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WO2010133740A1 - Cellule du spm génétiquement modifiée pour surexprimer la ngal et son utilisation en tant que médicament - Google Patents

Cellule du spm génétiquement modifiée pour surexprimer la ngal et son utilisation en tant que médicament Download PDF

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Publication number
WO2010133740A1
WO2010133740A1 PCT/ES2010/070334 ES2010070334W WO2010133740A1 WO 2010133740 A1 WO2010133740 A1 WO 2010133740A1 ES 2010070334 W ES2010070334 W ES 2010070334W WO 2010133740 A1 WO2010133740 A1 WO 2010133740A1
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genetically modified
cell according
cell
smf
polynucleotide
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Spanish (es)
Inventor
Georgina Hotter Corripio
Anna SOLA MARTÍNEZ
Michaela Jung
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Consejo Superior de Investigaciones Cientificas CSIC
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Consejo Superior de Investigaciones Cientificas CSIC
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0634—Cells from the blood or the immune system
    • C12N5/0644—Platelets; Megakaryocytes
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention falls within the field of biomedicine. Specifically, the present invention refers to a System cell
  • Phagocytic mononuclear or SMF preferably a monocyte or a macrophage, genetically modified to overexpress the lipocalin associated with neutrophil gelatinase (NGAL), to a method for obtaining it and its use for the preparation of a medicament for Ia prevention or treatment of damage caused by ischemia, ischemia followed by reperfusion or toxins, acute failure or rejection of an organ transplant.
  • NGAL neutrophil gelatinase
  • AAF acute renal failure
  • Normally, patients with clinical symptoms of renal failure are treated after the damage has developed, except in the case of patients who are going to have a kidney transplant. Since the disease is developed when the patient arrives at the hospital, it is urgently necessary to have healing routes by stimulating the regenerative and healing process in general.
  • a therapeutic approach aimed at reducing inflammation and kidney damage has been cell therapy with genetically modified macrophages.
  • the genetic alteration of the macrophages towards an anti-inflammatory phenotype through treatment with recombinant adenovirus to express IL-1 ra and its subsequent injection in inflammatory models of kidney disease can reduce infiltration, glomerular inflammation and even proteinuria ( Holdsworth et al. Lab Invest. 1984, 51: 172-180; Kitamura et al. Kidney Int. 2000, 57: 709-716; Kluth et al. Gene Ther. 2000, 7: 263-270).
  • NGAL is a 25 kDa protein from the lipocalin superfamily (Flower. FEBS Lett. 1994, 354: 7-11) that acts on proliferation in multiple cell types (Cowland et al. J Immunol. 2003, 171: 6630- 6639; Gwira et al. J Biol Chem. 2005, 280: 7875-7882). It is a protein expressed in tubular cells, which increases markedly in response to harmful stimuli such as ischemia or toxicity. It has also been described that improves the apoptosis of the tubular cell (Mishra et al. J Am Soc Nephrol. 2004, 15: 3073-3082).
  • the present invention relates to an SMF cell, preferably a monocyte or a macrophage, genetically modified to overexpress NGAL, a method for obtaining it and its use for the preparation of a medicament for the prevention or treatment of ischemia damage. , ischemia followed by reperfusion or toxins, an acute failure or an organ transplant rejection.
  • genetically modified macrophages to overexpress NGAL are capable of reducing inflammation and renal damage, in addition to inducing regeneration in an animal model of renal ischemia-reperfusion, when administered in the inflammatory phase of damage renal.
  • the invention therefore, provides a therapy that reduces inflammation and kidney damage, enhancing renal regeneration during the inflammatory phase of kidney damage.
  • This therapy can also be used in the prevention or treatment of ischemia damage, ischemia followed by reperfusion or caused by toxins, acute failure or rejection of transplantation of other organs.
  • the macrophage has its origin in the granulo-monocytic pluripotential progenitor cells (CGp-GM) of the bone marrow.
  • CSF colonial stimulation
  • the CGp-GM is differentiated into monocyte monopotential progenitor cell (CGm-M), which is differentiated in monoblast, and this, in turn, in promonocyte, the first cell Morphologically identifiable as a precursor of the macrophage and that already has some of its characteristics, such as adhesion to glass and phagocytic capacity.
  • promonocyte the first cell Morphologically identifiable as a precursor of the macrophage and that already has some of its characteristics, such as adhesion to glass and phagocytic capacity.
  • Circulating monocytes go through diafugdesis through the vascular endothelium, migrating to the tissues in which they will differentiate into macrophages, which, in turn, can occur in different functional states: residents, inflammatory and activated.
  • the set formed by spinal precursors, monocytes and tissue macrophages, is currently encompassed under the name of Phagocytic Mononuclear System (SMF).
  • SMF Phagocytic Mononuclear System
  • a first aspect of the invention relates to a cell of the isolated Phagocytic Mononuclear System (SMF) genetically modified characterized in that it comprises an exogenous polynucleotide (hereinafter, the polynucleotide of the invention) that has an identity with the SEQ ID NO: 1 selected from The list consisting of:
  • SMF Phagocytic Mononuclear System
  • SMF cell is capable of inducing renal regeneration during the inflammatory phase of renal damage.
  • Said capacity can be determined by conventional methods such as the tests described in the Example accompanying this description.
  • inflammatory phase refers to a first phase of renal damage characterized by an increase in inflammatory mediators, typically during the first 24 hours after an ischemic insult. In turn, this environment promotes kidney tissue damage by apoptosis and necrosis.
  • the regenerative phase begins with a change in the inflammatory environment.
  • the macrophage plays an important role in eliminating dead cells which, in turn, stimulate their change of phenotype towards anti-inflammatory and pro-proliferative, also allowing the induction of renal regeneration.
  • genetically modified includes here any SMF cell in which the genotype has been altered so that it comprises an exogenous experimentally introduced polynucleotide that has an identity with SEQ ID NO : 1 selected from the list consisting of:
  • SMF cell is capable of inducing renal regeneration during the inflammatory phase of renal damage.
  • polynucleotide and “nucleic acid” are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides (RNA or RNA) and deoxyribonucleotides (DNA or DNA).
  • the genetically modified SMF cell is characterized in that it comprises the exogenous polynucleotide with SEQ ID NO: 1, which corresponds to the nucleotide sequence of the human NGAL protein cDNA (Number of Genbank reference: NM_005564).
  • the genetically modified SMF cell is characterized in that It comprises the exogenous polynucleotide with SEQ ID NO: 2, of the mouse NGAL protein cDNA (Genbank reference number: NM_008491).
  • the genetically modified SMF cell is characterized in that it comprises the polynucleotide of the invention operatively linked to at least one control sequence of the list comprising:
  • a promoter that directs the transcription of said polynucleotide
  • b. a start signal of transcription c. a termination signal of the transcript
  • d. a polyadenylation signal e. a transcriptional activator.
  • Control sequence refers to polynucleotide sequences that affect the expression of the sequences to which they are linked. In eukaryotic cells, generally, said control sequences include promoters, termination signals, enhancers or silencers. It is intended that the term “control sequences” includes, at a minimum, all components whose presence is necessary for expression, and may also include additional components whose presence is advantageous.
  • Operaationally linked refers to a juxtaposition in which the components thus described have a relationship that allows them to function in the intended way.
  • a control sequence "operatively linked" to the polynucleotide is linked to it in such a way that expression of the coding sequence of the polynucleotide is achieved.
  • promoter refers to a region of the DNA located in position 5 'with respect to the starting point of the transcription and which is necessary or facilitates said transcription in an animal cell. This term includes, for example, but not limited to, constitutive promoters, specific cell or tissue promoters or inducible or repressible promoters.
  • the genetically modified SMF cell is characterized in that it comprises an exogenous polynucleotide that encodes the NGAL protein.
  • the genetically modified SMF cell is characterized in that it comprises an exogenous polynucleotide that encodes an amino acid sequence that has an identity with SEQ ID NO: 3 selected from the list consisting of:
  • SMF cell is capable of inducing renal regeneration during the inflammatory phase of renal damage.
  • Said capacity can be determined by conventional methods such as the tests described in the Example that accompanies this description.
  • amino acid sequence refers to a polymeric form of amino acids of any length, which may or may not be chemical or biochemically modified.
  • the genetically modified SMF cell is characterized in that it comprises an exogenous polynucleotide that encodes the amino acid sequence SEQ ID NO: 3, which corresponds to the amino acid sequence of the human NGAL protein (Genbank reference number: NP_005555).
  • the genetically modified SMF cell is characterized in that it comprises an exogenous polynucleotide that encodes the amino acid sequence SEQ ID NO: 4, which corresponds to the amino acid sequence of the mouse NGAL protein ( Genbank reference number: NP_032517).
  • NGAL lipocalin associated with neutrophil gelatinase
  • lipocalin-2 refer to a 25 kDa protein of the lipocalin superfamily.
  • identity refers to the proportion of identical nucleotides or amino acids between two nucleotide or amino acid sequences that are compared. The percentage of identity existing between two sequences can be easily verified by a person skilled in the art, for example, with the help of an appropriate computer program to compare sequences.
  • the SMF cell is a mammalian cell, and more, preferably, of a human. Even more preferably, the SMF cell is a monocyte or a macrophage.
  • Another aspect of the present invention refers to the use of the SFM cell, preferably, a monocyte or a macrophage, genetically modified as described above in this document (hereinafter, cell of the invention) for the preparation of a medicine.
  • SFM cell preferably, a monocyte or a macrophage, genetically modified as described above in this document (hereinafter, cell of the invention) for the preparation of a medicine.
  • gene therapy medication means any product obtained by means of a set of manufacturing processes intended to transfer, either in vivo or ex vivo, a prophylactic, diagnostic or therapeutic nucleic acid to animal cells, preferably human, and their subsequent expression in vivo.
  • Gene therapy drugs include, but are not limited to, naked nucleic acid, non-viral vectors, viral vectors or genetically modified cells.
  • the term "gene therapy medicine” refers to any medicine that comprises a modified SMF cell characterized in that it comprises the polynucleotide of the invention. They can be gene therapy drugs based on autologous cells (from the patient himself), such as allogeneic (from another human being) or xenogeneic (from animals).
  • tissue or organ is from the list comprising: kidney, liver, brain, heart, lung, stomach, intestine, colon, pancreas, bladder, uterus or skin. More preferably, the organ is the kidney.
  • ischemia refers to a temporary or permanent decrease in blood flow in a tissue or an organ, with the consequent decrease in oxygen supply.
  • the set of damage suffered by tissue or organ due to ischemia is known as "damage caused by ischemia.”
  • the term "reperfusion” refers to the restoration of the blood supply to a tissue or an organ that is ischemic as a result of a decrease in normal blood flow.
  • the recovery of blood flow restores the supply of oxygen and nutrients to the tissue, allowing the recovery of tissue or ischemic organ.
  • reperfusion itself it can injure the tissue or ischemic organ, causing what is known as “reperfusion damage”.
  • ischemia-reperfusion damage refers to all the damage suffered by a tissue or an organ due to a decrease in blood flow (ischemia) followed by a restoration of blood flow (reperfusion).
  • damage caused by toxins refers to the set of damages suffered by a tissue or an organ as a result of its exposure to toxins such as, but not limited to, antibiotics, anesthetics, chemotherapeutics, radiological contrasts, heavy metals, fungicides, pesticides, organic solvents, animal poisons, fungal or toxic toxins of endogenous origin.
  • a preferred embodiment of this aspect of the invention refers to the use of the cell of the invention for the preparation of a medicament for the prevention or treatment of damage caused by kidney ischemia or renal ischemia.
  • Renal ischemia consists of a temporary or permanent decrease in blood flow, with the consequent decrease in the oxygen supply to the kidney, as a consequence, for example, but not limited to a decrease in total blood volume, a redistribution of the blood or an obstruction .
  • the decrease in blood flow can be unilateral, when it affects only one kidney, or bilateral, when it affects the two kidneys.
  • the set of damages suffered by tissue or organ due to ischemia is known as "damage caused by renal ischemia.”
  • Another preferred embodiment of this aspect of the invention refers to the use of the cell of the invention for the preparation of a medicament for the prevention or treatment of damage caused by kidney ischemia-reperfusion or renal ischemia-reperfusion.
  • the term "renal ischemia reperfusion damage” refers to all the damage suffered by the kidney due to to a decrease in blood flow (renal ischemia) followed by a restoration of blood flow (reperfusion).
  • Another preferred embodiment of this aspect of the invention refers to the use of the cell of the invention for the preparation of a medicament for the prevention or treatment of the damage suffered by renal tissue or kidney as a result of its exposure to toxins such as, for example, but not limited to antibiotics, anesthetics, chemotherapeutics, radiological contrasts, heavy metals, fungicides, pesticides, organic solvents, animal poisons, fungal or toxic toxins of endogenous origin.
  • toxins such as, for example, but not limited to antibiotics, anesthetics, chemotherapeutics, radiological contrasts, heavy metals, fungicides, pesticides, organic solvents, animal poisons, fungal or toxic toxins of endogenous origin.
  • acute organic failure As a consequence of the damage caused by ischemia, ischemia reperfusion or toxins in an organ, an acute organic failure can occur.
  • acute failure refers to a clinical syndrome characterized by a sharp deterioration of the function of a certain organ.
  • tissue or organ is from the list comprising: kidney, liver, brain, heart, lung, stomach, intestine, colon, pancreas, bladder, uterus or skin. More preferably, the organ is the kidney.
  • acute renal failure As a result of damage caused by ischemia, ischemia-reperfusion or toxins in the kidney, acute renal failure (ARF) may occur.
  • Acute renal failure refers to a clinical syndrome characterized by an abrupt deterioration of renal function that results in a decrease in glomerular filtration and a accumulation of serum nitrogen products (such as, for example, urea or creatinine), alterations in the hydroelectrolytic balance and acid balance may also occur base.
  • the FRA can be classified into three main groups: functional FRA, parenq u matoso FRA or obstructive FRA.
  • a preferred embodiment of this aspect of the invention refers to the use of the cell of the invention for the preparation of a medicament for the prevention or treatment of an ARF.
  • tissue or organ is from the list comprising: kidney, liver, brain, heart, lung, stomach, intestine, colon, pancreas, bladder, uterus or skin. More preferably, the organ is the kidney.
  • the ARF associated with the damage caused by ischemia-reperfusion is one of the main causes for the initial delay in the function or rejection of a transplanted kidney.
  • a preferred embodiment of this aspect of the invention refers to the use of the cell of the invention for the preparation of a medicament for the prevention or treatment of rejection of a transplanted kidney.
  • Another aspect of the invention relates to a pharmaceutical composition comprising the cell of the invention.
  • Another aspect of the present invention relates to a pharmaceutical composition comprising the cell of the invention for the prevention or treatment of damage caused by ischemia, ischemia-reperfusion or toxins in a tissue or an organ.
  • a preferred embodiment of this aspect of the invention refers to a pharmaceutical composition comprising the cell of the invention for the prevention or treatment of damage caused by ischemia, ischemia-reperfusion or toxins in an organ of the list comprising: kidney, liver, brain, heart, lung, stomach, intestine, colon, pancreas, bladder, uterus or skin.
  • a more preferred embodiment of this aspect of the invention refers to a pharmaceutical composition comprising the cell of the invention for the prevention or treatment of damage caused by ischemia, ischemia-reperfusion or toxins in the kidney.
  • Another aspect of the present invention relates to a pharmaceutical composition comprising the cell of the invention for the prevention or treatment of an acute failure of an organ.
  • a preferred embodiment of this aspect of the invention refers to a pharmaceutical composition comprising the cell of the invention for the prevention or treatment of an acute failure of an organ of the list comprising: kidney, liver, brain, heart, lung , stomach, intestine, colon, pancreas, bladder, uterus or skin.
  • a more preferred embodiment of this aspect of the invention refers to a Pharmaceutical composition comprising the cell of the invention for the prevention or treatment of an ARF.
  • Another aspect of the present invention relates to a pharmaceutical composition comprising the cell of the invention for the prevention or treatment of rejection of a transplanted organ.
  • a preferred embodiment of this aspect of the invention refers to a pharmaceutical composition comprising the cell of the invention for the prevention or treatment of rejection of a transplanted organ of the list comprising kidney, liver, brain, heart, lung, stomach , intestine, colon, pancreas, bladder, uterus or skin.
  • a preferred embodiment of this aspect of the invention refers to a pharmaceutical composition comprising the cell of the invention for the prevention or treatment of rejection of a transplanted kidney.
  • the pharmaceutical composition as described above in this document also comprises a pharmaceutically acceptable carrier.
  • the pharmaceutical composition also comprises another active ingredient.
  • the pharmaceutical composition comprises together with a pharmaceutically acceptable carrier, in addition, another active ingredient.
  • active substance refers to any component that potentially provides a pharmacological activity or other different diagnostic effect , cure, mitigation, treatment or prevention of a disease, or that affects the structure or function of the body of the human being or other animals.
  • compositions can be formulated for administration in a variety of ways known in the state of the art.
  • compositions and / or their formulations may be administered to an animal and, more preferably, to a mammal, including a human, by a variety of routes, including, but not limited to parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrastromal, intraaricular, intrasynovial, intrathecal, intralesional, intraarterial, intracapsular, intracardiac, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular or topical.
  • routes including, but not limited to parenteral, intraperitoneal, intravenous, intradermal, epidural, intraspinal, intrastromal, intraaricular, intrasynovial, intrathecal, intralesional, intraarterial, intracapsular, intracardiac, intramuscular, intranasal, intracranial, subcutaneous, intraorbital, intracapsular or topical.
  • the dosage to obtain a therapeutically effective amount depends on a variety of factors, such as, for example, age, weight, sex or tolerance of the animal.
  • therapeutically effective amount refers to the amount of the pharmaceutically effective composition that produces the desired effect and, in general, will be determined among other causes, by the characteristics of said pharmaceutical composition and of the therapeutic effect to be achieved.
  • pharmaceutically acceptable “adjuvants” or “vehicles” that can be used in said compositions are the vehicles known in the state of the art.
  • Another aspect of the present invention relates to a method for obtaining the cell of the invention comprising:
  • step (a) transfecting the cell of step (a) with a nucleic acid comprising a polynucleotide, wherein said polynucleotide has an identity with SEQ ID NO: 1 selected from the list consists of:
  • the polynucleotide comprised in the nucleic acid transfected in step (b) of the method of the invention is SEQ ID NO: 1.
  • the polynucleotide comprised in the nucleic acid transfected in step (b) of the method of the invention is SEQ ID NO: 2.
  • the polynucleotide comprised in the nucleic acid transfected in step (b) is operatively linked to at least one control sequence of the list comprising:
  • a promoter that directs the transcription of said polynucleotide
  • b. a start signal of transcription c. a termination signal of the transcript
  • d. a polyadenylation signal e. a transcriptional activator.
  • transfect refers to introducing an exogenous nucleic acid into a eukaryotic cell.
  • the nucleic acid of step (b) of the method of the present invention can be introduced into the cell of the isolated SFM obtained in step (a), for example, but not limited, as a naked nucleic acid or by a vector.
  • vector or “gene transfer vector” refer to systems used in the process of transfection of an exogenous nucleic acid into a cell, thus allowing the vehiculation of the nucleic acid into the cell.
  • the SFM cell is contacted with a gene transfer vector, which comprises the nucleic acid comprising the polynucleotide of step (b), such that the nucleic acid is introduced into the cell under the appropriate conditions for said polynucleotide to be expressed inside the cell.
  • the vector can be viral or non-viral. There are numerous viral and non-viral vectors to introduce exogenous DNA into the stem cells that are well known to those skilled in the art.
  • Appropriate viral vectors for practicing this embodiment of the invention include, but are not limited to the following: adenoviral vectors, adeno-associated vectors, retroviral vectors, lentiviral vectors, alpha-viral vectors, herpesviral vectors and coronavirus-derived vectors.
  • Non-viral type vectors suitable for practicing this embodiment of the invention include, but are not limited to the following: gene gun, liposomes, polyamines, peptides, dendrimers, cationic glycopolymers, liposome-polycation complexes, proteins and gene transfer systems receiver-mediated
  • the transfection of the nucleic acid of step (b) is performed using an adenoviral vector.
  • the SMF cell obtained in step (a) is a mammalian cell, and more, preferably, of a human. Even more preferably, the SMF cell is a monocyte or a macrophage.
  • Figure 1 Shows the effect of the administration of genetically modified macrophages to overexpress NGAL on renal damage.
  • A Effect on the expression of the functional damage marker of the BUN kidney.
  • B Effect on the expression of the functional damage marker of the creatine kidney.
  • C Effect on histological damage analyzed in tissue sections stained with hematoxylin-eosin. Original magnification x 400.
  • Figure 2 It shows the effect of the administration of genetically modified macrophages to overexpress NGAL on proliferation and renal regeneration.
  • A Effect on the expression of the proliferation and renal regeneration marker Ki67.
  • B Effect on the expression of the creatine renal proliferation and regeneration marker.
  • C Effect on the expression of the regenerative markers PCNA and Statmina analyzed by immunofluorescence.
  • the PCNA is characterized by a nuclear staining of the cells during the late G1 phase and the S phase.
  • Statmina is a cytosolic protein that acts in the transition from the G2 phase to M (see arrows).
  • Original magnification x 400 is a nuclear staining of the cells during the late G1 phase and the S phase.
  • EXAMPLE 1 Genetically modified macrophages for the overexpression of NGAL to induce regeneration in an animal model of renal ischemia-reperfusion.
  • Rats of the strain Sprague Dawley were used, males weighing approximately 225-25Og (Charles River, France). All interventions were carried out under the supervision of the ethical committee of our institution and followed the guidelines of the European Union. The environmental conditions were kept constant, the temperature was 21-22 ° C, the relative humidity of 70% and the alternative light / dark cycles of 12h. The animals were fed a standard AO4 feed diet (Panlab, Barcelona) and water from the Barcelona ad libitum network.
  • the animals were anesthetized with Isoflurane, were placed supine and the body temperature was maintained between 36 and 37 ° C. After performing a middle laparotomy to access the kidney by carefully removing the intestinal package, bilateral ischemia was induced by clamping both renal arteriovenous pedicles for 45 minutes with a nontraumatic microvascular clamp. Subsequently the period of reperfusion, with the removal of the clamp and was verified visually with the observation of the return of blood flow to the kidney. The animal was then sutured and subcutaneous Buprex (4.16 ⁇ g / 100g weight) was administered. Animals subject to a sham operation were used as controls. During the entire operation process, the animals were well hydrated and the body temperature was maintained around 37 ° C.
  • the animals were housed under the control of a veterinarian. After 24h of reperfusion, the animal was sacrificed for the extraction of the kidneys and blood. The tissue was immediately stored in formalin for histological tests or frozen in carbonic snow and subsequently stored at - 80 0 C.
  • I / R NGAL. Animals subjected to I / R but with injection of 10x10 6 genetically modified macrophages to express NGAL per animal, by direct puncture of the inferior vena cava, 1 hour after the onset of reperfusion time.
  • Bgal I / R. Animals subjected to I / R but with injection of 10x10 6 macrophages with the control virus to express ⁇ -Galactosidase per animal, by direct puncture of the inferior vena cava, 1 hour after the start of reperfusion time .
  • DMEM Modified Eagle Medium
  • F-12 1 1 (volume / volume) with high glucose concentration, 15mM Hepes and stable glutamine, supplemented with 100 U / ml penicillin, 100 ⁇ g / ml streptomycin, 10%
  • GM-CSF granulocyte and monocyte colony stimulating factor
  • the cells were kept in teflon flashes, non-adherent, for 7 days for macrophage maturation.
  • the macrophages were separated by differential adhesion.
  • the cells were kept in an atmosphere with 5% CO2 in air, at 37 ° C.
  • NGAL adenovirus
  • Ad-NGAL adenovirus
  • Viraquest adenovirus
  • Ad-bgal ⁇ -Galactosidase
  • the titer of each virus was determined by plaque analysis in HeLa cells.
  • the efficiency of each transfection was measured by the determination of protein by ELISA in the cell culture supernatant.
  • Viral transfection was established with a multiplicity of infection (MOI) of 50. At 48 hours post-transfection, macrophages were collected in a tube and kept in PBS until subsequent infusion into the animal. Markers of functional kidney damage.
  • MOI multiplicity of infection
  • Blood urea nitrogen (BUN) and plasma creatinine were analyzed as markers of renal function using an ADVIA 2400 (Siemens Medical Diagnostics) of the Clinical Hospital of Barcelona.
  • the samples were incubated with secondary fluorescent antibodies to reveal staining with PCNA and Statmina in the tissue (rabbit anti-goat IgG conjugated with Alexa Fluor 488 for Statmina and goat anti-mouse IgG conjugated with Alexa Fluor 568 for PCNA; Molecular Probes) during two hours at room temperature in darkness.
  • the cuts were mounted with mowiol (Calbiochem) and the images were obtained by means of a Leica TCS NT laser confocal microscope (Leica Microsystems, Wetzlar, Germany) at an original magnification of x400. RT-PCR in real time.
  • RNA from the kidney samples was extracted using the TRIzol reagent according to the manufacturer's instructions (Invitrogen, Barcelona). Total RNA was extracted from the cells with the RNeasy mini Kit from Qiagen (Madrid) according to the manufacturer's instructions. RNA concentrations were calculated by absorbance determination at 260 nm. The integrity of the RNA thus obtained was examined by analysis of the 18S ribosomal RNA bands and
  • the expression of the genes analyzed was measured by real-time quantitative RT-PCR normalized with the constituent (or houskeeping) gene of glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
  • GPDH glyceraldehyde 3-phosphate dehydrogenase
  • Real-time RT-PCRs were carried out in a Bio-Rad Thermocycler (iCycler iQ Real-Time PCR detection System, Bio-Rad, Barcelona) and amplifications were made in 20 ⁇ l reactions with the RT-PCR Kit with SYBR-Green in two steps (Bio-Rad) according to the manufacturer's instructions.
  • the primers were the following: Ki-67: direct, SEQ ID NO: 5; reverse, SEQ ID NO: 6; PCNA: direct, SEQ ID NO: 7; reverse, SEQ ID NO: 8; NGAL: direct, SEQ ID NO: 9; reverse, SEQ ID NO: 10; TNF-a: direct, SEQ ID NO: 11; reverse, SEQ ID NO: 12; GAPDH: direct, SEQ ID NO: 13; reverse, SEQ ID NO: 14.
  • the data from the other cytokines (IL-1, IL-10, ⁇ L-4) were obtained using pre-validated primers from Qiagen (Barcelona, Spain) with reference numbers QT00181657, QT00106169 and QT00160678, respectively.
  • the cell supernatant was collected, centrifuged and stored at -80 0 C until use.
  • the 96-well ELISA plate was pre-incubated with Anti-mouse
  • Lipocalin-2 / NGAL Lipocalin-2 / NGAL (R&D Systems, Madrid), by adding 1 ⁇ l of the antibody's stock solution, diluted in 99 ⁇ l of 1: 100 carbonate buffer for 18 hours at 4 0 C. After washing the plate, the nonspecific junctions were blocked for 1 h at room temperature.
  • the data are shown as mean +/- the standard error of the mean (SEM), and the values of p> 0.05 were considered significant.
  • the statistical differences between groups were analyzed with an analysis of variance (ANOVA), and in case of significance the t-Student was applied.
  • FIG. 1C Histological analysis of renal tissue sections ( Figure 1C) reveals that the most affected area is in the kidney medulla. After 24 hours of reperfusion, severe interstitial edema, cell infiltration, necrosis, and the deposition of protein material in the tubular lumen are observed. However, treatment with macrophages previously modified to overexpress the NGAL preserves the integrity of the renal tissue. Macrophages treated with the control virus do not have this protective effect and a degree of damage similar to 24 hours of reperfusion is observed.
  • Figure 1C shows representative photos of the three main groups (Sham; I / R; I / R NGAL). Effect of the administration of genetically modified macrophages to overexpress NGAL on proliferation and regeneration.
  • Figure 2A shows the expression of Ki-67, a marker of cell proliferation and regeneration that is expressed during all phases of the cell cycle, except GO.
  • Ki-67 a marker of cell proliferation and regeneration that is expressed during all phases of the cell cycle, except GO.
  • the results of real-time RT-PCR show that after 24 hours of reperfusion Ki-67 is not expressed in the tubular epithelial cells of the kidney and therefore there is no regeneration at this time when the cells are probably dying by apoptosis induced by ischemia-reperfusion.
  • NGAL a marker of cell proliferation and regeneration that is expressed during all phases of the cell cycle, except GO.
  • the results of real-time RT-PCR show that after 24 hours of reperfusion Ki-67 is not expressed in the tubular epithelial cells of the kidney and therefore there is no regeneration at this time when the cells are probably dying by apoptosis induced by ischemia-reperfusion.
  • macrophages treated with the control virus did not have the ability to induce proliferation in
  • Figure 2B shows the expression of PCNA (proliferating cell nuclear antigen), a marker of cell proliferation and regeneration that has a peak of expression in the S phase of the cell cycle, and therefore in mitotically active cells.
  • PCNA proliferation factor receptor
  • the results of real-time RT-PCR show that it is only overexpressed in the group of macrophages overexpressing NGAL. Data are shown as mean +/- SEM, and p values> 0.05 were considered significant. Statistical differences between groups were analyzed with an ANOVA analysis, and in case of significance the t-Student was applied.
  • FIG. 2C shows the regeneration profile by immunofluorescence of PCNA and Statmina.
  • Statmina has been discovered as new proliferation marker in repair after an acute insult for renal ischemia. It is a cytosolic phosphoprotein and is associated with the induction of proliferation and the re-entry of cells in the cell cycle.
  • the PCNA interacts with various molecules, and affects cellular metabolism, DNA repair mechanisms and their synthesis.
  • Figure 3 shows the results of RT-PCR indicating the expression of both pro and anti-inflammatory cytokines after treatment with genetically modified macrophages with Ad-NGAL.
  • the results show a decrease in proinflammatory cytokines, represented by TNF- ⁇ and IL-1, when we administer macrophages treated with the NGAL carrier virus (in animals of the NGAL I / R group) compared to 24-hour inflammation levels. reperfusion in animals not treated (I / R) or treated with the control virus (I / R bgal).
  • anti-inflammatory cytokines represented by IL-10 and IL-4, show an increase after treatment with macrophages that overexpress NGAL.

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Abstract

La présente invention s'inscrit dans le domaine de la biomédecine. Plus spécifiquement, la présente invention concerne une cellule du système des phagocytes mononucléés ou SPM, de préférence un monocyte ou un macrophage, génétiquement modifiée pour surexprimer la lipocaline associée à la gélatinase des neutrophiles (NGAL, selon son sigle anglais), un procédé pour son obtention et son utilisation pour la préparation d'un médicament pour la prévention ou le traitement de dommages causés par l'ischémie, l'ischémie suivie de reperfusion ou causée par des toxines, l'insuffisance cardiaque aiguë ou le rejet de greffe d'un organe.
PCT/ES2010/070334 2009-05-19 2010-05-19 Cellule du spm génétiquement modifiée pour surexprimer la ngal et son utilisation en tant que médicament Ceased WO2010133740A1 (fr)

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ES200930183A ES2350078B1 (es) 2009-05-19 2009-05-19 Celula del smf modificada geneticamente para sobreexpresar ngal y su uso como medicamento
ESP200930183 2009-05-19

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Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006066587A1 (fr) * 2004-12-20 2006-06-29 Antibodyshop A/S Determination de la lipocaline neutrophile associee a la gelatinase (ngal) utile comme marqueur diagnostic dans les troubles renaux

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006066587A1 (fr) * 2004-12-20 2006-06-29 Antibodyshop A/S Determination de la lipocaline neutrophile associee a la gelatinase (ngal) utile comme marqueur diagnostic dans les troubles renaux

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
KITAMURA, M. ET AL.: "Adoptive transfer of nuclear factor-kappaB-inactive macrophages to the glomerulus.", KIDNEY INTERNATIONAL., vol. 57, no. 2, February 2000 (2000-02-01), pages 709 - 716 *
KLUTH, D.C. ET AL.: "Gene transfer into inflamed glomeruli using macrophages transfected with adenovirus.", GENE THERAPY., vol. 7, no. 3, February 2000 (2000-02-01), pages 263 - 270 *
MISHRA, J. ET AL.: "Amelioration of ischemic acute renal injury by neutrophil gelatinase-associated lipocalin.", JOURNAL OF THE AMERICAN SOCIETY OF NEPHROLOGY : JASN., vol. 15, no. 12, December 2004 (2004-12-01), pages 3073 - 3082 *
ROUDKENAR, M.H. ET AL.: "Neutrophil gelatinase-associated lipocalin acts as a protective factor against H(2)O(2) toxicity.", ARCHIVES OF MEDICAL RESEARCH., vol. 39, no. 6, August 2008 (2008-08-01), pages 560 - 566 *
VINUESA, E. ET AL.: "Lipocalin-2-induced renal regeneration depends on cytokines.", AMERICAN JOURNAL OF PHYSIOLOGY. RENAL PHYSIOLOGY, vol. 295, no. 5, November 2008 (2008-11-01), pages F1554 - F1562 *

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