WO2012052594A2 - Utilisation de dlk1 en tant qu'inhibiteur de l'angiogenèse - Google Patents

Utilisation de dlk1 en tant qu'inhibiteur de l'angiogenèse Download PDF

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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
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angiogenesis
dlk1
expression
gene construct
dlkl
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PCT/ES2011/070728
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WO2012052594A3 (fr
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Santiago LAMAS PELÁEZ
Patricia RODRÍGUEZ PÉREZ
María Ángeles HIGUERAS LÓPEZ
Jorge LABORDA FERNÁNDEZ
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Consejo Superior De Investigaciones Científicas (Csic)
Universidad De Castilla La Mancha
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Publication of WO2012052594A3 publication Critical patent/WO2012052594A3/fr

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    • 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.

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Abstract

La présente invention porte sur l'utilisation d'une construction génique qui comprend une séquence codant Dlk1 ou la protéine de recombinaison qui résulte de l'expression de ladite construction génique, pour la préparation d'un médicament pour l'inhibition de l'angiogenèse, préférablement de l'angiogenèse tumorale. Cette invention porte également sur l'utilisation d'un produit d'expression de Dlk1 ou de n'importe lequel de ses fragments en tant que biomarqueur pour la détermination de l'angiogenèse ou de son développement. Ainsi l'invention concerne également une méthode de détermination de l'angiogenèse pathologique ou de son développement, et l'utilisation, pour le diagnostic de l'angiogenèse pathologique, d'une trousse comprenant la séquence qui code pour dlk1 ou un quelconque de ses produits d'expression.
PCT/ES2011/070728 2010-10-21 2011-10-21 Utilisation de dlk1 en tant qu'inhibiteur de l'angiogenèse WO2012052594A2 (fr)

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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 *

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