WO2012052594A2 - USE OF Dlk1 AS AN ANGIOGENESIS INHIBITOR - Google Patents

USE OF Dlk1 AS AN ANGIOGENESIS INHIBITOR Download PDF

Info

Publication number
WO2012052594A2
WO2012052594A2 PCT/ES2011/070728 ES2011070728W WO2012052594A2 WO 2012052594 A2 WO2012052594 A2 WO 2012052594A2 ES 2011070728 W ES2011070728 W ES 2011070728W WO 2012052594 A2 WO2012052594 A2 WO 2012052594A2
Authority
WO
WIPO (PCT)
Prior art keywords
angiogenesis
dlk1
expression
gene construct
dlkl
Prior art date
Application number
PCT/ES2011/070728
Other languages
Spanish (es)
French (fr)
Other versions
WO2012052594A3 (en
Inventor
Santiago LAMAS PELÁEZ
Patricia RODRÍGUEZ PÉREZ
María Ángeles HIGUERAS LÓPEZ
Jorge LABORDA FERNÁNDEZ
Original Assignee
Consejo Superior De Investigaciones Científicas (Csic)
Universidad De Castilla La Mancha
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Consejo Superior De Investigaciones Científicas (Csic), Universidad De Castilla La Mancha filed Critical Consejo Superior De Investigaciones Científicas (Csic)
Publication of WO2012052594A2 publication Critical patent/WO2012052594A2/en
Publication of WO2012052594A3 publication Critical patent/WO2012052594A3/en

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/022—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from an adenovirus

Definitions

  • the present invention relates to the use of a gene construct comprising a sequence coding for Dlk1 or the recombinant protein that results from the expression of said gene construct for the preparation of a medicament for the inhibition of angiogenesis.
  • This use makes it possible to treat and prevent the formation of new blood vessels and is therefore useful for the treatment and prevention of pathogenic pathogenic processes such as tumor angiogenesis.
  • Angiogenesis consists in the formation of new branches of blood vessels from existing blood vessels. Angiogenesis is caused by the local destruction of the wall of preexisting blood vessels, the proliferation of endothelial cells, their migration and their organization in tubular structures around which the walls of blood vessels are formed. In adults, angiogenesis is very controlled and is only activated in processes such as wound repair, hypoxia and during the female reproductive cycle (Samaranayake et al. 2010. Hum Gene Ther 21: 381-396). The uncontrolled and / or excessive increase of angiogenesis is associated with various pathologies such as cancer, atherosclerosis, diabetic retinopathy, rheumatoid arthritis, psoriasis and macular degeneration.
  • angiogenesis inhibition is a very useful tool in antitumor therapy.
  • Antiangiogenic therapy in tumors has so far focused on the inhibition of vascular growth factor (VEGF) using neutralizing antibodies in front of it or in front of its receptors as antiangiogenic tools.
  • VEGF vascular growth factor
  • the Notch signaling pathway plays an important role in the formation of blood vessels.
  • the induction of endothelial expression of the Notch Delta-like 1 (DII1) ligand in postnatal arteriogenesis induced by ischemia in mice has been described (Limlaub et al. 2007. Cir Res 100: 363-371).
  • DII1 Notch Delta-like 1
  • Cir Res 100: 363-371 the use of blocking Notch receptor function using a soluble form of the Notchl receptor has proven useful for inhibition of angiogenesis in in vitro and in vivo studies in murine dermis after induction with VEGF and in murine xenografts of Breast and neuroblastoma tumors (Funahashi et al. 2008. Cancer Res 68: 4727-4735).
  • Dlkl a protein related to the Notch family "Delta-like homolog 1" (dlkl) in the inhibition of angiogenesis has not been described to date.
  • the Dlk1 gene codes for dlkl, a transmembrane protein that belongs to the family that contains repeats of EGF (epithelial growth factor) to which proteins such as Notch receptors and their ligands ⁇ Delta-Notch-Serrate family) also belong.
  • Dlkl also called Pref-1, "Fetal antigen-1” or pG2
  • pG2 fetal growth factor-1
  • pG2 fetal growth factor-1
  • Dlkl is highly expressed in the embryo and placenta during development, however, in adult tissues its expression is greatly reduced, even disappearing, with the exception of expression in ⁇ cells of the pancreas, bone marrow, pituitary and adrenal gland (Yevtod ⁇ yenko et al. 2006. Dev Dynamics 235: 1 1 15-1 123; Jensen et al. 1993. Hum Reprod 8: 635-641 ; Larsen et al. 1996. Lancet 347: 191; and Tornehave et al. 1996. Histochem Cell Biol 106: 535-542).
  • Dlkl participates in the differentiation control of various processes, including differentiation neuroendocrine, hepatocyte differentiation, hematopoiesis, osteogenesis and adipogenesis (WO9413701; Nueda et al 2007. J Mol Biol 367: 1281-1293). Dlk1 has also been related to the wound repair process since expression of Dlk1 has been detected in an undifferentiated mesenchymal tissue of the ear tissue repair zone of MRL and C57BL / 6 mice being the first where they are most Dlk1 expresses and those with the greatest tissue repair capacity (Samulewicz et al. 2002. Wound Repair Regen 10: 2 5-221).
  • Dlk1 In relation to the involvement of Dlk1 in angiogenesis, it has been described that in the embryo, the expression of Dlk1 has been related to the formation of embryonic blood vessels in mice since its expression has been seen in the developing endothelium of various blood vessels (for example in the cerebral arteries) as well as in the fetal blood vessels of the placenta (Yevtodiyenko et al. 2006. Dev Dynamics 235: 1 1 15-1 123). However, the use of overexpression of Dlk1 in the inhibition of angiogenesis has not been described to date.
  • the technical problem that the invention solves is an alternative medicine for the inhibition of angiogenesis obtained by using a gene construct comprising the sequence encoding Dlk1. Also part of the present invention is the use of the recombinant protein resulting from the expression of the gene construct comprising the sequence coding for Dlk1 for the preparation of a medicament for the inhibition of angiogenesis. In both cases the use is also contemplated for the inhibition of pathological processes that present with angiogenesis (pathological angiogenesis) such as cancer, atherosclerosis, diabetic retinopathy, rheumatoid arthritis, psoriasis and macular degeneration. Since there is currently no effective treatment for pathological angiogenesis, including tumor angiogenesis, an alternative that provides an effective treatment for such pathology is necessary.
  • pathological angiogenesis pathological angiogenesis
  • the invention describes the use of a gene construct comprising the sequence coding for Dlk1 or a functional biological equivalent thereof for the preparation of a medicament for the treatment of pathologies in which angiogenesis inhibition is necessary.
  • This use allows to prevent the formation of new blood vessels and therefore this new application is an alternative to improve the effectiveness of antiangiogenic treatments, including treatments against tumor angiogenesis.
  • Dlk1 was overexpressed in adult endothelium (hence differentiated cells) of animal models of angiogenesis through gene constructs.
  • the gene constructs used in the invention have been plasmids and adenoviruses in which the complementary DNA (cDNA) of Dlk1 has been fused to marker sequences that allow the selection of recombinants.
  • cDNA complementary DNA
  • In vitro tests have been carried out with Matrigel systems, ex vivo with aortic explants and in vivo with Matrigel plugs (supports) in animal models.
  • the use of the gene construct comprising the Dlk1 cDNA has been demonstrated in the inhibition of cell migration in reendothelialization processes (an important stage in angiogenesis, as noted above), specifically in healing processes of wounds ("wound healing") made in adult endothelial cells of aortaJ ' n vitro.
  • a first aspect of the invention relates to the use of a gene construct comprising the sequence coding for dlkl for the preparation of a medicament for the inhibition of angiogenesis.
  • dlkl also called in the literature “delta-like homolog (Drosophila) 1", “delta like homolog”, “secredeltin”, “preadipocyte factor T” fetal antigen 1 ",” Brevideltinin “,” DLK “,” FA1 “,” pG2 “,” SCP-1 “,” Pref-1 “,” PREF1 “and” ZOG "refers to a transmembrane protein that belongs to the Delta-Notch-Serrate signaling molecule family or its variants transcriptional sequence
  • the nucleotide sequence SEQ ID NO: 1 refers to the nucleotide sequence encoding Dlkl complementary DNA in Homo sapiens isoform a (accession number NM__003836.4)
  • amino acid sequence SEQ ID NO: 2 refers to the amino acid sequence of dlkl in Homo sapiens (accession number NP__003827.3).
  • Functional proteins originated from post-transcriptional modifications of the nucleotide sequence encoding SEQ ID NO: 1 are also part of the present invention, such as, but not limited to, the variants produced by cutting and splicing alternative, including: "delta-like 1 homolog isoform CRA b" (accession number EAW81713.1), “secredeltin” (accession number AAY4046.1), “brevideltinin” (accession number AAZ38943 .1) and 'brevideltinin truncated' (accession number AAZ66768.1) (see Table 1); as well as the gene constructs that encode them are also part of the invention.
  • Proteins with at least 70% identity with the amino acid sequence of SEQ ID NO: 2 are isoforms or amino acid sequences homologous to SEQ ID NO: 2 in Homo sapiens as well as in different animals, are also part of this invention as well as the nucleotide sequences that encode them.
  • the identity percentage has been chosen according to the Blastp program of the "National Center for Biotechnology Information" (NCBI) (http://www.ncbi.nlm.nih.gov/) (see Table 2).
  • % identity between two amino acid sequences, as understood in the present invention, refers to the number of amino acid positions over the total length of the sequence being compared, where all amino acids in that position are identical.
  • protein of the invention refers to functional proteins generated by alternative splicing of the sequence encoding SEQ ID NO: 1 or to proteins with at least 70% identity at the amino acid sequence SEQ ID NO: 2, or any of its fragments.
  • sequence of the invention refers to the nucleotide sequence that codes for the "protein of the invention”.
  • Dlk1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the dlkl protein, and which would comprise various variants from:
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, b) nucleic acid molecules whose chain would complement hybrid with the polynucleotide sequence of (a),
  • nucleic acid molecules whose sequence differs from (a) and / or (b) due to the degeneracy of the genetic code
  • nucleic acid molecules encoding a polypeptide comprising the aminoacidic sequence with an identity of at least 70%, 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 2; wherein the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the dlkl protein.
  • nucleic acid molecules encoding the isoforms or amino acid sequences homologous to SEQ ID NO: 2 with at least 70% identity with the amino acid sequence of SEQ ID NO: 2.
  • Gene construct means those genetic DNA constructs capable of transcribing to a polypeptide or fragment thereof, which code for the protein of the invention, hereinafter “gene construct of the invention”.
  • Said genetic construction of DNA would direct the in vitro or intracellular transcription of the dlkl sequence or fragment thereof, and comprises at least one of the following types of sequences: a) preferably double stranded nucleic acid, comprising less, the dlkl coding sequence for transcription in vitro, or intracellular, b) an expression cassette comprising nucleic acid operably linked to transcription control elements and optionally translation; c) a DNA nucleotide sequence, preferably double stranded, corresponding to a gene expression system or vector comprising the coding sequence of the dlkl sequence operably linked to at least one promoter that directs the transcription of said sequence of nucleotides of interest, and with other sequences necessary or appropriate for transcription and their appropriate regulation in time and place, for example, start and end signals, cut sites, polyadeny
  • medication refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, it refers to a composition capable of inhibiting angiogenesis.
  • Treatment refers to both therapeutic and prophylactic treatment or preventive measures. Those situations susceptible to treatment include those already associated with alterations as well as in those in which the alteration is prevented. An “alteration” is any condition that would benefit from treatment with the composition of the invention, as described herein.
  • the angiogenesis referred to in the present invention is that which consists in the formation of new branches of blood vessels (angiotubes or mycrovases in the present invention) from existing blood vessels that occurs under pathological conditions (pathological angiogenesis). Therefore, the present invention relates to the use of a gene construct comprising the sequence coding for dlkl for the preparation of a medicament for the inhibition of angiogenesis associated with pathologies that are selected from the list comprising: cancer, atherosclerosis , diabetic retinopathy, rheumatoid arthritis, psoriasis and macular degeneration.
  • the angiogenesis that is inhibited is tumor angiogenesis.
  • compositions of the present invention allow the transfection of the gene construct of the invention into a cell, in vivo or in vitro. Transfection could be carried out, but not limited to, direct transfection or vectors that facilitate access of the dlkl coding sequence into the cell. Therefore, in a preferred embodiment of the first aspect of the invention the gene construct of the invention comprises a vector that is selected from the list comprising, but not limited to, plasmid, bellow, artificial yeast chromosome (YACs), chromosome.
  • YACs artificial yeast chromosome
  • the gene constructs of the present invention can be conjugated with release peptides or other compounds to promote transport into the cell.
  • the gene construct comprises a plasmid as a vector.
  • the gene construct comprises an adenovirus as a vector.
  • the gene construct comprising the sequence that codes for dlkl is translated into the cell once it has been transfected and encoded for the protein that will ultimately perform the inhibition function inside the cell in the present invention.
  • the present invention also relates to the use of the recombinant protein resulting from the expression of the gene construct comprising the sequence coding for dlkl for the preparation of a medicament intended for the inhibition of angigenesis, and preferably of Tumor angigenesis.
  • said medicament that is, that which comprises the gene construct of the invention or the protein of the invention for the inhibition of angigenesis, further comprises at least one excipient and / or at least one pharmacologically acceptable carrier. .
  • excipient refers to a substance that helps the absorption of the medicament or pharmaceutical composition of the invention, stabilizes said pharmaceutical composition or aids in its preparation in the sense of giving it consistency or providing flavors that make it more pleasant.
  • the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as, for example, biblical calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
  • a “pharmacologically acceptable vehicle” refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants.
  • the carrier can be an inert substance or action analogous to any of the compounds of the present invention.
  • the function of the vehicle is to facilitate the incorporation of the expression product of the invention as well as other compounds, to allow a better dosage and administration or to give consistency and form to the pharmaceutical composition.
  • the presentation form is liquid, the vehicle is the diluent.
  • pharmaceutically acceptable refers to the compound referred to being allowed and evaluated so as not to cause damage to the organisms to which it is administered.
  • the invention is carried out by supplying an effective amount of the gene construct of the invention, the protein of the invention or a functional biological equivalent thereof in a tissue of an animal.
  • the composition provided by this invention can be provided by any route of administration, for which said composition is formulate in the appropriate pharmaceutical form to the route of administration chosen.
  • the amount of the gene construct of the invention or of the protein of the invention in said compositions is administered at a therapeutically effective concentration.
  • therapeutically effective concentration refers to the concentration of modulating agents calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said agents (and constructions) and the therapeutic effect to be achieved.
  • Pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.
  • Another aspect of the present invention relates to the use of at least one product of the expression of Dlk1, or any of its fragments as a biomarker for the determination of pathological angiogenesis or to determine the progression of pathological angiogenesis, in an isolated biological sample of a mammal, preferably human.
  • RNA such as, but not limited to, messenger RNA (mRNA) (sequence encoded by the cDNA of SEQ ID NO: 1), or any of its fragments; as well as that of any protein or any of its fragments resulting from the expression of SEQ ID NO: 1 of the present invention or having a homology with the RNA, protein or fragments thereof of at least 70%.
  • mRNA messenger RNA
  • biomarker in the present invention refers to a molecule that has a direct connection with the risk of pathological angiogenesis and serves to determine the disease status as well as to determine the progression of the disease.
  • the biological sample isolated from an organism such as, but not limited to, a human or other animal, may be a biological fluid or any cellular tissue of said organisms.
  • Diseases in which the alteration of dlkl activity can be diagnostic, and in particular pathological angigenesis, can be detected by measuring the amount of nucleic acids (DNA and / or RNA and / or mRNA) that code for dlkl, or the amount of dlkl protein that is expressed, compared to normal cells.
  • oligonucleotides can be done by methods well known in the state of the art (such as, but not limited to, probes with labeled nucleotides, DNA-DNA or DNA-RNA hybridization, PCR amplification using labeled nucleotides, RT- PCR). Methods for detecting Dlkl protein expression are also well known in the state of the art, such as poly or monoclonal antibodies, ELISA, radioimmunoassay (RIA), and FACS (fluorescence activated cell sorting).
  • methods well known in the state of the art such as, but not limited to, probes with labeled nucleotides, DNA-DNA or DNA-RNA hybridization, PCR amplification using labeled nucleotides, RT- PCR.
  • Methods for detecting Dlkl protein expression are also well known in the state of the art, such as poly or monoclonal antibodies, ELISA, radioimmunoassay (RIA), and FACS (fluorescence activated cell
  • a method for the determination of pathological angigenesis comprises, (a) detecting and / or quantifying in a biological sample isolated from a mammal, at least one product of Dlkl expression, or Any of its fragments.
  • a preferred embodiment refers to a method of determining pathological angigenesis, where step (b) also comprises comparing the data obtained in step (a) with data obtained from control samples to look for any significant deviation. Another more preferred embodiment further comprises step (c) attributing the significant deviation to the development of pathological angigenesis in said mammal.
  • control samples refers, for example, but not limited to a sample obtained from an individual. that does not develop a pathology associated with angiogenesis (healthy individual). This type of control sample is a negative control or negative control sample for pathological angiogenesis.
  • the term "significant deviation" as understood in the present invention refers to the variation of the expression of the nucleic acids encoding dlkl or the presence of the dlkl protein in the isolated sample, or a variation in the concentration of the protein of the invention in the isolated sample with respect to an isolated sample from a healthy individual where the difference is statistically significant.
  • the selection of the healthy individual is carried out by measuring the level of one or more common markers of pathological angiogenesis.
  • the common marker is known to one skilled in the art and is selected from the list comprising, but not limited to, VE-cadherins ("vascular endothelial-cadherin”), VEGF, fibroblast growth factor b (FGF-b), the soluble form of the VEGF receptor (VEGFR-1 or sFlt-1), angiopoietin (Ang-1) von W ⁇ llebrand factor, PECAM-1 ("platelet endothelial cell adhesion molecule-1”), IL-6, p53, factor VIII, CD105 glycoprotein, and GM and Gtlb gangliosides.
  • VE-cadherins vascular endothelial-cadherin
  • VEGF vascular endothelial-cadherin
  • FGF-b fibroblast growth factor b
  • VEGFR-1 or sFlt-1 the soluble form of the VEGF receptor
  • Ang-1 angiopoietin
  • PECAM-1 platelet
  • statically significant difference refers to the fact that there is statistical difference between the values compared, the statistical probability being at least greater than or less than 0.05 (p> 0.05 op> 0.05) and this being obtained according to the test statistic applicable to each case.
  • Another aspect of the invention comprises a method for determining the progression of pathological angiogenesis comprising,
  • step (a) determining a first concentration of at least one product of Dlkl expression, or any of its fragments in a biological sample isolated from a mammal; (b) determine a second concentration of said expression product of step (a) in an isolated biological sample of the same mammal subsequently taken from the sample of step (a), and (c) compare the second concentration obtained in step (b) with the first concentration obtained in step (a) to look for a significant deviation.
  • kits comprising the gene construct comprising the sequence coding for dlkl for the preparation of a medicament for the inhibition of angigenesis as well as the use of the kit comprising the recombinant protein resulting from the expression of the gene construct comprising the sequence coding for dlkl for the preparation of a medicament for the inhibition of angigenesis.
  • polynucleotide and “nucleic acid” are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides and deoxyribonucleotides.
  • peptide refers to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified.
  • Figure 1 It shows that Dlk1 expression delays cell migration in in vitro assays or wound healing. Assays were performed with a plasmid containing the human Dlk1 cDNA to verify the effect of dlkl overexpression on endothelial cell migration. A, it is shown that adult swine aorta cells that were transfected with a Dlk1 plasmid (Dlk1) have a significant delay in wound closure compared to cells in basal conditions (basal) or with cells expressing an empty control vector (pCMV6).
  • Dlk1 plasmid containing the human Dlk1 cDNA
  • A Adult porcine aorta endothelial cells transfected with Dlkl (Dlkl) cDNA were compared with untransfected cells (Basal) or, transfected with the empty plasmid pCMV6 (pCMV6) in both cells to which 5 ng / ml was added of VEGF directly to the culture medium (VEGF) or without adding (No VEGF).
  • B number of tubular structures that were formed in the presence of VEGF (white bars) and in the absence of VEGF (black bars).
  • Figure 3 It shows that Dlkl overexpression decreases angiogenesis in ex vivo assay in mouse aorta explants. Assays with collagen-embedded murine aorta rings were performed to verify the effect of dlkl overexpression on the formation of angiotubes in ex vivo systems of mouse aorta explants.
  • A scheme of the gene construct containing an adenovirus type5 (Ad type 5 dE1 / dE3).
  • CMV cytomegalovirus promoter
  • Dlk1 HA the sequence of Dlk1 referred to in SEQ ID NO: 1 with the insertion between nucleotide 1302 and 1303 of the hemagglutinin epitope sequence referred to in SEQ ID NO: 3
  • poly A tail poIyA
  • GFP green fluorescent protein
  • B collagen-embedded mouse aorta rings which were infected with empty adenovirus (Ad. Empty), adenovirus with GFP (Ad.GFP), or with adenovirus containing the human Dlk1 cDNA (Ad.Dlkl). Two trials are shown per experiment.
  • Figure 4 It shows that the overexpression of Dlk1 decreases the angiogenesis in MatrigeJ supports (or matrices) in vivo.
  • In vivo assays were performed by implanting matrix supports (or matrices) containing adenovirus in the subcutaneous tissue of mice to verify the effect of dlkl overexpression on inhibition of angiogenesis in in vivo systems.
  • As a negative angiogenesis control supports (matrices) were implanted with a Notch pathway inhibitor (DAPT).
  • DAPT Notch pathway inhibitor
  • the matrigel supports contained VEGF to activate angiogenesis, as a negative control a matrigel support was implanted with an adenovirus containing GFP and not containing VEGF.
  • A matrigel supports that were implanted subcutaneously in which macroscopically produced angiogenesis can be seen by visualization of a darker color corresponding to the coloration of the blood located in the formed vessels.
  • Ad.GFP adenovirus with GFP
  • Ad.DLK adenovirus comprising SEQ. ID. NO: 1
  • Ad.DLK-GFP adenovirus comprising SEQ. ID. NO: 1 and GFP
  • DAPT control with the DAPT inhibitor
  • DMSO control with DMSO.
  • B total hemoglobin content in implanted matrix holders.
  • Assays were performed with a plasmid containing the human Dlk1 cDNA to verify the effect of dlkl overexpression on the migration of adult endothelial cells of porcine and bovine aorta transfected with a plasmid containing the human cDNA and compared with untransfected cells. and transfected with the empty plasmid.
  • the plasmid used in this assay was pCMV6-XL4 ⁇ access sequence number AF067196 of the NCBI) to which it was linked by cloning in the region of MCS polilinkers between the targets of Not I, SEQ ID NO: 1.
  • this gene construct carries a hemagglutin (HA) epitope for localization of expression by anti-hemagglutinin antibodies.
  • the HA epitope (SEQ ID NO: 3) was located between nucleotides 1302 and 1303 of the Dlk1 cDNA used in the invention (SEQ ID NO 1).
  • EXAMPLE 2 Overexpression of Dlk1 inhibits the formation of angiotubes in Matrigel in vitro systems. To verify the in vitro inhibition of angiogenesis of a gene construct comprising the sequence encoding dlkl, a plasmid containing the human dlkl cDNA was used and the effect of dlkl overexpression on the formation of angiotubes in systems was checked. in vitro of matrigel.
  • Matrigel is the trade name (Beckton Dickinson and Company, BD TM) for a gelatinous mixture of proteins secreted by mouse tumor cells (sarcoma "Engelberth-Holm-Swarm", EHS) very rich in extracellular matrix proteins, being the largest laminin component, followed by collagen IV, proteoglycans and entactin / nidogen, among others.
  • EHS Engelbreth-Holm-Swarm
  • This mixture resembles the complex extracellular environment found in many tissues and is used in in vitro studies as a substrate of endothelial cells capable of forming tubular structures in a manner similar to the angiogenesis process that occurs in vivo.
  • the isolated endothelial cells were embedded in Matrigel and in the presence of culture medium supplemented or not with VEGF (5ng / ml) the formation of tubular networks was checked.
  • the plasmid used in this assay was pCMV6-XL4 (access sequence number AF067196 of the NCBI) to which it was attached by cloning in the region of MCS polilinkers between the targets of Not I, SEQ ID NO: 1.
  • this gene construct carries a hemagglutinin (HA) epitope useful for localization of expression by anti-HA antibodies.
  • the HA epitope (SEQ ID NO: 3) was located between nucleotides 1302 and 1303 of the Dlk1 cDNA used in the invention (SEQ ID NO 1).
  • angiotubes in adult endothelial cells of porcine and bovine aorta transfected with the empty plasmid pCMV6, with the Dlkl cDNA or with non-transfected cells was compared in either cells to which VEGF was added or without adding VEGF to the culture medium .
  • the number of angiotubes (tubular structures) that formed after transfecting the endothelial cells with the plasmid containing the human Dlk1 cDNA was significantly less than the number of angiotubes formed after transfection with the empty plasmid or in basal conditions, both in the presence of VEGF and in the absence of VEGF, both in porcine and bovine endothelium, so only the results of porcine aorta endothelial cells are shown (Figs. 2A and 2B).
  • EXAMPLE 3 Overexpression of Dik1 decreases angiogenesis in an ex vivo assay in mouse aortic explants.
  • a adenovirus containing the human dlkl cDNA and the effect of dlkl overexpression on the formation of angiotubes in ex vivo systems was verified for which mouse aorta rings embedded in type I collagen were used.
  • EXAMPLE 4 Overexpression of Dik1 decreases angiogenesis in matrigel supports in vivo.
  • Angiogenesis produced both macroscopically was observed by visualization of a darker color corresponding to the coloration of the blood located in the formed vessels (Fig. 4A) and by the assessment of the total hemoglobin content in the supports once they were removed from the site of implant (Fig. 4B).
  • adenoviruses containing the Dlk1 cDNA SEQ ID NO: 1
  • Ad.DLK1 -GFP Ad.DLK1 -GFP
  • the porcine or bovine aorta isolated and washed with PBS was treated with collagenase (0.03% weight / volume) (Sigma) for 15 minutes at 37 9 C. After centrifugation, the precipitates were resuspended in RPMI medium (Gibco) supplemented with 15% of (fetal calf serum) FCS (Gibco), 5% penicillin / streptomycin / fungizone (Gibco) and 5% heparin (Sigma) and grown in 25 cm 2 culture bottles. The purity of the preparations was analyzed by immunofluorescence using the anti-Factor VIII (von Willebrand) antibody (3 Obtaining Knock Out mice for Dlk1.
  • knock out mice was performed according to the existing literature (Raghumandan et al. 2008. Stem Cells Dev 17: 795-507). Briefly, the dlkl gene was silenced by inserting a Neomycin-resistant cassette that replaced 3.8 kbp of the endogenous allele, including the promoter and the first three exons of Dlk1. Said construct was transfected by electroporation to embryonic cells of mice of strain SvJ129. The chimeras were generated and established the relevant crosses to achieve the Dlk1 7 " mouse.
  • Genotype analysis was performed by" Southern blot "or PCR using the following primers of the sequences collected in SEQ ID NO: 4 (sense primer for Dlk1) , SEQ ID NO: 5 (sense primer for Neomycin) and SEQ ID NO: 6 (antisense primer that hybridizes with the complementary sequence in intron 3 of Dlk1): The phenotype analysis was performed after three crosses with strain C57BI / 6, and then crossings between heterozygotes They were used to generate homozygotes, heterozygotes and w ⁇ ld-type.
  • the cell preparation was first subjected to a negative selection using macrophage antibodies (CD16 / CD32) (1.6 ⁇ g / ml) (BD TM ) and anti-lgG-coated magnetic particles (6.6x10 5 particles / ml) (Invitrogen), and after 48 hours, to a double positive selection using the specific antibody for endothelial cells ICAM-2 (3.3 ⁇ g / ml) (BD TM) and magnetic particles.
  • the purity of the preparation was analyzed by flow cytometry using the ICAM-2 antibody (3 ⁇ g / ml) (BD TM).
  • Infections with adenovirus were performed at a concentration of 1 x 10 9 plaque forming units (pfu).
  • explants were incubated with adenoviruses for 24 hours before immersing them in the type I collagen matrix (SERVA).
  • SERVA type I collagen matrix
  • adenoviruses were mixed with matrigei supports and other components (VEGF and / or DAPT) (Biomol) before injecting them into the animals.
  • VEGF and / or DAPT Biomol
  • the mouse aorta was dissected and quickly transferred to cold PBS.
  • the fibro-fatty tissue was separated from the wall of the aorta avoiding damage, and was cut into segments of 1 mm in length.
  • a 96.40 ⁇ plate of the following type I collagen mixture (7.5 volumes of 2 mg / ml) (SERVA), 1 volume of 10x MEM medium (Gibco) was added to each well and 1.5 volumes of NaHC0 3 (15.6 mg / ml) and 0.1 volumes of 1 M NaOH to adjust the pH to 7.4. This mixture was allowed to solidify at 37 C for 10 minutes and before imbibing aortic rings.
  • the plate was incubated for another 10 minutes at 37 ° C and another 40 ⁇ of the collagen mixture was added to each explant. After another 10 minute incubation, 100 ⁇ of MCDB131 (Gibco) supplemented with 25 mM NaHCO 3 , 2.5% mouse serum (Source BioScience), 1% glutamine and 100 U / ml penicillin / streptomycin were added. Cultures were maintained at 37 Q C for 6 days.
  • adenovirus infections In cases of adenovirus infections, the clean aortas of adipose tissue were cut in half and incubated with 1 x 10 9 pfu of adenovirus in MCDB131 medium with low concentration serum (2%) for 24 hours. After this time, the infected aortas were washed to remove adenovirus residues, cut into rings of 1 mm in length and embedded in the type I collagen matrix. The explants were analyzed after 6 days of incubation using a microscope. fluorescence. Angiogenesis was evaluated by counting the number of microvessels (angiotubes) per ring.
  • Low growth factor Matrigel 500 ⁇ was injected and, depending on the case, adenovirus (1 x 10 9 pfu), DAPT (10 mg / kg weight), DMSO vehicle (dimethylsulfoxide) or VEGF (250 ng / ml), so subcutaneous in the upper abdomen of the anesthetized mouse.
  • adenovirus 1 x 10 9 pfu
  • DAPT 10 mg / kg weight
  • DMSO vehicle dimethylsulfoxide
  • VEGF 250 ng / ml
  • Inhibition of the Notch pathway in vivo was performed by adding to the Matrigel (500 ⁇ ) heparin (64 U / ml) and VEGF (250 ng / ml) mixture the DAPT compound at a concentration of (10 mg / kg weight) , before injecting it subcutaneously to the mouse.

Abstract

The invention relates to the use of a gene construction comprising a sequence encoding Dlk1 or the recombinant protein resulting from the expression of said gene construction for the preparation of a medicament for inhibiting angiogenesis, preferably tumoral angiogenesis. The invention also relates to the use of a product of the expression of Dlk1 or any of its fragments as a biomarker for determining angiogenesis or the progression thereof. The invention further relates to a method for determining pathological angiogenesis or the progression thereof, and to the use of a kit comprising the sequence encoding for dlk1 or any of its expression products for the diagnosis of pathological angiogenesis.

Description

Uso de Dlk1 como inhibidor de anqioqénesis  Use of Dlk1 as an anqiogenesis inhibitor
La presente invención se refiere al uso de una construcción géníca que comprende una secuencia codificante de Dlk1 o a la proteína recombinante que resulta de la expresión de dicha construcción génica para la preparación de un medicamento para la inhibición de la angiogénesis. Este uso permite tratar y prevenir la formación de nuevos vasos sanguíneos y por lo tanto es útil para el tratamiento y prevención de procesos angíogénicos patológicos tales como la angiogénesis tumoral. The present invention relates to the use of a gene construct comprising a sequence coding for Dlk1 or the recombinant protein that results from the expression of said gene construct for the preparation of a medicament for the inhibition of angiogenesis. This use makes it possible to treat and prevent the formation of new blood vessels and is therefore useful for the treatment and prevention of pathogenic pathogenic processes such as tumor angiogenesis.
ESTADO DE LA TÉCNICA ANTERIOR STATE OF THE PREVIOUS TECHNIQUE
La angiogénesis consiste en la formación de nuevas ramificaciones de vasos sanguíneos a partir de vasos sanguíneos existentes. La angiogénesis se produce por la destrucción local de la pared de los vasos sanguíneos preexistentes, la proliferación de las células endoteliales, la migración de las mismas y la organización de éstas en estructuras tubulares alrededor de las cuales se forman las paredes de los vasos sanguíneos. En adultos, la angiogénesis está muy controlada y se activa únicamente en procesos como la reparación de heridas, hipoxia y durante el ciclo reproductivo femenino (Samaranayake et al. 2010. Hum Gene Ther 21 :381 -396). El aumento descontrolado y/o excesivo de la angiogénesis está asociado a diversas patologías tales como cáncer, aterosclerosis, retinopatía diabética, artritis reumatoide, psoriasis y degeneración macular. Sin la activación de la angiogénesis, se ha descrito que la hipoxia en tumores sólidos no permite crecer al tumor y provoca la muerte de células tumorales (Karamysheva 2008. Biochemistry (Mosc.) 73:751 -762; y Folkman et al 1992. J Biol Chem 267:10931 -10934). Por todo esto, la inhibición de la angiogénesis es una herramienta de gran utilidad en la terapia antitumoral. La terapia antiangiogénica en tumores se ha centrado hasta la fecha en la inhibición del factor de crecimiento vascular (VEGF) utilizando anticuerpos neutralizantes frente al mismo o frente a sus receptores como herramientas antiangiogénicas. Sin embargo, se hace necesaria una alternativa ya que los anticuerpos neutralizantes frente a VEGF no producen ni una reducción del tumor ni un aumento de la supervivencia significativos, además de presentar un elevado coste de producción (Samaranayake et al. 2010. Hum Gene Ther 21 :381 -396). Angiogenesis consists in the formation of new branches of blood vessels from existing blood vessels. Angiogenesis is caused by the local destruction of the wall of preexisting blood vessels, the proliferation of endothelial cells, their migration and their organization in tubular structures around which the walls of blood vessels are formed. In adults, angiogenesis is very controlled and is only activated in processes such as wound repair, hypoxia and during the female reproductive cycle (Samaranayake et al. 2010. Hum Gene Ther 21: 381-396). The uncontrolled and / or excessive increase of angiogenesis is associated with various pathologies such as cancer, atherosclerosis, diabetic retinopathy, rheumatoid arthritis, psoriasis and macular degeneration. Without the activation of angiogenesis, it has been described that hypoxia in solid tumors does not allow the tumor to grow and causes the death of tumor cells (Karamysheva 2008. Biochemistry (Mosc.) 73: 751-762; and Folkman et al 1992. J Biol Chem 267: 10931-10934). For all this, angiogenesis inhibition is a very useful tool in antitumor therapy. Antiangiogenic therapy in tumors has so far focused on the inhibition of vascular growth factor (VEGF) using neutralizing antibodies in front of it or in front of its receptors as antiangiogenic tools. However, an alternative is necessary because the neutralizing antibodies against VEGF do not produce a significant reduction in the tumor or an increase in survival, besides presenting a high production cost (Samaranayake et al. 2010. Hum Gene Ther 21 : 381-396).
La vía de señalización de Notch juega un importante papel en la formación de vasos sanguíneos. Se ha descrito la inducción de la expresión endotelial del ligando de Notch Delta-like 1 (DII1 ) en la arteriogénesis postnatal inducida por isquemia en ratones (Limbourg et al. 2007. Cir Res 100:363-371 ). Por otra parte, el uso del bloqueo de la función del receptor Notch utilizando una forma soluble del receptor Notchl se ha demostrado útil para la inhibición de la angiogénesis en estudios in vitro e in vivo en dermis murina tras inducción con VEGF y en xenotransplantes murinos de tumores mamarios y de neuroblastoma (Funahashi et al. 2008. Cáncer Res 68:4727-4735). Sin embargo, no se ha descrito hasta la fecha el uso de una proteína relacionada con la familia Notch "Delta-like homolog 1 " (dlkl ) en la inhibición de la angiogénesis. El gen Dlk1 codifica para dlkl , una proteína transmembrana que pertenece a la familia que contiene repeticiones de EGF (factor de crecimiento epitelial) a la que también pertenecen proteínas como los receptores Notch y sus ligandos {familia Delta-Notch-Serrate). Dlkl (también llamado Pref-1 , "Fetal antigen-1 " o pG2) está muy expresado en el embrión y la placenta durante el desarrollo, sin embargo, en tejidos adultos su expresión se reduce considerablemente llegando incluso a desaparecer, con la excepción de la expresión en las células β del páncreas, la médula ósea, la pituitaria y la glándula adrenal (Yevtodíyenko et al. 2006. Dev Dynamics 235:1 1 15-1 123; Jensen et al. 1993. Hum Reprod 8:635-641 ; Larsen et al. 1996. Lancet 347:191 ; y Tornehave et al. 1996. Histochem Cell Biol 106:535-542). Dlkl participa en el control de diferenciación de diversos procesos, entre los que se incluye diferenciación neuroendocrina, diferenciación de hepatocítos, hematopoyesis, osteogénesís y adipogénesis (WO9413701 ; Nueda et al 2007. J Mol Biol 367:1281 -1293). Dlk1 también se ha relacionado con el proceso de reparación de heridas ya que se ha detectado expresión de Dlk1 en un tejido mesenquimal no diferenciado de la zona de reparación de tejido de la oreja de ratones MRL y C57BL/6 siendo en los primeros donde más se expresa Dlk1 y los que mayor capacidad de reparación tisular presentan (Samulewicz et al. 2002. Wound Repair Regen 10:2 5-221 ). En relación a la implicación de Dlk1 en angiogénesis, se ha descrito que en el embrión, la expresión de Dlk1 se ha relacionado con la formación de vasos sanguíneos embrionarios en ratón ya que se ha visto su expresión en el endotelio en desarrollo de diversos vasos sanguíneos (por ejemplo en las arterias cerebrales) así como en los vasos sanguíneos fetales de la placenta (Yevtodiyenko et al. 2006. Dev Dynamics 235:1 1 15-1 123). Sin embrago, no se ha descrito hasta la fecha el uso de la sobreexpresión de Dlk1 en la inhibición de la angiogénesis. The Notch signaling pathway plays an important role in the formation of blood vessels. The induction of endothelial expression of the Notch Delta-like 1 (DII1) ligand in postnatal arteriogenesis induced by ischemia in mice has been described (Limbourg et al. 2007. Cir Res 100: 363-371). On the other hand, the use of blocking Notch receptor function using a soluble form of the Notchl receptor has proven useful for inhibition of angiogenesis in in vitro and in vivo studies in murine dermis after induction with VEGF and in murine xenografts of Breast and neuroblastoma tumors (Funahashi et al. 2008. Cancer Res 68: 4727-4735). However, the use of a protein related to the Notch family "Delta-like homolog 1" (dlkl) in the inhibition of angiogenesis has not been described to date. The Dlk1 gene codes for dlkl, a transmembrane protein that belongs to the family that contains repeats of EGF (epithelial growth factor) to which proteins such as Notch receptors and their ligands {Delta-Notch-Serrate family) also belong. Dlkl (also called Pref-1, "Fetal antigen-1" or pG2) is highly expressed in the embryo and placenta during development, however, in adult tissues its expression is greatly reduced, even disappearing, with the exception of expression in β cells of the pancreas, bone marrow, pituitary and adrenal gland (Yevtodíyenko et al. 2006. Dev Dynamics 235: 1 1 15-1 123; Jensen et al. 1993. Hum Reprod 8: 635-641 ; Larsen et al. 1996. Lancet 347: 191; and Tornehave et al. 1996. Histochem Cell Biol 106: 535-542). Dlkl participates in the differentiation control of various processes, including differentiation neuroendocrine, hepatocyte differentiation, hematopoiesis, osteogenesis and adipogenesis (WO9413701; Nueda et al 2007. J Mol Biol 367: 1281-1293). Dlk1 has also been related to the wound repair process since expression of Dlk1 has been detected in an undifferentiated mesenchymal tissue of the ear tissue repair zone of MRL and C57BL / 6 mice being the first where they are most Dlk1 expresses and those with the greatest tissue repair capacity (Samulewicz et al. 2002. Wound Repair Regen 10: 2 5-221). In relation to the involvement of Dlk1 in angiogenesis, it has been described that in the embryo, the expression of Dlk1 has been related to the formation of embryonic blood vessels in mice since its expression has been seen in the developing endothelium of various blood vessels (for example in the cerebral arteries) as well as in the fetal blood vessels of the placenta (Yevtodiyenko et al. 2006. Dev Dynamics 235: 1 1 15-1 123). However, the use of overexpression of Dlk1 in the inhibition of angiogenesis has not been described to date.
La sobreexpresión de Dlk1 se ha probado hasta la fecha en adipogénesis pero no en angiogénesis. En adipogénesis se ha visto que la sobreexpresión de Dlk1 activa o inhibe la adipogénesis dependiendo del contexto celular, previniendo la adipogénesis en la línea celular 3T3-L1 pero activando la adipogénesis en la línea celular C3H10T1 /2 (Nueda et al. 2007. J Mol Biol 367:1281 -1293). Además, se ha sugerido la interacción de Dlk1 con Notchl in vivo y su participación en la cascada de señalización de Notchl en la regulación de los procesos de adipogénesis al expresar Dlk1 en una línea celular de ratón (Balb/c 14 que expresa Notchl pero no Dlk1 ) y comprobar que la expresión forzada Dik1 provoca una disminución en la actividad de Notch (Baladrón et al. 2005. Exp Cell Res 303:343-359). También se ha descrito el uso de la sobreexpresíón de Dlk1 en hematopoyesis. El uso de una fracción soluble de Dlk1 humano resulta útil para inhibir la diferenciación de las células madre hematopoyéticas, resultados que se han obtenido mediante transfección estable del cDNA de Dlk1 humano (W09731647). Overexpression of Dlk1 has been tested to date in adipogenesis but not in angiogenesis. In adipogenesis it has been found that overexpression of Dlk1 activates or inhibits adipogenesis depending on the cellular context, preventing adipogenesis in the 3T3-L1 cell line but activating adipogenesis in the C3H10T1 / 2 cell line (Nueda et al. 2007. J Mol Biol 367: 1281-1293). In addition, it has been suggested the interaction of Dlk1 with Notchl in vivo and its participation in the Notchl signaling cascade in the regulation of adipogenesis processes by expressing Dlk1 in a mouse cell line (Balb / c 14 expressing Notchl but not Dlk1) and verify that the forced expression Dik1 causes a decrease in Notch activity (Baladrón et al. 2005. Exp Cell Res 303: 343-359). The use of overexpression of Dlk1 in hematopoiesis has also been described. The use of a soluble fraction of human Dlk1 is useful for inhibiting the differentiation of hematopoietic stem cells, results that have been obtained by stable transfection of the human Dlk1 cDNA (W09731647).
EXPLICACIÓN DE LA INVENCIÓN EXPLANATION OF THE INVENTION
El problema técnico que resuelve la invención es un medicamento alternativo para la inhibición de la angiogénesis obtenido mediante el uso de una construcción génica que comprende la secuencia que codifica para Dlk1 . También forma parte de la presente invención el uso de la proteína recombinante resultante de la expresión de la construcción génica que comprende la secuencia que codifica para Dlk1 para la preparación de un medicamento para la inhibición de la angiogénesis. En ambos casos también se contempla el uso para la inhibición de procesos patológicos que cursan con angiogénesis (angiogénesis patológica) tales como cáncer, aterosclerosis, retinopatía diabética, artritis reumatoide, psoriasis y degeneración macular. Dado que en la actualidad no existe ningún tratamiento efectivo para la angiogénesis patológica, entre ella la angiogénesis tumoral, se hace necesaria una alternativa que proporcione un tratamiento efectivo para dicha patología. The technical problem that the invention solves is an alternative medicine for the inhibition of angiogenesis obtained by using a gene construct comprising the sequence encoding Dlk1. Also part of the present invention is the use of the recombinant protein resulting from the expression of the gene construct comprising the sequence coding for Dlk1 for the preparation of a medicament for the inhibition of angiogenesis. In both cases the use is also contemplated for the inhibition of pathological processes that present with angiogenesis (pathological angiogenesis) such as cancer, atherosclerosis, diabetic retinopathy, rheumatoid arthritis, psoriasis and macular degeneration. Since there is currently no effective treatment for pathological angiogenesis, including tumor angiogenesis, an alternative that provides an effective treatment for such pathology is necessary.
La invención describe el uso de una construcción génica que comprende la secuencia que codifica para Dlk1 o un equivalente biológico funcional de la misma para la preparación de un medicamento para el tratamiento de patologías en las que sea necesaria la inhibición de la angiogénesis. Este uso permite prevenir la formación de nuevos vasos sanguíneos y por lo tanto esta nueva aplicación es una alternativa para mejorar la efectividad de los tratamientos antiangiogénicos, incluidos los tratamientos frente a la angiogénesis tumoral. Con el fin de evaluar el potencial antiangiogénico de la secuencia que codifica para Dlk1 para la elaboración de un medicamento para prevenir la formación de vasos sanguíneos (angiogénesís), se procedió a sobreexpresar Dlk1 en endotelio adulto (por lo tanto células diferenciadas) de modelos animales de angiogénesís mediante construcciones génicas. Las construcciones génicas utilizadas en la invención han sido plásmidos y adenovirus en las que el DNA complementario (cDNA) de Dlk1 se ha fusionado a secuencias marcadoras que permiten la selección de los recombinantes. Se han realizado ensayos in vitro con sistemas de Matrigel, ex vivo con explantes de aorta e in vivo con "plugs" (soportes) de Matrigel en modelos anímales. Además, también se ha demostrado el uso de la construcción génica que comprende el cDNA de Dlk1 en la inhibición de la migración celular en procesos de reendotelizacion (una etapa importante en la angiogénesís, como se ha señalado anteriormente), concretamente en procesos de cicatrización de heridas ("wound healing") realizados en células endoteliales adultas de aortaJ'n vitro. The invention describes the use of a gene construct comprising the sequence coding for Dlk1 or a functional biological equivalent thereof for the preparation of a medicament for the treatment of pathologies in which angiogenesis inhibition is necessary. This use allows to prevent the formation of new blood vessels and therefore this new application is an alternative to improve the effectiveness of antiangiogenic treatments, including treatments against tumor angiogenesis. In order to evaluate the antiangiogenic potential of the sequence coding for Dlk1 for the preparation of a drug to prevent blood vessel formation (angiogenesis), Dlk1 was overexpressed in adult endothelium (hence differentiated cells) of animal models of angiogenesis through gene constructs. The gene constructs used in the invention have been plasmids and adenoviruses in which the complementary DNA (cDNA) of Dlk1 has been fused to marker sequences that allow the selection of recombinants. In vitro tests have been carried out with Matrigel systems, ex vivo with aortic explants and in vivo with Matrigel plugs (supports) in animal models. In addition, the use of the gene construct comprising the Dlk1 cDNA has been demonstrated in the inhibition of cell migration in reendothelialization processes (an important stage in angiogenesis, as noted above), specifically in healing processes of wounds ("wound healing") made in adult endothelial cells of aortaJ ' n vitro.
Así, un primer aspecto de la invención se refiere al uso de una construcción génica que comprende la secuencia que codifica para dlkl para la preparación de un medicamento para la inhibición de la angiogénesís. Thus, a first aspect of the invention relates to the use of a gene construct comprising the sequence coding for dlkl for the preparation of a medicament for the inhibition of angiogenesis.
El término "dlkl " también llamado en la literatura "delta-like homolog (Drosophila) 1", "delta like homolog", "secredeltin", "preadipocyte factor T "fetal antigen 1", "Brevideltinin", "DLK", "FA1 ", "pG2", "SCP-1 ", "Pref-1 ", "PREF1 " y "ZOG"; se refiere a una proteína transmembrana que pertenece a la familia de moléculas de señalización Delta-Notch-Serrate o a sus variantes transcrípcíonales. La secuencia de nucleotidos SEQ ID NO: 1 se refiere a la secuencia de nucleotidos que codifica el DNA complementario de Dlkl en Homo sapiens isoforma a (número de acceso NM__003836.4). La secuencia de aminoácidos SEQ ID NO: 2 se refiere a la secuencia de aminoácidos de dlkl en Homo sapiens (número de acceso NP__003827.3). Las proteínas (secuencias de aminoácidos) funcionales originadas a partir de modificaciones postranscripcionales de la secuencia de nucleótidos que codifica para la SEQ ID NO: 1 también forman parte de la presente invención, como por ejemplo, pero sin limitarnos, las variantes producidas por corte y empalme alternativo ("splicing"), entre ellas: "delta-like 1 homolog isoform CRA b" (número de acceso EAW81713.1 ), "secredeltin" (número de acceso AAY4046.1 ), "brevideltinin" (número de acceso AAZ38943.1 ) y "brevideltinin truncated' (número de acceso AAZ66768.1 ) (ver Tabla 1 ); así como también forman parte de la invención las construcciones génicas que las codifican. The term "dlkl" also called in the literature "delta-like homolog (Drosophila) 1", "delta like homolog", "secredeltin", "preadipocyte factor T" fetal antigen 1 "," Brevideltinin "," DLK "," FA1 "," pG2 "," SCP-1 "," Pref-1 "," PREF1 "and" ZOG "refers to a transmembrane protein that belongs to the Delta-Notch-Serrate signaling molecule family or its variants transcriptional sequence The nucleotide sequence SEQ ID NO: 1 refers to the nucleotide sequence encoding Dlkl complementary DNA in Homo sapiens isoform a (accession number NM__003836.4) The amino acid sequence SEQ ID NO: 2 refers to the amino acid sequence of dlkl in Homo sapiens (accession number NP__003827.3). Functional proteins (amino acid sequences) originated from post-transcriptional modifications of the nucleotide sequence encoding SEQ ID NO: 1 are also part of the present invention, such as, but not limited to, the variants produced by cutting and splicing alternative, including: "delta-like 1 homolog isoform CRA b" (accession number EAW81713.1), "secredeltin" (accession number AAY4046.1), "brevideltinin" (accession number AAZ38943 .1) and 'brevideltinin truncated' (accession number AAZ66768.1) (see Table 1); as well as the gene constructs that encode them are also part of the invention.
Tabla 1. Proteínas resultantes de las variantes de "splicing" de Dlk1 humana Table 1. Proteins resulting from the "splicing" variants of human Dlk1
Figure imgf000008_0001
Figure imgf000008_0001
Las proteínas con al menos un 70% de identidad con la secuencia de aminoácidos de la SEQ ID NO: 2 son isoformas o secuencias de aminoácidos homologas a la SEQ ID NO: 2 en Homo sapiens así como en diferentes animales, también forman parte de esta invención así como las secuencias de nucleótidos que las codifican. El porcentaje de identidad se ha elegido de acuerdo con el programa Blastp del "National Center for Biotechnology Information" (NCBI) (http://www.ncbi.nlm.nih.gov/) (ver Tabla 2). Proteins with at least 70% identity with the amino acid sequence of SEQ ID NO: 2 are isoforms or amino acid sequences homologous to SEQ ID NO: 2 in Homo sapiens as well as in different animals, are also part of this invention as well as the nucleotide sequences that encode them. The identity percentage has been chosen according to the Blastp program of the "National Center for Biotechnology Information" (NCBI) (http://www.ncbi.nlm.nih.gov/) (see Table 2).
Tabla 2. Porcentaje de identidad de la proteína dlkl en diferentes especies animales Especie (isoforma) H- Acceso % Identidad Table 2. Percentage of identity of the dlkl protein in different animal species Species (isoform) H- Access% Identity
Sus scrofa (B) NP_001041652.1 73  Sus scrofa (B) NP_001041652.1 73
Sus scrofa (C2) NP_001119573.1 80  Sus scrofa (C2) NP_001119573.1 80
Bos Taurus NP_776462.1 81  Bos Taurus NP_776462.1 81
Ovis aries (A) ADE59013.1 82  Ovis aries (A) ADE59013.1 82
Ovis aries (C2) ADE59014.1 79  Ovis aries (C2) ADE59014.1 79
Canis lupus familiaris XP_54798.2 88  Canis lupus familiaris XP_54798.2 88
Mus musculus NP_034182.2 85  Mus musculus NP_034182.2 85
Mus musculus (3) NP_001177633.1 81  Mus musculus (3) NP_001177633.1 81
Mus musculus (4) NP_001177634.1 75  Mus musculus (4) NP_001177634.1 75
Rattus norvegicus NP_446196.1 85  Rattus norvegicus NP_446196.1 85
El término "% de identidad" entre dos secuencias de aminoácidos, tal como se entiende en la presente invención, se refiere al número de posiciones aminoacídicas sobre la longitud total de la secuencia que se compara, donde todos los aminoácidos en esa posición son idénticos. The term "% identity" between two amino acid sequences, as understood in the present invention, refers to the number of amino acid positions over the total length of the sequence being compared, where all amino acids in that position are identical.
Por lo tanto, en la presente invención el término "proteína de la invención" se refiere a proteínas funcionales generadas por corte y empalme alternativo de la secuencia que codifica para la SEQ ID NO: 1 o a proteínas con al menos el 70% de identidad a la secuencia de aminoácido SEQ ID NO: 2, o a cualquiera de sus fragmentos. El término "secuencia de la invención" se refiere a la secuencia de nucleótidos que codifica para la "proteína de la invención". En el contexto de la presente invención, Dlk1 se define también por una secuencia de nucleótidos o polinucleótidos, que constituye la secuencia codificante de la proteína dlkl , y que comprendería diversas variantes procedentes de: Therefore, in the present invention the term "protein of the invention" refers to functional proteins generated by alternative splicing of the sequence encoding SEQ ID NO: 1 or to proteins with at least 70% identity at the amino acid sequence SEQ ID NO: 2, or any of its fragments. The term "sequence of the invention" refers to the nucleotide sequence that codes for the "protein of the invention". In the context of the present invention, Dlk1 is also defined by a nucleotide or polynucleotide sequence, which constitutes the coding sequence of the dlkl protein, and which would comprise various variants from:
a) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia aminoacídica de la SEQ ID NO: 2, b) moléculas de ácido nucleico cuya cadena complementaría híbrida con la secuencia polinucleotídica de (a), a) nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of SEQ ID NO: 2, b) nucleic acid molecules whose chain would complement hybrid with the polynucleotide sequence of (a),
c) moléculas de ácido nucleico cuya secuencia difiere de (a) y/o (b) debido a la degeneración del código genético, c) nucleic acid molecules whose sequence differs from (a) and / or (b) due to the degeneracy of the genetic code,
d) moléculas de ácido nucleico que codifican un polipéptido que comprende la secuencia amínoacídíca con una identidad de al menos un 70%, un 80%, un 90%, un 95%, un 98% o un 99% con la SEQ ID NO: 2; en las que el polipéptido codificado por dichos ácidos nucleicos posee la actividad y las características estructurales de la proteína dlkl . d) nucleic acid molecules encoding a polypeptide comprising the aminoacidic sequence with an identity of at least 70%, 80%, 90%, 95%, 98% or 99% with SEQ ID NO: 2; wherein the polypeptide encoded by said nucleic acids possesses the activity and structural characteristics of the dlkl protein.
e) moléculas de ácido nucleico que codifican para las isoformas o secuencias de aminoácidos homologas a la SEQ ID NO: 2 con al menos un 70% de identidad con la secuencia de aminoácidos de la SEQ ID NO: 2. e) nucleic acid molecules encoding the isoforms or amino acid sequences homologous to SEQ ID NO: 2 with at least 70% identity with the amino acid sequence of SEQ ID NO: 2.
Se entiende por "construcción génica" aquellas construcciones genéticas de DNA capaces de transcribirse a un polipéptido o fragmento del mismo, que codifican para la proteína de la invención, en adelante "construcción génica de la invención". Dicha construcción genética de DNA dirigiría la transcripción in vitro o intracelular de la secuencia de dlkl o fragmento de la misma, y comprende, al menos, uno de los siguientes tipos de secuencias: a) ácido nucleico preferentemente de doble cadena, que comprende, al menos, la secuencia codificante de dlkl para su transcripción in vitro, o intracelular, b) un cassette de expresión que comprende el ácido nucleico unido operativamente a elementos de control de la transcripción y opcionalmente de traducción; c) una secuencia de nucleótidos de DNA, preferentemente de doble cadena, correspondiente a un sistema o vector de expresión génica que comprende la secuencia codificante de la secuencia de dlkl operativamente enlazada con, al menos, un promotor que dirija la transcripción de dicha secuencia de nucleótidos de interés, y con otras secuencias necesarias o apropiadas para la transcripción y su regulación adecuada en tiempo y lugar, por ejemplo, señales de inicio y terminación, sitios de corte, señal de poliadenilación, origen de replicación, activadores transcripcionales {enhancers), silenciadores transcripcionales (silencers), etc, donde preferentemente el vector se selecciona de la lista que comprende: plásmido, bácmido, cromosoma artificial de levaduras (YACs), cromosoma artificial de bacterias (BACs), cromosoma artificial derivado del bacteriófago P1 (PACs), cósmido o virus con un origen de replicación heterólogo. "Gene construct" means those genetic DNA constructs capable of transcribing to a polypeptide or fragment thereof, which code for the protein of the invention, hereinafter "gene construct of the invention". Said genetic construction of DNA would direct the in vitro or intracellular transcription of the dlkl sequence or fragment thereof, and comprises at least one of the following types of sequences: a) preferably double stranded nucleic acid, comprising less, the dlkl coding sequence for transcription in vitro, or intracellular, b) an expression cassette comprising nucleic acid operably linked to transcription control elements and optionally translation; c) a DNA nucleotide sequence, preferably double stranded, corresponding to a gene expression system or vector comprising the coding sequence of the dlkl sequence operably linked to at least one promoter that directs the transcription of said sequence of nucleotides of interest, and with other sequences necessary or appropriate for transcription and their appropriate regulation in time and place, for example, start and end signals, cut sites, polyadenylation signal, origin of replication, transcriptional activators {enhancers), transcriptional silencers (silencers), etc., where preferably the vector is selected from the list comprising: plasmid, bacmid, artificial yeast chromosome (YACs), artificial bacterial chromosome (BACs), chromosome artificial derivative of bacteriophage P1 (PACs), cosmid or virus with a heterologous origin of replication.
El término "medicamento", tal y como se usa en esta memoria, hace referencia a cualquier sustancia usada para prevención, diagnóstico, alivio, tratamiento o curación de enfermedades en el hombre y los animales. En el contexto de la presente invención se refiere, a una composición capaz de inhibir la angiogénesis. The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, it refers to a composition capable of inhibiting angiogenesis.
"Tratamiento" se refiere a tanto el tratamiento terapéutico como el profiláctico o medidas preventivas. Aquellas situaciones susceptibles de tratamiento incluyen las ya asociadas con alteraciones así como en aquellas en las que se previene la alteración. Una "alteración" es cualquier condición que se beneficiaría del tratamiento con la composición de la invención, tal y como se describe en el presente documento.  "Treatment" refers to both therapeutic and prophylactic treatment or preventive measures. Those situations susceptible to treatment include those already associated with alterations as well as in those in which the alteration is prevented. An "alteration" is any condition that would benefit from treatment with the composition of the invention, as described herein.
El término "inhibición" como se usa aquí, se refiere principalmente a que el medicamento inhibe (disminuye) la formación de vasos sanguíneos y por tanto la angiogénesis. La angiogénesis a la que se refiere la presente invención es la que consiste en la formación de nuevas ramificaciones de vasos sanguíneos (angiotubos o mícrovasos en la presente invención) a partir de vasos sanguíneos existentes que se produce en condiciones patológicas (angiogénesis patológica). Por lo tanto, la presente invención se refiere al uso de una construcción géníca que comprende la secuencia que codifica para dlkl para la preparación de un medicamento para la inhibición de la angiogénesis asociada a patologías que se seleccionan de la lista que comprende: cáncer, aterosclerosis, retinopatía diabética, artritis reumatoide, psoriasis y degeneración macular. Preferiblemente la angiogénesis que se inhibe es la angiogénesis tumoral. The term "inhibition" as used herein, mainly refers to the drug inhibiting (decreasing) the formation of blood vessels and therefore angiogenesis. The angiogenesis referred to in the present invention is that which consists in the formation of new branches of blood vessels (angiotubes or mycrovases in the present invention) from existing blood vessels that occurs under pathological conditions (pathological angiogenesis). Therefore, the present invention relates to the use of a gene construct comprising the sequence coding for dlkl for the preparation of a medicament for the inhibition of angiogenesis associated with pathologies that are selected from the list comprising: cancer, atherosclerosis , diabetic retinopathy, rheumatoid arthritis, psoriasis and macular degeneration. Preferably the angiogenesis that is inhibited is tumor angiogenesis.
Las composiciones de la presente invención permiten la transfección de la construcción génica de la invención al interior de una célula, in vivo o in vitro. La transfección se podría llevar a cabo, pero sin limitamos a, transfección directa o vectores que faciliten el acceso de la secuencia codificante dlkl al interior de la célula. Por lo tanto, en una realización preferida del primer aspecto de la invención la construcción génica de la invención comprende un vector que se selecciona de la lista que comprende, pero sin limitarse a, plásmido, bácmido, cromosoma artificial de levaduras (YACs), cromosoma artificial de bacterias (BACs), cromosoma artificial derivado del bacteriófago P1 (PACs), cósmido, virus con un origen del replicacíón heterólogo, adenovirus, retrovirus, lentivirus, virus del Herpes simplex, o cualquiera de sus combinaciones. Así, por ejemplo, las construcciones génicas de la presente invención, pueden conjugarse con péptidos de liberación u otros compuestos para favorecer el transporte al interior de la célula. En una realización aún más preferida, la construcción génica comprende un plásmido como vector. En otra realización aún más preferida, la construcción génica comprende un adenovirus como vector. The compositions of the present invention allow the transfection of the gene construct of the invention into a cell, in vivo or in vitro. Transfection could be carried out, but not limited to, direct transfection or vectors that facilitate access of the dlkl coding sequence into the cell. Therefore, in a preferred embodiment of the first aspect of the invention the gene construct of the invention comprises a vector that is selected from the list comprising, but not limited to, plasmid, bellow, artificial yeast chromosome (YACs), chromosome. Artificial bacteria (BACs), artificial chromosome derived from bacteriophage P1 (PACs), cosmid, virus with an origin of heterologous replication, adenovirus, retrovirus, lentivirus, Herpes simplex virus, or any combination thereof. Thus, for example, the gene constructs of the present invention can be conjugated with release peptides or other compounds to promote transport into the cell. In an even more preferred embodiment, the gene construct comprises a plasmid as a vector. In another even more preferred embodiment, the gene construct comprises an adenovirus as a vector.
La construcción génica que comprende la secuencia que codifica para dlkl se traduce en el interior de la célula una vez se haya transfectado y codifica para la proteína que va a realizar la función de inhibición en última instancia en el interior de la célula en la presente invención. Por este motivo, la presente invención también se refiere al uso de la proteína recombínante que resulta de la expresión de la construcción génica que comprende la secuencia que codifica para dlkl para la preparación de un medicamento destinado a la inhibición de la angíogénesís, y preferentemente de la angíogénesís tumoral. En una realización preferida, dicho medicamento es decir, el que comprende la construcción génica de la invención o la proteína de la invención para la inhibición de la angíogénesís, además comprende, al menos, un excipiente y/o al menos, un vehículo farmacológicamente aceptable. Otra realización preferida se refiere a que dicho medicamento además comprende al menos otro principio activo. El término "excipiente" hace referencia a una sustancia que ayuda a la absorción del medicamento o composición farmacéutica de la invención, estabiliza dicha composición farmacéutica o ayuda a su preparación en el sentido de darle consistencia o aportar sabores que lo hagan más agradable. Así pues, los excipientes podrían tener la función de mantener los ingredientes unidos como por ejemplo almidones, azúcares o celulosas, función de endulzar, función de colorante, función de protección del medicamento como por ejemplo para aislarlo del aire y/o la humedad, función de relleno de una pastilla, cápsula o cualquier otra forma de presentación como por ejemplo el fosfato de calcio díbásico, función desintegradora para facilitar la disolución de los componentes y su absorción en el intestino, sin excluir otro tipo de excipientes no mencionados en este párrafo. The gene construct comprising the sequence that codes for dlkl is translated into the cell once it has been transfected and encoded for the protein that will ultimately perform the inhibition function inside the cell in the present invention. . For this reason, the present invention also relates to the use of the recombinant protein resulting from the expression of the gene construct comprising the sequence coding for dlkl for the preparation of a medicament intended for the inhibition of angigenesis, and preferably of Tumor angigenesis. In a preferred embodiment, said medicament, that is, that which comprises the gene construct of the invention or the protein of the invention for the inhibition of angigenesis, further comprises at least one excipient and / or at least one pharmacologically acceptable carrier. . Another preferred embodiment refers to said medicament further comprising at least one other active ingredient. The term "excipient" refers to a substance that helps the absorption of the medicament or pharmaceutical composition of the invention, stabilizes said pharmaceutical composition or aids in its preparation in the sense of giving it consistency or providing flavors that make it more pleasant. Thus, the excipients could have the function of keeping the ingredients together such as starches, sugars or cellulose, sweetening function, dye function, drug protection function such as to isolate it from air and / or moisture, function filling a tablet, capsule or any other form of presentation such as, for example, biblical calcium phosphate, a disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph.
Un "vehículo farmacológicamente aceptable" se refiere a aquellas sustancias, o combinación de sustancias, conocidas en el sector farmacéutico, utilizadas en la elaboración de formas farmacéuticas de administración e incluye, pero sin limitarse, sólidos, líquidos, disolventes o tensoactivos. El vehículo puede ser una sustancia inerte o de acción análoga a cualquiera de los compuestos de la presente invención. La función del vehículo es facilitar la incorporación del producto de expresión de la invención así como también de otros compuestos, permitir una mejor dosificación y administración o dar consistencia y forma a la composición farmacéutica. Cuando la forma de presentación es líquida, el vehículo es el diluyente. El término "farmacológicamente aceptable" se refiere a que el compuesto al que hace referencia esté permitido y evaluado de modo que no cause daño a los organismos a los que se administra. A "pharmacologically acceptable vehicle" refers to those substances, or combination of substances, known in the pharmaceutical sector, used in the preparation of pharmaceutical forms of administration and includes, but are not limited to, solids, liquids, solvents or surfactants. The carrier can be an inert substance or action analogous to any of the compounds of the present invention. The function of the vehicle is to facilitate the incorporation of the expression product of the invention as well as other compounds, to allow a better dosage and administration or to give consistency and form to the pharmaceutical composition. When the presentation form is liquid, the vehicle is the diluent. The term "pharmacologically acceptable" refers to the compound referred to being allowed and evaluated so as not to cause damage to the organisms to which it is administered.
La invención se lleva a cabo mediante el suministro de una cantidad eficaz de la construcción génica de la invención, la proteína de la invención o un equivalente biológico funcional de las mismas en un tejido de un animal. Por este motivo, la composición proporcionada por esta invención puede ser facilitada por cualquier vía de administración, para lo cual dicha composición se formulará en la forma farmacéutica adecuada a la vía de administración elegida. La cantidad de la construcción génica de la invención o de la proteína de la invención en dichas composiciones se administra a una concentración terapéuticamente efectiva. En el sentido utilizado en esta descripción, la expresión "concentración terapéuticamente efectiva" se refiere a la concentración de agentes moduladores calculada para producir el efecto deseado y, en general, vendrá determinada, entre otras causas, por las características propias de dichos agentes (y construcciones) y el efecto terapéutico a conseguir. Los adyuvantes y vehículos farmacéuticamente aceptables que pueden ser utilizados en dichas composiciones son los vehículos conocidos por los técnicos en la materia. The invention is carried out by supplying an effective amount of the gene construct of the invention, the protein of the invention or a functional biological equivalent thereof in a tissue of an animal. For this reason, the composition provided by this invention can be provided by any route of administration, for which said composition is formulate in the appropriate pharmaceutical form to the route of administration chosen. The amount of the gene construct of the invention or of the protein of the invention in said compositions is administered at a therapeutically effective concentration. In the sense used in this description, the term "therapeutically effective concentration" refers to the concentration of modulating agents calculated to produce the desired effect and, in general, will be determined, among other causes, by the characteristics of said agents (and constructions) and the therapeutic effect to be achieved. Pharmaceutically acceptable adjuvants and vehicles that can be used in said compositions are the vehicles known to those skilled in the art.
Otro aspecto de la presente invención se refiere al uso de al menos un producto de la expresión de Dlk1 , o cualquiera de sus fragmentos como biomarcador para la determinación de angiogénesis patológica o para determinar la progresión de angiogénesis patológica, en una muestra biológica aislada de un mamífero, preferiblemente humano. Another aspect of the present invention relates to the use of at least one product of the expression of Dlk1, or any of its fragments as a biomarker for the determination of pathological angiogenesis or to determine the progression of pathological angiogenesis, in an isolated biological sample of a mammal, preferably human.
El término "producto de expresión" se refiere a cualquier ARN como por ejemplo, pero sin limitarse, ARN mensajero (ARNm) (secuencia codificada por el cDNA de la SEQ ID NO: 1 ), o cualquiera de sus fragmentos; así como la de cualquier proteína o cualquiera de sus fragmentos que resulte de la expresión de la SEQ ID NO: 1 de la presente invención o posea una homología con el ARN, proteína o fragmentos de los mismos de al menos un 70%. The term "expression product" refers to any RNA such as, but not limited to, messenger RNA (mRNA) (sequence encoded by the cDNA of SEQ ID NO: 1), or any of its fragments; as well as that of any protein or any of its fragments resulting from the expression of SEQ ID NO: 1 of the present invention or having a homology with the RNA, protein or fragments thereof of at least 70%.
El término "biomarcador" en la presente invención se refiere a una molécula que tiene una conexión directa con el riesgo de padecer angiogénesis patológica y sirve para determinar el estado de la enfermedad así como para determinar la progresión de la misma. La muestra biológica aislada de un organismo, como por ejemplo, pero sin limitarse, un humano u otro animal, puede ser un fluido biológico o cualquier tejido celular de dichos organismos. Las enfermedades en las que la alteración de la actividad de dlkl puede ser diagnóstica, y en concreto la angíogénesís patológica, puede ser detectada midiendo la cantidad de ácidos nucleicos (ADN y/o ARN y/o mRNA) que codifican para dlkl , o la cantidad de proteína dlkl que se expresa, en comparación con células normales. La detección de los oligonucleótidos puede hacerse por métodos bien conocidos en el estado de la técnica (como por ejemplo, pero sin limitarse, sondas con nucleótidos marcados, hibridación ADN- ADN o ADN-ARN, amplificación por PCR empleando nucleótidos marcados, la RT-PCR). Procedimientos para detectar la expresión de la proteína Dlkl también son bien conocidos en el estado de la técnica, como por ejemplo anticuerpos poli o monoclonales, ELISA, radioinmunoensayo (RIA), y FACS (fluorescence activated ceíl sorting). The term "biomarker" in the present invention refers to a molecule that has a direct connection with the risk of pathological angiogenesis and serves to determine the disease status as well as to determine the progression of the disease. The biological sample isolated from an organism, such as, but not limited to, a human or other animal, may be a biological fluid or any cellular tissue of said organisms. Diseases in which the alteration of dlkl activity can be diagnostic, and in particular pathological angigenesis, can be detected by measuring the amount of nucleic acids (DNA and / or RNA and / or mRNA) that code for dlkl, or the amount of dlkl protein that is expressed, compared to normal cells. The detection of oligonucleotides can be done by methods well known in the state of the art (such as, but not limited to, probes with labeled nucleotides, DNA-DNA or DNA-RNA hybridization, PCR amplification using labeled nucleotides, RT- PCR). Methods for detecting Dlkl protein expression are also well known in the state of the art, such as poly or monoclonal antibodies, ELISA, radioimmunoassay (RIA), and FACS (fluorescence activated cell sorting).
Por tanto, en otro aspecto de la invención se describe un método para la determinación de angíogénesís patológica que comprende, (a) detectar y/o cuantificar en una muestra biológica aislada de un mamífero, al menos un producto de la expresión de Dlkl , o cualquiera de sus fragmentos. Therefore, in another aspect of the invention a method for the determination of pathological angigenesis is described which comprises, (a) detecting and / or quantifying in a biological sample isolated from a mammal, at least one product of Dlkl expression, or Any of its fragments.
Una realización preferida se refiere a un método de determinación de angíogénesís patológica, donde además comprende el paso (b) la comparación de los datos obtenidos en el paso (a) con datos obtenidos de muestras control para buscar alguna desviación significativa. Otra realización más preferida además comprende el paso (c) atribuir la desviación significativa al desarrollo de angíogénesís patológica en dicho mamífero. El término "muestras control" tal como se entiende en la presente invención se refiere, por ejemplo, pero sin limitarse, a una muestra obtenida de un individuo que no desarrolla una patología asociada a angiogénesis (individuo sano). Este tipo de muestra control, es un muestra control negativa o control negativo para angiogénesis patológica. El término "desviación significativa" tal como se entiende en la presente invención se refiere, a la variación de la expresión de los ácidos nucleicos que codifican para dlkl o la presencia de la proteína dlkl en la muestra aislada, o una variación en la concentración de la proteína de la invención en la muestra aislada respecto de una muestra aislada procedente de un individuo sano donde la diferencia sea estadísticamente significativa. La selección del individuo sano se lleva a cabo por medio de la medida del nivel de uno o más marcadores comunes de angiogénesis patológica. El marcador común es conocido por el experto en la materia y se selecciona de la lista que comprende, pero sin limitarse, VE-cadherínas ("vascular endotelial-cadherín"), VEGF, factor de crecimiento de fibroblastos b (FGF-b), la forma soluble del receptor de VEGF (VEGFR-1 o sFlt-1 ), angiopoyetina (Ang-1 ) factor de Von Wíllebrand, PECAM-1 ("platelet endotelial cell adhesión molecule-1 "), IL-6, p53, factor VIII, glicoproteína CD105, y los gangliósidos GM y Gtlb. En la presente invención "diferencia estadísticamente significativa" se refiere a que existen diferencia estadísticas entre los valores comparados, siendo la probabilidad estadística al menos mayor o menor que 0,05 (p>0,05 o p>0.05) y obteniéndose ésta según el test estadístico aplicable a cada caso. Otro aspecto de la invención comprende un método para determinar la progresión de angiogénesis patológica que comprende, A preferred embodiment refers to a method of determining pathological angigenesis, where step (b) also comprises comparing the data obtained in step (a) with data obtained from control samples to look for any significant deviation. Another more preferred embodiment further comprises step (c) attributing the significant deviation to the development of pathological angigenesis in said mammal. The term "control samples" as understood in the present invention refers, for example, but not limited to a sample obtained from an individual. that does not develop a pathology associated with angiogenesis (healthy individual). This type of control sample is a negative control or negative control sample for pathological angiogenesis. The term "significant deviation" as understood in the present invention refers to the variation of the expression of the nucleic acids encoding dlkl or the presence of the dlkl protein in the isolated sample, or a variation in the concentration of the protein of the invention in the isolated sample with respect to an isolated sample from a healthy individual where the difference is statistically significant. The selection of the healthy individual is carried out by measuring the level of one or more common markers of pathological angiogenesis. The common marker is known to one skilled in the art and is selected from the list comprising, but not limited to, VE-cadherins ("vascular endothelial-cadherin"), VEGF, fibroblast growth factor b (FGF-b), the soluble form of the VEGF receptor (VEGFR-1 or sFlt-1), angiopoietin (Ang-1) von Wíllebrand factor, PECAM-1 ("platelet endothelial cell adhesion molecule-1"), IL-6, p53, factor VIII, CD105 glycoprotein, and GM and Gtlb gangliosides. In the present invention "statistically significant difference" refers to the fact that there is statistical difference between the values compared, the statistical probability being at least greater than or less than 0.05 (p> 0.05 op> 0.05) and this being obtained according to the test statistic applicable to each case. Another aspect of the invention comprises a method for determining the progression of pathological angiogenesis comprising,
(a) determinar una primera concentración de al menos un producto de la expresión de Dlkl , o cualquiera de sus fragmentos en una muestra biológica aislada de un mamífero; (b) determinar una segunda concentración de dicho producto de expresión del paso (a) en una muestra biológica aislada del mismo mamífero tomada posteriormente a la muestra del paso (a), y (c) comparar la segunda concentración obtenida en el paso (b) con la primera concentración obtenida en el paso (a) para buscar una desviación significativa. (a) determining a first concentration of at least one product of Dlkl expression, or any of its fragments in a biological sample isolated from a mammal; (b) determine a second concentration of said expression product of step (a) in an isolated biological sample of the same mammal subsequently taken from the sample of step (a), and (c) compare the second concentration obtained in step (b) with the first concentration obtained in step (a) to look for a significant deviation.
También forma parte de la invención el uso de un kit que comprende la construcción génica que comprende la secuencia que codifica para dlkl para la preparación de un medicamento para la inhibición de la angíogénesis así como el uso del kit que comprende la proteína recombinante que resulta de la expresión de la construcción génica que comprende la secuencia que codifica para dlkl para la preparación de un medicamento para la inhibición de la angíogénesis. Also part of the invention is the use of a kit comprising the gene construct comprising the sequence coding for dlkl for the preparation of a medicament for the inhibition of angigenesis as well as the use of the kit comprising the recombinant protein resulting from the expression of the gene construct comprising the sequence coding for dlkl for the preparation of a medicament for the inhibition of angigenesis.
Los términos "polinucleótido" y "ácido nucleico" se usan aquí de manera intercambiable, refiriéndose a formas poliméricas de nucleótidos de cualquier longitud, tanto ribonucleótidos como desoxirribonucleótidos. The terms "polynucleotide" and "nucleic acid" are used interchangeably herein, referring to polymeric forms of nucleotides of any length, both ribonucleotides and deoxyribonucleotides.
Los términos "péptido", "oligopéptido", "polipéptido" y "proteína" se usan aquí de manera intercambiable, y se refieren a una forma poliméríca de aminoácidos de cualquier longitud, que pueden ser codificantes o no codificantes, química o bioquímicamente modificados. The terms "peptide", "oligopeptide", "polypeptide" and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which may be coding or non-coding, chemically or biochemically modified.
A lo largo de la descripción y las reivindicaciones la palabra "comprende" y sus variantes no pretenden excluir otras características técnicas, aditivos, componentes o pasos. Para los expertos en la materia, otros objetos, ventajas y características de la invención se desprenderán en parte de la descripción y en parte de la práctica de la invención. Las siguientes figuras y ejemplos se proporcionan a modo de ilustración, y no se pretende que sean limitativos de la presente invención. DESCRIPCION DE LAS FIGURAS Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following figures and examples are provided by way of illustration, and are not intended to be limiting of the present invention. DESCRIPTION OF THE FIGURES
Figura 1. Muestra que la expresión de Dlk1 retrasa la migración celular en ensayos in vitro óe cicatrización de heridas (wound healing). Se realizaron ensayos con un plásmído que contenía el cDNA de Dlk1 humana para comprobar el efecto de la sobreexpresión de dlkl en la migración de células endoteliales. A, se muestra que las células adultas de aorta porcina que fueron transfectadas con un plásmído de Dlk1 (Dlk1 ) presentan un retraso significativo en el cierre de las heridas comparadas con células en condiciones básales (basal) o con células que expresan un vector control vacío (pCMV6). B, Las células endoteliales aisladas de pulmón de ratones KO para Dlk1 (pérdida de función) presentan una migración significativamente mayor y una mayor capacidad de re-endotelización comparadas con ratones "wild-type" (WT). Figura 2. Muestra que la sobreexpresión de Dik1 inhibe la formación de angiotubos en sistemas in vitro de Matrigel. Se realizaron ensayos con un plásmído que contenía el cDNA de Dlk1 humana para comprobar el efecto de la sobreexpresión de dlkl en la formación de angiotubos en sistemas in vitro de matrigel. A, Las células endoteliales adultas de aorta porcina transfectadas con el cDNA de Dlkl (Dlkl ) se compararon con células sin transfectar (Basal) o, transfectadas con el plásmido pCMV6 vacío (pCMV6) tanto en células a las que se añadió 5 ng/ml de VEGF directamente al medio de cultivo (VEGF) o sin añadir (No VEGF). B, número de estructuras tubulares que se formaron en presencia de VEGF (barras blancas) y en ausencia de VEGF (barras negras). Figure 1. It shows that Dlk1 expression delays cell migration in in vitro assays or wound healing. Assays were performed with a plasmid containing the human Dlk1 cDNA to verify the effect of dlkl overexpression on endothelial cell migration. A, it is shown that adult swine aorta cells that were transfected with a Dlk1 plasmid (Dlk1) have a significant delay in wound closure compared to cells in basal conditions (basal) or with cells expressing an empty control vector (pCMV6). B, The isolated lung endothelial cells of KO mice for Dlk1 (loss of function) exhibit significantly greater migration and greater re-endothelialization capacity compared to "wild-type" (WT) mice. Figure 2. Shows that overexpression of Dik1 inhibits the formation of angiotubes in Matrigel in vitro systems. Assays were performed with a plasmid containing the human Dlk1 cDNA to verify the effect of dlkl overexpression on the formation of angiotubes in in vitro matrigel systems. A, Adult porcine aorta endothelial cells transfected with Dlkl (Dlkl) cDNA were compared with untransfected cells (Basal) or, transfected with the empty plasmid pCMV6 (pCMV6) in both cells to which 5 ng / ml was added of VEGF directly to the culture medium (VEGF) or without adding (No VEGF). B, number of tubular structures that were formed in the presence of VEGF (white bars) and in the absence of VEGF (black bars).
Figura 3. Muestra que la sobreexpresión de Dlkl disminuye la angiogénesis en ensayo ex vivo en explantes de aorta de ratón. Se realizaron ensayos con anillos de aorta murina embebidos en colágeno para comprobar el efecto de la sobreexpresión de dlkl en la formación de angiotubos en sistemas ex vivo de explantes de aorta de ratón. A, esquema de la construcción génica que contiene un adenovirus tipo5 (Ad tipo 5 dE1/dE3). CMV, promotor de citomegalovirus; Dlk1 HA, la secuencia de Dlk1 a la que se refiere la SEQ ID NO: 1 con la inserción entre el nucleótido 1302 y 1303 de la secuencia del epítopo de hemaglutinina a la que se refiere la SEQ ID NO: 3; cola poli A (poIyA); y "green fluorescente protein" (GFP). B, anillos de aorta de ratón embebidos en colágeno los cuales se infectaron con adenovirus vacío (Ad. Empty), adenovirus con GFP (Ad.GFP), o con adenovirus que contenía el cDNA de Dlk1 humana (Ad.Dlkl ). Se muestran dos ensayos por experimento. C, número de mícrovasos que se formaron en presencia de adenovirus vacío {Ad. Empty), adenovirus con GFP (Ad.GFP), o con adenovirus que contenía el cDNA de Dlk1 humana (Ad.Dlkl ). Figure 3. It shows that Dlkl overexpression decreases angiogenesis in ex vivo assay in mouse aorta explants. Assays with collagen-embedded murine aorta rings were performed to verify the effect of dlkl overexpression on the formation of angiotubes in ex vivo systems of mouse aorta explants. A, scheme of the gene construct containing an adenovirus type5 (Ad type 5 dE1 / dE3). CMV, cytomegalovirus promoter; Dlk1 HA, the sequence of Dlk1 referred to in SEQ ID NO: 1 with the insertion between nucleotide 1302 and 1303 of the hemagglutinin epitope sequence referred to in SEQ ID NO: 3; poly A tail (poIyA); and "green fluorescent protein" (GFP). B, collagen-embedded mouse aorta rings which were infected with empty adenovirus (Ad. Empty), adenovirus with GFP (Ad.GFP), or with adenovirus containing the human Dlk1 cDNA (Ad.Dlkl). Two trials are shown per experiment. C, number of mycrovases that formed in the presence of empty adenovirus {Ad. Empty), adenovirus with GFP (Ad.GFP), or with adenovirus containing the human Dlk1 cDNA (Ad.Dlkl).
Figura 4. Muestra que ia sobreexpresión de Dlk1 disminuye ia angiogénesis en soportes (o matrices) de MatrigeJ in vivo. Se realizaron ensayos in vivo mediante la implantación de soportes (o matrices) de matrígel que contenían adenovirus en el tejido subcutáneo de ratones para comprobar el efecto de la sobreexpresión de dlkl en la inhibición de la angiogéneis en sistemas in vivo. Como control negativo de angiogénesis, se implantaron soportes (matrices) con un inhibidor de la ruta de Notch (DAPT). Los soportes de matrigel contenían VEGF para activar la angiogénesis, como control negativo se implantó un soporte de matrigel con un adenovirus que contenía GFP y que no contenía VEGF. A, soportes de matrigel que se implantaron subcutáneamente en los que se aprecia la angiogénesis producida macroscópicamente por visualización de un color más oscuro que corresponde a la coloración de la sangre localizada en los vasos formados. Ad.GFP, adenovirus con GFP; Ad.DLK, adenovirus que comprende la SEQ. ID. NO: 1 ; Ad.DLK-GFP, adenovirus que comprende la SEQ. ID. NO: 1 y GFP; DAPT, control con el inhibidor DAPT; y DMSO, control con DMSO. B, contenido total de hemoglobina en los soportes de matrígel implantados. EJEMPLOS Figure 4. It shows that the overexpression of Dlk1 decreases the angiogenesis in MatrigeJ supports (or matrices) in vivo. In vivo assays were performed by implanting matrix supports (or matrices) containing adenovirus in the subcutaneous tissue of mice to verify the effect of dlkl overexpression on inhibition of angiogenesis in in vivo systems. As a negative angiogenesis control, supports (matrices) were implanted with a Notch pathway inhibitor (DAPT). The matrigel supports contained VEGF to activate angiogenesis, as a negative control a matrigel support was implanted with an adenovirus containing GFP and not containing VEGF. A, matrigel supports that were implanted subcutaneously in which macroscopically produced angiogenesis can be seen by visualization of a darker color corresponding to the coloration of the blood located in the formed vessels. Ad.GFP, adenovirus with GFP; Ad.DLK, adenovirus comprising SEQ. ID. NO: 1; Ad.DLK-GFP, adenovirus comprising SEQ. ID. NO: 1 and GFP; DAPT, control with the DAPT inhibitor; and DMSO, control with DMSO. B, total hemoglobin content in implanted matrix holders. EXAMPLES
Los siguientes ejemplos específicos que se proporcionan en este documento de patente sirven para ilustrar la naturaleza de la presente invención. Estos ejemplos se incluyen solamente con fines ilustrativos y no han de ser interpretados como limitaciones a la invención que aquí se reivindica. Por tanto, los ejemplos descritos más adelante ilustran la invención sin limitar el campo de aplicación de la misma. EJEMPLO 1. La expresión de Dlk1 retrasa la migración celular en ensayos in vitro de cicatrización de heridas (wound healing). The following specific examples provided in this patent document serve to illustrate the nature of the present invention. These examples are included for illustrative purposes only and should not be construed as limitations on the invention claimed herein. Therefore, the examples described below illustrate the invention without limiting its scope of application. EXAMPLE 1. Dlk1 expression delays cell migration in in vitro trials of wound healing.
Se valoró el uso de una construcción génica que comprende el cDNA de Dlk1 humano en la migración celular de reparación de heridas dado que una fase importante de la angiogénesis es la migración del endotelio para formar los nuevos vasos sanguíneos. The use of a gene construct comprising the human Dlk1 cDNA in wound repair cell migration was assessed since an important phase of angiogenesis is the migration of the endothelium to form the new blood vessels.
Se realizaron ensayos con un plásmido que contenía el cDNA de Dlk1 humana para comprobar el efecto de la sobreexpresión de dlkl en la migración de células endoteliales adultas de aorta porcina y bovina transfectadas con un plásmido que contenía el cDNA humano y se comparó con células sin transfectar y transfectadas con el plásmido vacío.  Assays were performed with a plasmid containing the human Dlk1 cDNA to verify the effect of dlkl overexpression on the migration of adult endothelial cells of porcine and bovine aorta transfected with a plasmid containing the human cDNA and compared with untransfected cells. and transfected with the empty plasmid.
El plásmido utilizado en este ensayo fue el pCMV6-XL4 {secuencia de acceso número AF067196 del NCBI) al que se unió mediante clonación en la región de polilinkers MCS entre las dianas de Not I, la SEQ ID NO: 1 . Además esta construcción génica lleva un epítopo de hemaglutína (HA) para la localización de la expresión mediante anticuerpos anti-hemaglutinina. El epítopo de HA (SEQ ID NO: 3) se situó entre los nucleótidos 1302 y 1303 del cDNA de Dlk1 utilizado en la invención la (SEQ ID NO 1 ). Se observó que las células endoteliales adultas tanto de aorta porcina como bovina transfectadas con el plásmido que comprendía Dlk1 (Dlk1 ) presentaron un retraso significativo en el cierre de las heridas comparadas con células en condiciones básales o con las células que expresaban un vector control, por lo que se muestran únicamente los resultados de las células endoteliales de aorta porcina (Fig. 1 A). The plasmid used in this assay was pCMV6-XL4 {access sequence number AF067196 of the NCBI) to which it was linked by cloning in the region of MCS polilinkers between the targets of Not I, SEQ ID NO: 1. In addition, this gene construct carries a hemagglutin (HA) epitope for localization of expression by anti-hemagglutinin antibodies. The HA epitope (SEQ ID NO: 3) was located between nucleotides 1302 and 1303 of the Dlk1 cDNA used in the invention (SEQ ID NO 1). It was observed that adult endothelial cells of both porcine and bovine aorta transfected with the plasmid comprising Dlk1 (Dlk1) presented a significant delay in wound closure compared with cells in basal conditions or with cells expressing a control vector, by which shows only the results of porcine aorta endothelial cells (Fig. 1 A).
Además, se procedió a evaluar la implicación de la ausencia de expresión de Dlk1 en la migración de endotelio utilizando ratones en los que la expresión del gen Dlk1 se eliminó por completo (ratones knock-out o KO). Para ello se utilizaron células endoteliales aisladas de pulmón de los ratones KO para Dlk1 (que presentaban ausencia de expresión del gen Dlk1 ) y se observó que presentaban una migración significativamente mayor y una mayor capacidad de re-endotelización comparadas con ratones que expresaban Dlk1 (ratones "wild- type'O WT) (Fig. 1 B). In addition, the implication of the absence of Dlk1 expression in endothelial migration was evaluated using mice in which the expression of the Dlk1 gene was completely eliminated (knock-out or KO mice). For this, endothelial cells isolated from the lungs of the KO mice for Dlk1 were used (which had no expression of the Dlk1 gene) and it was observed that they presented significantly greater migration and a greater capacity for re-endothelization compared to mice expressing Dlk1 (mice "wild-type'O WT) (Fig. 1 B).
EJEMPLO 2. La sobreexpresión de Dlk1 inhibe ia formación de angiotubos en sistemas in vitro de Matrigel. Para comprobar la inhibición in vitro de la angiogénesis de una construcción génica que comprendía la secuencia que codifica para dlkl , se utilizó un plásmido que contenía el cDNA humano de dlkl y se comprobó el efecto de la sobreexpresión de dlkl en la formación de angiotubos en sistemas in vitro de matrigel. EXAMPLE 2. Overexpression of Dlk1 inhibits the formation of angiotubes in Matrigel in vitro systems. To verify the in vitro inhibition of angiogenesis of a gene construct comprising the sequence encoding dlkl, a plasmid containing the human dlkl cDNA was used and the effect of dlkl overexpression on the formation of angiotubes in systems was checked. in vitro of matrigel.
Matrigel es el nombre comercial (Beckton Dickinson and Company, BD™) para una mezcla gelatinosa de proteínas secretadas por células de tumor de ratón (sarcoma "Engelberth-Holm-Swarm", EHS) muy rica en proteínas extracelulares de matriz, siendo el mayor componente laminina, seguido de colágeno IV, proteoglicanos y entactina/nidogen, entre otros. Esta mezcla se asemeja al ambiente extracelular complejo encontrado en muchos tejidos y es utilizada en estudios in vitro como substrato de células endotelíales capaces de formar estructuras tubulares de manera similar al proceso de angiogénesis que ocurre in vivo. En la presente invención, las células endotelíales aisladas se embebieron en Matrigel y en presencia de medio de cultivo suplementado o no con VEGF (5ng/ml) se comprobó la formación de redes tubulares. Matrigel is the trade name (Beckton Dickinson and Company, BD ™) for a gelatinous mixture of proteins secreted by mouse tumor cells (sarcoma "Engelberth-Holm-Swarm", EHS) very rich in extracellular matrix proteins, being the largest laminin component, followed by collagen IV, proteoglycans and entactin / nidogen, among others. This mixture resembles the complex extracellular environment found in many tissues and is used in in vitro studies as a substrate of endothelial cells capable of forming tubular structures in a manner similar to the angiogenesis process that occurs in vivo. In the present invention, the isolated endothelial cells were embedded in Matrigel and in the presence of culture medium supplemented or not with VEGF (5ng / ml) the formation of tubular networks was checked.
El plásmido utilizado en este ensayo fue el pCMV6-XL4 (secuencia de acceso número AF067196 del NCBI) al que se unió medíante clonación en la región de polilinkers MCS entre las dianas de Not I, la SEQ ID NO: 1 . Además esta construcción génica lleva un epítopo de hemaglutinína (HA) útil para la localización de la expresión mediante anticuerpos anti-HA. El epítopo de HA (SEQ ID NO: 3) se situó entre los nucleótidos 1302 y 1303 del cDNA de Dlk1 utilizado en la invención la (SEQ ID NO 1 ). Se comparó la generación de angiotubos en células endotelíales adultas de aorta porcina y bovina transfectadas con el plásmido pCMV6 vacío, con el cDNA de Dlkl o con células sin transfectar, tanto en células a las que se añadió VEGF o sin añadir VEGF al medio de cultivo. El número de angiotubos (estructuras tubulares) que se formaron tras transfectar las células endotelíales con el plásmido que contenía el cDNA humano de Dlk1 fue significativamente menor que el número de angiotubos formados tras transfección con el plásmido vacío o en condiciones básales, tanto en presencia de VEGF y en ausencia de VEGF, tanto en endotelio porcino como bovino por lo que se muestran únicamente los resultados de las células endotelíales de aorta porcina (Figs. 2A y 2B). The plasmid used in this assay was pCMV6-XL4 (access sequence number AF067196 of the NCBI) to which it was attached by cloning in the region of MCS polilinkers between the targets of Not I, SEQ ID NO: 1. In addition, this gene construct carries a hemagglutinin (HA) epitope useful for localization of expression by anti-HA antibodies. The HA epitope (SEQ ID NO: 3) was located between nucleotides 1302 and 1303 of the Dlk1 cDNA used in the invention (SEQ ID NO 1). The generation of angiotubes in adult endothelial cells of porcine and bovine aorta transfected with the empty plasmid pCMV6, with the Dlkl cDNA or with non-transfected cells, was compared in either cells to which VEGF was added or without adding VEGF to the culture medium . The number of angiotubes (tubular structures) that formed after transfecting the endothelial cells with the plasmid containing the human Dlk1 cDNA was significantly less than the number of angiotubes formed after transfection with the empty plasmid or in basal conditions, both in the presence of VEGF and in the absence of VEGF, both in porcine and bovine endothelium, so only the results of porcine aorta endothelial cells are shown (Figs. 2A and 2B).
EJEMPLO 3. La sobreexpresión de Dik1 disminuye ia angiogénesis en ensayo ex vivo en explantes de aorta de ratón. Para comprobar la inhibición ex vivo de la angiogénesis de una construcción génica que comprendía la secuencia que codifica para dlkl , se utilizó un adenovírus que contenía el cDNA humano de dlkl y se comprobó el efecto de la sobreexpresion de dlkl en la formación de angiotubos en sistemas ex vivo para lo cual se utilizaron anillos de aorta de ratón embebidos en colágeno tipo I. El adenovirus que contenía bajo el promotor del CMV (cítomegalovirus) la secuencia de Dlkl a la que se refiere la SEQ ID NO: 1 , tenía entre el nucleótido 1302 y 1303 insertada la secuencia del epítopo de hemaglutinina a la que se refiere la SEQ ID NO: 3 seguido de una cola poli A y de GFP para su identificación (Fig. 3 A). El adenovirus era de primera generación tipo 5 deficiente en la región E1 (sin capacidad de replícación) y en la región E3 (con capacidad de incorporación de material genético de hasta 8,2 Kb) (dE1 /dE3). EXAMPLE 3. Overexpression of Dik1 decreases angiogenesis in an ex vivo assay in mouse aortic explants. To verify the ex vivo inhibition of angiogenesis of a gene construct comprising the sequence encoding dlkl, a adenovirus containing the human dlkl cDNA and the effect of dlkl overexpression on the formation of angiotubes in ex vivo systems was verified for which mouse aorta rings embedded in type I collagen were used. The adenovirus contained under the promoter of the CMV (cytomegalovirus) the Dlkl sequence referred to in SEQ ID NO: 1, had between the nucleotide 1302 and 1303 inserted the hemagglutinin epitope sequence referred to in SEQ ID NO: 3 followed by a tail poly A and GFP for identification (Fig. 3 A). The adenovirus was first-generation type 5 deficient in the E1 region (without capacity for replication) and in the E3 region (with the ability to incorporate genetic material up to 8.2 Kb) (dE1 / dE3).
La sobreexpresion de Dlkl en los anillos de aorta mediante adenovirus disminuyó significativamente la formación de angiotubos, comparando con explantes de aorta infectados con adenovirus que expresaban GFP o un vector control (Figs. 3B y 3C). Overexpression of Dlkl in the aortic rings by adenovirus significantly decreased the formation of angiotubes, compared with adenovirus-infected aortic explants expressing GFP or a control vector (Figs. 3B and 3C).
EJEMPLO 4. La sobreexpresion de Dik1 disminuye ia angiogénesis en soportes de matrigel in vivo. EXAMPLE 4. Overexpression of Dik1 decreases angiogenesis in matrigel supports in vivo.
Para comprobar la inhibición in vivo de la angiogénesis de una construcción génica que comprendía la secuencia que codifica para dlkl , se utilizó el adenovirus explicado previamente (Fig. 3 A) que contenía el cDNA humano de dlkl y se comprobó el efecto de la sobreexpresion de dlk en la formación de angiotubos en sistemas in vivo para lo cual se utilizaron soportes de matrigel implantados subcutáneamente en ratones. To verify the inhibition in vivo of the angiogenesis of a gene construct comprising the sequence encoding dlkl, the adenovirus previously explained (Fig. 3 A) containing the human dlkl cDNA was used and the effect of overexpression of dlk in the formation of angiotubes in in vivo systems for which matrigel supports implanted subcutaneously in mice were used.
Se observó la angiogénesis producida tanto macroscópicamente por visualización de un color más oscuro que corresponde a la coloración de la sangre localizada en los vasos formados (Fig. 4A) como mediante la valoración del contenido total de hemoglobina en los soportes una vez se retiraron del sitio de implante (Fig. 4B). En el caso de los adenovirus que contenían el cDNA de Dlk1 (SEQ ID NO: 1 ) (Ad.DLK1 -GFP), se observó una presencia significativamente menor de angiogéneis comparado con los adenovirus control y similar a la producida por el inhibidor de la ruta e señalización de Notch DAPT {Figs. 4A y 4B). Angiogenesis produced both macroscopically was observed by visualization of a darker color corresponding to the coloration of the blood located in the formed vessels (Fig. 4A) and by the assessment of the total hemoglobin content in the supports once they were removed from the site of implant (Fig. 4B). In the case of adenoviruses containing the Dlk1 cDNA (SEQ ID NO: 1) (Ad.DLK1 -GFP), a significantly lower presence of angiogenesis was observed compared to the control adenoviruses and similar to that produced by the inhibitor of route and signaling of Notch DAPT {Figs. 4A and 4B).
MATERIAL Y MÉTODOS EMPLEADOS. MATERIAL AND METHODS USED.
Obtención de células endoteliales de aorta porcina y bovina Obtaining endothelial cells of porcine and bovine aorta
La aorta porcina o bovina aislada y lavada con PBS se sometió a tratamiento con colagenasa (0.03 % peso/volumen) (Sigma) durante 15 minutos a 379 C. Tras la centrifugación, los precipitados se resuspendieron en medio RPMI (Gibco) complementado con 15% de (suero fetal de ternera) FCS (Gibco), 5% penicilina/estreptomicina/fungizona (Gibco) y 5% heparina (Sigma) y cultivados en botellas de cultivo de 25 cm2. La pureza de las preparaciones se analizó por ínmunofluorescencia usando el anticuerpo antí-Factor VIII (von Willebrand) (3
Figure imgf000024_0001
Obtención de los ratones Knock Out para Dlk1. The porcine or bovine aorta isolated and washed with PBS was treated with collagenase (0.03% weight / volume) (Sigma) for 15 minutes at 37 9 C. After centrifugation, the precipitates were resuspended in RPMI medium (Gibco) supplemented with 15% of (fetal calf serum) FCS (Gibco), 5% penicillin / streptomycin / fungizone (Gibco) and 5% heparin (Sigma) and grown in 25 cm 2 culture bottles. The purity of the preparations was analyzed by immunofluorescence using the anti-Factor VIII (von Willebrand) antibody (3
Figure imgf000024_0001
Obtaining Knock Out mice for Dlk1.
La construcción de los ratones knock out (KO) fue realizada conforme a la bibliografía existente (Raghumandan et al. 2008. Stem Cells Dev 17:795-507). Brevemente, el gen de dlkl fue silenciado al insertar un cassette resistente a Neomicina que sustituyó 3.8 kbp del alelo endógeno, incluyendo el promotor y los tres primeros exones de Dlk1 . Dicha construcción fue transfectada por electroporación a células embrionarias de ratones de la cepa SvJ129. Las quimeras fueron generadas y establecidos los cruces pertinentes para conseguir el ratón Dlk17" . El análisis del genotipo fue realizado por "Southern blot" o PCR usando los siguientes cebadores de las secuencias recogidas en SEQ ID NO: 4 (cebador sentido para Dlk1 ), SEQ ID NO: 5 (cebador sentido para Neomicina) y SEQ ID NO: 6 (cebador antisentido que híbrida con la secuencia complementaria en el intrón 3 de Dlk1 ): El análisis del fenotipo se realizó después de tres cruces con la cepa C57BI/6, y seguidamente, los cruces entre heterocigotos fueron usados para generar los homocigotos, heterocigotos y wíld-type. The construction of knock out mice (KO) was performed according to the existing literature (Raghumandan et al. 2008. Stem Cells Dev 17: 795-507). Briefly, the dlkl gene was silenced by inserting a Neomycin-resistant cassette that replaced 3.8 kbp of the endogenous allele, including the promoter and the first three exons of Dlk1. Said construct was transfected by electroporation to embryonic cells of mice of strain SvJ129. The chimeras were generated and established the relevant crosses to achieve the Dlk1 7 " mouse. Genotype analysis was performed by" Southern blot "or PCR using the following primers of the sequences collected in SEQ ID NO: 4 (sense primer for Dlk1) , SEQ ID NO: 5 (sense primer for Neomycin) and SEQ ID NO: 6 (antisense primer that hybridizes with the complementary sequence in intron 3 of Dlk1): The phenotype analysis was performed after three crosses with strain C57BI / 6, and then crossings between heterozygotes They were used to generate homozygotes, heterozygotes and wíld-type.
Obtención de células endoteliales de pulmón de ratones KO. Obtaining lung endothelial cells from KO mice.
Una vez extraídos los pulmones del ratón KO para Dlk-1 , estos se lavaron en medio Ham-F12 (Gibco) y se disgregaron mecánicamente antes de someterlos a tratamiento con colagenasa (0.1 % peso/volumen) durante 1 hora a 37eC. El sobrenadante de la centrifugación se eliminó y el precipitado se cultivó durante 24h a 37s C. La preparación celular se sometió primero a una selección negativa usando anticuerpos frente a macrófagos (CD16/CD32) (1 ,6 μg/ml) (BD™) y anti-lgG-recubierto de partículas magnéticas (6,6x105 partículas/ml) (Invitrogen), y después de 48 horas, a una doble selección positiva usando el anticuerpo específico para células endoteliales ICAM-2 (3,3 μg/ml) (BD™) y partículas magnéticas. La pureza de la preparación se analizó por citometría de flujo usando el anticuerpo ICAM-2 (3 μg/ml) (BD™). Once the lungs of the KO mouse for Dlk-1 were removed, they were washed in Ham-F12 medium (Gibco) and mechanically disintegrated before being treated with collagenase (0.1% weight / volume) for 1 hour at 37 ° C. The centrifugal supernatant was removed and the precipitate was cultured for 24 hours at 37 s C. The cell preparation was first subjected to a negative selection using macrophage antibodies (CD16 / CD32) (1.6 μg / ml) (BD ™ ) and anti-lgG-coated magnetic particles (6.6x10 5 particles / ml) (Invitrogen), and after 48 hours, to a double positive selection using the specific antibody for endothelial cells ICAM-2 (3.3 μg / ml) (BD ™) and magnetic particles. The purity of the preparation was analyzed by flow cytometry using the ICAM-2 antibody (3 μg / ml) (BD ™).
Ensayo de "wound healing". "Wound healing" trial.
Las células endoteliales adultas de aorta porcina, bovina, y de pulmón de ratón se cultivaron en monocapa en presencia de 10% FBS (20% en el caso de las células de pulmón). Usando una punta estéril de pipeta, se realizaron incisiones en la monocapa y la velocidad y tiempo de migración de las células al área de la "herida" o re-endotelización se monitorizó de forma continua durante 24 horas usando un microscopio de contraste de fases (10x objetivo) acoplado a una cámara monocromática Hamamatsu CCD C9100-02. En el caso de las células que expresan el vector control pCMV6 o Dlk1 , las transfecciones se realizaron 24 horas antes de realizar las incisiones. La cuantifícación se realizó midiendo el área que quedaba por cerrar al tiempo del cierre de la primera "herida". Adult endothelial cells of porcine, bovine, and mouse lung aorta were cultured in monolayer in the presence of 10% FBS (20% in the case of lung cells). Using a sterile pipette tip, incisions were made in the monolayer and the speed and time of migration of the cells to the "wound" area or re-endothelialization was monitored continuously for 24 hours using a phase contrast microscope ( 10x objective) coupled to a Hamamatsu CCD C9100-02 monochrome camera. In the case of cells expressing the control vector pCMV6 or Dlk1, transfections were performed 24 hours before making the incisions. The quantification was performed measuring the area that remained to close at the time of the closure of the first "wound."
Transfección de células endoteliales con ei plásmido pCMV6. Transfection of endothelial cells with plasmid pCMV6.
Todas las transfeccíones se realizaron usando Lipofectamina 2000 (Invitrogen), siguiendo las condiciones recomendadas por la compañía. Las células endoteliales de aorta bovina y/o porcina al 60-80% de confluencia se transfectaron con 0.25 de plásmido del vector control pCMV6 o de Dlk1 , y se usaron en los distintos ensayos 24 horas post-transfección. All transfections were performed using Lipofectamine 2000 (Invitrogen), following the conditions recommended by the company. Bovine and / or porcine aorta endothelial cells at 60-80% confluence were transfected with 0.25 plasmid from the control vector pCMV6 or Dlk1, and used in the various tests 24 hours post-transfection.
Ensayo de angiogénesis in vitro con sistemas de matrigei. In vitro angiogenesis assay with matrigei systems.
Las células endoteliales adultas aisladas de aorta porcina y/o bovina (3x104 células por pocilio en placas de 96 pocilios) se embebieron en 40 μΙ de matriz extracelular de Matrigei (BD™ Bioscience) en presencia de 10% suero y suplementadas o no, según el caso, con 5 ng/ml de VEGF (R&D Systems). Después de 4-6 horas de incubación a 37s C, el número de estructuras tubulares (angiotubos) fue cuantifícado usando el programa Angioquant (MATLAB) y confirmado mediante contaje visual. Adult endothelial cells isolated from porcine and / or bovine aorta (3x10 4 cells per well in 96-well plates) were embedded in 40 μΙ of Matrigei extracellular matrix (BD ™ Bioscience) in the presence of 10% serum and supplemented or not, as appropriate, with 5 ng / ml of VEGF (R&D Systems). After 4-6 hours of incubation at 37 s C, the number of tubular structures (angiotubes) was quantified using the Angioquant program (MATLAB) and confirmed by visual counting.
Infección con adenovirus. Infection with adenovirus.
Las infecciones con adenovirus se realizaron a una concentración de 1 x109 unidades formadoras de placas (ufp). En el caso de los anillos de aorta, los explantes se incubaron con los adenovirus durante 24 horas antes de embeberlos en la matriz de colágeno tipo I (SERVA). En los ensayos de angiogénesis in vivo, los adenovirus se mezclaron con los soportes de matrigei y demás componentes (VEGF y/o DAPT) (Biomol) antes de inyectarlos a los anímales. Ensayo de angiogénesis ex vivo con expiantes de anillos de aorta. Infections with adenovirus were performed at a concentration of 1 x 10 9 plaque forming units (pfu). In the case of aortic rings, explants were incubated with adenoviruses for 24 hours before immersing them in the type I collagen matrix (SERVA). In the in vivo angiogenesis assays, adenoviruses were mixed with matrigei supports and other components (VEGF and / or DAPT) (Biomol) before injecting them into the animals. Ex vivo angiogenesis test with expiants of aortic rings.
La aorta de ratón se diseccionó y transfirió rápidamente a PBS frío. El tejido fibroadiposo se separó de la pared de la aorta evitando dañarla, y se troceó en segmentos de 1 mm de longitud. Usando puntas de pipetas previamente enfriadas, se añadió a cada pocilio de una placa de 96, 40 μΙ de la siguiente mezcla de colágeno tipo I (7.5 volúmenes de 2 mg/ml) (SERVA), 1 volumen de medio 10x MEM (Gibco) y 1 .5 volúmenes de NaHC03 (15.6 mg/ml) y 0.1 volúmenes de NaOH 1 M para ajustar el pH a 7.4. Dicha mezcla se dejó solidificar a 37e C durante 10 minutos antes de embeber los anillos de aorta. Tras este tiempo, se procedió a incubar la placa otros 10 minutos a 37- C y se añadieron otros 40 μΙ de la mezcla de colágeno a cada explante. Tras otra incubación de 10 minutos, se añadieron 100 μΙ de MCDB131 (Gibco) suplementado con 25 mM NaHCO3, 2.5% de suero de ratón (Source BioScience), 1 % de glutamina y 100 U/ml penicilina/estreptomicina. Los cultivos se mantuvieron a 37Q C durante 6 días. The mouse aorta was dissected and quickly transferred to cold PBS. The fibro-fatty tissue was separated from the wall of the aorta avoiding damage, and was cut into segments of 1 mm in length. Using precooled pipette tips, a 96.40 μΙ plate of the following type I collagen mixture (7.5 volumes of 2 mg / ml) (SERVA), 1 volume of 10x MEM medium (Gibco) was added to each well and 1.5 volumes of NaHC0 3 (15.6 mg / ml) and 0.1 volumes of 1 M NaOH to adjust the pH to 7.4. This mixture was allowed to solidify at 37 C for 10 minutes and before imbibing aortic rings. After this time, the plate was incubated for another 10 minutes at 37 ° C and another 40 μΙ of the collagen mixture was added to each explant. After another 10 minute incubation, 100 μΙ of MCDB131 (Gibco) supplemented with 25 mM NaHCO 3 , 2.5% mouse serum (Source BioScience), 1% glutamine and 100 U / ml penicillin / streptomycin were added. Cultures were maintained at 37 Q C for 6 days.
En los casos de infecciones con adenovirus, las aortas limpias de tejido adiposo se cortaron por la mitad y se incuban con 1 x109 ufp de adenovirus en medio MCDB131 con suero a baja concentración (2%) durante 24 horas. Después de este tiempo, las aortas infectadas se lavaron para eliminar restos de adenovirus, se cortaron en anillos de 1 mm de longitud y se embebieron en la matriz de colágeno tipo I. Los explantes se analizaron después de 6 días de incubación usando un microscopio de fluorescencia. La angiogénesis se evaluó contando el número de microvasos (angiotubos) por anillo. In cases of adenovirus infections, the clean aortas of adipose tissue were cut in half and incubated with 1 x 10 9 pfu of adenovirus in MCDB131 medium with low concentration serum (2%) for 24 hours. After this time, the infected aortas were washed to remove adenovirus residues, cut into rings of 1 mm in length and embedded in the type I collagen matrix. The explants were analyzed after 6 days of incubation using a microscope. fluorescence. Angiogenesis was evaluated by counting the number of microvessels (angiotubes) per ring.
Ensayo de angiogénesis in vivo con Piugs de matrigel. Angiogenesis assay in vivo with matrigel Piugs.
Se inyectó Matrigel bajo en factores de crecimiento (500 μΙ) suplementado con heparína (64 U/ml) y según los casos, adenovirus (1 x109 ufp), DAPT (10 mg/Kg peso), vehículo DMSO (dimetilsulfóxido) ó VEGF (250 ng/ml), de forma subcutánea en el abdomen superior del ratón anestesiado. Después de 10 días, la angiogénesis in vivo se determinó analizando el contenido de hemoglobina de los plugs (soportes) mediante método colorímétrico (TMB, Sigma). Low growth factor Matrigel (500 μΙ) supplemented with heparin (64 U / ml) was injected and, depending on the case, adenovirus (1 x 10 9 pfu), DAPT (10 mg / kg weight), DMSO vehicle (dimethylsulfoxide) or VEGF (250 ng / ml), so subcutaneous in the upper abdomen of the anesthetized mouse. After 10 days, in vivo angiogenesis was determined by analyzing the hemoglobin content of the plugs (supports) by colorimetric method (TMB, Sigma).
Inhibición de ia ruta de señalización de Notch con DAPT. Inhibition of the Notch signaling path with DAPT.
La inhibición de la ruta de Notch in vivo se realizó añadiendo a la mezcla de Matrigel (500 μΙ) heparina (64 U/ml) y VEGF (250 ng/ml) el compuesto DAPT a una concentración de (10 mg/Kg peso), antes de inyectarla subcutáneamente al ratón. Inhibition of the Notch pathway in vivo was performed by adding to the Matrigel (500 μΙ) heparin (64 U / ml) and VEGF (250 ng / ml) mixture the DAPT compound at a concentration of (10 mg / kg weight) , before injecting it subcutaneously to the mouse.

Claims

REIVINDICACIONES
1. Uso de una construcción génica que comprende la secuencia que codifica para dlkl para la preparación de un medicamento para la inhibición de la angiogénesis. 1. Use of a gene construct comprising the sequence encoding dlkl for the preparation of a medicament for the inhibition of angiogenesis.
2. Uso según la reivindicación 1 , donde la angiogénesis es angiogénesis tu moral. 2. Use according to claim 1, wherein the angiogenesis is your moral angiogenesis.
3. Uso según cualquiera de las reivindicaciones 1 ó 2 donde la construcción génica comprende un vector que se selecciona de la lista que comprende: plásmido, bácmido, cromosoma artificial de levaduras (YACs), cromosoma artificial de bacterias (BACs), cromosoma artificial derivado del bacteriófago P1 (PACs), cósmico, virus con un origen de replicación heterólogo, adenovirus, retrovirus, lentivírus virus del Herpes simplex, o cualquiera de sus combinaciones. 3. Use according to any of claims 1 or 2 wherein the gene construct comprises a vector that is selected from the list comprising: plasmid, bacmid, artificial yeast chromosome (YACs), artificial bacterial chromosome (BACs), derived artificial chromosome of the bacteriophage P1 (PACs), cosmic, virus with an origin of heterologous replication, adenovirus, retrovirus, lentivirus Herpes simplex virus, or any combination thereof.
4. Uso según cualquiera de las reivindicaciones 1 a 3 donde la construcción génica comprende un plásmido como vector. 4. Use according to any one of claims 1 to 3 wherein the gene construct comprises a plasmid as a vector.
5. Uso según cualquiera de las reivindicaciones 1 a 3 donde la construcción génica comprende un adenovirus como vector. 5. Use according to any of claims 1 to 3 wherein the gene construct comprises an adenovirus as a vector.
6. Uso de la proteína recombinante que resulta de la expresión de la construcción génica descrita en cualquiera de las reivindicaciones 1 a 5 para la preparación de un medicamento para la inhibición de la angiogénesis. 6. Use of the recombinant protein resulting from the expression of the gene construct described in any of claims 1 to 5 for the preparation of a medicament for the inhibition of angiogenesis.
7. Uso de la proteína recombinante según la reivindicación 6 donde la angiogénesis es angiogénesis tumoral. 7. Use of the recombinant protein according to claim 6 wherein the angiogenesis is tumor angiogenesis.
8. Uso según cualquiera de las reivindicaciones 1 a 7, donde el medicamento además comprende al menos un excipiente y/o al menos un vehículo farmacéuticamente aceptable. 8. Use according to any of claims 1 to 7, wherein the medicament further comprises at least one excipient and / or at least one pharmaceutically acceptable carrier.
9. Uso según cualquiera de las reivindicaciones 1 a 8, donde el medicamento además comprende al menos otro principio activo. 9. Use according to any of claims 1 to 8, wherein the medicament further comprises at least one other active ingredient.
10. Uso según cualquiera de las reivindicaciones 1 a 9, donde el medicamento se presenta en una forma adaptada a la administración por vía oral, parenteral o intradérmica. 10. Use according to any one of claims 1 to 9, wherein the medicament is presented in a form adapted to oral, parenteral or intradermal administration.
1 1. Uso de al menos un producto de la expresión de Dlk1 , o de cualquiera de sus fragmentos como biomarcador para la determinación de angiogénesis o para determinar la progresión de angiogénesis patológica, en una muestra biológica aislada de un mamífero. 1 1. Use of at least one product of the expression of Dlk1, or of any of its fragments as a biomarker for the determination of angiogenesis or to determine the progression of pathological angiogenesis, in an isolated biological sample of a mammal.
12. Método para la determinación de angiogénesis patológica que comprende, a) detectar y/o cuantificar en una muestra biológica aislada de un mamífero, al menos un producto de la expresión de Dlk1 , o cualquiera de sus fragmentos. 12. Method for the determination of pathological angiogenesis comprising, a) detecting and / or quantifying in a biological sample isolated from a mammal, at least one product of the expression of Dlk1, or any of its fragments.
13. Método según la reivindicación 12, donde además comprende el paso b) comparar los datos obtenidos en el paso (a) con datos obtenidos con al menos una muestra control. 13. Method according to claim 12, wherein further comprising step b) comparing the data obtained in step (a) with data obtained with at least one control sample.
14. Método según la reivindicación 13, donde además comprende el paso c) de atribuir la desviación significativa obtenida en el paso (b) al desarrollo de angiogénesis patológica en dicho mamífero. 14. Method according to claim 13, further comprising step c) of attributing the significant deviation obtained in step (b) to the development of pathological angiogenesis in said mammal.
15. Método para determinar la progresión de angiogénesis patológica que comprende, a) Determinar una primera concentración de al menos un producto de la expresión de Dlk1 , o cualquiera de sus fragmentos en una muestra biológica aislada de un mamífero. 15. Method for determining the progression of pathological angiogenesis comprising, a) Determine a first concentration of at least one product of the expression of Dlk1, or any of its fragments in a biological sample isolated from a mammal.
b) Determinar una segunda concentración de dicho producto de expresión del paso (a) en una muestra biológica aislada del mismo mamífero tomada posteriormente a la muestra del paso (a), y c) Comparar la segunda concentración obtenida en el paso (b) con la primera concentración obtenida en el paso (a) para buscar una desviación significativa.  b) Determine a second concentration of said expression product of step (a) in an isolated biological sample of the same mammal subsequently taken from the sample of step (a), and c) Compare the second concentration obtained in step (b) with the First concentration obtained in step (a) to find a significant deviation.
16. Uso de un kit que comprende una construcción génica que comprende la secuencia que codifica para dlkl o cualquiera de sus productos de expresión para el diagnóstico de la angiogénesis patológica. 16. Use of a kit comprising a gene construct comprising the sequence encoding dlkl or any of its expression products for the diagnosis of pathological angiogenesis.
PCT/ES2011/070728 2010-10-21 2011-10-21 USE OF Dlk1 AS AN ANGIOGENESIS INHIBITOR WO2012052594A2 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ES201031547A ES2381161B1 (en) 2010-10-21 2010-10-21 USE OF DLK1 AS AN ANGIOGENESIS INHIBITOR.
ESP201031547 2010-10-21

Publications (2)

Publication Number Publication Date
WO2012052594A2 true WO2012052594A2 (en) 2012-04-26
WO2012052594A3 WO2012052594A3 (en) 2014-12-04

Family

ID=45975665

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/ES2011/070728 WO2012052594A2 (en) 2010-10-21 2011-10-21 USE OF Dlk1 AS AN ANGIOGENESIS INHIBITOR

Country Status (2)

Country Link
ES (1) ES2381161B1 (en)
WO (1) WO2012052594A2 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5644031A (en) * 1992-12-11 1997-07-01 The United States Of America As Represented By The Department Of Health And Human Services Delta-like gene expressed in neuroendocrine tumors
US20090326205A1 (en) * 2006-11-10 2009-12-31 Liv Tech Inc. ANTI-HUMAN Dlk-1 ANTIBODY SHOWING ANTI-TUMOR ACTIVITY IN VIVO

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5644031A (en) * 1992-12-11 1997-07-01 The United States Of America As Represented By The Department Of Health And Human Services Delta-like gene expressed in neuroendocrine tumors
US20090326205A1 (en) * 2006-11-10 2009-12-31 Liv Tech Inc. ANTI-HUMAN Dlk-1 ANTIBODY SHOWING ANTI-TUMOR ACTIVITY IN VIVO

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YEVTODIYENKO A. ET AL.: 'Dlkl Expression Marks Developing Endothelium and Sites of Branching Morphogenesis in the Mouse Embryo and Placenta.' DEVELOPMENTAL DYNAMICS. vol. 235, 2006, pages 1115 - 1123 *
YIN D. ET AL.: 'DLK1 increased expression in gliomas and associated with oncogenicActivities.' ONCOGENE vol. 25, 2006, pages 1852 - 1861 *

Also Published As

Publication number Publication date
ES2381161A1 (en) 2012-05-23
ES2381161B1 (en) 2013-04-18
WO2012052594A3 (en) 2014-12-04

Similar Documents

Publication Publication Date Title
ES2750550T3 (en) TALEN-based gene correction
ES2564794T3 (en) R-spondins as modulators of angiogenesis and vasculogenesis
US7799896B2 (en) Methods of inhibiting tumor cell proliferation
JP5802195B2 (en) Compositions and methods for modulating stem cells and uses thereof
Yang et al. A cilium-independent role for intraflagellar transport 88 in regulating angiogenesis
US10925976B2 (en) Smooth muscle specific inhibition for anti-restenotic therapy
US20110189097A1 (en) Use of WNT inhibitor to inhibit angiogenesis in the CNS
Beck et al. Egr-1 regulates expression of the glial scar component phosphacan in astrocytes after experimental stroke
CA3020344A1 (en) Tdp-43 mitochondrial localization inhibitor for the treatment of neurodegenerative disease
US20020187936A1 (en) Methods of treating liver disease and liver damage with growth hormone and foxM1B
WO2018152103A1 (en) Myomerger polypeptide, nucleic acid molecules, cells, and related methods
ES2957688T3 (en) Double and inducible suicidal gene construction and its use in gene therapy
ES2559106B1 (en) Method of activating the expression of the Pitx2 gene to promote muscle regeneration
US20040109844A1 (en) Methods of treating age-related defects and diseases
AU2011256098A1 (en) Method for reducing expression of downregulated in renal cell carcinoma in malignant gliomas
US10258672B2 (en) Compositions and methods of treating root avulsion injury
S Lee Regulation of V-ATPase expression in mammalian cells
ES2381161B1 (en) USE OF DLK1 AS AN ANGIOGENESIS INHIBITOR.
US10772938B2 (en) Compositions and methods of increasing longevity or treating cellular stress
JP4488720B2 (en) Apoptosis-related proteins and uses thereof
EP2221066A1 (en) Use of VgII3 activity modulator for the modulation of adipogenesis
US20210244828A1 (en) Miri26-5p for treating motor neuron diseases
ES2620702B2 (en) Gene construction and its use in the treatment of cardiac fibrosis
WO2023205741A1 (en) Chimeric efferocytic receptors improve apoptotic cell clearance and alleviate inflammation
WO2005082412A1 (en) Drug for treating and preventing cancer

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11833901

Country of ref document: EP

Kind code of ref document: A2

122 Ep: pct application non-entry in european phase

Ref document number: 11833901

Country of ref document: EP

Kind code of ref document: A2