WO2013036610A2 - Compositions comprenant le mg53 et procédés de traitement et de prévention de lésions au niveau des voies respiratoires - Google Patents

Compositions comprenant le mg53 et procédés de traitement et de prévention de lésions au niveau des voies respiratoires Download PDF

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WO2013036610A2
WO2013036610A2 PCT/US2012/053920 US2012053920W WO2013036610A2 WO 2013036610 A2 WO2013036610 A2 WO 2013036610A2 US 2012053920 W US2012053920 W US 2012053920W WO 2013036610 A2 WO2013036610 A2 WO 2013036610A2
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injury
polypeptide
nucleic acid
lung
cell
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PCT/US2012/053920
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WO2013036610A3 (fr
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Jianjie Ma
Noah Weisleder
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University Of Medicine And Dentistry Of New Jersey
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Priority to US14/343,560 priority Critical patent/US20150110778A1/en
Priority to EP12829354.5A priority patent/EP2753363A4/fr
Priority to CN201280056362.1A priority patent/CN104066452A/zh
Publication of WO2013036610A2 publication Critical patent/WO2013036610A2/fr
Publication of WO2013036610A3 publication Critical patent/WO2013036610A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates to compositions and methods of use thereof for the modulation of respiratory function.
  • eukaryotic cells To maintain cellular homeostasis, eukaryotic cells must conserve the integrity . of their plasma membrane through active recycling and repair in response to various sources of damage. For example, in response to external damage and internal degeneration, the cells of the body must repair the membrane surrounding the each individual cell in order to maintain their function and the health of the organism.
  • Repair of damage to the plasma membrane is an active and dynamic process that requires several steps, including participation of molecular sensor(s) that can detect acute injury to the plasma membrane, nucleation of intracellular vesicles at the injury site and vesicle fusion to enable membrane patch formation. It has been demonstrated that entry of extracellular calcium is involved in the fusion of intracellular vesicles to the plasma membrane, however, the molecular machinery involved in sensing the damaged membrane signal and the nucleation process for repair-patch formation have not been fully resolved.
  • vesicles are normally found within the cell, but upon damage to the cell membrane, these vesicles move to the damage site and form a patch to maintain the cell integrity. Without this essential function, the cell can die and the cumulative effect of this cellular injury can eventually result in dysfunction of the tissue or organ.
  • Acute respiratory distress syndrome is a syndrome of severe acute lung injury (ALI) characterized by widespread lung inflammation and increased pulmonary vascular permeability.
  • the causes for ARDS/ALI include direct and indirect lung injuries.
  • Direct lung injuries may include pneumonia, inhalation of gastric contents, near drowning, inhalation injury, and pulmonary contusion.
  • Indirect injuries result from sepsis, severe trauma, massive blood transfusion, and drug overdose (Ware, atthay, 2000).
  • Sepsis and pneumonia are the most common insults, seen in 86% and 29% of ARDS, respectively (Ferguson, Frutos-Vivar, Esteban, et al., 2007).
  • ARDS Advanced age and cigarette smoking are additional risk factors for ARDS (Iribarren, Jacobs, Sidney, Gross, Eisner, 2000). ARDS presents rapidly following the inciting injury; 50% of patients who will develop ARDS do so within 24 hours, and 85% of patients who will develop ARDS do so within 72 hours (Roupie, Lepage, Wysocki, Fagon, Chastre, Dreyfuss, et al., 1999). The incidence of ALI ARDS has been unclear, possibly due to changing definitions of the disorders.
  • a DS is associated with a variety of symptoms, including dyspnea, hypoxemia, diffuse bilateral pulmonary infiltrates, and decreased lung infiltrates (Udobi, Childs, Touijer, 2003). Patients develop tachycardia, cyanosis, course rales, and respiratory distress and are usually treated in the intensive care unit (ICU). Treatment is focused primarily on supportive care and prevention of complications.
  • the cornerstone of treatment is mechanical ventilation to ensure adequate gas exchange (Krishnan, Brower, 2000). While mechanical ventilation is a life-sustaining therapy, extended use may perpetuate or exacerbate the existing lung injury and lead to a complication known as ventilator-associated lung injury (VALI) (Krishnan, Brower, 2000. Tremblay, Slutsky, 1998; International consensus conferences in intensive care medicine: ventilator-associated lung injury in ARDS, 1998).
  • VALI ventilator-associated lung injury
  • ventilator-injured lung cells are shown to undergo plasma membrane stress damage, as indicated by increased propidium-iodide label uptake.
  • the underlying injury is characterized by a three-phase pathophysiology (Udobi, Childs, Touijer, 2003; Tomashefski, 1990).
  • Udobi Childs, Touijer, 2003; Tomashefski, 1990.
  • the exudative phase occurring during the first week after onset, the alveolar epithelium and vascular endothelium are damaged, resulting in the leakage of water, protein, inflammatory cells, and red blood cells into the interstitium and alveolar lumen.
  • diffuse alveolar damage occurs as a result of an amplified pro-inflammatory response.
  • the inflammatory mediators including lactate dehydrogenase (LDH), tumor necrosis factor (TNF), interleukins- 1B, 6, and 10, platelet-activating factor, neuropeptide substance P, chemokines, and cytokines (Cepkova, Matthay, 2006; Zhou, Chen, Chen, Zhang, Andersson, 2010; Puneet, Moochhala, Bhatia, 2005), are seen in increased levels in the bronchialveolar lavage (BAL) fluid in ARDS patients (Chen, Lee, Liu, Wang, Wang, Perng, 2005; Minamino & Komuro, 2006).
  • the presence of inflammatory mediators is considered a biomarker of the disease (Tzouvelekis, Pneumatikos, Bouros, 2005).
  • the inflammatory cascade induces neutrophil-dependent injury to alveolar type I and type II cells (Cepkova, matthay, 2006).
  • Type I cells account for 90% of alveolar cells, across which gas exchange occurs.
  • Type II cells produce surfactant, which plays a role in preventing alveolar collapse. Damage to alveolar cells thus results in reduced gas exchange, reduced surfactant production, and alveolar collapse (Minamino & Komuro, 2006). To our knowledge, there is no effective treatment targeting this inflammation-induced alveolar damage.
  • targeting membrane repair may be an alternative therapy for ARDS.
  • facilitating membrane repair would address the underlying injury to the lung at one or more of the following three timepoints.
  • cell membrane resealing following the initial injury may prevent the inflammatory cascade that leads to ARDS in the first place. If the damage has already led to the exudative phase of ARDS, when diffuse alveolar damage has already been induced, membrane repair may lessen the total alveolar necrosis. Decreasing cell damage at this point, during the exudative phase, may prevent the disease from progressing into the proliferative and/or fibrotic phases.
  • MG53 Mitsugumin 53
  • TRHV172 muscle-specific tripartite motif family protein
  • MG53 protein can increase the capacity of various cell types to reseal their membranes following injury when the MG53 protein is applied outside of the cell.
  • MG53 provides a unique potential for a therapeutic agent targeting cell membrane repair in pulmonary disorders such as ARDS.
  • ARDS can be confirmed by histopathological examination, lung edema (greater wet- to-dry ratio), and increased levels of LDH, TNF-a and IL-6 in the bronchoalveolar lavage fluid (BALF) of mice (Li, Li, Zhou, Song, Liang, Huang, 2010).
  • BALF bronchoalveolar lavage fluid
  • the present invention relates to the surprising and unexpected discovery of proteins and processes involved in the protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • nucleic acids and polypeptides encoded from nucleic acids of the invention, which, alone or in combination with other components, can modulate tissue repair and function.
  • compositions for example, polypeptides, nucleic acids encoding cytoplasmic, nuclear, membrane bound, and secreted polypeptides; as well as vectors, host cells, antibodies, recombinant proteins, pseudopeptides, fusion proteins, chemical compounds, and methods for producing the same.
  • the present invention also relates to compositions useful as therapeutics for treating and/or prevention of respiratory cell and/or tissue damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • compositions of the invention comprise MG53 polypeptides, and nucleic acids encoding MG53 polypeptides, for example, the protein of SEQ ID NO. 1 and MG53 receptor polypeptides, and mutants, homologs, fragments, truncations, pseudopeptides, peptide analogs, and peptidomimetics as well as proteins and compounds that can modulate the activity of MG53 or intermolecular interactions of MG53 with MG53 receptor polypeptides, for example, CSN6, kinesin, caveolin-3 (“CaV3”) (SEQ ED NO. 8), periaxin, and mye I in-basic-protein.
  • CSN6, kinesin, caveolin-3 CaV3
  • the invention relates to compositions comprising a polypeptide of the invention in combination with an agent that exerts its effects, synergistically, with the activity of an MG53 polypeptide or MG53 receptor polypeptide.
  • the modulating agents include, for example, a bioactive agent, phosphotidylserine; zinc, for example, in the form of a zinc salt, zinc carrier or zinc conjugate; notoginsing; and an oxidizing agent.
  • the invention relates to compositions and methods related to the treatment and/or prevention of lung tissue damage.
  • the invention encompasses, for example, the administration of an effective amount of a therapeutic composition of the invention for the prevention and/or treatment of cell or tissue damage, such as those occurring in subjects suffering due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention relates to nucleic acids, including interfering nucleic acids, and polypeptides encoding MG53 and/or MG53 interacting proteins, for example, CSN6, kinesin, caveolin-3 (SEQ ID NO. 8), periaxin, and myelin-basic-protein, mutants, truncations, fragments, homologs, pseudopeptides and peptidomimetics, as well as compounds that can modulate their activity or their intermolecular interactions with MG53, Therefore, in additional aspects, the present invention encompasses methods for the targeting of caveolin-3 (SEQ ID NO. 8) gene expression, activity, and/or intermolecular interactions for the treatment and/or prevention of a disease or disorder in a subject, for example, injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • caveolin-3 SEQ ID NO. 8
  • the invention relates to methods of screening and identifying agents useful as therapeutics for the treatment or prevention of injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention encompasses agents, peptides, nucleic acids, or chemical compounds that are agonists of the expression and/or activity and/or phosphorylation of at least one of MG53 or CaV3.
  • the invention encompasses agents, peptides, nucleic acids, or chemical compounds that are antagonists of the expression or activity, for example, by phosphorylation, of the mitochondrial permeability transition pore (MPTP).
  • MPTP mitochondrial permeability transition pore
  • the invention relates to methods of treating and/or preventing due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof, comprising the administration of an effective amount of an agonist of the expression and/or activity of at least one of MG53 or CaV3.
  • the invention provides a use of an agent that modulates at least one of G53 activity, MG53 expression, or the MG53 mediated membrane repair in a lung cell for the manufacture of a medicament for treating and/or preventing lung injury, said medicament comprising a therapeutically effective amount of the agent and a pharmaceutically acceptable carrier.
  • the invention provides a use of a MG53 polypeptide or
  • MG53 nucleic acid in the manufacture of a medicament for treating and/or preventing a lung injury, said medicament comprising a therapeutically effective amount of MG53 polypeptide or MG53 nucleic acid and a pharmaceutically acceptable excipient.
  • the invention provides use of an agent that modulates at least one of MG53 activity, MG53 expression, or the MG53 mediated membrane repair in a lung cell in a method for treating and/or preventing lung injury, said method comprising administering an effective amount of a therapeutic composition comprising the agent and a pharmaceutically acceptable excipient, wherein the composition is effective in treating lung injury.
  • the invention relates to the use of a MG53 polypeptide or
  • MG53 nucleic acid in a method for treating a lung injury, said method comprising administering an effective amount of a therapeutic composition comprising the MG53 polypeptide or MG53 nucleic acid and a pharmaceutically acceptable excipient, wherein the composition is effective in treating lung injury.
  • the agent is at least one of an MG53 polypeptide; an
  • the effective amount of the agent which may comprise or consist of an MG53 polypeptide or MG53 nucleic acid of the invention, can be from 0.1 mg/kg and 1000 mg/kg body weight/day.
  • the agonist of MG53 activity comprises at least one of phosphotidyl serine, zinc or zinc salt, Zn-l-hydroxypyridine-2-thine (Zn-HPT), notoginsing, an oxidizing agent, thimerosal, or combination thereof.
  • the agent is a stem ceil capable of differentiation into a lung cell, wherein the stem cell has been modified such that it demonstrates enhanced activity or expression of MG53.
  • the lung injury comprises damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the lung injury comprises lung cell or lung tissue injury due to direct injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the MG53 polypeptide has the amino acid sequence of at least one of SEQ ID NOs.: 1 , 3, 5, 9, 10, 1 1, 12, 13, 14, 15, or 16 or bioactive portion thereof.
  • the MG53 nucleic acid has the nucleotide sequence of at least one of SEQ ID NOs.: 2, 4, or 6.
  • compositions of the invention can be administered extracellularly or system ically in combination with a pharmaceutically acceptable excipient, wherein the composition is effective in treating or preventing injury of lung tissue.
  • the present invention relates to a method of screening for a compound useful for treating lung injury comprising the steps of: providing a lung cell expressing a nucleic acid encoding a polypeptide having an amino acid sequence with at least 70% homology to an MG53 polypeptide; providing a library of test compounds; treating the cell with the test compound; inducing damage to the membrane of the lung cell; measuring for a change in MG53 expression, activity, or amount and the ability of the cell to repair damage to a membrane; and comparing the treated cell versus an untreated cell, wherein an agent that is capable of modulating MG53 expression, activity or amount is indicative of an agent useful for treating lung injury.
  • FIG. 1 MG53 is a muscle specific member of the TRIM protein family.
  • FIG. 2 Illustrates an exemplary domain comparison of some homologous proteins that contain one or more of the conserved tripartite motifs which are present in MG53.
  • MG53 is unique in its ability to translocate to an injury site at the cell membrane following multiple forms of insult and mediate repair of the damaged membrane - a function which is not exhibited by the other TRIM family proteins listed. While these TRIM proteins all contain similar domains and/or are expressed in striated muscle, none fully recapitulate the domain organization of MG53.
  • FIG. 3 MG53 contains unique TRIM and SPRY motifs and is predominantly expressed in muscle cells.
  • A Diagram of motif structure of MG53. From the results of cDNA cloning and homology searches, several motif sequences are detected in MG53 as shown. The sequences of rabbit and mouse MG53 cDNAs have been deposited in the databases under accession numbers AB231473 and AB231474, respectively.
  • B Western blot analysis shows the specific expression of MG53 in skeletal and cardiac muscles. Lysate (20 ⁇ g total protein per lane) from mouse tissues (lung, kidney, skeletal muscle, liver, heart, brain) were analyzed using anti-mouse MG53 polyclonal antibody.
  • C Immunofluorescence staining of longitudinal transverse sections from mouse skeletal muscle cells. Scale bar is 125 ⁇ .
  • FIG. 4 MG53 can protect against membrane damage in airway cells.
  • MG53 can repair membrane damage in human airway epithelial cells.
  • Human C38 airway epithelial cells were transfected with GFP-MG53 (left) and then subjected to mechanical membrane damage by microelectrode needle penetration (arrow). MG53 can translocate to membrane repair sites in airway epithelial cells (right). Translocation of GFP-MG53 to the injury site (arrow) was monitored by live cell confocal microscopy.
  • Treatment with 0.005% saponin to permeablize the membrane also results in translocation to the plasma membrane (right). In both cases, cell membrane damage resulted in the translocation of MG53 to the plasma membrane.
  • BAL fluid was obtained from mice 24 h after LPS injection challenge by lavage via the tracheal cannula with PBS. Mice were treated with either saline or the indicated dose of MG53. Total number of cells obtained following lung lavage is shown.
  • Each column indicates the mean ⁇ S.E.M of eight mice. Statistically significant differences from the normal group is indicated by M , p ⁇ 0.05 (one-way ANOVA/Dunnett's post-hoc test).
  • FIG. 6 MG53 prevents breakdown of the barrier function of the lung following injury.
  • BAL fluid was obtained from mice 24 h after LPS injection challenge by lavage via the tracheal cannula with PBS. Mice were treated with either saline or the indicated dose of MG53. Total protein concentration was obtained following lung lavage and is shown. Each column indicates the mean ⁇ S.E.M of eight mice. Statistically significant differences from the normal group is indicated by , p ⁇ 0.01 ; Statistically significant differences from the vehicle group is indicated by * , p ⁇ 0.05**, p ⁇ 0.01 by (one-way ANOVA/Dunnett's post-hoc test).
  • FIG. 7 MG53 reduces membrane permeability following lung injury.
  • BAL fluid was obtained from mice 24 h after LPS injection challenge by lavage via the tracheal cannula with PBS. Mice were treated with either saline or the indicated dose of MG53. The LDH activity in the BALF following lung lavage is shown. Each column indicates the mean ⁇ S.E.M of eight mice. Statistically significant differences from the normal group is indicated by m , pO.01 ; Statistically significant differences from the vehicle group is indicated by *, p ⁇ 0.05**, p ⁇ 0.01 by (one-way ANOVA/Dunnett's post-hoc test).
  • FIG. 8 MG53 reduces the inflammatory response following lung injury.
  • BAL fluid was obtained from mice 24 h after LPS injection challenge by lavage via the tracheal cannula with PBS. Mice were treated with either saline or the indicated dose of MG53. The IL-6 protein levels in the BALF following lung lavage is shown. Each column indicates the mean ⁇ S.E.M of eight mice. Statistically significant differences from the normal group is indicated by , p ⁇ 0.01 ; Statistically significant differences from the vehicle group is indicated by * , p ⁇ 0.05**, pO.Oi by (one-way ANOVA/Dunnett's post-hoc test).
  • FIG. 9 MG53 reduces edema following lung injury.
  • Lung tissue was obtained 24 h after LPS challenge to mice. Mice were treated with either saline or the indicated dose of MG53. The whole lung wet:dry weight ratio was used to establish if edema was present in the tissue following injury.
  • Each column indicates the mean ⁇ S.E.M of six mice. Statistically significant differences from the normal group is indicated by # , p ⁇ 0.05; Statistically significant differences from the vehicle group is indicated by **, p ⁇ 0.01 by (oneway ANOVA Dunnett's post-hoc test).
  • FIG. 10 MG53 can prevent LPS-induced degradation of lung structure.
  • FIG. 11 MG53 can prevent LPS-induced breakdown of lung mucosal thickness.
  • Lung tissue was obtained 24 h after LPS challenge to mice. Mice were treated with either saline or the indicated dose of MG53. After H&E staining, the lung mucosal thickness was evaluated under light microscopy. Each column indicates the mean ⁇ S.E.M of six mice. Statistically significant differences from the normal group is indicated by *, p ⁇ 0.05 by (Mann- Whitney Rank test). There was a dose dependent improvement in the mucosal thickness with MG53 treatment.
  • SEQ ID NO: 1 human MG53 polypeptide sequence
  • SEQ ID NO: 2 human MG53 nucleotide sequence
  • SEQ ID NO: 3 (mouse MG53 polypeptide sequence);
  • SEQ ID NO: 4 (mouse MG53 nucleotide sequence);
  • SEQ ED NO: 5 (rabbit MG53 polypeptide sequence);
  • SEQ ED NO: 6 (rabbit MG 3 nucleotide sequence);
  • SEQ ID NO: 7 mutant human MG53 polypeptide sequence
  • SEQ ED NO: 8 human Cav3 polypeptide sequence
  • SEQ ED NO: 9 (opossum MG53 polypeptide sequence);
  • SEQ ID NO: 10 (dog MG53 polypeptide sequence);
  • SEQ ED NO: 11 (chimp MG53 polypeptide sequence);
  • SEQ ED NO: 12 rhesus monkey MG53 polypeptide sequence
  • SEQ ID NO: 13 (bovine MG53 polypeptide sequence);
  • SEQ ID NO: 14 (rat MG53 polypeptide sequence);
  • SEQ ED NO: 15 (Xenopus laevis MG53 polypeptide sequence); [0067] SEQ ED NO: 16 (Xenopus tropicalis MG53 polypeptide sequence);
  • SEQ ID NO: 17 TAT HIV-1 polypeptide sequence
  • SEQ ID NO: 18 (shRNA MG53 - sense);
  • SEQ ID NO: 19 shRNA MG53 - antisense
  • SEQ ID NO: 20 (shRNA MG53);
  • SEQ ED NO: 21 (shRNA MG53);
  • SEQ ID NO: 22 shRNA CaV3 - sense
  • SEQ ID NO: 23 shRNA CaV3 - antisense
  • SEQ ID NO: 24 shRNA CaV3 - sense
  • SEQ ID NO: 25 shRNA CaV3 - antisense
  • MG53 can provide a significant protective response in airway cells to minimize damage in tissues due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof, and therefore, the targeting and modulating MG53 gene expression, polypeptide synthesis, activity or protein-protein interactions represent a novel therapeutic intervention for the treatment and/or prevention of, for example, ARDS, VALI and many other forms of lung injury.
  • the invention is related, in part, to the surprising and unexpected discovery of recombinant nucleic acid sequences and related polypeptides (See, SEQ ID NOs.: 1-15), which are capable of facilitating the treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • MG53 mitsugumin53
  • TAM tri-partite motif
  • ARDS is associated with a variety of symptoms, including dyspnea, hypoxemia, diffuse bilateral pulmonary infiltrates, and decreased lung infiltrates (Udobi, Childs, Touijer, 2003). Patients develop tachycardia, cyanosis, course rales, and respiratory distress and are usually treated in the intensive care unit (ICU). Treatment is focused primarily on supportive care and prevention of complications. The cornerstone of treatment is mechanical ventilation to ensure adequate gas exchange (Krishnan, Brower, 2000). While mechanical ventilation is a life-sustaining therapy, extended use may perpetuate or exacerbate the existing lung injury and lead to a complication known as ventilator-associated lung injury (VALI) (Krishnan, Brower, 2000.
  • VALI ventilator-associated lung injury
  • MG53 muscle-specific TRIM-family protein
  • TAM72 muscle-specific TRIM-family protein
  • biopolymer compositions described herein are referred to generally as “MG53 nucleic acids” or “MG53 polynucleotides,” wherein any corresponding encoded polypeptides can be referred to as “MG53 polypeptides” or “MG53 proteins.”
  • MG53 is used generally to refer to MG53 related and/or MG53-derived biopoiymers as explicitly, implicitly, or inherently described herein.
  • the cells of the body In response to external damage and internal degeneration, the cells of the body must repair the membrane surrounding the each individual cell in order to maintain their function and the health of the organism. Defects in the ability of the cell to repair external membranes or be refractory to oxidative stress have been linked to many diseases, such as neurodegenerative diseases (Parkinson's Disease), heart attacks, ischemia, hypoxia, heart failure and muscular dystrophy. In addition, the muscle weakness and atrophy associated with various diseases, as well as the normal aging process, has been linked to altered membrane repair and/or oxidative stress.
  • membrane damage and oxidative stress occurs in many other pathologic states outside of chronic disease, for example, UV exposure, minor cuts, dermal abrasion, surgical incisions and ulcers, ischemia, reperfusion, hypoxia in both diabetic and otherwise healthy patients all involve some component of damage to cellular membranes and oxidative stress.
  • these cells In order for these cells to repair their membranes in response to acute damage they make use of small packets of membrane that are inside of the cell, referred to as vesicles. These vesicles are normally found within the cell, but upon damage to the cell membrane, these vesicles move to the damage site and form a patch to maintain the cell integrity.
  • the cell can die and the cumulative effect of this cellular injury can eventually result in dysfunction of the tissue or organ. It is contemplated that the present invention provides compositions and methods for treating and/or preventing the detrimental effects of cell/tissue damage, in particular, the protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof,
  • the present invention relates to MG53 nucleic acids, and MG53 polypeptides encoded from nucleic acids of the invention, which, alone or in combination with other components, can modulate the process of cell membrane repair and protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof, and the associated pathologic events that can occur as a result, in a broad range of cell and tissue types.
  • the invention encompasses compositions comprising, for example, MG53 polypeptides, MG53 nucleic acids encoding recombinant MG53 polypeptides; as well as vectors, and host cells comprising the same; antibodies, pseudopeptides, fusion proteins, chemical compounds, and methods for producing the same.
  • the present invention also relates to compositions useful as therapeutics for treating and/or prevention of protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • this aspect of the invention comprises compositions of the invention together with a pharmaceutically acceptable excipients. Exemplary excipients, which are suitable for use in any embodiment of the invention, are described herein.
  • the compositions of the invention may additionally include another biologically active agent that complements or synergizes the activity of the compositions of the invention.
  • the therapeutic compositions of the invention comprise MG53 polypeptides, and nucleic acids encoding MG53 polypeptides, for example, the protein of SEQ ID NO. 1 and MG53 polypeptide mutants, homologs, fragments, truncations, pseudopeptides, peptide analogs, and peptidomimetics, as well as nucleic acids (e.g., small RNAs or antisense R As), polypeptides, and compounds that can modulate the activity of MG53 or intermolecular interactions of MG53 with its receptors (i.e., direct or indirect binding or interacting proteins), for example, caveolin-3 (SEQ ID NO. 8).
  • nucleic acids e.g., small RNAs or antisense R As
  • polypeptides e.g., small RNAs or antisense R As
  • compounds that can modulate the activity of MG53 or intermolecular interactions of MG53 with its receptors i.e., direct or indirect binding or interacting
  • the invention includes a composition of the invention in combination with an agent that modulates, synergistically, the activity of an MG53 polypeptide.
  • the modulating agents include, for example, phosphotidylserine; zinc, for example, in the form of a zinc salt, zinc carrier or zinc conjugate; notoginsing; or an oxidizing agent.
  • the invention relates to methods for the treatment or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention comprises the administration of an effective amount of a therapeutic composition of the invention to an individual, wherein the composition is effective for the protection or repair of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention comprises a method for the treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof, comprising the steps of performing treatment on an individual and administering an effective amount of a therapeutic composition of the invention to the individual either prior to the inury event, substantially contemporaneously, or subsequent to the injury event.
  • the method can also include the addition of an agent that modulates the activity or expression of at least one MG53-binding protein, such as but not limited to caveolin-3.
  • the invention relates to interfering and antisense nucleic acids that modulate the expression of MG53 or an MG53 receptor.
  • MG53 receptor includes polypeptides that interact directly and/or indirectly with MG53, and include, for example, caveolin-3 (SEQ ID NO. 8), PI3K, Akt, GS 3p, and/or ERK 1/2, as well as compounds that can modulate their activity or their intermolecular interactions with MG53.
  • the present invention encompasses methods for the targeting of caveolin-3, gene expression, activity, and/or intermolecular interactions for the treatment and/or prevention of a disease or disorder in a subject, for example, treatment or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention encompasses methods of screening and identifying agents from a library of agents useful as therapeutics for the treatment or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention encompasses providing a library of chemical compounds and screening for binding, modulation of activity, and/or expression of MG53 and/or CaV3, wherein the agent represents a candidate useful as a therapeutic or research tool for the treatment/prevention, and/or study of damage to airway cells and/or tissue from injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • agents identified according to the methods of the invention include those that are highly specific for the target polypeptide, and therefore, will have few "off target” effects. In general, agents having little or no non-specific effects will demonstrate fewer negative side- effects in vivo.
  • the invention encompasses agents that are peptides, nucleic acids, or chemical compounds which are antagonists of the expression or activity, for example, by phosphorylation, of the mitochondrial permeability transition pore (MPTP).
  • MPTP mitochondrial permeability transition pore
  • the invention relates to methods of treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof, comprising the administration of an effective amount of an agonist of the expression and/or activity of at least one of MG53 or CaV3 or an antagonist of the expression or activity of the mitochondrial permeability transition pore (MPTP).
  • MPTP mitochondrial permeability transition pore
  • the invention encompasses an isolated or recombinant nucleic acid encoding a polypeptide, which comprises a combination of amino acid and/or peptide components (i.e., structural components or amino acid domains), which when combined together, result in a polypeptide having the activity as described herein.
  • the components comprise a RING zinc finger- binding domain (or alternately, RING finger zinc-binding domain), a B-box domain, a Leucine zipper coiled-coil domain, a phospholipid binding domain, a redox sensitive amino acid, an E3-ligase domain, and a SPRY domain, wherein the components are covalently joined contiguously in a single polypeptide, and wherein the polypeptide facilitates treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • nucleic acids encoding the respective amino acid or peptide domains can be cloned from any desired parental gene and combined into a single contiguous using standard molecular biological techniques.
  • the invention encompasses novel polypeptides formed by expressing genes or cDNA constructs formed by combining nucleotides encoding amino acid or peptide components from other members of the TRIM family, for example (by accession number) TRIM1 (NMJH2216, NM 052817); TRIM2 (AF220018); TRIM3 (AF045239); TRTM4 (AF220023); TRIMS (AF220025); TRIM6 (AF220030); TRIM7 (AF220032); TRIM8 (AF281046); TRJM9 (AF220036); TRIM10 (Y07829); TRTMl 1 (AF220125); TRIM13 (AF220127, NM_001007278); TRIM14 (NM_014788,
  • the invention comprises an isolated or recombinant polypeptide encoded by nucleic acids of the invention, having a RING finger zinc-binding domain, a B-box domain, a Leucine zipper coiled-coil domain, a phospholipid binding domain, a redox sensitive amino acid, an E3-ligase domain, a SPRY domain, and optionally a calcium binding domain, wherein the components are covalently joined contiguously in a single polypeptide, and wherein the polypeptide facilitates treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the present description highlights the important amino acid structural components or features for creating polypeptides able to facilitate treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof (i.e., a RING finger zinc-binding domain, a B-box domain, Leucine zipper coiled-coil domain, a phospholipid binding domain, redox sensitive amino acid, E3-ligase domain, SPRY domain).
  • RING finger zinc-binding domains a B-box domains, Leucine zipper coiled-coil domains, a phospholipid binding domains, redox sensitive amino acids, E3-ligase domains, SPRY domains, and calcium binding domains may vary between evolutionarily related proteins as well as unrelated proteins, as indicated above, there exists a number of genes belonging to the TRIM family, which includes MG53, which contain one or all of the above structural components or domains.
  • these domains may be readily cloned from the gene or cDNA of a TRIM family member, and grafted or cloned into the framework of another TRIM family gene (i.e., MG53) using well known techniques in molecular biology in order to create novel proteins. Also, because it is generally recognized that evolutionarily conserved amino acid sequences will function similarly, it is within the abilities of those skilled in the art to generate additional proteins in accordance with the instant teachings, and to assess the ability of the recombinant proteins to facilitate membrane repair without undue experimentation. As such, recombinant proteins assembled from the domains of the TRIM family members, for example, those identified above, is expressly contemplated as being within the scope of the invention.
  • the invention encompasses an isolated or recombinant nucleic acid encoding an MG53 polypeptide as set forth in SEQ ED NOs.: 1, 3, 5, 7, 8, 9-15, and/or a homolog, or fragment thereof wherein the polypeptide treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention relates to compositions comprising a polypeptide of the invention in combination with at least one other agent, which is capable of facilitating treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the agent acts synergistically, via direct or indirect interaction with the polyptide of the invention, to facilitate treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • agents such as phosphotidylserine, zinc, oxidizing agents, and plant extracts can modulate the membrane repair activity of the polypeptides of the invention.
  • any of the polypeptide-containing compositions encompassed by the invention may also comprise, in combination, an effective amount of at least one of a phospholipid; a zinc containing agent; an oxidizing agent; a plant extract or a combination thereof.
  • the phospholipid is phosphytidylserine.
  • the zinc containing agent is a zinc ionophore, for example, Zn-l-hydroxypyridine-2-thine (Zn-HPT).
  • the oxidizing agent is thimerosal.
  • the plant extract is notoginsing extract.
  • the invention relates to a composition
  • a composition comprising an isolated or recombinant polypeptide of the invention in combination with a pharmaceutically acceptable carrier.
  • the composition may further comprise, in combination, an effective amount of at least one of a phospholipid; a zinc containing agent; an oxidizing agent; a plant extract or a combination thereof.
  • the phospholipid is phosphytidylserine.
  • the zinc containing agent is a zinc ionophore, for example, Zn-l-hydroxypyridine-2-thine (Zn-HPT).
  • the oxidizing agent is thimerosal.
  • the plant extract is notoginsing extract.
  • the present invention also relates to the surprising and unexpected finding that polypeptides of the invention can facilitate treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the repair mechanism is mediated by the oxidative- induced formation of polypeptide oligomers, e.g., dimers, through the coiled-coil domain in the protein, which contains a leucine zipper protein-protein interaction motif.
  • Zn zinc
  • MG53-mediated membrane resealing zinc (Zn) is required for MG53-mediated membrane resealing, and the presence of additional Zn can improve the activity of MG53; an extract from the plant notoginseng can also improve the function of MG53 in membrane resealing; and MG53 requires its endogenous E3-ligase activity to produce membrane repair following acute damage.
  • Zn zinc
  • Polypeptides suitable for extracellular administration include, for example, MG53, MG53 fusion proteins, for example, MG53 fused with cell penetrating peptides, for example, HTV- TAT protein (See WO 2008/054561, which is incorporated herein by reference).
  • certain embodiments of this aspect comprise therapeutic compositions comprising polypeptides of the invention, for example, MG53, in combination with a pharmaceutically acceptable carrier, wherein the therapeutic composition is administered systemically, and wherein the systemically administered composition is effective in facilitating treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the therapeutic compositions of the invention further comprise, in combination with a polypeptide of the invention, one or more additional ingredients, including a phospholipid; a zinc containing agent; an oxidizing agent; a plant extract or a combination thereof, which have a synergistic effect on the activity of the polypeptides of the invention.
  • the therapeutic of the invention may comprise one or more biologically active ingredients such as, Analgesics, Antacids, Antianxiety Drugs, Antiarrhythmics, Antibacterials, Antibiotics, Anticoagulants ' and Thrombolytics, Anticonvulsants, Antidepressants, Antidiarrheais, Antiemetics, Antifungals, Antihistamines, Antihypertensives, Anti -Inflammatories, Antineoplastics, Antipsychotics, Antipyretics, Antivirals, Barbiturates, Beta-Blockers, Bronchodiiators, Cold Cures, Corticosteroids, Cough Suppressants, Cytotoxics, Decongestants, Diuretics, Expectorants, Hormones, Hypoglycemics (Oral), Immunosuppressives, Laxatives, Muscle Relaxants, Sedatives, Sex Hormones, Sleeping Drugs, Tranquilizer, Vitamins or a combination thereof.
  • the invention relates to methods of treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof comprising the steps of administering to an individual an effective amount of a nucleic acid encoding a polypeptide of the invention, for example, MG53, homologs, fragments, and derivatives thereof, wherein the polypeptide is effective in the treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof, in vitro, i vivo or ex vivo.
  • the invention relates to methods of treating and/or preventing a disease or pathological condition related to treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof, comprising administering to an individual an effective amount of a composition comprising a nucleic acid encoding a polypeptide of the invention, for example, MG53, homolog, fragment or derivative thereof, in combination with a pharmaceutically acceptable carrier, wherein the composition is effective in treating and/or preventing cell myocardial cell or tissue damage.
  • the disease or pathological condition related to treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof comprising administering to an individual an effective amount of a composition comprising a nucleic acid encoding a polypeptide of the invention, for example, MG53, homolog, fragment or derivative thereof, in combination with a pharmaceutically acceptable carrier, wherein the composition
  • nucleic acids or polypeptides of the invention may be delivered or administered in any pharmaceutically acceptable form, and in any pharmaceuticaly acceptable route as described in further detail below.
  • compositions comprising nucleic acids and/or polypeptides of the invention can be delivered systemically or administered directly to a cell or tissue for the treatment and/or prevention of lung cell or tissue damage.
  • the nucleic acids and/or polypeptides of the invention comprise a carrier moiety that improves bioavailability, drug half-life, efficacy or potency.
  • the invention relates to an isolated or recombinant membrane repair polypeptide complex.
  • MG53 polypeptides demonstrate the ability to interact (e.g., bind non-covelently) and form complexes, either directly or indirectly, with a number other cellular proteins, in particular, CaV3.
  • the invention comprises a recombinant chimeric nucleic acid comprising, in a single open reading frame, a polynucleotide encoding an MG53 polypeptide linked in a contiguous nucleic acid to another polynucleotide encoding another polypeptide, for example, CaV3.
  • the chimera may comprise a plurality of polynucleotides encoding any combination of MG53, CaV3 or other proteins linked in a single contiguous nucleic acid, which is comprised within a single open reading frame.
  • the translation results in a single polypeptide contining functional domains of one or more of G53 and/or CaV3, wherein the chimeric protein complex is able to facilitate treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention further comprises a method of treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof, comprising administering to a cell an effective amount of a nucleic acid encoding a chimeric polypeptide of the invention, wherein the complex is capable of treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention includes a method of treating or preventing disease or pathological condition related to cell or tissue damage comprising administering to an individual an effective amount of isolated chimeric polypeptide of the invention, wherein the chimeric polypeptide complex is capable of ameliorating the effects of the disease or pathological condition.
  • the invention relates to methods of treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof comprising modulating the expression level or activity of at least one of MG53 and CaV3.
  • the invention relates to methods of screening for compounds that are effective for the treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof, by contacting at least one of MG53 or CaV3, with a test compound; and measuring the binding of the test compound, and/or the activity of MG53 or CaV3, and/or the measuring the effects on airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the recombinant membrane repair polypeptides can be produced in prokaryotic cells or eukaryotic cells, for example, mammalian cells and then secreted into the extracellular solution through protein engineering, an approach that should produce large quantities of functional protein.
  • the invention relates to nucleic acids, including interfering nucleic acids that hybridize to a nucleic acid encoding MG53 or CaV3, mutants, truncations, fragments, or homologs thereof, for treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • therapeutic compositions can be administered in any suitable pharmaceutical form as described herein or as commonly known in the art.
  • the invention provides an isolated nucleic acid encoding polypeptide molecules, for example, MG53 gene hybridizing nucleic acid molecules, and nucleic acids encoding MG53 polypeptides having at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% identity to the nucleic acids disclosed in SEQ ID NOS: 2, 4, and 6.
  • the isolated nucleic acid molecules of the invention will hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of an MG53 nucleic acid sequence.
  • the invention also includes an isolated nucleic acid that encodes an MG53 polypeptide, or a fragment, homolog, analog, fusion protein, or derivative thereof.
  • the nucleic acid can encode a polypeptide at least 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% identity to a polypeptide comprising the amino acid sequences of SEQ ID NOS: 1 , 3, 5, 7, 8, and 9-15.
  • the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS: 2, 4, and 6.
  • an oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of an MG53 nucleic acid (e.g., SEQ ID NOS: 2, 4, and 6) or a complement of said oligonucleotide.
  • an MG53 nucleic acid e.g., SEQ ID NOS: 2, 4, and 6
  • polypeptides for example, MG53 polypeptides (SEQ ID NOS: 1, 3, 5, 7, 8, and 9-15).
  • the polypeptides e.g., MG53 polypeptides, include an amino acid sequence that is substantially identical to the amino acid sequence of a human MG53 polypeptide (SEQ ID NO.:l).
  • the invention comprises the use of a therapeutic composition comprising an effective amount of an agent that modulates at least one of MG53 activity, MG53 expression, or the MG53 signaling cascade in a lung cell in the manufacture of a medicament for the treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the use of the therapeutic composition can comprise an effective amount of from 0.1 mg/kg and 1000 mg/kg body weight/day.
  • the therapeutic agent is at least one of an MG53 polypeptide; an MG53 receptor polypeptide; a nucleic acid encoding an MG53 polypeptide; a nucleic acid encoding an MG53 receptor polypeptide; an inhibitory or antisense RNA specific for a nucleic acid encoding MG53, an MG53 receptor, caveolin-3, or an agonist or antagonist of MG53, an MG53 receptor, or caveolin-3.
  • the agonist of MG53 acitvity comprises at least one of phosphotidylserine, zinc or zinc salt, Zn-l-hydroxypyridine- 2-thine (Zn-HPT), notoginsing, an oxidizing agent, thimerosal, or combination thereof.
  • the polypeptide can have the amino acid sequence of at least one of SEQ ID NOs.: 1 , 3, 5, 9, 10, 1 1, 12, 13, 14, 15, or 16 or bioactive portion thereof.
  • the agent is a stem cell capable of differentiation into a lung cell, and wherein the stem cell has been modified such that it demonstrates enhanced activity or expression of MG53.
  • the therapeutic composition can further comprise a pharmaceutically acceptable carrier or excipient.
  • the injury comprises treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention also includes the use of a therapeutic composition comprising an effective amount of an MG53 polypeptide or MG53 nucleic acid, and a pharmaceutically acceptable excipient, in the manufacture of a medicament for the treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • the invention also features antibodies that immunoseiectively-bind to polypeptides, for example, MG53, polypeptides, or fragments, homologs, analogs, pseudopeptides, peptidomimetics or derivatives thereof.
  • the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier.
  • the therapeutic can be a nucleic acid, e.g., a MG53 nucleic acid, for example, a peptide nucleic acid, a cDNA, or RNA, such as for example, a small inhibitory RNA; a membrane repair polypeptidefor example, MG53; or an antibody specific for a MG53 polypeptide.
  • the invention includes, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
  • the invention includes a method of producing a polypeptide by culturing a cell that includes an endogenous or exogenously expressed nucleic acid encoding a membrane repair polypeptide, for example a MG53 nucleic acid, under conditions allowing for expression of the polypeptide encoded by the DNA. If desired, the polypeptide can then be recovered.
  • the invention includes a method of producing a polypeptide by culturing a cell that contains an endogenous nucleic acid encoding a polypeptide, for example a MG53 nucleic acid, disposed upstream or downstream of an exogenous promoter.
  • the exogenous promoter is incorporated into a host cell's genome through homologous recombination, strand break or mismatch repair mechanisms.
  • the invention includes a method of detecting the presence of a polypeptide of the invention, for example, an MG53 polypeptide, in a sample.
  • a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
  • the complex is detected, if present, thereby identifying the membrane repair polypeptide, for example, an MG53 polypeptide, within the sample.
  • the invention also includes methods to identify specific ceil or tissue types based on their expression of a nucleic acid encoding a polypeptide of the invention, for example a MG53 polypeptide or a related fusion polypeptide, thereof.
  • the invention includes fusion proteins comprising a "tag" or indicator portion and, for example, an MG53 portion.
  • the tag or indicator portion can be a peptide adapted for purification purposes, for example, FLAG tag, 6xHis tag, Maltose- Binding Protein (MBP) tag, or the like.
  • the tag peptide comprises a peptide adapted for providing a signal such as an antibody epitope or a fluorescent peptide.
  • Still other aspects include the fusion of the MG53 with a peptide that is adapted for mediating subcellular localization or translocation across a cellular membrane, for example, a TAT fusion protein from the HTV virus to facilitate cell penetration or a modified cellular localization tag to couple MG53 to particular cellular organelles.
  • Also included in the invention is a method of detecting the presence of a nucleic acid molecule of the invention in a sample by contacting the sample with a nucleic acid probe or primer, and detecting whether the nucleic acid probe or primer bound to a nucleic acid encoding, for example, an MG53 polypeptide.
  • the invention provides a method for modulating the activity of a polypeptide of the invention, for example, an MG53 polypeptide, by contacting a cell that includes the MG53 polypeptide, with a compound that binds to the MG53 polypeptide in an amount sufficient to modulate the activity of said polypeptide.
  • the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
  • a therapeutic of the invention in the manufacture of a medicament for treating or preventing disorders or syndromes including, e.g., cardiovascular disease, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, aortic stenosis, atrial septal defect (ASD), atrioventricular (A-V) canal defect, ductus arteriosus, pulmonary stenosis, subaortic stenosis, ventricular septal defect (VSD), valve diseases, hypercoagulation, ischemia/reperfusion injury, hypoxic injury, oxidative damage, age-related tissue degeneration, surgically related lesions, heart failure, secondary pathologies caused by heart failure and hypertension, hypotension, angina pectoris, myocardial infarction, tuberous sclerosis, heart attacks, heart failure, muscular dystrophy, stroke, and/or other pathologies and disorders of the like.
  • cardiovascular disease e.g., cardiovascular disease, cardiomyopathy, atherosclerosis, hypertension, congenital heart defects, a
  • the therapeutic composition of the invention comprises, in certain embodiments, for example, a nucleic acid encoding a MG53; a nucleic acid that binds a nucleic acid encoding MG 3; an MG53 polypeptide, peptide analog, pseudopeptide or peptidomimetic based thereon; a small molecule modulator of MG53 or an MG53 protein- protein interaction; or a MG53 -specific antibody or biologically-active derivatives or fragments thereof.
  • MG53 mediates the treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof. Therefore, targeting the expression and/or activity of these nucleic acids, polypeptides, and homoiogs thereof will allow for a novel treatment of various acute and chronic diseases and conditions related to lung damage.
  • the invention comprises therapeutic compositions useful as a surgical adjuvant, in any of the embodiments described herein, the surgical adjuvant composition of the invention can be used or applied as a stand alone therapeutic directly to the surgical site or it can be integrally associated with a surgical or medical implement, for example, the therapeutic of the invention may be conjugated to a polymer- based stent, tube or other implantable device, such that the therapeutic diffuses to the site of action in a controlled manner to accelerate healing and/or to minimize trauma from an invasive surgical procedure.
  • the therapeutic composition of the invention is applied as, for example, a film or coating to the medical implement such that the therapeutic diffuses into the blood stream or surrounding tissues and/or wears away, and is thereby delivered directly to the site of tissue damage; minimizing or ameliorating the amount of damage that occurs due to the use of the surgical implement or procedure.
  • the therapeutic composition of the invention can be in any pharmaceutically acceptable form and administered by any pharmaceutically acceptable route, for example, the therapeutic composisition can be administered as an oral dosage, either single daily dose or unitary dosage form, for the treatment and/or protection of airway cells and/or tissue from damage due to injury to the lungs or complications of underlying respiratory, cardiovascular or infectious diseases, or any combination thereof.
  • Such pharmaceutically acceptable carriers and excipients and methods of administration will be readily apparent to those of skill in the art, and include compositions and methods as described in the USP-NF 2008 (United States Pharmacopeia/National Formulary), which is incorporated herein by reference in its entirety.
  • phrases "pharmaceutically or pharmacologically acceptable” refer to molecular entities and compositions that do not produce an adverse, allergic or other undesirable reaction when administered to an animal, or a human, as appropriate.
  • pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
  • the active compounds will generally be formulated for parenteral administration, e.g., formulated for injection via the intravenous, intraarthricular, intrathecal, intramuscular, sub-cutaneous, intra-lesional, or even intraperitoneal routes.
  • parenteral administration e.g., formulated for injection via the intravenous, intraarthricular, intrathecal, intramuscular, sub-cutaneous, intra-lesional, or even intraperitoneal routes.
  • the preparation of an aqueous composition that contains a marker antibody, conjugate, inhibitor or other agent as an active component or ingredient will be known to those of skill in the art in light of the present disclosure.
  • such compositions can be prepared as injectibles, either as liquid solutions or suspensions; solid forms suitable for using to prepare solutions or suspensions upon the addition of a liquid prior to injection can also be prepared; and the preparations can also be emulsified.
  • the invention relates to nucleic acids, including interfering nucleic acids, and polypeptides encoding membrane repair interacting proteins and/or MG53 interacting proteins, and homologs thereof; pseudopeptides and peptidomimetics; as well as compounds that can modulate the activity of membrane repair polypeptides or MG53 or their intermolecular interactions.
  • compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the polypeptides can be used as immunogens to produce antibodies specific for the invention, and as vaccines. They can also be used to screen for potential agonist and antagonist compounds.
  • a cDNA encoding polypeptides of the invention for example, MG53 or CaV3, may be useful in gene therapy when administered to a subject in need thereof.
  • the compositions of the present invention will have efficacy for treatment of patients suffering from the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the invention further includes a method for screening for predisposition to a disorder or syndrome including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the method includes contacting a test agent to a nucleic acid or polypeptide encoding MG53 or CaV3, and determining if the test agent binds to said target. Binding of the test agent to a nucleic acid or polypeptide encoding MG53 or CaV3, indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
  • Also within the scope of the invention is a method for screening for a modulator of activity, or of latency or predisposition to disorders or syndromes including, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
  • the test animal expresses a recombinant polypeptide encoded by a nucleic acid of the invention.
  • Expression or activity of a polypeptide of the invention is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly-expresses the polypeptide of the invention and is not at increased risk for the disorder or syndrome.
  • the expression of polypeptides of the invention in both the test animal and the control animal is compared. A change in the activity of the polypeptide in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.
  • the invention includes a method for determining the presence of or predisposition to a disease associated with dysfunctional or altered levels of a nucleic acid or polypeptide for MG53 or CaV3, in a subject (e.g., a human subject).
  • the method includes measuring the amount of the a nucleic acid or polypeptide for MG53 or CaV3, in a test sample from the subject and comparing the amount of the nucleic acid or polypeptide in the test sample to the amount of the nucleic acid or polypeptide present in a control sample.
  • An alteration in the level of the nucleic acid or polypeptide in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
  • the predisposition includes, e.g., the diseases and disorders disclosed above and/or other pathologies and disorders of the like.
  • the expression levels of the new polypeptides of the invention can be used in a method to screen for various disorders as well as to determine the stage of particular disorders.
  • the invention can be used in a method to identity the cellular receptors of MG53 and downstream effectors of the invention by any one of a number of techniques commonly employed in the art. These include but are not limited to the two- hybrid system, affinity purification, co-precipitation with antibodies or other specific- interacting molecules.
  • MG53 receptor relates generally to any protein or fragment thereof capable of undergoing binding to a MG53 protein.
  • the modulation of MG53 activity is accomplished by, for example, the use of or modulation of, for example, MG53 binding partners, i.e., factors that directly or indirectly bind to MG53, and enhance or neutralize its biological activities, and include, for example, neutralizing anti-MG53 antibodies, caveolin-3, anti-caveolin-3 antibodies, pseudopeptides, peptide analogs or peptidomimetics that bind and disrupt MG53 or CaV3 activity or intermolecular interactions; or the use of nucleotide sequences derived from MG53 or CaV3 genes, including coding, non-coding, and/or regulatory sequences to modulate expession by, for example, antisense, ribozyme, and/or triple helix approaches.
  • MG53 binding partners i.e., factors that directly or indirectly bind to MG53, and enhance or neutralize its biological activities
  • MG53 binding partners i.e., factors that directly or indirectly bind to MG53, and enhance or neutralize its
  • the present invention features a nucleic acid molecule, such as a decoy RNA, dsRNA, siR A, shRNA, micro RNA, aptamers, antisense nucleic acid molecules, which down regulates expression of a sequence encoding MG53 proteins, and/or MG53 receptors, for example, caveolin-3.
  • a nucleic acid molecule of the invention has an endonuclease activity or is a component of a nuclease complex, and cleaves an MG53 and/or CaV3 mRNA.
  • a nucleic acid molecule of the invention comprises between 12 and 100 bases complementary to RNA having a nucleic acid sequence encoding a member selected from the group of MG53 or CaV3. In another embodiment, a nucleic acid molecule of the invention comprises between 14 and 24 bases complementary to RNA having a nucleic acid sequence encoding a member selected from the group of MG53 or CaV3. In any embodiment described herein, the nucleic acid molecule can be synthesized chemically according to methods well known in the art.
  • the present invention provides a kit comprising a suitable container, th active agent capable of inhibiting membrane repair polypeptide activity, expression or binding in a pharmaceutically acceptable form disposed therein, and instructions for its use.
  • the invention relates to a method for diagnosing or monitoring disorder or disease or progression comprising detecting for the presence of a nucleotide polymorphism in the membrane repair gene, for example, MG53 gene, associated with the disease, through the detection of the expression level of a member selected from the group of MG53 or CaV3.
  • MG53 or MG53 receptor gene includes the 5' UTR, 3' UTR, promoter sequences, enhancer sequences, intronic and exonic DNA of the gene as well as the mRNA or cDNA sequence.
  • the MG53 or MG53 receptor gene polymorphisms associated with tissue repair disorders, and hence useful as diagnostic markers according to the methods of the invention may appear in any of the previously named nucleic acid regions. Techniques for the identification and monitoring of polymorphisms are known in the art and are discussed in detail in U.S. Patent No: 6,905,827 to Wohlgemuth, which is incorporated herein by reference in its entirety for all purposes.
  • Certain aspects of the invention encompass methods of detecting gene expression or polymorphisms with one or more DNA molecules wherein the one or more DNA molecules has a nucleotide sequence which detects expression of a gene corresponding to the oligonucleotides depicted in the Sequence Listing.
  • the oligonucleotide detects expression of a gene that is differentially expressed.
  • the gene expression system may be a candidate library, a diagnostic agent, a diagnostic oligonucleotide set or a diagnostic probe set.
  • the DNA molecules may be genomic DNA, RNA, protein nucleic acid (PNA), cDNA or synthetic oligonucleotides. Following the procedures taught herein, one can identify sequences of interest for analyzing gene expression or polymorphisms. Such sequences may be predictive of a disease state.
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs thereof.
  • the nucleic acid molecule may be single-stranded or double-stranded, but preferably is comprised double-stranded DNA.
  • the invention relates to diagnostic oligonucleotides and diagnostic oligonucleotide set(s), for which a correlation exists between the health status of an individual, and the individual's expression of RNA or protein products corresponding to the nucleotide sequence. In some instances, only one oligonucleotide is necessary for such detection.
  • RNA or protein products may be identified by any means capable of detecting expression or a polymorphism of RNA or protein products, including but not limited to differential expression screening, PCR, RT-PCR, SAGE analysis, high- throughput sequencing, microarrays, liquid or other arrays, protein-based methods (e.g., western blotting, proteomics, mass-spectrometry, and other methods described herein), and data mining methods, as further described herein.
  • protein-based methods e.g., western blotting, proteomics, mass-spectrometry, and other methods described herein
  • data mining methods as further described herein.
  • the various aspects and embodiments are also directed to genes which encode homologs, orthologs, and paralogs of other membrane repair proteins, membrane repair polypeptide binding proteins, and membrane repair polypeptide receptor genes and include all isoforms, splice variants, and polymorphisms.
  • Those additional genes can be analyzed for target sites using the methods described for MG53 and MG53 receptor proteins, and/or genes. Thus, the inhibition and the effects of such inhibition of the other genes can be performed as described herein.
  • down-regulate it is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more proteins, or activity of one or more proteins, is reduced below that observed in the absence of the nucleic acid molecules of the invention.
  • inhibition or down-regulation with enzymatic nucleic acid molecule preferably is below that level observed in the presence of an enzymatically inactive or attenuated molecule that is able to bind to the same site on the target RNA, but is unable to cleave that RNA.
  • inhibition or down-regulation with antisense oligonucleotides is preferably below that level observed in the presence of, for example, an oligonucleotide with scrambled sequence or with mismatches.
  • inhibition or down-regulation of genes with the nucleic acid molecule of the instant invention is greater in the presence of the nucleic acid molecule than in its absence.
  • up-regulate is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more protein subunits, or activity of one or more protein subunits is greater than that observed in the absence of the nucleic acid molecules of the invention.
  • the expression of a gene can be increased in order to treat, prevent, ameliorate, or modulate a pathological condition caused or exacerbated by an absence or low level of gene expression.
  • the invention relates to a method for treating or preventing ischemic reperfusion or hypoxic injury to myocardial tissue by up-regulating the expression, and/or activity of an MG53 and/or MG53 receptor gene.
  • module is meant that the expression of the gene, or level of RNAs or equivalent RNAs encoding one or more proteins, or activity of one or more proteins is up- regulated or down-regulated, such that the expression, level, or activity is greater than or less than that observed in the absence of the nucleic acid molecules of the invention.
  • RNA RNA sequences including but not limited to a segment encoding a polypeptide.
  • Complementarity refers to the ability of a nucleic acid to form hydrogen bond(s) with another RNA sequence by either traditional Watson -Crick or other non- traditional types.
  • RNA is meant a molecule comprising at least one ribonucleotide residue.
  • ribonucleotide or “2'-OH” is meant a nucleotide with a hydroxyl group at the 2' position of a D-ribo-furanose moiety.
  • nucleotide is meant a heterocyclic nitrogenous base in N-glycosidic linkage with a phosphorylated sugar.
  • Nucleotides are recognized in the art to include natural bases (standard), and modified bases well known in the art. Such bases are generally located at the ⁇ position of a nucleotide sugar moiety. Nucleotides generally comprise a base, sugar and a phosphate group.
  • the nucleotides can be unmodified or modified at the sugar, phosphate and/or base moiety, (also referred to interchangeably as nucleotide analogs, modified nucleotides, non-natural nucleotides, non-standard nucleotides and other; see for example, Usman and McSwiggen, supra; Eckstein et al., International PCT Publication No. WO 92/07065; Usman et al., International PCT Publication No. WO 93/15187; Uhlman & Peyman, supra all are hereby incorporated by reference herein).
  • modified nucleic acid bases known in the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22, 2183.
  • nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5- methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • 6-methyluridine 6-methyluridine
  • propyne quesosine, 2- thiouridine, 4-thiouridine, wybutosine, wybutoxosine, 4-acetyltidine, 5- (carboxyhydroxymethyl)uridme, 5'-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1- methylinosine, 2,2-dimethylguanosine, 3-methylcytidine, 2-methyladenosine, 2- methylguanosine, N6-methyladenosine, 7-methylguanosine, 5-methoxyaminomethyl-2- thiouridine, 5-methylaminomethyluridine, 5-methylcarbonyhnethyluridine, 5- methyloxyuridine, 5-methyl-2-thiouridine, 2-methylthio-N6-isopentenyladenosine, beta-D- mannosylque
  • modified bases in this aspect is meant nucleotide bases other than adenine, guanine, cytosine and uracil at position or their equivalents; such bases can be used at any position, for example, within the catalytic core of an enzymatic nucleic acid molecule and/or in the substrate-binding regions of the nucleic acid molecule.
  • antisense nucleic acid it is meant a non-enzymatic nucleic acid molecule that binds to target RNA by means of RNA-RNA or RNA-DNA or RNA-PNA (protein nucleic acid; Egholm et al., 1993 Nature 365, 566) interactions and alters the activity of the target RNA (for a review, see Stein and Cheng, 1993 Science 261, 1004 and Woolf et al., U.S. Pat. No. 5,849,902).
  • antisense molecules are complementary to a target sequence along a single contiguous sequence of the antisense molecule.
  • an antisense molecule can bind to substrate such that the substrate molecule forms a loop or hairpin, and/or an antisense molecule can bind such that the antisense molecule forms a loop or hairpin.
  • the antisense molecule can be complementary to two (or even more) non-contiguous substrate sequences or two (or even more) non-contiguous sequence portions of an antisense molecule can be complementary to a target sequence or both.
  • antisense DNA can be used to target RNA by means of DNA-RNA interactions, thereby activating RNase H, which digests the target RNA in the duplex.
  • the antisense oligonucleotides can comprise one or more RNAse H activating region, which is capable of activating RNAse H cleavage of a target RNA.
  • Antisense DNA can be synthesized chemically or expressed via the use of a single stranded DNA expression vector or equivalent thereof.
  • dsRNAs Long double-stranded RNAs
  • dsRNAs can be used to silence the expression of target genes in a variety of organisms and cell types (e.g., worms, fruit flies, and plants).
  • RNAi RNA interference pathway
  • the dsRNAs get processed into 20-25 nucleotide (nt) small interfering RNAs (siRNAs) by an RNase ⁇ -like enzyme called Dicer (initiation step).
  • siRNAs assemble into endoribonuclease- containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process.
  • RISCs RNA-induced silencing complexes
  • the siRNA strands subsequently guide the RISCs to complementary RNA molecules, where they cleave and destroy the cognate RNA (effecter step). Cleavage of cognate RNA takes place near the middle of the region bound by the siRNA strand.
  • introduction of long dsRNA >30 nt
  • initiates a potent antiviral response exemplified by nonspecific inhibition of protein synthesis and RNA degradation.
  • the mammalian antiviral response can be bypassed, however, by the introduction or expression of siRNAs.
  • RNAs small interfering RNAs targeted against HIV-1 rev transcripts in human cells. Nature Biotechnol. 20:500-505; Miyagishi M, and Taira K. (2002).
  • U6-promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells. Nature Biotechnol. 20:497-500; Paddison PJ, Caudy AA, Bernstein E, Hannon GJ, and Conklin DS. (2002).
  • Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes & Dev. 16:948-958; Paul CP, Good PD, Winer I, and Engelke DR. (2002).
  • vectors any nucleic acid-based technique used to deliver a desired nucleic acid, for example, bacterial plasmid, viral nucleic acid, HAC, BAC, and the like.
  • nucleic acid molecules of the instant invention can be used to treat diseases or conditions discussed above.
  • the subject can be treated, or other appropriate cells can be treated, as is evident to those skilled in the art, individually or in combination with one or more drugs under conditions suitable for the treatment.
  • double stranded RNA or “dsRNA” is meant a double stranded RNA that matches a predetermined gene sequence that is capable of activating cellular enzymes that degrade the corresponding messenger RNA transcripts of the gene.
  • dsRNAs are referred to as short intervening RNA (siRNA) and can be used to inhibit gene expression (see for example Elbashir et a1hack 2001 , Nature, 41 1, 494-498; and Bass, 2001, Nature, 41 1, 428- 429).
  • double stranded RNA or “dsRNA” as used herein refers to a double stranded RNA molecule capable of RNA interference "RNAi", including short interfering RNA “siRNA” see for example Bass, 2001 , Nature, 41 1, 428-429; Elbashir et al., 2001, Nature, 41 1, 494-498; and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No.
  • cell is used in its usual biological sense, and does not refer to an entire multicellular organism.
  • the cell can, for example, be in vivo, in vitro or ex vivo, e.g., in cell culture, or present in a multicellular organism, including, e.g., birds, plants and mammals such as humans, cows, sheep, apes, monkeys, swine, dogs, and cats.
  • the cell can be prokar otic (e.g., bacterial eel!) or eukaryotic (e.g., mammalian or plant cell).
  • Oligonucleotides are synthesized using protocols known in the art as described in Caruthers et al., 1992, Methods in Enzymology 21 1 , 3 19, Thompson et al., International PCT Publication No. WO 99/54459, Wincott et al., 1995, Nucleic Acids Res. 23, 2677 2684, Wincott et al., 1997, Methods Mol. Bio., 74, 59, Brennan et al, 1998, Biotechnol Bioeng., 61, 33 45, and Brennan, U.S. Pat. No. 6,001,311. All of these references are incorporated herein by reference.
  • nucleic acid molecules of the present invention can be synthesized separately and joined together post-synthetically, for example by ligation (Moore et al., 1992, Science 256, 9923; Draper et al., International PCT publication No. WO 93/23569; Shabarova et al., 1991 , Nucleic Acids Research 19, 4247; Bellon et al., 1997, Nucleosides & Nucleotides, 16, 951 ; Bellon et al., 1997, Bioconjugate Chem. 8, 204).
  • nucleic acid molecules of the present invention can be modified extensively to enhance stability by modification with nuclease resistant groups, for example, 2'-amino, 2'-C-a11yl, 2'-flouro, 2 -O-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31 , 163).
  • nuclease resistant groups for example, 2'-amino, 2'-C-a11yl, 2'-flouro, 2 -O-methyl, 2'-H (for a review see Usman and Cedergren, 1992, TIBS 17, 34; Usman et al., 1994, Nucleic Acids Symp. Ser. 31 , 163).
  • Nucleic acid molecules having chemical modifications that maintain or enhance activity are provided. Such nucleic acid is also generally more resistant to nucleases than unmodified nucleic acid. Nucleic acid molecules are preferably resistant to nucleases in order to function as effective intracellular therapeutic agents. Improvements in the chemical synthesis of RNA and DNA (Wincott et al., 1 95 Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods in Enzymology 21 1, 3-19 (incorporated by reference herein) have expanded the ability to modify nucleic acid molecules by introducing nucleotide modifications to enhance their nuclease stability as described above.
  • nucleic acid-based molecules of the invention can lead to better treatment of the disease progression by affording the possibility of combination therapies (e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules and/or other chemical or biological molecules).
  • combination therapies e.g., multiple antisense or enzymatic nucleic acid molecules targeted to different genes, nucleic acid molecules coupled with known small molecule inhibitors, or intermittent treatment with combinations of molecules and/or other chemical or biological molecules.
  • the treatment of subjects with nucleic acid molecules can also include combinations of different types of nucleic acid molecules.
  • the invention features modified nucleic acid molecules with phosphate backbone modifications comprising one or more phosphorothioate, phosphorodithioate, methylphosphonate, morpholino, amidate carbamate, carboxymethyl, acetamidate, polyamide, sulfonate, sulfonamide, sulfamate, formacetal, thioformacetal, and/or alkylsilyl, substitutions.
  • nucleic acid e.g., antisense and ribozyme
  • such modifications can enhance shelf-life, half- life in vitro, bioavailability, stability, and ease of introduction of such oligonucleotides to the target site, including e.g., enhancing penetration of cellular membranes and conferring the ability to recognize and bind to targeted cells.
  • nucleic Acid Molecules [00171] Administration of Nucleic Acid Molecules. Methods for the delivery of nucleic acid molecules are described in Akhtar et al., 1992, Trends Cell Bio., 2, 139; and Delivery Strategies for Antisense Oligonucleotide Therapeutics, ed. Akhtar, 995 which are both incorporated herein by reference. Sullivan et al., PCT WO 94/02595, further describes the general methods for delivery of enzymatic RNA molecules. These protocols can be utilized for the delivery of virtually any nucleic acid molecule.
  • Nucleic acid molecules can be administered to cells by a variety of methods known to those familiar to the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres.
  • the nucleic acid/vehicle combination is locally delivered by direct injection or by use of an infusion pump.
  • Other routes of delivery include, but are not limited to oral (tablet or pill form) and/or intrathecal delivery (Gold, 1997, Neuroscience, 76, 1153-1 158).
  • the molecules of the instant invention can be used as pharmaceutical agents.
  • Pharmaceutical agents prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) a disease state in a subject.
  • the negatively charged polynucleotides of the invention can be administered (e.g., RNA, DNA or protein) and introduced into a subject by any standard means, with or without stabilizers, buffers, and the like, to form a pharmaceutical composition.
  • RNA, DNA or protein e.g., RNA, DNA or protein
  • standard protocols for formation of liposomes can be followed.
  • the compositions of the present invention can also be formulated and used as tablets, capsules or elixirs for oral administration; suppositories for rectal administration; sterile solutions; suspensions for injectable administration; and the other compositions known in the art.
  • the present invention also includes pharmaceutically acceptable formulations of the compounds described.
  • formulations include salts of the above compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or subject, preferably a human.
  • systemic administration is meant in vivo systemic absorption or accumulation of drugs in the blood stream followed by distribution throughout the entire body. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell (i.e., a cell to which the negatively charged polymer is desired to be delivered to).
  • pharmacological compositions injected into the blood stream should be soluble.
  • Administration routes which lead to systemic absorption include, without limitations: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.
  • the use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation which can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful.
  • compositions or formulation that allows for the effective distribution of the nucleic acid molecules and other biomolecules of the instant invention in the physical location most suitable for their desired activity.
  • agents suitable for formulation with the nucleic acid molecules of the instant invention include: PEG conjugated nucleic acids, phospholipid conjugated nucleic acids, nucleic acids containing lipophilic moieties, phosphorothioates, P- glycoprotein inhibitors (such as Pluronic P85) which can enhance entry of drugs into various tissues, for example the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin.
  • biodegradable polymers such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after implantation (Emerich, DF et al, 1999, Cell Transplant, 8, 47- 58) Alkermes, Inc. Cambridge, Mass.; and loaded nanoparticles, such as those made of polybutylcyanoacrylate, which can deliver drugs across the blood brain barrier and can alter neuronal uptake mechanisms (Prog Neuropsychopharmacol Biol Psychiatry, 23, 941-949, 1999).
  • Other non-limiting examples of delivery strategies, including CNS delivery of nucleic acid molecules include material described in Boado et al., 1998, J. Pharm.
  • the invention also features the use of the composition comprising surface- modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long- circulating liposomes or stealth liposomes).
  • Nucleic acid molecules of the. invention can also comprise covalently attached PEG molecules of various molecular weights. These formulations offer a method for increasing the accumulation of drugs in target tissues. This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem.
  • compositions prepared for storage or administration which include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (A. R. Gennaro edit. 1985) hereby incorporated by reference herein.
  • preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p- hydroxybenzoic acid.
  • antioxidants and suspending agents can be used.
  • an effective amount, pharmaceutically effective dose, therapeutically effective amount, or pharmaceutically effective amount is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the. symptoms) of a disease state or pathological condition.
  • the effective amount depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors which those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 1000 mg kg body weight day of active ingredients is administered dependent upon potency of the negatively charged polymer.
  • effective amounts of the compositions of the invention encompass those amounts utilized in the examples to facilitate the intended or desired biological effect.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds that exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the formulations can be administered orally, topically, parenterally, by inhalation or spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles.
  • parenteral as used herein includes percutaneous, subcutaneous, intravascular (e.g., intravenous), intramuscular, or intrathecal injection or infusion techniques and the like.
  • a pharmaceutical formulation comprising a nucleic acid molecule of the invention and a pharmaceutically acceptable carrier.
  • One or more nucleic acid molecules of the invention can be present in association with one or more non-toxic pharmaceutically acceptable carriers and/or diluents and/or adjuvants, and if desired other active ingredients.
  • compositions of the invention can be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsion, hard or soft capsules, or syrups or elixirs.
  • compositions intended for oral use can be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions can contain one or more such sweetening agents, flavoring agents, coloring agents or preservative agents in order to provide pharmaceutically elegant and palatable preparations.
  • Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients that are suitable for the manufacture of tablets.
  • excipients can be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, corn starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc.
  • the tablets can be uncoated or they can be coated by known techniques. In some cases such coatings can be prepared by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
  • a time delay material such as glyceryl monosterate or glyceryl distearate can be employed.
  • Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin or olive oil.
  • Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions.
  • excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydropropyl-methylceliulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents can be a naturally-occurring phosphatide, for example, lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene sorbito
  • the aqueous suspensions can also contain one or more preservatives, for example ethyl, or n-propyl p- hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.
  • preservatives for example ethyl, or n-propyl p- hydroxybenzoate
  • coloring agents for example ethyl, or n-propyl p- hydroxybenzoate
  • flavoring agents for example ethyl, or n-propyl p- hydroxybenzoate
  • sweetening agents such as sucrose or saccharin.
  • Oily suspensions can be formulated by suspending the active ingredients in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin.
  • the oily suspensions can contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol.
  • Sweetening agents and flavoring agents can be added to provide palatable oral preparations.
  • These compositions can be preserved by the addition of an anti-oxidant such as ascorbic acid.
  • Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives.
  • compositions of the invention can also be in the form of oil-in- water emulsions.
  • the oily phase can be a vegetable oil or a mineral oil or mixtures of these.
  • Suitable emulsifying agents can be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol, anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate.
  • the emulsions can also contain sweetening and flavoring agents.
  • Syrups and elixirs can be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol, glucose or sucrose. Such formulations can also contain a demulcent, a preservative and flavoring and coloring agents.
  • the pharmaceutical compositions can be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension can be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents that have been mentioned above.
  • the sterile injectable preparation can also be a sterile injectable solution or suspension in a non-toxic parentally acceptable diluent or solvent, for example as a solution in 1,3-butanediol.
  • Suitable vehicles and solvents that can be employed are water, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are conventionally employed as a solvent or suspending medium.
  • any bland fixed oil can be employed including synthetic mono-or diglycerides.
  • fatty acids such as oleic acid find use in the preparation of injectables.
  • Nucleic acid molecules of the invention can also be administered in the form of suppositories, e.g., for rectal administration of the drug or via a catheter directly to the bladder itself. These compositions can be prepared by mixing the drug with a suitable non- irritating excipient that is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter and polyethylene glycols.
  • Nucleic acid molecules of the invention can be administered parenterally in a sterile medium. The drug, depending on the vehicle and concentration used, can either be suspended or dissolved in the vehicle.
  • adjuvants such as local anesthetics, preservatives and buffering agents can be dissolved in the vehicle.
  • Dosage unit forms generally contain between from about 1 mg to about 5000 mg of an active ingredient. It is understood that the specific dose level for any particular patient or subject depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy,
  • the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
  • the composition can also be administered to a subject in combination with other therapeutic compounds to increase the overall therapeutic effect. The use of multiple compounds to treat an indication can increase the beneficial effects while reducing the presence of side effects.
  • nucleic acid molecules of the instant invention can be expressed within cells from eukaryotic promoters (e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon et al., 1991, Proc. Natl. Acad. Sci. USA, 88, 10591 5; ashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3 15; Dropulic et al., 1992, J. Virol., 66, 1432 41 ; Weerasinghe et al., 1991, J.
  • eukaryotic promoters e.g., Izant and Weintraub, 1985, Science, 229, 345; McGarry and Lindquist, 1986, Proc. Natl. Acad. Sci., USA 83, 399; Scanlon e
  • the invention features an expression vector comprising a nucleic acid sequence encoding at least one of the nucleic acid molecules of the instant invention.
  • the nucleic acid sequence encoding the nucleic acid molecule of the instant invention is operably linked in a manner which allows expression of that nucleic acid molecule.
  • RNA polymerase I eukaryotic RNA polymerase I
  • polymerase II RNA polymerase ⁇
  • poly III RNA polymerase III
  • Transcripts from pol II or pol III promoters are expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type depends on the nature of the gene regulatory sequences (enhancers, silencers, etc.) present nearby.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Elroy-Stein and Moss, 1990, Proc. Natl. Acad. Sci.
  • nucleic acid molecules such as ribozymes expressed from such promoters can function in mammalian cells (e.g. Kashani-Sabet et al., 1992, Antisense Res. Dev., 2, 3 15; Ojwang et al., 1992, Proc. Natl. Acad. Sci.
  • the invention features an expression vector comprising nucleic acid sequence encoding at least one of the nucleic acid molecules of the invention, in a manner which allows expression of that nucleic acid molecule.
  • the expression vector comprises in one embodiment; a) a transcription initiation region; b) a transcription termination region; c) a nucleic acid sequence encoding at least one said nucleic acid molecule; and wherein said sequence is operably linked to said initiation region and said termination region, in a manner which allows expression and/or delivery of said nucleic acid molecule.
  • a further object of the present invention is to provide a kit comprising a suitable container, the therapeutic of the invention in a pharmaceutically acceptable form disposed therein, and instructions for its use.
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence encoding an MG53 polypeptide, or MG53 receptor polypeptide.
  • the term "complementary” refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule
  • binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect.
  • fragments are defined as sequences of at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, and are at most some portion less than a full length sequence.
  • Host cells as described herein may include a cell that might be used to carry a heterologous nucleic acid, or expresses a peptide or protein encoded by a heterologous nucleic acid.
  • a host cell can contain genes that are not found within the native (non- recombinant) form of the cell, genes found in the native form of the cell where the genes are modified and re-introduced into the cell by artificial means, or a nucleic acid endogenous to the cell that has been artificially modified without removing the nucleic acid from the cell.
  • a host cell may be eukaryotic or prokaryotic.
  • a "host cell” can also be one in which the endogenous genes or promoters or both have been modified to produce one or more of the polypeptide components of the complex of the invention.
  • Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound.
  • nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the proteins of the invention under stringent, moderately stringent, or low stringent conditions. See e.g.
  • Nucleic acid derivatives and modifications include those obtained by gene replacement, site-specific mutation, deletion, insertion, recombination, repair, shuffling, endonuclease digestion, PCR, subcloning, and related techniques.
  • homologs can be naturally occurring, or created by artificial synthesis of one or more nucleic acids having related sequences, or by modification of one or more nucleic acid to produce related nucleic acids.
  • Nucleic acids are homologous when they are derived, naturally or artificially, from a common ancestor sequence (e.g., orthologs or paralogs). If the homology between two nucleic acids is not expressly described, homology can be inferred by a nucleic acid comparison between two or more sequences. If the sequences demonstrate some degree of sequence similarity, for example, greater than about 30%, 40%, 50%, 60%, 70%, 80%, or 90% at the primary amino acid structure level, it is concluded that they share a common ancestor.
  • genes are homologous if the nucleic acid sequences are sufficiently similar to allow recombination and/or hybridization under low stringency conditions.
  • hybridization refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under low, medium, or highly stringent conditions, including when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • “conservative mutations” also include the substitution, deletion or addition of nucleic acids that alter, add or delete a single amino acid or a small number of amino acids in a coding sequence where the nucleic acid alterations result in the substitution of a chemically similar amino acid.
  • Amino acids that may serve as conservative substitutions for each other include the following: Basic: Arginine (R), Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E), Asparagine (N), Glutamine (Q); hydrophilic: Glycine (G), Alanine (A), Valine (V), Leucine (L), Isoleucine (I); Hydrophobic: Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Sulfur- containing: Methionine (M), Cysteine (C).
  • sequences that differ by conservative variations are generally homologous.
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • a mammalian expression vector for suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
  • a polynucleotide can be a DNA molecule, a cDNA molecule, genomic DNA molecule, or an RNA molecule.
  • a polynucleotide as DNA or RNA can include a sequence wherein T (thymidine) can also be U (uracil). If a nucleotide at a certain position of a polynucleotide is capable of forming a Watson-Crick pairing with a nucleotide at the same position in an anti-parallel DNA or RNA strand, then the polynucleotide and the DNA or RNA molecule are complementary to each other at that position.
  • the polynucleotide and the DNA or RNA molecule are substantially complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hybridize with each other in order to effect the desired process.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • transformation is meant a permanent or transient genetic change induced in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell).
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular ceil type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987.
  • lymphoid-specific promoters (Caiame and Eaton, 1988. Adv. Immunol. 43: 235- 275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.
  • pancreas-specific promoters Eslund, et al., 1985. Science 230: 912-916
  • mammary gland-specific promoters e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166
  • Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the alpha- fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546).
  • the nucleic acids encoding an MG53 polypeptide or MG53 receptor can be present as: one or more naked DNAs; one or more nucleic acids disposed in an appropriate expression vector and maintained episomally; one or more nucleic acids incorporated into the host cell's genome; a modified version of an endogenous gene encoding the components of the complex; one or more nucleic acids in combination with one or more regulatory nucleic acid sequences; or combinations thereof.
  • the nucleic acid may optionally comprise a linker peptide or fusion protein component, for example, His-Tag, FLAG-Tag, Maltose Binding Protein (MBP)-Tag, fluorescent protein, GST, TAT, an antibody portion, a signal peptide, and the like, at the 5' end, the 3' end, or at any location within the ORF.
  • a linker peptide or fusion protein component for example, His-Tag, FLAG-Tag, Maltose Binding Protein (MBP)-Tag, fluorescent protein, GST, TAT, an antibody portion, a signal peptide, and the like, at the 5' end, the 3' end, or at any location within the ORF.
  • the nucleic acid of the invention comprises a polynucleotide encoding soluble portions of MG53 or an MG53 receptor. Any of the embodiments described herein, can be achieved using standard molecular biological and genetic approaches well known to those of ordinary skill in the art.
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method by procedures well known in the art.
  • CaCl 2 method such as E. coli
  • MgC ⁇ , bCl, liposome, or liposome-protein conjugate can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
  • Thes examples are not limiting on the present invention; numerous techniques exist for transfecting host cells that are well known by those of skill in the art and which are contemplated as being within the scope of the present invention.
  • the host is a eukaryote
  • methods of transfection with DNA include calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors, as well as others known in the art, may be used.
  • the eukaryotic cell may be a yeast cell (e.g., Saccharomyces cerevisiae) or may be a mammalian cell, including a human cell. For long- term, high-yield production of recombinant proteins, stable expression is preferred.
  • the present invention encompasses therapeutic methods utiltizing host cells, and stem cells modified according to the methods of the invention, which can be used in transplantation and/or adoptive cellular therapeutic approaches.
  • a stem cell for example, a lung stem ceil is isolated from a host, wherein the stem cell is capable of differentiating into a lung cell, and wherein the isolated stem cell is modified such that it demonstrates a modulated, for example, enhanced, MG53 activity, MG53 gene expression, or modulated MG53 signalling cascade.
  • the stem cell is contacted with an agent, for example, an MG53 polypeptide, MG53 nucleotide or agent that enhances the MG53 signalling cascade in lung cells.
  • an agent for example, an MG53 polypeptide, MG53 nucleotide or agent that enhances the MG53 signalling cascade in lung cells.
  • the modified stem cell can then be cultured in vitro, and subsequently administered to an individual in need thereof, for example, a patient that has sustained lung damage due to acute injury or various disease states.
  • antibody refers to immunoglobuiin molecules and immunologically active portions of immunoglobulin (Ig) molecules, i.e., molecules that contain an antigen-binding site that specifically binds (immunoreacts with) an antigen, comprising at least one, and preferably two, heavy (H) chain variable regions (abbreviated herein as VH), and at least one and preferably two light (L) chain variable regions (abbreviated herein as VL).
  • H heavy chain variable regions
  • VL light chain variable regions
  • Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab, Fab' and F(ab')2 fragments, and an Fab expression library.
  • VH and VL regions can be further subdivided into regions of hypervariability, termed “complementarity determining regions” ("CDR"), interspersed with regions that are more conserved, termed “framework regions” (FR).
  • CDR complementarity determining regions
  • FR framework regions
  • the extent of the framework region and CDR's has been precisely defined (see, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, which are incorporated herein by reference).
  • Each VH and VL is composed of three CDR's and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • antibody molecules obtained from humans relates to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule.
  • Certain classes have subclasses as well, such as IgGi, IgG 2 , and others.
  • the light chain may be a kappa chain or a lambda chain. Reference herein to antibodies includes a reference to all such classes, subclasses and types of human antibody species.
  • Antibodies can be prepared from the intact polypeptide or fragments containing peptides of interest as the immunizing agent.
  • a preferred antigenic polypeptide fragment is 15-100 contiguous amino acids of MG53, or MG53 receptor protein.
  • the peptide is located in a non-transmembrane domain of the polypeptide, e.g., in an extracellular or intracellular domain.
  • An exemplary antibody or antibody fragment binds to an epitope that is accessible from the extracellular milieu and that alters the functionality of the protein.
  • the present invention comprises antibodies that recognize and are specific for one or more epitopes of MG53, or MG53 receptor protein, variants, portions and/or combinations thereof.
  • antibodies of the invention may target and interfere with the MG53/MG53 receptor interaction to inhibit signaling.
  • Monoclonal antibodies can be obtained by injecting mice or rabbits with a composition comprising an antigen, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B lymphocytes, fusing the lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • Monoclonal antibodies can be isolated and purified from hybridoma cultures by techniques well known in the art.
  • the antibody can be recombinantly produced, e.g., produced by phage display or by combinatorial methods. Phage display and combinatorial methods can be used to isolate recombinant antibodies that bind to membrane repair polypeptide, MG53, membrane repair polypeptide binding protein, MG53 binding protein, membrane repair polypeptide receptor, and/or MG53 receptor proteins or fragments thereof (as described in, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Fuchs et al. (1991) Bio/Technology 9: 1370-1372; Hay et al.
  • Human monoclonal antibodies can also be generated using transgenic mice carrying the human immunoglobulin genes rather than the mouse system. Splenocytes from these transgenic mice immunized with the antigen of interest are used to produce hybridomas that secrete human mAbs with specific affinities for epitopes from a human protein (see, e.g., Wood et al. International Application WO 91/00906; Lonberg, N. et al.
  • a therapeutically useful antibody to the components of the complex of the invention or the complex itself may be derived from a "humanized" monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementarity determining regions (CDRs) from heavy and light variable chains of the mouse immunoglobulin into a human variable domain, then substituting human residues into the framework regions of the murine counterparts .
  • the antibodies can also be derived from human antibody fragments isolated from a combinatorial immunoglobulin library; see, for example, Barbas et al, Methods: A Companion to Methods in Enzymology 2, 1 19, 1991.
  • chimeric antibodies can be obtained by splicing the genes from a mouse antibody molecule with appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological specificity; see, for example, Takeda et al., Nature 314: 544-546, 1985.
  • a chimeric antibody is one in which different portions are derived from different animal species.
  • Anti-idiotype technology can be used to produce monoclonal antibodies that mimic an epitope.
  • An anti-idiotypic monoclonal antibody made to a first monoclonal antibody will have a binding domain in the hypervariable region that is the "image" of the epitope bound by the first monoclonal antibody.
  • techniques used to produce single chain antibodies can be used to produce single chain antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
  • Antibody fragments that recognize specific epitopes, e.g., extracellular epitopes can be generated by techniques well known in the art.
  • Such fragments include Fab fragments produced by proteolytic digestion, and Fab fragments generated by reducing disulfide bridges.
  • the monoclonal antibodies, fragments thereof, or both may be unlabelled or labeled with a therapeutic agent.
  • agents can be coupled directly or indirectly to the monoclonal antibody by techniques well known in the art, and include such agents as drugs, radioisotopes, lectins and toxins.
  • the dosage ranges for the administration of monoclonal antibodies are large enough to produce the desired effect, and will vary with age, condition, weight, sex, age and the extent of the condition to be treated, and can readily be determined by one skilled in the art. Dosages can be about 0.1 mg kg to about 2000 mg/kg.
  • the monoclonal antibodies can be administered intravenously, intraperitoneally, intramuscularly, and/or subcutaneously.
  • At least one epitope encompassed by the antigenic peptide is a region of MG53, or MG53 receptor that is located on the surface of the protein, e.g., a hydrophilic region.
  • a hydrophobicity analysis of the protein sequence will indicate which regions of a polypeptide are particularly hydrophilic and, therefore, are likely to encode surface residues useful for targeting antibody production.
  • hydropathy plots showing regions of hydrophilicity and hydrophobicity may be generated by any method well known in the art, including, for example, the Kyte Doolittle or the Hopp Woods methods, either with or without Fourier transformation. See, e.g., Hopp and Woods, 1981, Proc.
  • Antibodies that are specific for one or more domains within an antigenic protein, or derivatives, fragments, analogs or homologs thereof, are also provided herein.
  • a protein of the invention, or a derivative, fragment, analog, homolog or ortholog thereof, may be utilized as an immunogen in the generation of antibodies that immunospecifically bind these protein components.
  • Fully human antibodies essentially relate to antibody molecules in which the entire sequence of both the light chain and the heavy chain, including the CD s, arise from human genes. Such antibodies are termed "human antibodies", or “fully human antibodies” herein.
  • Human monoclonal antibodies can be prepared by the trioma technique; the human B- cell hybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) and the EBV hybridoma technique to produce human monoclonal antibodies (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • Human monoclonal antibodies may be utilized in the practice of the present invention and may be produced by using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by transforming human B-cells with Epstein Barr Virus in vitro (see Cole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R. Liss, Inc., pp. 77-96).
  • human antibodies can also be produced using additional techniques, including phage display libraries (Hoogenboom and Winter, J. Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol, 222:581 (1991)).
  • human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos.
  • Human antibodies may additionally be produced using transgenic nonhuman animals which are modified so as to produce fully human antibodies rather than the animal's endogenous antibodies in response to challenge by an antigen.
  • the endogenous genes encoding the heavy and light immunoglobulin chains in the nonhuman host have been incapacitated, and active loci encoding human heavy and light chain immunoglobulins are inserted into the host's genome.
  • the human genes are incorporated, for example, using yeast artificial chromosomes containing the requisite human DNA segments.
  • An animal which provides all the desired modifications is then obtained as progeny by crossbreeding intermediate transgenic animals containing fewer than the full complement of the modifications.
  • the preferred embodiment of such a nonhuman animal is a mouse, and is termed the XenomouseTM as disclosed in PCT publications WO 96/33735 and WO 96/34096.
  • a therapeutically effective amount of a therapeutic agent as described herein relates generally to the amount needed to achieve a therapeutic objective. As noted above, this may be a binding interaction between the antibody and its target antigen that, in certain cases, interferes with the functioning of the target, and in other cases, promotes a physiological response.
  • the amount required to be administered will furthermore depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 500 mg/kg body weight.
  • Antibodies specifically binding a protein of the invention, as well as other molecules identified by the screening assays disclosed herein, can be administered for the treatment of various disorders in the form of pharmaceutical compositions. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components are provided, for example, in Remington: The Science And Practice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et al., editors) Mack Pub.
  • the active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly ⁇ (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano- particles, and nanocapsules) or in macroemulsions.
  • colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano- particles, and nanocapsules
  • macroemulsions for example, liposomes, albumin microspheres, microemulsions, nano- particles, and nanocapsules
  • the formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
  • Sustained-release preparations can be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No.
  • copolymers of L-glutamic acid and gamma-ethy!-L-glutamate non- degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.
  • LUPRON DEPOTTM injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate
  • poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • An agent for detecting an analyte protein is an antibody capable of binding to an analyte protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal.
  • An intact antibody, or a fragment thereof e.g., Fab or F(ab)2 can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling of the probe or antibody by coupling (i e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling of the probe or antibody by reactivity with another reagent that is directly labeled.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. Included within the usage of the term "biological sample”, therefore, is blood and a fraction or component of blood including blood serum, blood plasma, or lymph. That is, the detection method of the invention can be used to detect an analyte mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
  • in vitro techniques for detection of an analyte mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of an analyte protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence.
  • In vitro techniques for detection of an analyte genomic DNA include Southern hybridizations. Procedures for conducting immunoassays are described, for example in "ELISA: Theory and Practice: Methods in Molecular Biology", Vol. 42, J. R. Crowther (Ed.) Human Press, Totowa, N.J., 1995; "Immunoassay", E. Diamandis and T.
  • in vivo techniques for detection of an analyte protein include introducing into a subject a labeled anti-an analyte protein antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques intracavity, or transdermally, alone or with effector cells.
  • Preparations for administration of the therapeutic of the invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles including fluid and nutrient replenishers, electrolyte replenishers, and the like.
  • Preservatives and other additives may be added such as, for example, antimicrobial agents, anti-oxidants, chelating agents and inert gases and the like.
  • compositions suitable for administration typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference.
  • Such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used.
  • the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, intraperitoneal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, CremophorTM. (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (e.g., magnesium stearate, talc or silica); disintegrants (e.g., potato starch or sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulphate).
  • binding agents e.g., pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers e.g., lactose, microcrystalline cellulose or calcium hydrogen phosphate
  • lubricants e.g., magnesium stearate, talc or silica
  • disintegrants e.g., potato starch
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups, or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid).
  • suspending agents e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats
  • emulsifying agents e.g., lecithin or acacia
  • non-aqueous vehicles e.g., almond oil, oily esters, eth
  • compositions may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • buccal administration the compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebu!iser, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethan- e, carbon dioxide or other suitable gas.
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
  • the compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
  • the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
  • Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. .
  • Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated, to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • the nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see, e.g., U.S. Pat. No. 5,328,470) or by stereotactic injection (see, e.g., Chen, et al., 1994. Proc. Natl. Acad. Sci. USA 91 : 3054- 3057).
  • the pharmaceutical preparation of the gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
  • the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • MG53 a muscle specific TRIM family protein.
  • MG53 was isolated using a previously established an immuno-proteomic approach that allows identification of novel proteins involved in myogenesis, Ca signaling and maintenance of membrane integrity in striated muscle cells. Briefly, this approach uses a monoclonal antibody library containing -6500 clones that was generated from mice immunized with triad-enriched membranes from rabbit skeletal muscle. Antibodies of interest were selected based on the z-line staining patterns of striated muscle sections observed under an immunofluorescence microscope. The target-proteins were purified through antibody-affinity column, and partial amino acid sequences of the purified proteins were obtained.
  • the complete cDNA coding for the target gene was isolated from a skeletal muscle cDNA library. Homologous gene screening was then used to search for the presence of different isoforms of the identified genes in other excitable tissues. Finally, transgenic or knockout mouse models were generated to study the in vivo physiological function of genes of interest.
  • MG53 contains the prototypical tri-partite motifs that include a Ring, B-box and Coiled-Coil (RBCC) moieties, as well as a SPRY domain at the carboxyl-terminus (Figs. 1, 2, and 3 A).
  • the SPRY domain is a conserved sequence first observed in the ryanodine receptor Ca 2+ release channel in the sarcoplasmic reticulum of excitable cells.
  • TRIM a conserved primary structure with these TRIM sub-family proteins.
  • MG53 mediates vesicle trafficking in muscle cells. Although there is no membrane-spanning segment or lipid-modification motif in its primary structure, MG53 appears to be primarily restricted to membrane structures in skeletal muscle. Immunohistochemical analysis revealed specific labeling for MG53 in the sarcolemma membrane and intracellular vesicles (Fig. 3D). MG53 is a muscle-specific protein that contains TRIM and SPRY motifs. In previous studies we have established a monoclonal antibody (mAb) library that targets proteins associated with the triad junction in skeletal muscle.
  • mAb monoclonal antibody
  • MG53 an antigen named MG53 with a molecular size of 53 kilodaltons (kDa), which was recognized by mAb5259.
  • MG53 was partially purified from rabbit skeletal muscle by an immunoaffinity column conjugated with mAb5259, and subjected to amino acid sequencing. Based on the obtained partial amino acid sequences, cDNAs encoding MG53 were isolated from rabbit and mouse skeletal muscle libraries. Genomic library search identified the corresponding MG53 gene on the human 16pl 1.2 locus. The predicted amino acid sequences for MG53 in several species are shown in Fig. 1.
  • MG53 contains the prototypical TRIM signature sequence of RBCC plus a SPRY domain at the carboxyl-terminus, and thus belongs to the TRIM/RBCC family (Fig. 1). Of the approximately 60 TRIM family members so far identified in the mammalian genomes, 15 members carry a similar SPRY domain following the RBCC domain, and MG53 shows a conserved primary structure with these TRIM sub-family proteins (Fig. 2). However, surprisingly and unexpectedly our studies indicate that MG53 is the only TRIM family protein of those in Fig. 2 that demonstrate membrane repair function.
  • MG53 is a muscle specific TRIM family protein that displays a unique subcellular distribution pattern for a TRIM family protein.
  • Futher studies showed that the membrane repair capacity of MG53 could be active in cells outside of its native muscle context.
  • GFP-MG53 could translocate to injury sites in cultured human airway epithelial cells following microelectrode needle penetration (Fig. 4A) or damage to the membrane induced by exposure to saponin detergent (Fig. 4B).
  • MG53 Prevention of membrane damage to lung epithelial cell permeability by MG53 could also protect against structural and functional changes to the lung following this injury.
  • One important aspect contributing to lung pathology following injury is the inflammatory response.
  • MG53 treatment could provide dose dependent improvment in the release of an important inflammatory cytokine, interlukin-6 (IL-6) , indicating that MG53 application can decrease lung inflammatory response following injury (Fig. 8).
  • IL-6 interlukin-6
  • This prevention of inflammation can also improve the degree of edema that appears in the lung following injury (Fig, 9), which is an indication that MG53 should be able to improve gas exchange in an injured lung. This would be important for treating the life threatening symptoms of lung injury.
  • C2C12 murine myoblast cell line used for all studies was purchased from the American Type Culture Collection (Manassas, VA). Cells were grown in a humidified environment at 37°C and 5% C0 2 in DMEM medium for C2C12 or Ham's F12 medium for CHO cells supplemented with 10% fetal bovine serum, 100 units/ml penicillin and 100 ug/ml streptomycin. In order to induce myotube differentiation, C2C 12 myoblasts were grown to confluence and the medium was switched to DMEM containing 2% horse serum, penicillin (100 U/ml), streptomycin (100 ⁇ g ml).
  • C2C12 myoblasts or CHO cells were plated at 70% confluence in glass-bottom dishes. After 24 hours, cells were transfected with plasmids described above using GeneJammer reagent (Stratagene). Cells were visualized by live cell confocal imaging at 24-48 hours after transfection or at times indicated for individual experiments. In some experiments, C2C12 myoblasts were allowed to differentiate into myotubes for the indicated time before observation.
  • Plasmids construction The full-length mouse MG53 cDNA and associated truncation mutants were generated by PC using the primers described in supplemental table 1.
  • the PCR-amplified cDNA was inserted into pCMS-EGFP vector (Invitrogen) at Nhe I/Xba I sites.
  • pCMS-EGFP vector Invitrogen
  • GFP-TRIM, GFP-SPRY, MG53-GFP, TRIM-GFP and SPRY-GFP PCR products were inserted into pEGFP-C l at the Xhol/Xbal sites, or pEGFP- N l at the Xhol/Kpnl sites.
  • mice Mouse model of lung injury - BALB/c mice were randomly grouped into 5 groups. The animals were approximately 18-20 g old when at the initiation of the experiment. LPS (Sigma) was dissolved (9 mg) in 7 ml 0.9% saline. Test articles and reference drug were administered according to the following dosage protocols: For group 3, administration of MG53 (6 mg/kg) was started 2 h, 6 h, 12 h and 18h after LPS injection through caudal vein. For group 4, mice were received an i.v injection of MG53 (1.2 mg/kg) 6 h after LPS challenge. For group 5, mice were received an i.v injection of MG53 (6 mg kg) 6 h after LPS challenge.
  • mice were handled in one of two ways: 8 mice for BALF collection and 6 mice for edema measurements by wet/dry weight ratios.
  • BALF collection - Lungs were lavaged via the tracheal cannula with 0.5 ml of PBS. The course was repeated again and approximately 1.2 mL fluid (BALF) was obtained from each lung. The total protein in BALF was measured by Bradford assay. Total cells and neutrophil cell number in BALF was measured using Wright-Giemsa staining. LDH and IL-6 levels in BALF was measured using commecially avaiable colometric assay kits.

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Abstract

La présente invention concerne des compositions et des procédés de traitement et/ou de protection des cellules et/ou des tissus des voies respiratoires de dommages dus à des lésions au niveau des poumons ou de complications de maladies respiratoires, cardiovasculaires ou infectieuses sous-jacentes, ou toute combinaison de ceux-ci.
PCT/US2012/053920 2006-07-11 2012-09-06 Compositions comprenant le mg53 et procédés de traitement et de prévention de lésions au niveau des voies respiratoires WO2013036610A2 (fr)

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US14/343,560 US20150110778A1 (en) 2006-07-11 2012-09-06 Compositions comprising mg53 and methods for the treatment and prevention of airway injury
EP12829354.5A EP2753363A4 (fr) 2011-09-07 2012-09-06 Compositions comprenant le mg53 et procédés de traitement et de prévention de lésions au niveau des voies respiratoires
CN201280056362.1A CN104066452A (zh) 2011-09-07 2012-09-06 治疗和预防气道损伤的含mg53组合物及方法

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CN107629123A (zh) * 2017-09-07 2018-01-26 北京大学 一种抗mg53蛋白的纳米抗体及应用
WO2018024110A1 (fr) * 2016-08-01 2018-02-08 北京大学 Mutant de mg53, son procédé de préparation et ses utilisations
WO2018170796A1 (fr) * 2017-03-22 2018-09-27 Tsinghua University Trim72 en tant que cible thérapeutique potentielle pour la sla par l'ubiquitination de la protéine fus mutante
US20200179482A1 (en) * 2018-12-07 2020-06-11 Ohio State Innovation Foundation Composition for and method of facilitating corneal tissue repair
CN112940098A (zh) * 2016-04-06 2021-06-11 牡丹江友搏药业有限责任公司 一种mg53突变体及其制备方法和应用
US11197912B2 (en) 2020-04-14 2021-12-14 Ohio State Innovation Foundation Prevention and treatment of viral infection and viral infection-induced organ failure
EP4003388A1 (fr) * 2019-07-25 2022-06-01 Ohio State Innovation Foundation Composition et procédé pour améliorer la performance tissulaire

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KR20100099249A (ko) * 2007-12-04 2010-09-10 유니버시티 오브 메디신 앤드 덴티스트리 오브 뉴 저지 세포막 재봉합을 조절하기 위한 조성물 및 방법
CN101797375B (zh) * 2009-12-02 2013-01-23 北京大学 Mg53蛋白预防和/或治疗心脏缺血/再灌损伤的用途

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CN112940098A (zh) * 2016-04-06 2021-06-11 牡丹江友搏药业有限责任公司 一种mg53突变体及其制备方法和应用
JP2019530433A (ja) * 2016-08-01 2019-10-24 北京大学Ｐｅｋｉｎｇ Ｕｎｉｖｅｒｓｉｔｙ Ｍｇ５３突然変異体、その作製方法、およびその使用
WO2018024110A1 (fr) * 2016-08-01 2018-02-08 北京大学 Mutant de mg53, son procédé de préparation et ses utilisations
US11306296B2 (en) 2016-08-01 2022-04-19 Hope Medicine (Nanjing) Co., Ltd. MG53 mutants, methods of making the same, and uses thereof
JP7144402B2 (ja) 2016-08-01 2022-09-29 ホープ・メディシン（ナンジン）・カンパニー・リミテッド Ｍｇ５３突然変異体、その作製方法、およびその使用
WO2018170796A1 (fr) * 2017-03-22 2018-09-27 Tsinghua University Trim72 en tant que cible thérapeutique potentielle pour la sla par l'ubiquitination de la protéine fus mutante
CN107629123A (zh) * 2017-09-07 2018-01-26 北京大学 一种抗mg53蛋白的纳米抗体及应用
CN107629123B (zh) * 2017-09-07 2020-08-25 北京大学 一种抗mg53蛋白的纳米抗体及应用
US20200179482A1 (en) * 2018-12-07 2020-06-11 Ohio State Innovation Foundation Composition for and method of facilitating corneal tissue repair
EP4003388A1 (fr) * 2019-07-25 2022-06-01 Ohio State Innovation Foundation Composition et procédé pour améliorer la performance tissulaire
EP4003388A4 (fr) * 2019-07-25 2024-05-22 Ohio State Innovation Foundation Composition et procédé pour améliorer la performance tissulaire
US11197912B2 (en) 2020-04-14 2021-12-14 Ohio State Innovation Foundation Prevention and treatment of viral infection and viral infection-induced organ failure
EP4135744A4 (fr) * 2020-04-14 2024-01-10 Ohio State Innovation Foundation Prévention et traitement d'une défaillance d'organe induite par une infection virale

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