU2004202064A1|2004-06-10|Methods for Altering Cardiac Cell Phenotype

---

US8933048B2|2015-01-13|Methods of treating cardiomyopathy

---

EP2044199B1|2012-11-14|Extended antegrade epicardial coronary infusion of adeno-associated viral vectors comprisingserca2a for gene therapy

---

HU0300923A2|2003-07-28|Methods and compositions useful for modulation of angiogenesis using protein kinase raf and ras

---

JP6461085B2|2019-01-30|SDF-1 delivery for treating ischemic tissue

---

US20170049856A1|2017-02-23|Sdf-1 delivery for treating advanced ischemic cardiomyopathy

---

WO2015153357A1|2015-10-08|Compositions and methods for improving cardiac function

同族专利:

---

公开号 | 公开日

---

JP2013503202A|2013-01-31|

---

US8883756B2|2014-11-11|

---

WO2011026041A9|2011-07-21|

---

JP5856059B2|2016-02-09|

---

CN102740894A|2012-10-17|

---

EP2473196A4|2015-01-07|

---

US20120289586A1|2012-11-15|

---

MX357779B|2018-07-24|

---

AU2010286511A1|2012-04-12|

---

WO2011026041A2|2011-03-03|

---

US20120289585A1|2012-11-15|

---

CA2772610C|2018-01-23|

---

US20140135383A1|2014-05-15|

---

CN102740894B|2015-07-15|

---

MX2012002610A|2012-08-23|

---

EP2473196B1|2017-05-31|

---

EP2473196A2|2012-07-11|

---

CA2772610A1|2011-03-03|

---

US8513213B2|2013-08-20|

---

US20130303597A1|2013-11-14|

---

BR112012004395A2|2016-11-16|

---

AU2010286511B2|2016-05-26|

---

US20120283315A1|2012-11-08|

---

US9844581B2|2017-12-19|

---

US8513007B2|2013-08-20|

---

BR112012004395B8|2021-05-25|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US3773919A|1969-10-23|1973-11-20|Du Pont|Polylactide-drug mixtures|

---

AU650085B2|1990-11-13|1994-06-09|Immunex Corporation|Bifunctional selectable fusion genes|

---

US5197985A|1990-11-16|1993-03-30|Caplan Arnold I|Method for enhancing the implantation and differentiation of marrow-derived mesenchymal cells|

---

AU6953394A|1993-05-21|1994-12-20|Targeted Genetics Corporation|Bifunctional selectable fusion genes based on the cytosine deaminase gene|

---

US5602301A|1993-11-16|1997-02-11|Indiana University Foundation|Non-human mammal having a graft and methods of delivering protein to myocardial tissue|

---

CN100569297C|1995-02-28|2009-12-16|加利福尼亚大学董事会|Gene transfer-mediated vascularization therapy|

---

US6121246A|1995-10-20|2000-09-19|St. Elizabeth's Medical Center Of Boston, Inc.|Method for treating ischemic tissue|

---

US5980887A|1996-11-08|1999-11-09|St. Elizabeth's Medical Center Of Boston|Methods for enhancing angiogenesis with endothelial progenitor cells|

---

DE69827021T2|1997-01-29|2005-02-24|Cornell Research Foundation, Inc.|AT VARIOUS PLACES ADVERTISING ADENOVIRUS VECTOR FOR INDUCING ANGIOGENESIS|

---

MXPA00003885A|1997-10-22|2004-04-23|Inst Genetics Llc|Chimeric polypeptides containing chemokine domains.|

---

PT998486E|1997-06-13|2007-08-21|Genentech Inc|Protein recovery by chromatography followed by filtration upon a charged layer|

---

US20020031803A1|1997-06-20|2002-03-14|Cooper Mark J.|Expression system for production of therapeutic proteins|

---

US20030129750A1|1998-02-05|2003-07-10|Yitzhack Schwartz|Homing of donor cells to a target zone in tissue using active therapeutics or substances|

---

IL137616D0|1998-02-06|2001-07-24|Collateral Therapeutics Inc|Variants of vegf-a|

---

US6676937B1|1998-03-09|2004-01-13|Caritas St. Elizabeth's Medical Center Of Boston Inc.|Compositions and methods for modulating vascularization|

---

CA2322559C|1998-03-09|2012-07-17|St. Elizabeth's Medical Center|Compositions and methods for modulating vascularization|

---

AT525477T|1998-03-30|2011-10-15|Northwest Biotherapeutics Inc|THERAPEUTIC AND DIAGNOSTIC APPLICATIONS BASED ON THE ROLE OF CXCR-4 IN TUMOR GENESIS|

---

US20020107196A1|1998-07-21|2002-08-08|Smithkline Beecham Corporation|Method for inducing chemotaxis in endothelial cells by administering stromal cell derived factor-1alpha|

---

IL125532D0|1998-07-27|1999-03-12|Yeda Res & Dev|Hematopoietic cell composition for use in transplantation|

---

WO2000006086A2|1998-07-31|2000-02-10|The Trustees Of Columbia University In The City Of New York|Use of inhibitors of the activation of cxcr4 receptor by sdf-1 in treating rheumatoid arthritis|

---

WO2000015285A1|1998-09-14|2000-03-23|Mirus Corporation|A process for delivering nucleic acids to cardiac tissue|

---

DE19844479C1|1998-09-28|2000-04-13|Siemens Ag|Integrated memory with a differential sense amplifier|

---

EP1013770A1|1998-12-23|2000-06-28|Université Louis Pasteur de Strasbourg|Non-viral transfection vector|

---

DE19900743A1|1999-01-12|2000-07-13|Aventis Pharma Gmbh|New complexing proteins|

---

WO2000041732A1|1999-01-19|2000-07-20|The Children's Hospital Of Philadelphia|Hydrogel compositions for controlled delivery of virus vectors and methods of use thereof|

---

US20050153451A1|1999-02-26|2005-07-14|Wolff Jon A.|Intravascular delivery of non-viral nucleic acid|

---

AU3376300A|1999-02-26|2000-09-14|University Of Pittsburgh|Bone marrow transplantation for hepatic regeneration and repair|

---

EP1041148A1|1999-04-02|2000-10-04|Mogen International N.V.|Pathogen inducible promoter|

---

WO2000059941A1|1999-04-08|2000-10-12|The General Hospital Corporation|Purposeful movement of human migratory cells away from an agent source|

---

US7125856B1|1999-04-15|2006-10-24|St. Elizabeth's Medical Center Of Boston, Inc.|Angiogenic growth factors for treatment of peripheral neuropathy|

---

US6358697B2|1999-04-21|2002-03-19|Children's Hospital Medical Center|Intracellular pharmaceutical targeting|

---

US7399751B2|1999-11-04|2008-07-15|Sertoli Technologies, Inc.|Production of a biological factor and creation of an immunologically privileged environment using genetically altered Sertoli cells|

---

AU4801501A|2000-05-26|2001-11-29|Transgene S.A.|Complex for transferring an anionic substance of interest into a cell|

---

JP5414958B2|2000-06-05|2014-02-12|ザ・トラスティーズ・オブ・コランビア・ユニバーシティー・イン・ザ・シティー・オブ・ニューヨーク|Identification of human bone marrow-derived endothelial progenitor cells and use of human bone marrow-derived endothelial progenitor cells to improve the function of cardiomyocytes after ischemic injury|

---

AU8469501A|2000-07-31|2002-02-13|New York Medical College|Methods and compositions for the repair and/or regeneration of damaged myocardium|

---

KR20020059609A|2000-08-04|2002-07-13|벤슨 로버트 에이치.|Vascular endothelial growth factor 2|

---

US20040009940A1|2000-10-20|2004-01-15|Coleman Michael E.|Gene delivery formulations and methods for treatment of ischemic conditions|

---

US7402567B2|2000-10-27|2008-07-22|The Regents Of The University Of California|Treatment of disease by inducing cell apoptosis|

---

US20020094327A1|2000-11-05|2002-07-18|Petersen Bryon E.|Targeting pluripotent stem cells to tissues|

---

WO2002040646A1|2000-11-14|2002-05-23|Universite Libre De Bruxelles|Generation and use of dendritic cells|

---

CA2430401A1|2000-12-01|2002-06-06|Schering Corporation|Uses of mammalian genes and related reagents|

---

AR027161A1|2001-05-15|2003-03-19|Bio Sidus S A|METHOD FOR INDUCTING NEOVASCULAR PROLIFERATION AND TISSULAR REGENERATION|

---

US7399740B2|2001-06-28|2008-07-15|Yeda Research And Development Co. Ltd.|Poly-Glu,Tyr for neuroprotective therapy|

---

KR20040023724A|2001-08-07|2004-03-18|기린 비루 가부시키가이샤|Process for Preparing Hematopoietic Stem Cells|

---

CA2460184A1|2001-09-14|2003-03-27|Stem Cell Therapeutics Inc.|Prolactin induced increase in neural stem cell numbers and therapeutical use thereof|

---

US7402724B2|2002-01-04|2008-07-22|Mayo Foundation For Medical Education And Research|Longevity and PAPP-A|

---

AU2002226683A1|2002-01-17|2003-07-30|Cardio Incorporated|Complex therapy for tissue regeneration|

---

US7396537B1|2002-02-28|2008-07-08|The Trustees Of The University Of Pennsylvania|Cell delivery patch for myocardial tissue engineering|

---

KR20050013531A|2002-03-15|2005-02-04|디파트먼트 오브 베테랑스 어페어스 오피스 오브 더 제네럴 카운셀 |Methods and Compositions Using Cellular Asialodeterminants and Glycoconjugates for Targeting Cells to Tissues and Organs|

---

US20030199464A1|2002-04-23|2003-10-23|Silviu Itescu|Regeneration of endogenous myocardial tissue by induction of neovascularization|

---

US20050271639A1|2002-08-22|2005-12-08|Penn Marc S|Genetically engineered cells for therapeutic applications|

---

US20040037811A1|2002-08-22|2004-02-26|The Cleveland Clinic Foundation|Stromal cell-derived factor-1 mediates stem cell homing and tissue regeneration in ischemic cardiomyopathy|

---

US20040161412A1|2002-08-22|2004-08-19|The Cleveland Clinic Foundation|Cell-based VEGF delivery|

---

FR2843697B1|2002-08-26|2005-12-09|Sod Conseils Rech Applic|HETEROCARPINE, A PROTEIN OF VEGETABLE ORIGIN WITH ANTICANCER PROPERTIES|

---

JP2004099471A|2002-09-05|2004-04-02|Cardio Corp|Medicine for treating cardiac infarction and cardiac failure|

---

AU2003300332A1|2002-10-08|2004-05-04|American National Red Cross|Method for enriching adherent monocyte populations|

---

US20040258669A1|2002-11-05|2004-12-23|Dzau Victor J.|Mesenchymal stem cells and methods of use thereof|

---

JP4672370B2|2002-12-05|2011-04-20|ケースウエスタンリザーブユニバーシティ|Cell-based treatment of ischemia|

---

EP1600511A4|2003-02-19|2006-08-23|Dnavec Research Inc|Method of treating ischemic disease|

---

AU2003902037A0|2003-04-24|2003-05-15|Mclachlan, Craig|Method for tissue growth|

---

US20050104072A1|2003-08-14|2005-05-19|Slater David B.Jr.|Localized annealing of metal-silicon carbide ohmic contacts and devices so formed|

---

US7396680B2|2003-10-31|2008-07-08|Cornell Research Foundation, Inc.|Stem cell-specific promoters and their use|

---

IL158868D0|2003-11-13|2004-05-12|Yeda Res & Dev|Methods of generating and using stem cells enriched with immature primitive progenitor|

---

US8071364B2|2003-12-24|2011-12-06|Transgenrx, Inc.|Gene therapy using transposon-based vectors|

---

JP5269411B2|2004-06-21|2013-08-21|ザクリーブランドクリニックファウンデーション|CCR ligand for homing stem cells|

---

EP1803464A4|2004-09-17|2009-09-09|Cellgentech Inc|External preparation for treating skin ulcer|

---

CA2581652C|2004-09-27|2013-10-29|Valentis, Inc.|Formulations and methods for treatment of inflammatory diseases|

---

US20060105950A1|2004-10-25|2006-05-18|Caritas St. Elizabeth's Medical Center Of Boston, Inc.|Morphogen compositions and use thereof to treat wounds|

---

WO2006083394A2|2004-12-21|2006-08-10|Ethicon, Inc.|Postpartum cells derived from placental tissue, and methods of making, culturing, and using the same|

---

US7951632B1|2005-01-26|2011-05-31|University Of Central Florida|Optical device and method of making|

---

GB2424312B|2005-03-14|2010-03-03|Denso Corp|Method of forming an ohmic contact in wide band semiconductor|

---

KR20130086057A|2005-09-16|2013-07-30|크리 인코포레이티드|Methods of processing semiconductor wafers having silicon carbide power devices thereon|

---

EP2264741B1|2006-01-10|2021-03-10|Cree, Inc.|Silicon carbide dimpled substrate|

---

US20070173471A1|2006-01-24|2007-07-26|Losordo Douglas W|Morphogen compositions and methods of use thereof to treat heart disorders|

---

US7405195B2|2006-03-27|2008-07-29|Natural Beauty Bio-Technology Limited|Cosmetic compositions|

---

EP2142206B1|2007-03-30|2014-07-30|The Cleveland Clinic Foundation|SDF-1 for use in the treatment of ischemic peripheral vascular disorders|

---

US20100272679A1|2007-12-14|2010-10-28|Penn Marc S|Compositions and methods of promoting wound healing|

---

MX357779B|2009-08-28|2018-07-24|Cleveland Clinic Found|Sdf-1 delivery for treating ischemic tissue.|

---

US20130252876A1|2010-09-15|2013-09-26|The Cleveland Clinic Foundation|Compositions and method for promoting musculoskeletal repair|

---

US20120068161A1|2010-09-16|2012-03-22|Lee Keon-Jae|Method for forming graphene using laser beam, graphene semiconductor manufactured by the same, and graphene transistor having graphene semiconductor|

---

JP5569376B2|2010-12-07|2014-08-13|住友電気工業株式会社|Manufacturing method of semiconductor device|US20050271639A1|2002-08-22|2005-12-08|Penn Marc S|Genetically engineered cells for therapeutic applications|

---

EP2142206B1|2007-03-30|2014-07-30|The Cleveland Clinic Foundation|SDF-1 for use in the treatment of ischemic peripheral vascular disorders|

---

US20100272679A1|2007-12-14|2010-10-28|Penn Marc S|Compositions and methods of promoting wound healing|

---

MX357779B|2009-08-28|2018-07-24|Cleveland Clinic Found|Sdf-1 delivery for treating ischemic tissue.|

---

US9308277B2|2010-02-25|2016-04-12|Mesoblast International Sàrl|Protease-resistant mutants of stromal cell derived factor-1 in the repair of tissue damage|

---

US20130295064A1|2010-10-14|2013-11-07|University Of Central Florida Research Foundation, Inc.|Cardiac induced pluripotent stem cells and methods of use in repair and regeneration of myocardium|

---

US20130136729A1|2011-11-11|2013-05-30|University of Virginia Patent Foundation, d/b/a University of Virginia Licensing & Ventures Group|Compositions and methods for targeting and treating diseases and injuries using adeno-associated virus vectors|

---

EP2968436A4|2013-03-15|2016-10-26|Juventas Therapeutics Inc|The use of sdf-1 to mitigate scar formation|

---

WO2014145199A2|2013-03-15|2014-09-18|The Cleveland Clinic Foundation|Retrograde delivery of sdf-1 for treatment of myocardial infarction|

---

CN105264071B|2013-03-15|2020-05-19|克利夫兰临床医学基金会|SDF-1 delivery for treatment of ischemic tissue|

---

US20160278810A1|2013-05-31|2016-09-29|Mark Edmund Richey|Vaginal surgical apparatus|

---

US10166044B1|2013-05-31|2019-01-01|Freshwater Bay Industries, Llc|Apparatus for repositioning the vagina, cervix, uterus and pelvic floor and method to secure same|

---

WO2015168145A1|2014-04-28|2015-11-05|Penn Marc S|Sdf-1 delivery for treating advanced ischemic cardiomyopathy|

---

EP3139945A4|2014-05-08|2017-11-29|Wake Forest University Health Sciences|Methods of treating incontinence and other sphincter deficiency disorders|

---

EP3237622B1|2014-12-23|2021-02-24|Ilya Pharma AB|Methods for wound healing|

---

CA3004742A1|2015-11-11|2017-05-18|Intrexon Corporation|Compositions and methods for expression of multiple biologically active polypeptides from a single vector for treatment of cardiac conditions and other pathologies|

---

US11229682B2|2017-03-30|2022-01-25|Wake Forest University Health Sciences|Methods of treatment for kidney disease|

法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

---

2018-03-27| B15K| Others concerning applications: alteration of classification|Ipc: A61K 38/19 (2006.01), A61K 9/00 (2006.01), A61K 31 |

---

2018-04-10| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2019-01-29| B07E| Notice of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|Free format text: NOTIFICACAO DE ANUENCIA RELACIONADA COM O ART 229 DA LPI |

---

2019-11-12| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2020-04-22| B06A| Notification to applicant to reply to the report for non-patentability or inadequacy of the application [chapter 6.1 patent gazette]|

---

2020-10-13| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-01-12| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 10 (DEZ) ANOS CONTADOS A PARTIR DE 12/01/2021, OBSERVADAS AS CONDICOES LEGAIS. |

---

2021-05-25| B16C| Correction of notification of the grant|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 30/08/2010 OBSERVADAS AS CONDICOES LEGAIS. PATENTE CONCEDIDA CONFORME ADI 5.529/DF |

优先权:

---

申请号 | 申请日 | 专利标题

---

US23777509P| true| 2009-08-28|2009-08-28|

---

US61/237,775|2009-08-28|

---

US33421610P| true| 2010-05-13|2010-05-13|

---

US61/334,216|2010-05-13|

---

PCT/US2010/047175|WO2011026041A2|2009-08-28|2010-08-30|Sdf-1 delivery for treating ischemic tissue|

---