Patent Application
20200239862
The present invention relates to a pharmaceutical composition for preventing or treating heart failure. Specifically, the present invention relates to a gene construct that comprises a nucleotide sequence encoding a SERCA2a protein or a fragment thereof and a nucleotide sequence encoding a CCN5 protein or a fragment thereof, and to a pharmaceutical composition for preventing or treating heart failure which comprises the gene construct as an active ingredient.
Heart failure (HF) is a disease in which complex symptoms occur due to structural and functional impairment in the ventricular pump function that fills or ejects blood. Heart failure is a disease with a higher mortality rate than cancer, and the mortality rate thereof within 5 years of diagnosis is 50% or higher. The number of heart failure patients is estimated at 38 million people all over the world, and prevalence thereof is also increasing with aging. However, there is no fundamental cure for heart failure, and current treatments can only slow down progression of the disease. Therefore, medical care thereof incurs a lot of treatment costs, which is a big burden on patients (Braunwald E. Lancet, 2015; 385: 812-824).
Heart failure is caused by a wide range of heart diseases that result from heart conditions, genetic defects, and systemic diseases. Diseases related to onset of heart failure typically include ischemic, hypertensive, and cardiac valvular diseases, and also include primary cardiomyopathy, secondary cardiomyopathy including amyloidosis, congenital heart diseases, pericardial diseases, and the like, which develop due to genetic or acquired causes (Maron B J. et al., Circulation., 2006; 113: 1807-1816). Heart failure, which develops from various heart diseases, results in pathological cardiac remodeling that leads to structural alteration and dysfunction of the heart. Specifically, regarding the remodeling, there are remodeling at the cellular level, caused by alterations in size, shape, and function of cardiomyocytes, and remodeling at the tissue level, caused by heart tissue fibrosis resulting from excessive accumulation of extracellular matrix (ECM). Such pathological cardiac remodeling involves disease-related changes in transcriptional, signaling, structural, electrophysiological, and functional roles in cardiomyocytes.
The myocardial extracellular matrix (ECM) is a sophisticated structure that supports the mechanical function for efficient contraction and relaxation of cardiomyocytes, and plays a role in facilitating adequate transfer of force, electrical signal transmission, intercellular communication, exchange of metabolites, and the like in a microenvironment within the cardiac muscle. Increased stress, damage, and disease in the heart wall result in progression of fibrosis in the extracellular matrix, thereby causing damage to the motor function, such as contraction and relaxation, of cardiomyocytes and myofibers (Li A H. et al., Circ Res., 2014; 114(5): 916-27).
In addition, pathological remodeling results in dysfunction in contraction and relaxation of the cardiac muscle, and disappearance of cardiomyocytes. Hypertrophy and death of cardiomyocytes at the cellular level affect myocardial excitation-contraction coupling and also result in pathological changes in the molecular mechanism that regulates cardiomyocyte contraction, cell survival, mitochondrial function related to energy metabolism, and oxidative stress (Koitabashi N., et al., Nat. rev. Cardiol., 2012; 9:147-157).
Cardiac tissues undergo pathological structural alterations such as facilitating generation and fibrosis of myofibroblasts, stiffening of vascular smooth muscle, dysfunction in vascular endothelial cells, and inflammatory actions of immune cells. Such disease progression at the cellular level leads to remodeling at the tissue level through integrated processes such as hypertrophy and death of cardiomyocytes, loss of blood vessels, fibrosis, inflammation, metabolic dysfunction, and electrophysiological remodeling, thereby causing heart failure (Burchfield J S et al., Circulation. 2013; 128: 388-400).
Neurohormonal blockers have been used for last 40 years as a treatment for heart failure patients. However, despite efficacy of heart failure treatments to relieve symptoms and decrease overload stress in the heart, prognosis of heart failure patients is very poor, with their mortality reaching 50% after 5 years of onset and 90% after 10 years of onset. In addition, in a case where heart failure progresses severely, there is no treatment method other than cardiac assist device and heart transplantation. Therefore, there is an urgent need for development of fundamental cure for heart failure and new therapies to restore the heart.
In relation to new therapies, the areas where development of treatments is actively made in recent years include drugs, cell therapies, miRNAs, and gene therapies, which regulate disease-related signaling systems. Among the above treatments, some drugs have been reported to be effective in animal heart failure disease models or in small-scale phase II clinical trials (Braunwald E. Lancet, 2015; 385: 812-824, VonLueder T G. et al., Nat. Rev. Cardiol., 2015; 12: 730-740).
In particular, various preclinical trials for treatment of heart diseases in animals have been conducted by virtue of understanding at the gene level on the disease mechanism of heart diseases, discovery of therapeutic genes, design and packaging techniques for gene vehicles, rapid development of delivery techniques, and the like (Gorski P A, et al. Cell Metabol. 2015; 21: 183-194).
In recent years, numerous studies have reported that delivery of SERCA2a gene in heart failure animal models resulted in increased survival rate as well as increased cardiac contractility. However, in a phase IIb clinical trial with a recombinant adeno-associated virus expressing SERCA2a, AAV-SERCA2a, in which a large number of clinical patients as many as 250 participates, gene therapies for heart failure did not show valid clinical benefits unlike the experimental results in pigs, sheep, dogs, and even primates (Rincon M Y et al., Cardiovas. Res., 2015; 108: 4-20).
Even in development of drugs for treatment of heart failure, cases have been reported where new drugs, which have shown a therapeutic effect in phase II clinical trials, show less than expected efficacy or end in failure in phase III clinical trials (Vaduganathan M, et al., Nat. Rev. Cardiol., 2013; 10: 85-97).
Current treatments for heart failure focus on relieving symptoms and decreasing overload stress in the heart. However, despite efficacy of the treatments, prognosis of heart failure patients is very pessimistic, with their mortality reaching 50% after 5 years of onset and 90% after 10 years of onset. It has been found that among the disease mechanisms which have been newly understood in the area of developing treatments for heart failure, cardiac remodeling, involving fibrosis that directly affects the cardiac pump function, and damage of cardiomyocytes interact with each other so that they are intimately linked to affect development, progression, and prognosis of diseases, and form a vicious cycle.
Therefore, since treatment of a single drug target or disease mechanism may cause insufficient results in treatment of heart failure, there is a need for studies on a new complex treatment to achieve functional recovery at the cellular level and to treat cardiac histological remodeling.
The present inventors have studied to develop an effective treatment for heart failure, and as a result, have identified that a gene construct, an expression vector, and a recombinant virus, each of which comprises a nucleotide sequence encoding a SERCA2a protein or a fragment thereof and a nucleotide sequence encoding a CCN5protein or a fragment thereof, exhibit a synergistic therapeutic effect on dysfunction caused by heart failure in a mouse model, in which the heart failure has been induced by multiple etiologies, thereby completing the present invention.
In an aspect of the present invention, there is provided a gene construct, comprising (i) a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and (ii) a nucleotide sequence encoding a CCN5 protein or a fragment thereof.
In another aspect of the present invention, there is provided a recombinant expression vector loaded with the gene construct.
In yet another aspect of the present invention, there is provided a recombinant virus comprising the gene construct.
In still yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating heart failure, comprising, as an active ingredient, the gene construct, the recombinant expression vector, or the recombinant virus.
In still yet another aspect of the present invention, there is provided a method for preventing or treating heart failure, comprising a step of administering the pharmaceutical composition to a subject.
In still yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating heart failure, comprising an expression vector loaded with a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and an expression vector loaded with a nucleotide sequence encoding a CCN5 protein or a fragment thereof.
In still yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating heart failure, comprising a recombinant virus that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.
In still yet another aspect of the present invention, there is provided a method for preventing or treating heart failure, comprising a step of administering, to a subject, (i) an expression vector loaded with a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and (ii) an expression vector loaded with a nucleotide sequence encoding a CCN5 protein or a fragment thereof.
In still yet another aspect of the present invention, there is provided a method for preventing or treating heart failure, comprising a step of administering, to a subject, (i) a recombinant virus that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and (ii) a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.
A common feature of heart failure is that cardiomyocytes disappear and fibrosis of heart tissue progresses, and this structural remodeling of the heart causes dysfunction in the cardiac pump. However, disappearance of cardiomyocytes and fibrosis of cardiac tissue may vary depending on the stage of disease progression, and may interact with each other to show a vicious cycle. Therefore, simultaneously treating disappearance of cardiomyocytes and fibrosis of cardiac tissue may be the most effective treatment.
The pharmaceutical composition for preventing and treating heart failure, of the present invention, is such that a SERCA2a protein and a CCN5 protein are simultaneously expressed. The pharmaceutical composition can exert a synergistic therapeutic effect through prevention of disappearance of cardiomyocytes and increased activity thereof achieved by the SERCA2a protein as well as prevention of fibrosis of cardiac cells and tissues achieved by the CCN5 protein, and thus can be effectively used for prevention or treatment of heart failure that is a complex disease induced by various etiologies.
In an aspect of the present invention, there is provided a gene construct comprising (i) a nucleotide sequence encoding SERCA2a protein or a fragment thereof; and (ii) a nucleotide sequence encoding CCN5 protein or a fragment thereof. Here, the nucleotide sequence may be in the form of mRNA.
As used herein, the term “SERCA2a protein,” which is an abbreviation of sarcoplasmic reticulum calcium ATPase 2a, refers to a protein that functions to cause reuptake of calcium into the sarcoplasmic reticulum using ATP energy. Specifically, the SERCA2a protein may have the amino acid sequence represented by SEQ ID NO: 1. In addition, the nucleotide sequence encoding the SERCA2a protein may be the sequence represented by SEQ ID NO: 2 or SEQ ID NO: 13.
In addition, the fragment of the SERCA2a protein may be one obtained by truncation of a portion of the N-terminus and/or C-terminus of the wild-type SERCA2a as long as the fragment maintains activity of the SERCA2a protein. Specifically, the fragment of the SERCA2a protein may be one obtained by truncation of 1 to 100, 1 to 50, 1 to 20, or 1 to 10 amino acids from the N-terminus or C-terminus.
As used herein, the term “CCN5 protein” refers to a matricellular protein belonging to the CCN family that plays various roles in regulation of cellular functions such as vascular disease induction, angiogenesis, tumorigenesis, fibrosis disease induction, cell differentiation, and survival. The CCN5 protein, unlike other CCN family proteins, has no C-terminal domain and is also called WISP-2, HICP, Cop1, CTGF-L, or the like. In addition, the CCN5 protein consists of a single polypeptide chain having 250 amino acids. Owing to a 22-amino acid secretory leader sequence at the N-terminus, the CCN5 protein is secreted out of a cell and functions as a signaling protein.
Specifically, the CCN5 protein may have the amino acid sequence represented by SEQ ID NO: 3. In addition, the nucleotide sequence encoding the CCN5 protein may be the sequence represented by SEQ ID NO: 4 or SEQ ID NO: 14.
In addition, the fragment of the CCN5 protein may be one obtained by truncation of a portion of the N-terminus and/or C-terminus of the wild-type CCN5 as long as the fragment maintains activity of the CCN5 protein. Specifically, the fragment of the CCN5 protein may be one obtained by truncation of 1 to 30, 1 to 20, 1 to 10, or 1 to 5 amino acids from the N-terminus or C-terminus.
The gene construct may further comprise a self-cleavage sequence located between the nucleotide sequence (i) and the nucleotide sequence (ii). The self-cleavage sequence may be a 2A peptide sequence derived from positive-stranded RNA viruses such as Picornaviridae, Iflaviruses, Tetraviridae, and Discistroviridae, or a 2A peptide sequence derived from double-stranded RNA viruses such as Rotaviruses, Cypoviruses, and Totiviridae (Garry A Luke, et al Journal of General Virology, 2008; 89: 1036-1042).
A nucleotide sequence encoding a 2A peptide derived from porcine teschovirus-1, Thosea asigna virus, equine rhinitis A virus, or foot-and-mouth disease virus, which is typically widely used in research, may be present between the nucleotide encoding the SERCA2a protein or a fragment thereof and the nucleotide encoding the CCN5 protein or a fragment thereof. Specifically, the self-cleavage sequence may be, but is not limited to, a nucleotide sequence encoding a 2A peptide derived from porcine teschovirus-1. In addition, the self-cleavage sequence may be the nucleotide sequence represented by SEQ ID NO: 6.
The nucleotide sequence encoding the 2A peptide derived from porcine teschovirus-1 may be a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 5. In addition, the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 5 may be the nucleotide sequence represented by SEQ ID NO: 6.
The nucleotide sequence encoding the 2A peptide derived from Thosea asigna virus may be a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 7. In addition, the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 7 may be the nucleotide sequence represented by SEQ ID NO: 8.
The nucleotide sequence encoding the 2A peptide derived from equine rhinitis A virus may be a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 9. In addition, the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 9 may be the nucleotide sequence represented by SEQ ID NO: 10.
The nucleotide sequence encoding the 2A peptide derived from foot-and-mouth disease virus may be a nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 11. In addition, the nucleotide sequence encoding the amino acid sequence represented by SEQ ID NO: 11 may be the nucleotide sequence represented by SEQ ID NO: 12.
Except for the 2A peptide sequence, which is a representative self-cleavage sequence, a variety of self-cleavage sequences exist, including Picornaviridae 2A sequences (for example, derived from Encephalomyocaditis virus, Theiler's murine encephalomyelitis virus, Theiler's-like virus, Saffold virus, equine rhinitis B virus, bovine rhinovirus, Ljungan virus, Seneca Valley virus, duck hepatitis virus) present in mammals, and 2A sequences derived from Iflaviruses (for example, infectious flacherie virus, Ectropisobliqua picorna-like virus, Perina nuda picorna-like virus), Tetraviruses (for example, Euprosterna elaeasa virus, Providence virus), or Dicistroviridae (for example, cricket paralysis virus, Drosophila C virus, Acute bee paralysis virus, Kashmir bee virus, Israeli acute bee paralysis virus) present in insects, among positive-stranded RNA viruses. In addition, among double-stranded RNA viruses, examples thereof may include Rotaviral 2A sequences (for example, derived from porcine rotavirus A, Bovine rotavirus C, Human rotavirus C, adult diarrhea virus) present in mammals, Cypoviral 2A sequences (for example, derived from Bombyx mori cytoplasmic polyhedrosis virus, Lymantria dispar cypovirus, Dendrolimus punctatus cypovirus, Operophtera brumata cypovirus) present in insects, and Totiviridae 2A sequences (for example, derived from infectious myonecrosis virus) present in Penaeid Shrimp. As such, various 2A sequences may exist, and these are not limited to the above.
In addition, in the gene construct, the nucleotide sequences (i) and (ii) may be contained, in 5′ to 3′ direction, in the order of (i)-(ii). When SERCA2a-P2A-CCN5, an embodiment of the gene construct, is expressed in a cell, the SERCA2a protein may be inserted into the sarcoplasmic reticulum membrane, and the CCN5 protein may be secreted out of the cell. In addition, when CCN5-P2A-SERCA2a, an embodiment of the gene construct of the present invention, is expressed in a cell, the SERCA2a protein may be inserted into the sarcoplasmic reticulum membrane, and the CCN5 protein may be secreted out of the cell.
In addition, the gene construct of (i) or (ii) may contain a promoter sequence operatively linked thereto.
As used herein, the term “operatively linked” refers to functional linkage between a nucleotide expression regulatory sequence (such as promoter, signal sequence, or array of transcription factor binding sites) and other nucleotide sequences. The regulatory sequence regulates transcription and/or translation of the other nucleotide sequences.
Specifically, a promoter linked to a nucleotide sequence encoding a SERCA2a protein or CCN5 protein may operate in animal cells, preferably in mammalian cells, to regulate transcription of the SERCA2a gene or the CCN5 gene. The promoter may include promoters derived from mammalian viruses and promoters derived from mammalian cell genomes. In addition, the promoter includes a synthetic promoter (synthetic muscle- and cardiac-restricted promoter, SPC5-12) obtained by combination of genomic sequences of mammalian cells, intended to increase muscle- and heart-specific expression. The promoter may operate specifically in cardiac cells, and may also operate in any cells.
In an embodiment, the promoter may be linked in the form of i) promoter-SERCA2a-P2A-promoter-CCN5, ii) promoter-CCN5-P2A-SERCA2a, iii) promoter-SERCA2a-P2A-CCN5, or iv) promoter-CCN5-P2A-SERCA2a.
The promoter may be any one selected from the group consisting of cytomegalovirus (CMV) promoter, adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, HSV tk promoter, RSV promoter, EF1 alpha promoter, metallothionein promoter, beta-actin promoter, human IL-2 gene promoter, human IFN gene promoter, human IL-4 gene promoter, human lymphotoxin gene promoter, and human GM-CSF gene promoter, as well as synthetic muscle- and cardiac-restricted promoter (SPC5-12). However, the promoter is not limited thereto. Specifically, the promoter may be CMV promoter.
In addition, the gene construct of the present invention may be delivered into a cell using liposomes. Liposomes are formed automatically by phospholipids dispersed in the aqueous phase, and liposomes containing SERCA2a gene and CCN5 gene may interact with cells, through a mechanism such as endocytosis, adsorption to cell surface, or fusion with plasma cell membrane, thereby delivering the SERCA2a gene and the CCN5 gene into a cell.
In the present invention, when a pharmaceutical composition of the present invention is prepared based on a viral vector containing a gene construct, a method of administering the pharmaceutical composition may be performed according to virus infection methods known in the art. In addition, in the present invention, when the gene construct is contained in a naked recombinant DNA molecule or a plasmid, a microinjection method, a liposome-mediated transfection method, a DEAE-dextran treatment method, and a gene bombardment method may be used to introduce a gene into cells.
In another aspect of the present invention, there is provided a recombinant expression vector loaded with the gene construct.
As used herein, the term “expression vector” refers to a recombinant vector capable of expressing a target protein in a target host cell, the recombinant vector being a gene construct that contains essential regulatory elements operatively linked to a gene insert so that the gene insert is expressed.
In addition, the expression vector may contain a signal sequence in order for cells to facilitate protein secretion. Specific initiation signals may also be required for efficient translation of an inserted nucleic acid sequence. These signals contain the ATG start codon and contiguous sequences. Expression efficiency may be increased by introduction of an appropriate transcription- or translation-enhancing element.
The expression vector may be any one selected from the group consisting of plasmid vectors and cosmid vectors.
The plasmid vector may include, but is not limited to, commercially available plasmids such as pUC18, pBAD, and pIDTSAMRT-AMP.
In yet another aspect of the present invention, there is provided a recombinant virus, comprising the gene construct.
The virus may be any one selected from the group consisting of adenovirus, adeno-associated virus (AAV), retrovirus, lentivirus, herpes simplex virus, vaccinia virus, and the like. Specifically, the virus may be, but is not limited to, adeno-associated virus.
The adenovirus is widely used as a gene transfer vector because of its mid-sized genome, ease of manipulation, high titer, wide target-cell range, and excellent infectivity. Its genome may be flanked by 100 to 200 bp of inverted terminal repeat (ITR) which is an essential cis element for DNA replication and packaging. The adenovirus may further contain E1 regions (E1A and E1B) of the genome which encode proteins involved in viral DNA replication.
Among adenovirus vectors, replication-incompetent adenoviruses lacking the E1 regions may be used. On the other hand, E3 region is deleted from conventional adenovirus vectors to provide a site for insertion of a foreign gene.
Thus, a gene construct comprising the SERCA2a gene and the CCN5 gene, of the present invention, may be inserted into the deleted E1 regions (E1A region and/or E1B region, preferably E1B region) or E3 region. In an embodiment, the gene construct comprising the SERCA2a gene and the CCN5 gene may be inserted into the E3 region.
In addition, since up to approximately 105% of the wild-type genome can be packaged in adenovirus, about 2 kb may be additionally packaged in adenovirus. Thus, a foreign sequence to be inserted into adenovirus may be additionally linked to the adenoviral genome.
Adenovirus has 42 different serotypes and subgroups A to F. In an embodiment, the adenovirus vector of the present invention may be obtained from adenovirus type 5 belonging to subgroup C. Biochemical and genetic information on adenovirus type 5 is well known.
Foreign genes to be delivered by adenovirus replicate in the same way as episomes, and therefore, have very low genotoxicity to host cells.
The retrovirus is widely used as a gene transfer vector because the retrovirus is capable of inserting its gene into the host genome and delivering a large amount of foreign genetic material, and has a broad spectrum of cells it can infect. In order to construct a retroviral vector, the gene construct comprising the SERCA2a gene and the CCN5 gene may be inserted into the retroviral genome instead of the retroviral sequence to produce a replication-incompetent virus. In order to produce virions, a packaging cell line, which expresses gag, pol, and env genes, and does not express long terminal repeat (LTR) and Ψ sequence, may be constructed and used.
The adeno-associated virus (AAV) is suitable as the gene delivery system of the present invention because it is capable of infecting non-dividing cells and has capacity to infect various types of cells. Details of construction and use of AAV vectors are disclosed in U.S. Pat. Nos. 5,139,941 and 4,797,368. Typically, AAV having a gene construct that contains SERCA2a gene and CCN5 gene may be produced by co-transformation of a plasmid containing the gene construct that contains SERCA2a gene and CCN5 gene, which is flanked by two AAV terminal repeats, and an expression plasmid containing the wild-type AVV coding sequence that lacks the terminal repeats.
Vectors derived from the vaccinia virus, the lentivirus, or the herpes simplex virus may also be used to deliver, into a cell, the CCN5 gene and the target nucleotide sequence to be delivered.
In still yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating heart failure, comprising, as an active ingredient, a gene construct, a recombinant expression vector, or a recombinant virus, of the present invention.
When a pharmaceutical composition of the present invention is made into preparations, examples of a pharmaceutically acceptable carrier to be contained therein may include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
A dosage form of the pharmaceutical composition may vary depending on method of use, and may be made into injections.
A dose of the pharmaceutical composition of the present invention is desirably determined in consideration of the patient's age, sex, condition, degree of absorption of active ingredients in the body, inactivation rate, and drugs used in combination; and when the pharmaceutical composition is a virus, the pharmaceutical composition may be administered in an amount of 1.0×103 to 1.0×1020 viral genomes per day on an adult basis. Specifically, the pharmaceutical composition of the present invention may be administered in an amount of 1.0×103 to 1.0×1020, 1.0×108 to 1.0×1016, 1.0×1012 to 1.0×1015, or 1.0×1013 to 1.0×1014 viral genomes per day on an adult basis.
In addition, when the pharmaceutical composition is a plasmid vector, the pharmaceutical composition may be administered at a concentration of 0.1 μg/1 μl to 1 mg/1 μl per day on an adult basis. In addition, when the pharmaceutical composition is a plasmid vector, the dose may include 0.1 ml, 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml or higher, and include all values and ranges therebetween.
In still yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating heart failure, comprising, as an active ingredient, SERCA2a protein and CCN5 protein.
A pharmaceutical composition of the present invention is parenterally administered, and the parental administration includes intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, method for direct injection into tissue, and the like.
As used herein, the term “acceptable carrier” refers to some or all of the following substances and includes those suitable for a particular dose: solvents, diluents, liquid vehicles, dispersants, suspension adjuvants, surfactants, isotonic agents, thickeners, emulsifiers, preservatives, solid binders, lubricants, or the like. Alfanso R. Gennaro, Remington's Pharmaceutical Sciences, 19th edition, 1995, Macna Publishing Co. Easton, Pa. presents various carriers for use in pharmaceutical compositions with known techniques and compositions. Examples of pharmaceutical composition of pharmaceutically acceptable carriers include, but are not limited to, the following. Glucose, sucrose sugar, starch such as corn starch and potato starch, cellulose and derivatives thereof such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; tragacanth in powder form; malt; gelatin; talc; excipients such as cocoa butter, suppository wax, peanut butter, cottonseed oil, safflower oil, sesame oil, olive oil, and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; buffers such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free distilled water; isotonic saline; Ringer's solution; ethyl alcohol and phosphate buffered water, sodium lauryl sulfate and magnesium stearate, colorants, colorants, releasing agents, coating agents, sweeteners, flavoring agents and fragrances, antioxidants, and the like may be contained at the compound manufacturer's discretion.
In still yet another aspect of the present invention, there is provided a method for preventing or treating heart failure, comprising a step of administering the pharmaceutical composition to a subject.
Here, the subject may be a mammal, preferably a human. Specifically, the subject may be a human or a non-human mammal that is suffering from or may be at risk of heart failure.
In still yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating heart failure, comprising an expression vector loaded with a gene construct that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and an expression vector loaded with a gene construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.
The SERCA2a protein or a fragment thereof is as described above for the gene construct. The CCN5 protein or a fragment thereof is as described above for the gene construct.
In addition, the gene construct may contain a promoter sequence operatively linked thereto.
Specifically, a promoter linked to a nucleotide sequence encoding a CCN5 protein or a fragment thereof may operate, preferably in animal cells, and more preferably in mammalian cells, to regulate transcription of the CCN5 gene. The promoter includes promoters derived from the mammalian viruses and promoters derived from mammalian cell genomes. The promoter may operate specifically in cardiac cells, and may also operate in any cells.
The promoter is as described above, and may specifically be a CMV promoter.
In still yet another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating heart failure, comprising a recombinant virus containing a gene construct that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and a recombinant virus containing a gene construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.
The gene construct that contains the nucleotide sequence encoding the SERCA2a protein or a fragment thereof and the gene construct that contains the nucleotide sequence encoding the CCN5 protein or a fragment thereof are as described for the “pharmaceutical composition for preventing or treating heart failure, comprising an expression vector loaded with a gene construct that contains a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and an expression vector loaded with a gene construct that contains a nucleotide sequence encoding a CCN5 protein or a fragment thereof.”
In still yet another aspect of the present invention, there is provided a method for preventing or treating heart failure, comprising the steps of (i) administering, to a subject, an expression vector loaded with a nucleotide sequence encoding a SERCA2a protein or a fragment thereof; and (ii) administering, to the subject, an expression vector loaded with a nucleotide sequence encoding a CCN5 protein or a fragment thereof.
Here, the subject may be a mammal, preferably a human. Specifically, the subject may be a human or another mammal that is suffering from or may be at risk of heart failure.
The administration steps (i) and (ii) may be carried out simultaneously. In addition, after the administration step (i) or (ii), the remaining administration step may be carried out at a time interval.
In still yet another aspect of the present invention, there is provided a method for preventing or treating heart failure, comprising the steps of (i) administering a recombinant virus that contains a nucleotide sequence encoding a SERCA2a protein; and (ii) administering a recombinant virus that contains a nucleotide sequence encoding a CCN5 protein.
Here, the subject may be a mammal, preferably a human. Specifically, the subject may be a human or another mammal that is suffering from or may be at risk of heart failure.
The administration steps (i) and (ii) may be carried out simultaneously. In addition, after the administration step (i) or (ii), the remaining administration step may be carried out at a time interval.
In still yet another aspect of the present invention, there is provided a use of a gene construct of the present invention for preventing or treating heart failure.
In still yet another aspect of the present invention, there is provided a use of a recombinant expression vector of the present invention for preventing or treating heart failure.
In still yet another aspect of the present invention, there is provided a use of a recombinant virus of the present invention for preventing or treating heart failure.
In still yet another aspect of the present invention, there is provided a use of a gene construct of the present invention for preparation of a pharmaceutical composition for preventing or treating heart failure.
In still yet another aspect of the present invention, there is provided a use of a recombinant expression vector of the present invention for preparation of a pharmaceutical composition for preventing or treating heart failure.
In still yet another aspect of the present invention, there is provided a use of a recombinant virus of the present invention for preparation of a pharmaceutical composition for preventing or treating heart failure.
Hereinafter, the present invention will be described in detail by way of experimental examples and examples. However, the following experimental examples and examples are only for illustrating the present invention, and the present invention is not limited to the following preparation examples and examples.
The gene constructs, PTR-CMV-SERCA2a, pTR-CMV-CCN5, pTR-CMV-SERC2a-P2A-CCN5, and pTR-CMV-CCN5-P2A-SERC2a, were constructed to express CCN5 protein and SERCA2a protein, either individually or simultaneously (
The SERCA2a moiety consists of the entire cDNA sequence of human SERCA2a protein. The next linked P2A moiety is a self-cleavage site derived from porcine teschovirus-1 and consists of a nucleotide sequence encoding 22 amino acids. Lastly, the CCN5 moiety consists of the entire cDNA sequence of human CCN5 protein.
pTR-CMV-SERCA2a-P2A-CCN5 recombinant plasmid was completed by removing the luciferase moiety from pTR-CMV-luciferase vector and inserting the SERCA2a-P2A-CCN5 gene construct in place thereof. The protein produced by the recombinant plasmid is divided into the SERCA2a protein and the CCN5 protein by self-cleavage between the 21st amino acid, glycine, and the 22nd amino acid, proline, at the P2A site. The SERCA2a protein may remain in the endoplasmic reticulum membrane and perform its intrinsic function. In addition, the CCN5 protein may migrate into the endoplasmic reticulum and then be secreted out of the cell in the form in which the signal peptide is cleaved, thereby performing its intrinsic function. In contrast, pTR-CMV-CCN5-P2A-SERCA2a recombinant plasmid was also constructed by removing the luciferase moiety from pTR-CMV-luciferase vector and inserting the CCN5-P2A-SERCA2a gene construct in place thereof. Again, the protein produced thereby is divided into the SERCA2a protein and the CCN5 protein by the same function of the P2A sequence.
In order to produce self-complementary adeno-associated virus (AAV, serotype 9), human CCN5 gene and SERCA2a gene were cloned into pds-AAV2-EGFP vector. In order to improve virus packaging and viral delivery efficiency, eGFP sequence was removed during AAV vector construction. Recombinant AAV was constructed using 293T cells. AAV particles in a cell culture were collected and precipitated with ammonium sulfate. The resultant was purified by ultracentrifugation using iodixanol gradient. The AAV particles were enriched through several dilution and enrichment processes in such a manner that iodixanol is exchanged with lactated Ringer's solution using centrifugation. The AAV concentration was quantified using quantitative RT-PCR and SDS-PAGE.
The 293T cell line having expressed the gene was homogenized in a solution, pH 7.0, containing 40 mM imidazole, 10 mM NaF, 1 mM EDTA, 300 mM sucrose, and 0.5 mM DTT, and 500 μg of lysate was added to an uptake reaction buffer, pH 7.0, of 100 mM KCl, 5 mM MgCl2, 5 mM NaN3, 0.5 M EGTA, and 40 mM imidazole. Uptake experiments were performed using pCa 6 (0.0185 μmol) of calcium containing radioisotopes. Treatment with 1 μM Ruthenium red (Sigma Aldrich) was performed and the resultant was allowed to stand at temperature of 37° C. for 3 minutes. Then, reaction was allowed to start while performing treatment with 5 mM K-oxalate and Mg-ATP (Sigma Aldrich). 500 μl of the reaction product was filtered through a 0.45 μm filter (Millipore) up to 4 minutes at 1-minute intervals, and the count per minute (cpm) was measured using a scintillation counter (Beckman).
Cells or cardiac tissues were homogenized in a minimal volume of a 50 mM Tris-HCl solution, pH 7.4, to which a broad-spectrum protease inhibitor cocktail (Calbiochem) had been added. Proteins were separated by SDS-PAGE and transferred to polyvinylidene fluoride membrane (Schleicher & Schuell). After being blocked with 5% (w/v) skim milk for 1 hour and washed with TBST, the membrane was allowed to react with SERCA2a antibody (21st Century Biochemical), CCN5 antibody (Sigma Aldrich), and GAPDH antibody (Sigma-Aldrich). The membrane was then reacted with horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch, WestGrove, Pa., USA) and developed using a chemiluminescent substrate (Dogen). The resultant was photographed and quantified using LAS software.
Cardiac tissues were taken from animal models and then fixed with 10% (w/v) formalin at room temperature for 5 days. Then, washing with PBS was performed. Each sample was embedded in paraffin and the tissue block was cut into 7μm thick sections. In order to check degree of cardiac fibrosis and infarction, the resultant was stained with Picrosirius red (Sigma Aldrich) and Masson-Trichrome (Sigma Aldrich). Then, observations were made under an optical microscope.
Mice were anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg), and echocardiography was conducted. Recording was performed through 2-dimensional imaging and M-mode tracking function, and fractional shortening and ventricular size ratio were determined (GE Vivid Vision).
Measurement of hemodynamics in vivo was performed using a 1.2 Fr pressure-volume conductance catheter (Scisense Inc., Ontario, Canada). Mice were anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg), intubated through tracheostomy, and ventilation was performed by adjusting mechanical air flow to 7 μl/g of tidal volume and 120 breaths per minute. A pressure-volume (PV) catheter was placed in the left ventricle and the pressure-volume data was analyzed using IOX2 software (emka TECHNOLOGIES).
In order to observe expression of the SERCA2a protein and the CCN5 protein by a recombinant plasmid of the present invention, pTR-CMV-SERA2a, pTR-CMV-CCN5, or pTR-CMV-SERCA2a-P2A-CCN5 prepared in Experimental Example 1 was delivered into cultured cells using lipofectamine. The obtained cells and cultures were subjected to Western blotting in the same manner as in Experimental Method 2, to check expression of SERCA2a, CCN5, and GAPDH proteins.
As a result, it was identified that in a case of being expressed by a single promoter, the SERCA2a protein is retained in the cytoplasm and the CCN5 protein is secreted out of the cell (
In addition, in order to compare protein expression level and activity of pTR-CMV-SERC2a-P2A-CCN5 and pTR-CMV-CCN5-P2A-SERC2a prepared in Experimental Example 1 were delivered into cultured cells using lipofectamine. The obtained cells and cultures were subjected to calcium uptake assay in the same manner as in Experimental Method 1.
As a result, no significant difference was observed in terms of expression level between the SERCA2a protein and the CCN5 protein, and the cells having expressed pTR-CMV-CCN5-P2A-SERC2a exhibited low calcium reuptake activity (
This is due to the characteristic that the CCN5 protein is a secreted protein, suggesting that when the SERCA2a protein and the CCN5 protein are expressed in the order of SERCA2a-CCN5, both proteins are expressed in their normal structure, whereas when the SERCA2a protein and the CCN5 protein are expressed in the order of CCN5-SERCA2a, the SERCA2a protein, which is translated following the CCN5 protein, may be produced as an abnormal protein having topology that is opposite to its original topology.
I. Identification of Heart Failure Therapeutic Effect
In order to identify a heart failure therapeutic effect of a recombinant virus containing the gene construct that contains a nucleotide sequence encoding the SERCA2a protein and a nucleotide sequence encoding the CCN5 protein and, of the present invention, the mice, in which heart failure has been induced by multiple etiologies, were used.
For the heart failure-induced mice, an ischemia-reperfusion injury-induced heart failure model, a transverse aortic constriction-induced heart failure model, and an angiotensin II-induced heart failure model were produced. The heart failure-induced mice were divided into AAV9-Control, AAV9-SERCA2a administration group, AAV9-CCN5 administration group, and AAV9-SERCA2a-P2A-CCN5 administration group, and their heart failure therapeutic effects were compared.
8- to 10-week-old B6C3F1 mice were anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg), and subjected to surgery. Ischemia was induced by placing polyethylene 10 (PE10) tubing with a diameter of 1 mm on top of the left anterior descending coronary artery and performing tying. After 30 minutes, reperfusion was induced by performing untying and removing the PE10 tubing. Simultaneously with inducing reperfusion, each mouse was injected, via the tail vein, with 1×1011 viral genomes (vgs) of AAV9-Control, AAV9-CCN5, AAV9-SERCA2a, or AAV9-SERCA2a+AAV9-CCN5. After 4 weeks, myocardial function measurement and histological analysis were performed (
First, in order to identify whether effective delivery of the viral genome is made, expression of the SERCA2a protein and the CCN5 protein was checked through western blotting in the same manner as in Experimental Method 2.
As a result, it was identified that as compared with the AAV9-Control administration group, expression of the CCN5 protein was significantly increased in the AAV9-CCN5 administration group, and expression of the SERCA2a protein was significantly normalized in the AAV9-SERCA2a administration group. In addition, it was identified that in the AAV9-CCN5 and AAV9-SERCA2a combination administration group, expression of the CCN5 protein and the SERCA2a protein was significantly increased (
In addition, hearts were extracted from the mice, in which heart failure has been induced by ischemia-reperfusion injury, and photographed. Tissue staining was performed in the same manner as in Experimental Method 3.
As a result, the AAV9-CCN5 administration group, the AAV9-SERCA2a administration group, and the AAV9-CCN5 and AAV9-SERCA2a combination administration group exhibited a significantly decreased infarct area size, as compared with the infarct area in the AAV9-Control administration group. In particular, the AAV9-CCN5 and AAV9-SERCA2a combination administration group exhibited a remarkably decreased infarct area size (
Echocardiography and hemodynamics were performed to identify whether morphological changes in heart tissue actually affect heart contractility.
In order to measure fractional shortening (FS), a parameter of cardiac contractility, echocardiography was performed in the same manner as in Experimental Method 4. As a result, it was identified that the fractional shortening, which has been decreased by ischemia-reperfusion injury, was significantly restored in the AAV9-CCN5 administration group, the AAV9-SERCA2a administration group, and the AAV9-CCN5 and AAV9-SERCA2a combination administration group. In particular, the AAV9-CCN5 and AAV9-SERCA2a combination group exhibited remarkably increased fractional shortening (
In addition, hemodynamics measurement was conducted in the same manner as in Experimental Method 5, to accurately analyze cardiac contractility. As a result, the end-systolic pressure volume relationship (ESPVR), a parameter of systolic myocardial strength, was decreased by ischemia-reperfusion injury. The AAV9-SERCA2a administration group exhibited significantly increased ESPVR, and the AAV9-CCN5 administration group did not exhibit significantly increased ESPVR. This is the same phenomenon as known in the study of CCN5, supporting the fact that CCN5 does not affect heart failure. In addition, the AAV9-CCN5 and AAV9-SERCA2a combination group exhibited synergistically increased ESPVR (
In addition, it was identified that the end-diastolic pressure volume relationship (EDPVR), a parameter of diastolic myocardial strength, was increased upon ischemia-reperfusion injury. The AAV9-CCN5 administration group exhibited significantly decreased EDPVR, and the AAV9-SERCA2a administration group did not exhibit significantly decreased EDPVR. This is the same experimental result as previous studies, supporting the fact that the SERCA2a protein does not affect diastolic heart failure. In addition, the AAV9-CCN5 and AAV9-SERCA2a combination administration group exhibited remarkably decreased EDPVR (
Based on these data, it was identified that a synergistic therapeutic effect is obtained by combination administration of AAV9-CCN5 and AAV9-SERCA2a.
8- to 10-week-old B6C3F1 mice were anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg), and subjected to surgery. 2 to 3 mm of the proximal sternum was longitudinally dissected to ensure a visual field of the aortic arch. Thereafter, a 27-gauge needle was used to make a connection between the innominate artery and the carotid artery. Then, tying was performed and the needle was removed, thereby causing a constriction of transverse aorta. 8 Weeks after induction of heart failure with the constriction, each mouse was injected, via the tail vein, with 1×1011 vgs of AAV9-Control, AAV9-CCN5, AAV9-SERCA2a, or AAV9-SERCA2a-P2A-CCN5. After 8 weeks, myocardial function measurement and histological analysis were performed (
First, in order to identify whether effective delivery of the viral genome was made, expression of the SERCA2a protein and the CCN5 protein was checked through western blotting in the same manner as in Experimental Method 2. As a result, it was identified that as compared with the AAV9-Control administration group, expression of the CCN5 protein was significantly increased in the AAV9-CCN5 administration group, and expression of the SERCA2a protein was significantly increased in the AAV9-SERCA2a administration group. In addition, it was identified that even in the AAV9-SERCA2a-P2A-CCN5 administration group, expression of the CCN5 protein and the SERCA2a protein was significantly increased (
Hearts were extracted from the mice, in which heart failure had been induced by ischemia-reperfusion injury, and heart tissues were stained with Masson-trichrome in the same manner as in Experimental Method 3. As a result, it was identified that the AAV9-Control administration group exhibited an increased cardiac internal dimension size; and it was identified that the AAV9-CCN5 administration group, the AAV9-SERCA2a administration group, and the AAV9-SERCA2a-P2A-CCN5 administration group exhibited a decreased cardiac internal dimension size. In particular, it was identified that the AAV9-SERCA2a-P2A-CCN5 administration group exhibited a remarkably decreased internal dimension size. In addition, it was identified that the AAV9-CCN5 administration group, the AAV9-SERCA2a administration group, and the AAV9-SERCA2a-P2A-CCN5 administration group exhibited a decrease in degree of cardiac fibrosis caused by heart failure (
Echocardiography and hemodynamics analysis were performed to identify whether morphological changes in heart tissue actually affect heart contractility.
In order to measure fractional shortening (FS), a parameter of cardiac contractility, echocardiography was performed in the same manner as in Experimental Method 4. As a result, it was identified that the fractional shortening, which has been decreased by aortic constriction, was significantly restored in the AAV9-CCN5 administration group, the AAV9-SERCA2a administration group, and the AAV9-CCN5 and AAV9-SERCA2a combination administration group. In particular, the AAV9-SERCA2a-P2A-CCN5 administration group exhibited remarkably increased fractional shortening (
In addition, hemodynamics analysis was performed in the same manner as in Experimental Method 5, to accurately analyze cardiac contractility. As a result, the end-systolic pressure volume relationship (ESPVR), a parameter of systolic myocardial strength, was decreased by aortic constriction. The AAV9-SERCA2a administration group exhibited significantly restored ESPVR, and the AAV9-CCN5 administration group did not exhibit significantly restored ESPVR. In addition, the AAV9-SERCA2a-P2A-CCN5 administration group exhibited remarkably increased ESPVR (
In addition, it was identified that the end-diastolic pressure volume relationship (EDPVR), a parameter of diastolic myocardial strength, was increased upon ischemia-reperfusion injury. The AAV9-CCN5 administration group exhibited significantly decreased EDPVR, and the AAV9-SERCA2a administration group did not exhibit significantly decreased EDPVR. In addition, the AAV9-SERCA2a-P2A-CCN5 administration group exhibited remarkably decreased EDPVR (
8- to 10-week-old B6C3F1 mice were anesthetized by intraperitoneal injection of ketamine (95 mg/kg) and xylazine (5 mg/kg), and heart failure was induced by subcutaneous infusion of angiotensin II (Ang II). Angiotensin II was infused subcutaneously for 2 weeks at a concentration of 3 mg/kg per day using a small osmotic pump (Alzet 1002, Alzet). 2 Weeks after induction of heart failure with Ang II, each mouse was injected, via the tail vein, with 1×1011 viral genomes (vgs) of AAV9-Control, AAV9-CCN5, AAV9-SERCA2a, or AAV9-SERCA2a-P2A-CCN5. After 4 weeks, myocardial function measurement and histological analysis were performed (
First, in order to identify whether effective delivery of the viral genome is made, expression of the SERCA2a protein and the CCN5 protein was checked through western blotting in the same manner as in Experimental Method 2.
As a result, it was identified that as compared with the AAV9-Control administration group, expression of the CCN5 protein was significantly restored in the AAV9-CCN5 administration group, and expression of the SERCA2a protein was significantly restored in the AAV9-SERCA2a administration group. In addition, it was identified that even in the AAV9-SERCA2a-P2A-CCN5 administration group, expression of the CCN5 protein and the SERCA2a protein was significantly increased (
Hearts were extracted from the mice, in which heart failure had been induced by infusion of angiotensin II, and heart tissues were stained with Masson-trichrome in the same manner as in Experimental Method 3.
As a result, it was identified that the AAV9-Control administration group exhibited an increased cardiac internal dimension size; and it was identified that the AAV9-CCN5 administration group, the AAV9-SERCA2a administration group, and the AAV9-SERCA2a-P2A-CCN5 administration group exhibited a decreased cardiac internal dimension size. In particular, it was identified that the AAV9-SERCA2a-P2A-CCN5 administration group exhibited a remarkably decreased lumen size (
In addition, echocardiography was performed in the same manner as in Example 6, to measure the inner wall thickness of the heart. Changes in the inner wall thickness of the heart were quantified and graphically represented.
As a result, it was identified that the inner wall thickness of the heart was decreased by infusion of angiotensin II, and it was identified that the AAV9-CCN5 administration group, the AAV9-SERCA2a administration group, and the AAV9-SERCA2a-P2A-CCN5 administration group exhibited a significantly restored increase in inner wall thickness of heart (
Echocardiography and hemodynamics were performed to identify whether morphological changes in heart tissue actually affect heart contractility.
In order to measure fractional shortening (FS), a parameter of cardiac contractility, echocardiography was performed in the same manner as in Experimental Method 4. As a result, it was identified that the fractional shortening, which has been decreased by aortic constriction, was significantly restored in the AAV9-CCN5 administration group, the AAV9-SERCA2a administration group, and the AAV9-CCN5 and AAV9-SERCA2a combination administration group. In particular, the AAV9-SERCA2a-P2A-CCN5 administration group exhibited remarkably restored fractional shortening (
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2017-0127442 | Sep 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2018/011521 | 9/28/2018 | WO | 00 |
