EP1366155A1 - Procedes et agents pour modifier l'angiogenese humaine - Google Patents

Procedes et agents pour modifier l'angiogenese humaine

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Publication number
EP1366155A1
EP1366155A1 EP02744892A EP02744892A EP1366155A1 EP 1366155 A1 EP1366155 A1 EP 1366155A1 EP 02744892 A EP02744892 A EP 02744892A EP 02744892 A EP02744892 A EP 02744892A EP 1366155 A1 EP1366155 A1 EP 1366155A1
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Prior art keywords
nucleic acid
acid molecule
arna
sequence
protein
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EP02744892A
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German (de)
English (en)
Inventor
Herwig Brunner
Brigitte Koch-Pelster
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KOCH-PELSTER, DR., BRIGITTE
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Fraunhofer Gesellschaft zur Forderung der Angewandten Forschung eV
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Publication of EP1366155A1 publication Critical patent/EP1366155A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to RNA components of a metallo-ribonucleoprotein morphogen from leukocytes, processes for their preparation and characterization and nucleic acid complexes comprising these RNA components and processes for their use, in particular processes and agents for modifying angiogenesis and Methods and agents for tumor cell control.
  • Angiogenesis is the formation of new blood vessels by sprouting capillaries from an existing vascular system (Hertig, 1935).
  • the neovascularization that takes place in the context of angiogenesis takes place in several partial steps (Folkman and Shing, 1992).
  • the basement membrane surrounding the blood vessels is broken down with the help of various proteolytic enzymes, for example collagenase, and the extracellular matrix is also fragmented in the perivascular space.
  • the endothelial cell is then differentiated, the cell morphology changing and pseudopodia associated therewith being formed.
  • Angiogenic stimuli cause the exposed endothelial cells to migrate in the direction of a chemotactic stimulus. The endothelial cells then proliferate.
  • pathological angiogenic processes include, for example, diabetic retinopathy, the various forms of rheumatoid arthritis and the formation of hemangiomas.
  • pathological processes are caused by the fact that angiogenetically stimulating factors, such as, for example, metalloproteases, are in excess of their inhibitors, for example TIMP (tissue inhibitor of metalloproteases).
  • TIMP tissue inhibitor of metalloproteases
  • Targeted inhibition of individual angiogenesis factors attempts to inhibit such pathological angiogenic processes (Adamis et al., 1996).
  • Tumor angiogenesis is a special case of pathogenic angiogenesis (Folkman 1971, Folkman 1995b).
  • Angiotropin a metallo-ribonucleoprotein morphogen isolated from ischemic heart muscle tissue, wound fluid and the supernatants of serum-free mass cultures of lectin-stimulated porcine leucocytes, is known to be a mediator of angiogenetic processes (Wissler and Renner 1981, Wissler 1982, Wissler 1984) , The angiogenic efficacy of angiotropin has been demonstrated using standard in vivo and in vitro test systems. For example, a test on corioallantoic membranes of the chicken embryo (CAM test) showed that after administration of angiotropin there was increased capillarization in this membrane (Wissler 1982, Noll 1998, Kuhn 1998).
  • stage II ie after about four to seven days, long bipolar cytoplasmic extensions with tubular structures were observed that organized themselves spatially. There were one or two large transparent vacuoles with movable granules inside the endothelial cells. If the endothelial cells were still stimulated with angiotropin-containing medium, they went into stage III. The vacuoles disappeared and irregularly shaped particles appeared outside the cell body and the pseudopods. If stage II endothelial cells were further cultivated with new medium which did not contain angiotropin, the cell morphological changes regressed after 24 hours until the cells returned to their original appearance.
  • angiotropin caused a reversible change in the cell morphology and thus the phenotype of endothelial cells. It is also important that angiotropin had no mitogenic effect on endothelial cell cultures and therefore only differentiation, but none Proliferation of the endothelial cells induced (Höckel et al. 1986, Höckel et al. 1987, Höckel 1988).
  • angiotropin stimulated the migration of endothelial cells, but not the migration of 3T3 fibroblasts (Höckel et al. 1987, Höckel 1988). This means that angiotropin has a specific effect on endothelial cells. Furthermore, it could be demonstrated that angiotropin has nuclease activity and can inhibit translation in in vitro translation assays (Seibt 1998, Noll 1998).
  • Angiotropin is a metal-containing ribonucleotide polypeptide (RNP) complex that could be isolated extracellularly. Copper, calcium, sodium and potassium ions have been detected as metal ions (Wissler et al., 1986). The copper ion is presumably present in a divalent form (Kuhn et al., 1996, Kuhn 1998). The sequencing of the protein component ARP (angipotropin related protein) showed that it is 100% homologous to calgranulin C (Kuhn 1998).
  • ARP angipotropin related protein
  • the protein belongs to the family of SlOO proteins and has already been isolated from porcine granulocytes without an RNA component, in which it forms 8% of the total cytosolic protein (Dell 'Angelica et al., 1994). It is a protein with 91 amino acids and a molecular weight of 10614 daltons. Like other SlOO proteins, calgranulin C has two calcium binding motifs known as EF hand structures (Kretsinger 1980, Isobe et al. 1981; Moncrief et al. 1990; Klingman & Hilt 1988; Schfer & Walkermann 1996).
  • RNPs biologically active metal-containing, in particular copper, zinc or calcium-containing ribonucleopolypeptides (RNPs) as non-mitogenic morphogens for blood vessels of a defined primary structure, as well as methods for producing and obtaining these RNPs.
  • RNPs biologically active metal-containing, in particular copper, zinc or calcium-containing ribonucleopolypeptides
  • a partial sequence of the porcine angiotropin RNA sequence and the amino acid sequence of the protein component are described.
  • RNA component (ARNA) of porcine angiotropin in particular the sequences ARNA II, ARNA III, ARNA IV and ARNA V.
  • RNA sequences associated with porcine angiotropin in particular the ARNA I sequence and the ARNA VI sequence.
  • RNA sequences associated with porcine angiotropin In view of the experimentally proven importance of angiotropin for angiogenesis in the mammalian organism, in particular with regard to the use of this morphogen for the targeted modulation of pathogenic angiogenic processes in humans, for example for the treatment of human diseases, it is desirable to understand the RNA sequences of human angiotropin to isolate and characterize.
  • the publications known in the prior art do not provide any insight into the molecular mechanisms that underlie the angiogenic activity of angiotropin. In particular, in particular, it is not known what function the RNA component specifically has.
  • angiotropin in human medicine, however, it is desirable to understand these molecular mechanisms precisely in order to be able to specifically modulate the angiogenic activity of angiotropin, that is to say to induce or to increase or to inhibit it as required ,
  • the technical problem on which the present invention is based is therefore to provide methods and means for isolating and characterizing RNA sequences for human angiotropin, and further methods and means based on these methods and means, with the aid of which, in particular in the human body, physiologically desired angiogenesis.
  • Processes, for example in wound healing, can be specifically induced or strengthened, or pathological angiogenesis processes, for example in diabetic retinopathy or in tumor formation, can be specifically inhibited.
  • nucleic acid molecule which is suitable as a functional component of a biologically active metallo-ribonucleoprotein complex, selected from the group consisting of:
  • nucleic acid molecule which is obtainable by reverse transcription of placenta Total RNA with ARNA I-specific primers, in particular with the primers shown in SEQ ID No. 6 and / or 7, and a fragment thereof;
  • nucleic acid molecule comprising at least one of the nucleotide sequences shown in SEQ ID Nos. 1 to 5 and 8 to 15 and a fragment thereof;
  • nucleic acid molecule which can be obtained by substitution, addition, inversion and / or deletion of one or more bases of a nucleic acid molecule according to a) to b);
  • nucleic acid molecule which hybridizes with a nucleic acid molecule according to a) to c) and a fragment thereof
  • nucleic acid molecule which is complementary to a nucleic acid molecule according to a) to b), and a fragment thereof.
  • the invention also solves this problem by providing nucleic acid complexes which contain these nucleic acid molecules and which combine the nucleic acid molecule according to the invention together with at least one metal ion, the nucleic acid molecule according to the invention together with at least one metal ion and at least one protein (also referred to here as metal-containing ribonucleotide polypeptides) or contain the nucleic acid molecule according to the invention together with a protein (also referred to here as ribonucleotide polypeptides).
  • Such nucleic acid complexes, in particular metal-containing ribonucleotide polypeptides are suitable for a large number of diagnostic and therapeutic applications are outstandingly suitable.
  • the technical problem was solved in a preferred embodiment by the human RNA sequence ARNA I associated with human angiotropin using the RT-PCR method using the porcine primers ARNA I (forward) and ARNA I (reverse) from the Total RNA was isolated from human placenta, an angiogenetically extremely active tissue.
  • this human ARNA-I sequence contains a 42 nucleotide sequence segment which was not detected in the porcine ARNA I sequence .
  • MFOLD program a computer model of the secondary structure of the human ARNA I sequence was created and compared with the secondary structure of all known ARNA sequences, including the porcine ARNA sequences.
  • RNA sequences with ribozyme activity into target cells of the angiotropin complex, for example endothelial cells
  • these RNA sequences are provided with the sequences which form the hairpin loop in the ARNA sequences using known methods of genetic engineering and then, using these RNA sequences, the ARP component and metal ions, ternary complexes are formed in vitro which are introduced into the cells to be treated.
  • the proven hairpin loop of the ARNA sequences according to the invention offers the possibility, using the nucleic acids according to the invention containing them, to isolate further proteins, for example other SlOO proteins, which, like ARP, can bind to such hairpin structures and as potential transport molecules for ARNA sequences or for nucleic acids modified according to the invention, which are naturally not associated with the angiotropin complex, can be used.
  • further proteins for example other SlOO proteins, which, like ARP, can bind to such hairpin structures and as potential transport molecules for ARNA sequences or for nucleic acids modified according to the invention, which are naturally not associated with the angiotropin complex, can be used.
  • ternary angiotropin complexes ie metal-containing ribonucleotide proteins
  • ARNA I sequences CuCl 2
  • ARP Corioallantois membrane
  • the results of the CAM assays showed an overall RNA-dependent regulatory mechanism for angiotropin-induced angiogenesis.
  • ARNA I sense or sense RNA
  • II copper
  • ternary complexes reconstituted with the ARNA I (antisense or antisense) sequence (ARNA I (as)) not only showed no angiogenic activity, but could even inhibit angiogenic processes in the test system.
  • the human ARNA I (s) sequence as an active ingredient for a therapeutic agent for inducing angiogenesis, for example in the context of wound healing, while in a further preferred embodiment the human ARNA I (as) Sequence as an active ingredient for a therapeutic agent for inhibiting patho- general angiogenic processes, especially tumor angiogenesis, can be used.
  • the ARNA I (s) or (as) sequence according to the invention can be used together with metal ions and / or proteins, for example calgranulin C, or its components.
  • a nucleic acid which can be associated with human angiotropin and which is the human ARNA I sequence is provided for the first time.
  • the human ARNA I sequence was according to the invention from a human total placenta RNA by means of the RT-PCR method using the primers ARNA I (forward) (SEQ ID No. 6) and ARNA I specific for the porcine ARNA I sequence (reverse) (SEQ ID No. 7) isolated and cloned.
  • ARNA I forward
  • ARNA I specific for the porcine ARNA I sequence reverse
  • a second sequence called human pseudo-ARNA I sequence was isolated and cloned.
  • ARNA sequences are understood to mean nucleic acids, in particular RNAs, which can be isolated extracellularly and in native form under suitable conditions together with a protein, in particular ARP (angiotropin-related protein) or calgranulin C or similar proteins, and Metal ions, in particular copper, calcium, sodium or potassium ions, form a ternary complex, in particular an angiotropin complex, which has angiogenic activity.
  • ARP angiotropin-related protein
  • calgranulin C or similar proteins calgranulin C or similar proteins
  • Metal ions in particular copper, calcium, sodium or potassium ions, form a ternary complex, in particular an angiotropin complex, which has angiogenic activity.
  • An ARNA I sequence is used in connection with the present invention a nucleic acid, in particular an RNA, understood that, in methods for the purification of angiotropin, for example using lectin-induced porcine leukocytes as the starting material, after various purification steps as the only known ARNA sequence in the after the last purification step obtained fraction, which still has angiogenic activity, is specifically enriched and is therefore assumed to be the RNA form which is incorporated into the angiotropin complex.
  • angiogenic activity means that a substance has the ability to induce or intensify the formation of new blood vessels by sprouting capillaries from an already existing vascular system.
  • human ARNA I sequence When sequencing the human ARNA I sequence, it was found according to the invention that it contains a sequence section which is completely identical in terms of its length and its sequence to the porcine ARNA I sequence isolated from lectin-stimulated porcine leukocytes.
  • the human ARNA I sequence also contains a human-specific section comprising 42 nucleotides, which was not detected in the porcine sequence and is shown in SEQ ID No. 1.
  • the first 19 nucleotides of these 42 nucleotides have special human specificity and are shown in both orientations in SEQ ID No. 14 and 15. Since the identified human ARNA I sequence did not contain a poly (A) tail at the 3 'end, the orientation in which the native sequence is intra- or extracellular.
  • SEQ ID No. 2 shows the sequence of SEQ ID No. 1 in an anti-sense orientation.
  • SEQ ID No. 3 shows the complete nucleotide sequence of the human ARNA I molecule in sense orientation.
  • SEQ ID No. 4 shows the complete sequence in antisense orientation.
  • the region comprising 42 nucleotides in sense orientation (SEQ ID No. 1) is located at the 3 ′ region of the human ARNA-I sequence (SEQ ID No. 3, positions 164 to 205).
  • this nucleic acid according to the invention which is presumably a pre-mRNA or a section of a larger pre-mRNA, consists of two different sections and is shown in SEQ ID No. 5 and schematically in FIG. 3. These two sections of the pseudo-ARNA I sequence are probably intron (ARNA I (as)) and exon (L27a exon II) sequences.
  • the 5 'section of the pseudo-ARNA I sequence (SEQ ID No. 5) comprises the first 63 nucleotides (SEQ ID No. 11) of the 5' end of the human ARNA I (antisense) sequence (SEQ ID No.
  • the first (ie 5 'located) 23 nucleotides of the 63 nucleotides mentioned correspond to the first 23 nucleotides of the porcine ARNA I (s) sequence. This is due to the self-complementarity of the 5 'and 3' ends of the human ARNA I (as). The same applies to ARNA I (s). Nucleotides 43 to 49 (inclusive) are part of the intron sequence of the pseudo-ARNA-I sequence.
  • the 19 nucleotides 24 to 42 (each inclusive) of the 63 nucleotides of the human ARNA I (as) sequence mentioned represent the human-specific antisense nucleotide sequence in SEQ ID No. 2. therefore the last (i.e. 3 'positions 50 to 63 in SEQ ID No. 4 or 11) are 14 nucleotides (ie: 5'-GCU AAC AAA GUU UU-3') of the 63 nucleotide ARNA I (as) - Sequence section (SEQ ID No. 11) within the human pseudo-ARNA I sequence homologous to a section of the second exon of the pre-mRNA of the human ribosomal L27a protein. This 14 nucleotide section of the ARNA I (as) sequence could therefore be part of the 3 'splice site of this pre-mRNA.
  • a further section of the pseudo-ARNA I sequence comprises the ARNA (as) sequence with the nucleotides 64 to 5 located 5 'to the right of the 63 nucleotide sequence 205 and the L27a exon I sequence located 5 'therefrom.
  • Ribosomal proteins are generally known to be one of the oldest proteins in terms of developmental biology and that the genes on which they are based are highly conserved. It is also known from the human ribosomal L27a protein that the mRNA of this protein in carcinoma cells is strongly overexpressed compared to the mRNA of other ribosomal proteins, so that it can be assumed that this protein is involved in an increased cell proliferation (Frigerio et al 1995). Although the gene underlying the pseudo-ARNA I sequence cannot be assigned a clear function at the present time, it can be assumed that it is very likely that it is a pseudogen of the ribosomal L27a gene is. Pseudogenes are found particularly in gene families whose products are often needed.
  • the partial sequence of the ARNA I sequence contained therein is obviously conserved across all species as a putative intron sequence, since this sequence region is also contained in the porcine ARNA I sequence.
  • the pseudo-ARNA I sequence supports the assumption that the ARNA I sequence is the intron sequence of a longer pre-mRNA and is spliced from it in a circular form.
  • the potential generation of a circular ARNA I (s) is shown in FIG. 3.
  • nucleic acid is therefore selected, selected from the group consisting of:
  • a nucleic acid which can be obtained by reverse transcription of total placenta RNA with ARNA I-specific primers, in particular with the nucleotide sequences shown in SEQ ID No. 6 and / or No. 7, or a fragment thereof;
  • nucleic acid comprising at least one nucleotide sequence shown in SEQ ID Nos. 1, 3, 5, 8, 10, 12 or / and 14;
  • nucleic acid which is complementary to a nucleic acid according to a) to b), in particular a nucleic acid with at least one of the nucleic acids shown in SEQ ID No. 2, 4, 9, 11, 13 or / and 15 set nucleotide sequence, or a fragment thereof;
  • nucleic acid which can be obtained by substitution, addition, inversion and / or deletion of one or more bases of a nucleic acid according to a) to c);
  • nucleic acid which hybridizes due to the degeneration of the genetic code with a nucleic acid according to a) to d) or a fragment thereof.
  • the nucleic acid can be single-stranded or double-stranded.
  • the nucleic acid can be a DNA sequence, for example part of a genomic DNA sequence or a cDNA sequence, or an RNA sequence, for example a mRNA sequence or a part thereof.
  • a DNA sequence in the RNA sequences shown, the uracil bases marked with U must be replaced by T, that is to say thymine bases, and vice versa.
  • the nucleic acid can be part of a longer native nucleic acid molecule.
  • the nucleic acid can exist both as a circular and as a linear nucleic acid molecule.
  • the nucleic acids, shown in SEQ ID Nos. 1 to 15, are detected by the invention in both orientations 5 'to 3' and 3 'to 5' and as a sense and anti-sense strand.
  • the nucleic acid can be of natural origin, that is, it can be isolated both extracellularly and intracellularly from cells, tissues or organs of a mammalian organism be of synthetic origin.
  • the nucleic acid can be associated with metal ions, in particular copper, sodium, potassium or calcium ions, and also with proteins, such as calgranulin C or ARP.
  • the invention also encompasses modified nucleic acids which are obtainable, for example, by substitution, addition, inversion and / or deletion of one or more bases of a nucleic acid according to the invention, that is to say also nucleic acids which are referred to as mutants, derivatives or functional equivalents of a nucleic acid according to the invention can.
  • the sequence of the nucleic acids can, for example, be specifically modified in order to provide suitable restriction cleavage sites within the nucleic acid sequence or to remove unnecessary nucleic acid sequences or restriction cleavage sites.
  • the nucleic acids according to the invention are inserted into plasmids and subjected to mutagenesis or a sequence change by recombination using standard microbiology / molecular biology methods.
  • the present invention also encompasses nucleic acids which hybridize with one of the nucleic acids described above according to a) to d).
  • nucleic acid which hybridizes with a nucleic acid according to a) to d) used in connection with the present invention means a nucleic acid which hybridizes under moderately stringent conditions with a nucleic acid according to a) to d).
  • hybridization with a radioactive gene probe in a hybridization solution (25% formamide; 5 x SSPE; 0.1% SDS; 5 x Denhardt's solution; 50 ⁇ g / ml herring sperm DNA; for the composition of the individual components see Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition (1989), Cold Spring Harbor Laboratory Press, NY, USA) for 20 hours at 37 ° C.
  • unspecifically bound probe is washed by washing the filter twice in 2 x SSC / 0.1% SDS removed at 42 ° C.
  • the filters are preferably washed with 0.5 x SSC / 0.1% SDS, particularly preferably with 0.1 x SSC / 0.1% SDS at 42 ° C.
  • a model of the secondary structure was modeled from this nucleic acid using the MFOLD program.
  • the MFOLD program version 2 (Zuker 1989; Jaeger et al. 1989; Jaeger et al. 1990) provides information about the structures of nucleic acids with the lowest free enthalpy.
  • models of the secondary structures were created in order to determine possible structural homologies between these nucleic acids and thus to characterize essential functional areas of the nucleic acids.
  • the computer modeling according to the invention of the secondary structure of the human ARNA I (s) sequence shown in FIG. 1 showed that, due to its large number of sequence-complementary regions, this nucleic acid forms a highly symmetrical, double-stranded secondary structure over large sequence sections, in particular by means of two cloverleaf structures is marked.
  • the second cloverleaf structure differs from the first in that it contains the open 5 '-3' end of the nucleic acid molecule.
  • the sequence of the 3 'end of the human ARNA I sequence is complementary to the sequence of the 5' end. This sequence complementarity also made it possible to amplify the human ARNA I sequence with only one primer.
  • a comparison of the secondary structure created with that of other known ARNA sequences showed that all ARNA sequences have the common consensus sequence 5'-CUG-3 ', in particular the sequences shown in SEQ ID No. 12 and 13, each of which only occurs in a sequence orientation and is always in the area of a hairpin loop.
  • the hairpin loop found in the known ARNA sequences presumably represents the binding domain of these ARNA sequences to ARP or calgranulin C or a similar protein.
  • the consensus sequence 5'-CUG-3 'contained in the hairpin loop is located in a sequence section which Has homologies to the sequence of the hammerhead ribozyme.
  • the hammerhead ribozyme is a component of self-splicing, circular, viral RNA molecules that replicate either alone or as a function of helper viruses (Haseloff and Gerlach 1988).
  • the conserved consensus sequence 5'-CUGANGA (N) X GAAAC of the hammerhead ribozyme is partly also in the human ARNA I sequence (sense orientation) (5'-CUG GAU UGA AAA G -3 ', SEQ ID no. 8) included. This indicates that the ARNA I molecule, like the other ARNA molecules, contains in this sequence section, in addition to the protein binding domain, a further functional region which has ribozyme activity.
  • the invention therefore also relates to a nucleic acid which, in its secondary structure, forms a hairpin loop with the consensus sequence 5'-CUG-3 ', in particular with one of the sequences shown in SEQ ID Nos. 12 and 13.
  • the invention further relates to a nucleic acid whose hairpin loop formed in the secondary structure with one of the sequences shown in SEQ ID Nos. 12 and 13 mediates the binding of the nucleic acid to a protein, in particular an angiotropin-related protein molecule.
  • the present invention also relates to a binding domain of nucleic acids, in particular ARNA nucleic acids, for the first time, for binding to proteins, in particular ARP or calgranulin C or similar proteins, which is characterized by the formation of a characteristic hairpin loop with the consensus occurring only in a sequence orientation.
  • Sequence 5'-CUG-3 ' in particular a sequence selected from SEQ ID No. 12 or 13, the latter possibly having a higher specificity for the protein binding domain ARP or Cal C.
  • ARP or calgranulin C or the like Proteins are molecules for the inter- and / or intracellular transport of the various RNA components associated with the angiotropin complex. According to the invention, this offers the possibility of using the angiotropin protein constituent ARP or similar proteins, in particular other S100 proteins, also for RNA molecules, which are not naturally associated with the angiotropin complex, as transport molecules.
  • RNA molecules with enzymatic activity such as, for example, ribozymes, or nucleic acids, for example, RNA molecules, which are the transcription products of genes of potential therapeutic or diagnostic interest.
  • ARNA-specific sequences according to the invention which, under suitable conditions, lead to the formation of a hairpin loop for binding to ARP or calgranulin C or similar proteins, in particular other SlOO proteins, which corresponds to that detected in ARNA sequences.
  • Those nucleic acids which have an ARNA-specific hairpin structure for binding to ARP or similar transport proteins are preferably produced using conventional synthetic or genetic engineering processes (cf. Sambrook et al. 1989).
  • the attached hairpin loop sequence can optionally be modified such that it has a stronger homology to the consensus sequence of hammerhead ribozymes than the hairpin loop of the human ARNA I sequence according to the invention. If the nucleic acid to be transferred should not have any ribozyme activity, the sequence for forming a hairpin loop can be modified in such a way that the nucleotides relevant for the ribozyme activity are exchanged for nucleotides which lead to a blocking of the ribozyme activity. Such sequence modifications can be carried out with the aid of customary genetic engineering methods, for example methods of in vitro mutagenesis.
  • the nucleic acids produced or modified in this way can be specifically introduced into cells and / or tissues that target natural cells. len or target tissue for angiotropin, for example endothelial cells. If the nucleic acids produced or modified in this way are to be introduced specifically into other target cells, other proteins, for example other SlOO proteins, which may have different target cell specificities, are used to form ternary complexes. According to the invention, those proteins which enable binding to the nucleic acid can be used for the association of the nucleic acid with the protein. These can be proteins that have the same binding domain to RNAs as ARP or Cal C.
  • proteins can bind to an RNA which has either the sequence sequence 5 '-CUG-3' or the sequence sequence of SEQ ID No. 12 or SEQ ID No. 13 within a hairpin loop.
  • These proteins can also have additional sequence domains, that is to say different from ARP or Cal C. These further sequence domains can mediate a target cell specificity that differs from ARP and Cal C.
  • the nucleic acid complex formed can dock specifically to the desired cells, penetrate them and then develop the desired activity.
  • the nucleic acid complex formed can be introduced in vivo or in vitro into any eukaryotic cells, cell cultures or tissues, but especially cells and tissues from mammals and birds.
  • nucleic acids or genes are switched off, for example via a ribozyme activity of the introduced nucleic acids, via an anti-sense mechanism or a cosuppression effect. It is also possible to introduce genes which were not previously present in these cells or whose function was impaired in these cells. For example, targeted tumor therapy is possible in that target cells are recognized and combated by these specifically recognizing nucleic acid complexes.
  • the method according to the invention is also particularly suitable for somatic gene therapy. Genes can also be introduced into cells that act as reporter genes, for example in order to label or detect such cells.
  • RNA molecules are generally very sensitive to hydrolysis, which leads to a relatively short half-life of the molecules.
  • efficiency with conventional methods for transfecting RNA molecules was very low.
  • several methods are described in the prior art which can be used to overcome the problems mentioned.
  • a chemical modification of the ribose-phosphate framework can stabilize an RNA so that it shows greater resistance to hydrolysis (Perreault et al., 1991; Yang et al. 1992; Heidenreich et al. 1993), in particular 2 ' -OH groups by other functional groups, for example amino, fluoro-, O-allyl or O-methyl groups can be substituted.
  • RNA molecules can be introduced intracellularly (Akhtar et al. 1991).
  • nucleic acids modified according to the invention Before the nucleic acids modified according to the invention are introduced into target cells, they can therefore optionally be chemically modified by, for example, one or more functional 2'-OH groups of the sugar-phosphate skeleton against other functional groups, for example an amino, Fluoro-, O-allyl or O-methyl group are exchanged to stabilize the nucleic acid.
  • the nucleic acids complexed with metal ions and / or proteins can optionally be introduced into target cells using liposomes in order to improve the efficiency of the transport into the desired target cells.
  • the invention also captures post-transcriptional modifications occurring naturally in cells.
  • the present invention therefore relates to a method for transporting a nucleic acid molecule into eukaryotic cells, cell cultures or tissues, in particular cells and tissues from mammals and birds, in vitro or in vivo, comprising the following steps: - Modification of the nucleic acid molecule to be transported, which is of course not related to the angiotropin complex and / or similar complexes, by adding sequences of a nucleic acid molecule according to the invention which, under suitable conditions, form a hairpin loop with the Consensus sequence 5'-CUG-3 'are suitable;
  • the present invention relates to a method for transporting a nucleic acid molecule into a eukaryotic cell, the protein being an ARP molecule, calgranulin C or a similar protein, in particular a protein having the binding motif of ARP or calgranulin C for the nucleic acid molecule is.
  • metal ions such as Cu 2+ , Mg 2+ , Ca 2+ , Na + , K + or Zn 2+ ions are used to produce the ternary complex.
  • the modified nucleic acid molecule is a molecule with enzymatic activity.
  • the invention also provides for the ARNA to be specifically labeled for diagnostic purposes.
  • radioactive label with digoxigenin or with fluorescent markers.
  • digoxigenin with digoxigenin or with fluorescent markers.
  • in situ hybridizations can be carried out, which would allow cellular localization of the ARNA after cellular uptake.
  • An ARNA labeled with fluorescent substances can be tracked microscopically both temporally and spatially in cultured cells after their cellular uptake.
  • a particularly preferred embodiment of the present invention therefore sees the use of ARNA-specific sequences, that is to say the nucleic acids according to the invention, in particular sequences which, under suitable conditions, are capable of forming a hairpin structure containing the consensus sequence 5'-CUG-3 ' are for the modification of nucleic acids that are not naturally associated with the angiotropin complex for the production of diagnostics and therapeutic agents.
  • the nucleic acids modified in this way can be used as diagnostics or therapeutic agents for specific cells or tissues by binding them to ARP or calgranulin C or similar transport molecules and introducing them into the target cells to be treated via these transport molecules.
  • diagnostic agents are understood to mean any substances that specifically determine the presence of conditions, Can recognize processes or substances or their absence and in particular can draw conclusions about disease states. Diagnostics often have recognizing and marking functions.
  • therapeutic agents is understood to mean in particular those substances which can be used either prophylactically or accompanying the disease in order to avoid, alleviate or eliminate disease states.
  • diseases are also understood to mean conditions such as unnatural states of mind, pregnancies, signs of aging, developmental disorders or the like.
  • a further preferred embodiment of the present invention therefore provides the use of ARNA-specific sequences, that is to say the nucleic acids according to the invention, in particular sequences which, under suitable 'conditions, form the consensus sequence 5'-CUG-3', in particular a sequence shown in SEQ ID No. 12 or 13 containing hairpin structure are able to isolate proteins which, similar to ARP, can bind to such hairpin structures.
  • Such proteins can be, for example, other representatives of the SlOO proteins, which may have different cell specificities than ARP.
  • the function of the human ARNA I sequence according to the invention was tested in vivo using using the chorioallantoic membrane test on incubated chicken egg.
  • the chorioallantoic membrane test is a simple test system for examining neovascularization processes. An angiogenic effect can easily be demonstrated by the formation of blood vessels.
  • Angiotropin RNA synthesized in vitro was reconstituted with ARP (calgranulin C) and CuCl 2 to a ternary angiotropin complex and applied to the exposed membrane.
  • the reconstituted ternary angiotropin complexes or the in vitro synthesized ARNA I (sense or antisense) sequences had a toxic effect on the chicken embryos over a wide concentration range. Within the non-lethal concentration range, the results of the CAM assays showed an RNA-dependent regulatory mechanism for angiotropin-induced angiogenesis. According to the invention, it was surprisingly found that not only the reconstituted ternary angiotropin complex comprising ARNA I (s) RNA, copper (II) ions and native porcine calgranulin C showed angiogenic activity, but also the ARNA I (s) sequence in combination with copper (II) ions, but without the protein component calgranulin C. It was also surprisingly found that ternary complexes containing ARNA I (antisense) sequences (ARNA I (as)) not only showed no angiogenic activity, but in the test system could even inhibit angiogenesis.
  • the ARNA I (s) sequence is used as an active ingredient for producing a therapeutic agent for inducing angiogenic processes, in particular for wound healing.
  • the ARNA I (s) molecule can be used in transplant surgery in order to accelerate the integration of the transplant through the increased vascularization and to avoid the formation of ischemic necrosis.
  • the ARNA I (s) molecule can be used to induce the neovascularization of coronary arteries. This avoids coronary angioplasty using a balloon catheter or by means of a bypass operation.
  • the ARNA I (s) sequence can be used either in combination with copper (II) ions and calgranulin C or only in combination with copper (II) ions, that is to say without the calgranulin C protein.
  • the ARNA I (as) sequence is used as an active ingredient for producing a therapeutic agent for inhibiting pathogenic angiogenic processes, in particular in the case of hemangiomas or in tumor angiogenesis.
  • the ARNA I (as) sequence is preferably in the form of a ternary complex, that is to say in combination with calgranulin C and copper (II) ions. In a preferred embodiment, this is ternary complex along with potassium and sodium ions.
  • the nucleic acids intended for use as an active ingredient for modulating angiogenic processes can also be chemically modified by, for example, one or more functional 2′-OH groups of the sugar-phosphate structure against other functional groups, for example an amino, fluorine or oxygen group -Allyl or 0-methyl group are exchanged to stabilize the nucleic acid.
  • the nucleic acids complexed with metal ions and / or proteins can also be introduced into the target cells using liposomes in order to improve the efficiency of the transfection.
  • the present invention therefore also relates to a method for inducing angiogenic processes in tissues of a mammalian organism, comprising the following steps:
  • the present invention also relates to a method for inhibiting angiogenic processes in tissues of a mammalian organism, comprising the following steps: - Production of a ternary complex by complexing a nucleic acid molecule according to the invention in antisense orientation with metal ions and optionally a protein in vitro; and
  • the present invention further relates to methods and uses resulting from FIG. 2, by means of which the molecular functions of the RNA component ARNA I associated with the angiotropin complex can be used in the differentiation of endothelial cells to capillary cells in the context of angiogenesis ,
  • the ARNA I (s) sequence present in the native angiotropin is partially complementary to the intron sequence of the pseudo-ARNA I sequence.
  • the antisense transcript of the pseudo-ARNA I gene therefore probably represents the precursor transcript for ARNA I (s).
  • This antisense transcript is transcribed analogously to a pre-mRNA and contains the ARNA I (s) as an intron.
  • ARNA I (s) form, possibly in circular form, is then spliced from this transcript.
  • the potential generation of the ARNA I (s) sequence is shown in FIG. 3.
  • the ARNA I (s) sequence is presumably circular.
  • the complete metallo-RNP complex angiotropin is constituted by association of the ARNA I (s) molecule with ARP and Cu 2+ and / or optionally zinc, calcium, potassium and / or sodium ions. If necessary, this grain plex secretes from the cell, diffuses to the endothelial cells and reaches the interior of the endothelial cells by means of endocytosis or via a receptor. There the single-stranded ARNA I (s) sequence is attached to homologous regions of the pre-mRNA or mRNA of the L27a gene, so that it can no longer function as a transcription template for the synthesis of the ribosomal L27a protein.
  • the nucleic acid molecules according to the invention which in particular enable binding to the mRNA of the L27a gene, for example the sequences of SEQ ID Nos. 1 to 5, 10 and 11 inhibition of the ribosomal L27a protein is a key site.
  • the mRNA of this protein is strongly overexpressed in carcinoma cells compared to the mRNA of other ribosomal proteins, so that there is a direct connection between this protein and an increased cell proliferation.
  • angiotropin complexes comprising ARNA I sequences into these cells.
  • the ARNA I sequences lead to a direct inhibition of L27a translation in the carcinoma cells and thus to an inhibition of the proliferation of these cells.
  • the invention therefore also relates to methods for reducing the proliferation of cells and / or for premature initiation of differentiation of these cells, a nucleic acid molecule of the present invention, in particular a nucleic acid molecule, being shown in one of SEQ ID Nos. 1, 2, 3, 4 , 5, 10 and / or 11, a vector containing these nucleic acid molecules, a host cell containing these molecules or vectors, a complex containing these nucleic acid molecules and / or an antibody against this complex is introduced into the cell, thereby the Activity and / or expression of the L27a gene or gene product encoded thereby inhibited or reduced, and thus proliferation the cell is reduced or inhibited and, if necessary, the differentiation is initiated prematurely.
  • a nucleic acid molecule of the present invention in particular a nucleic acid molecule, being shown in one of SEQ ID Nos. 1, 2, 3, 4 , 5, 10 and / or 11, a vector containing these nucleic acid molecules, a host cell containing these molecules or vectors, a
  • the cells are strongly proliferating cells, in particular stem cells, tumor cells and cells which, for example, are degenerate due to pathogenic processes and proliferate strongly, but also endothelial cells .
  • the cells are pathologically altered cells, for example cells which arise in psoriasis (type of psoriasis).
  • the cells are nerve cells.
  • a protein in the nucleic acid complex of the aforementioned type which on the one hand binds the nucleic acid molecule but on the other hand also has specificity with regard to the target cell to be transformed.
  • a molecule can be calgranulin C or ARP or a similar protein.
  • a similar protein in connection with the present invention is a protein which enables binding to a nucleic acid molecule of the present invention, in particular of the type defined above, in particular has a binding domain of calgranulin C or ARP to the nucleic acid molecule. According to the invention, this protein has recognition sites which do not allow specific recognition, binding and penetration of specific target cells to be transformed all cells of the treated organism are recorded.
  • the invention also relates to vectors containing a nucleic acid sequence of the present invention, optionally together with regulatory elements, elements which enable replication in a host, elements which enable the selection of cells transformed with the vector or other elements known in the art.
  • the vector can be, for example, a cosmid, phage or plasmid.
  • the invention also relates to host cells containing such vectors.
  • host cells can be bacterial, plant, animal or human cells, for example primate cells, mouse cells, hamster cells or also insect cells.
  • the invention also encompasses nucleic acid complexes comprising at least one nucleic acid molecule of the present invention, either together with at least one metal ion and / or a protein.
  • the protein can be, for example, from the family of SlOO proteins (Dell'Ange lica et al., loc. cit.), in particular a human or porcine calgranulin C or angiotropin-related protein molecule.
  • a S100 protein is understood to mean a protein with two calcium binding motifs, which are referred to as EF hand structures, in particular a protein with an amino acid sequence identity of at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, 97% and 99% to human ARP.
  • Ca 2+ , Cu 2+ , Na + , K + , Zn 2+ or Mg + ions can be used as the metal ion, for example.
  • a nucleic acid complex according to the invention comprising a nucleic acid molecule and a protein or its constituent, for example oligopeptide or polypeptide, is also referred to as a ribonucleotide polypeptide and, if this contains metal ions, as a metal-containing ribonucleotide polypeptide.
  • Such complexes can have chemical, radioactive or fluorescent markings, also with a cytotoxic effect.
  • the invention also relates to antibodies, in particular monoclonal or polyclonal antibodies, which can specifically recognize and bind one of the aforementioned nucleic acid complexes.
  • the invention also relates to diagnostic or pharmaceutical compositions containing at least one of the above-mentioned agents, such agents comprising the nucleic acids, vectors, host cells, nucleic acid according to the invention.
  • agents comprising the nucleic acids, vectors, host cells, nucleic acid according to the invention.
  • compositions also optionally contain additives or auxiliaries, such as pharmaceutically acceptable carriers, colorants, flavorings, sweeteners, thickeners, release agents, acidifying agents, lubricants, preservatives or the like.
  • the pharmaceutical compositions can be used to specifically influence the angiomorphogenesis and the vascular state of a tissue of the body of organisms, to transmit genetic information into cells, to selectively change the nucleic acid content of cells, to induce angiogenic processes and / or to heal wounds Inhibition of angiogenic processes and / or to combat tumors.
  • the nucleic acid complexes according to the invention also have a cell-selective morphogenic effect in vitro on isolated primary and / or cloned blood capillary endothelial cells in culture for the non-mitogenic induction of the change in the cell phenotype from the confluent state or / and for the non-mitogenic change in the spatiotemporal supracellular organization of the cells to three-dimensional ones organoid capillary-like structures in culture.
  • they are specific Chemotropic effect on blood vessels in vivo, suitable for inducing a directional growth of blood vessels in vivo and for inducing neovascularization of tissues through directed ingrowth of blood vessels.
  • compositions provided according to the invention contain, for example, the nucleic acids and / or nucleic acid complexes according to the invention.
  • they can be administered subcutaneously, intracutaneously, intramuscularly or intravenously.
  • the compositions according to the invention can be used topically or systemically.
  • the diagnostic compositions can be used, for example, to diagnose changes in blood vessel growth.
  • nucleic acids, nucleosides or nucleotides for example for deaminating adenosine and deoxiadenosine, for the hydrolysis of inosine and deoxiinosine, for the hydrolysis of guanosine, for deaminating guanine, for the hydrolysis of phosphoric acid diesters, for the hydrolysis of phosphoric acid monoesters, for the hydrolysis of nucleic acid substrates as well as for the production of nucleic acids in vivo and in vitro.
  • the uses according to the invention are were to carry out magnesium 2+ ions.
  • the above-mentioned agents can be used to produce adenosine, deoxiadenosine, guanosine, deoxiguanosine, inosine, adenine, guanine, hypoxanthine or xanthine.
  • the invention relates to the use of the abovementioned agents for the abovementioned purposes, in particular for the prophylaxis or curing of diseases, and to the use of the abovementioned agents for the production of medicaments for achieving the abovementioned purposes, in particular for the prophylaxis or curing of diseases.
  • the invention also relates to the use of SlOO proteins, in particular ARP, calgranulin C, or the nucleic acid complexes of the abovementioned invention as a transport molecule for nucleotide sequences, in particular RNA molecules.
  • FIG. 1 shows the secondary structure of the human ARNA I sequence (antisense) created with the aid of the MFOLD program.
  • FIG. 2 shows in schematic form the mechanism of ARNA I (s) -induced translation inhibition via an antisense mechanism.
  • This antisense Mechanism leads to differentiation of endothelial cells, whereby capillaries are formed.
  • FIG. 3 schematically shows the structure of the pseudo-ARNA I sequence according to the invention (top: sense strand, bottom: anti-sense strand).
  • sequence listing (all sequences are shown in 5 'to 3' orientation) includes:
  • SEQ ID No. 1 shows 42 nucleotides of the human, specific ARNA I sequence which were not detected in the corresponding porcine ARNA I sequence (sense orientation).
  • SEQ ID No. 2 shows 42 nucleotides of the human, specific ARNA-I sequence from SEQ ID No. 1 in anti-sense orientation.
  • SEQ ID No. 3 shows the complete, 205 nucleotide sequence of the human ARNA I sequence, which comprises the 163 nucleotide sequence part, which is identical to the sequence of the porcine ARNA I sequence, in sense orientation.
  • SEQ ID No. 4 shows the complete, 205 nucleotide sequence of the human ARNA I sequence from SEQ ID No. 3 in the anti-sense orientation.
  • SEQ ID No. 5 shows the sequence of the human pseudo-ARNA I sequence (sense orientation) comprising 235 nucleotides, including the ARNA I nucleotides 1 to 63 present at the 5 'end in the anti-sense orientation.
  • SEQ ID No. 6 shows the sequence of the primer ARNA I (forward) used to isolate the human ARNA I sequence.
  • SEQ ID No. 7 shows the sequence of the primer ARNA I (reverse) used to isolate the human ARNA I sequence.
  • SEQ ID No. 8 shows a section of the human ARNA-I sequence (sense orientation) with potential ribozyme activity.
  • SEQ ID No. 9 shows the sequence of SEQ ID No. 8 in anti-sense orientation.
  • SEQ ID No. 10 shows the 63 nucleotides of human ARNA I mentioned in SEQ ID No. 5 in sense orientation.
  • SEQ ID No. 11 shows the 63 nucleotides of human ARNA I mentioned in SEQ ID No. 5 in an anti-sense orientation.
  • SEQ ID No. 12 shows the consensus area located in the hairpin loop area between ARNA I (s) and ARNA II (as) sequences in sense orientation.
  • SEQ ID No. 13 shows the sequence of SEQ ID No. 12 in an anti-sense orientation.
  • SEQ ID No. 14 shows the region of SEQ ID No. 1 with 19 nucleotides with special human specificity in sense orientation.
  • SEQ ID No. 15 shows the 19 nucleotides, particularly human-specific region of SEQ ID No. 2 in an antisense orientation.
  • RNA from human placenta was used using the primer ARNA I (forward) (SEQ ID No. 6), which contains an Eco Rl interface, and the primer ARNA I (reverse) (SEQ ID No. 7), which contains a Barn HI restriction site, subjected to an RT-PCR.
  • the amplification products obtained were cleaved with the restriction enzymes Eco RI and Barn HI and subjected to gel electrophoretic purification on a 2.5% agarose gel. After the fragments had been purified, they were cloned into the vector pUC 19, which had previously been cleaved with Eco RI and Barn HI.
  • sequence of the insertions in the cloning vector was determined. Sequencing was carried out using the Dye Terminator Cycle Sequencing Kit and the ABI PRISM 377A DNA sequencer. The complete and partial sequences of ARNA I (s), ARNA I (as) and pseudo-ARNA I obtained in this way are shown in SEQ ID No. 1 to SEQ ID No. 5 and 8 to 15. In Fi 3, the pseudo-ARNA I sequence is shown schematically. The upper area represents the 5'-3 'orientation of a sense transcript from the pseudo-ARNA I in pre-mRNA form, that is to say the potential L27a exon I and the potential intron, comprising the ARNA I nucleotides No.
  • Nucleotides 50-63 of ARNA I (as) represent a common sequence section between ARNA I (as) and the region of the pseudo-ARNA I transcript that is homologous to the 5 'end of exon II of L27a.
  • the 3 'splice point of the pre-mRNA of L27a or possibly also of the pseudo-ARNA I (s) sequence lies in the area marked with the double arrow.
  • the antisense transcript of the pseudo-ARNA I (lower area) is the corresponding intron area ARNA I (s) 142 to 1 and ARNA I (s) 205 to 143, which serves as a template for the generation of a circular ARNA I (s) - Sequence serves.
  • the potential circularization of this sequence is shown by means of the arrow shown.
  • the arrowhead indicates the position of the 5 'and the arrow end the position of the 3' splice point of ARNA I (s) within the antisense transcript the pseudo-ARNA I sequence.
  • Nucleotides 143-156 of ARNA I (s) can hybridize against this exon from the position of the 3 'splice site of exon II of the mRNA or pre-mRNA of the L27a gene.
  • the cloned ARNA I sequences were used as templates for in vitro RNA synthesis.
  • blunt-end DNA templates were created via PCR, which enabled the subsequent RNA transcript to be terminated in a defined manner.
  • These DNA templates contained a promoter sequence for the T7 RNA polymerase at the 5 'end and the desired DNA sequence whose RNA was to be synthesized at the 3' terminal to the promoter sequence.
  • RNA synthesis was carried out using highly concentrated T7 RNA polymerase according to the method of Milligan and Uhlenbeck (1989), but the RNA synthesis was carried out over 18 h. After synthesis, the RNA was purified using DNase treatment and phenol / chloroform treatment. After the RNA had been purified using microconcentrations from Amicon (optionally also replaced by ethanol filling), the RNA synthesized in this way could be used for the in vitro reconstitution of ternary angiotropin complexes.
  • RNA synthesized in Example 2 was used to reconstitute ternary angiotropin complexes. puts. Intracellularly isolated and purified porcine calgranulin C from porcine granulocytes was used as the protein component. CuCl 2 was also used. Since the concentration ratio between RNA and calgranulin C has not yet been clearly quantified experimentally (Kuhn 1998), the ternary complexes were reconstituted using different concentrations. After a 1 hour incubation at 37 ° C the complexes were diluted 1: 100 with buffer solution.
  • the CAM test was carried out as described by Noll (1998) and Kuhn (1998).
  • FIG. 2 shows schematically the cellular mechanism of the translation inhibition induced by native human ARNA I (s) by means of an antisense mechanism.
  • the ARNA I (s) generated in leucocytes 50 represents the antisense transcript of the Pseudo-ARNA I gene with the ARNA l (s) sequence shown in black as the intron sequence.
  • This intron sequence comprises the complete sequence of ARNA I (s) (SEQ ID No. 3), which is therefore a cellular splice Mechanism is subject.
  • the gene product of the pseudo-ARNA gene is therefore not or not only on the sense strand, but rather on the antisense strand.
  • the antisense transcript is thus the functional transcript of this gene.
  • the antisense transcript of the pseudo-ARNA I gene represents the precursor transcript for ARNA I (s).
  • ARNA I (s) By association of the spliced, presumably circularized ARNA I (s) (15) with ARP 18 and Cu 2+ ions, the complete metallo according to the invention becomes -RNP complex angiotropin 20 constituted. If necessary, the metallo-RNP complex angiotropin (designated 20) is secreted into the extracellular space 30 and diffuses to the endothelial cells 40.
  • the ARNA I (s) 15 hybridizes against the pre-mRNA 60 or mRNA of the L27a gene or directly against the L27a gene at the transcriptional level. This inhibits the synthesis of the ribosomal L27a protein.
  • the aforementioned system can be used as a model system for cell-selective RNA transfer and the stabilization of RNA in antisense and ribozyme strategies.
  • the invention therefore also covers endothelium-specific transfection kits.
  • the invention makes it possible to heal wounds with regard to the ARNA I (s) sequence and the nucleic acid complexes containing them, to heal coronary coronary artery diseases and to carry out improved transplantation surgery.
  • the ARNA I (s) sequence according to the invention and nucleic acid complexes containing them can also be used to induce the growth of nerve cells.
  • the ARNA I (as) sequence and the nucleic acid complexes containing it it is possible to inhibit tumor angiogenesis and to carry out gene therapies for example for hemangiomas.

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Abstract

L'invention concerne des procédés et des agents utilisés pour modifier l'angiogenèse humaine. Les agents utilisés dans ces procédés peuvent être des acides nucléiques, des complexes d'acides nucléiques (angiotropine) ou des anticorps agissant à leur encontre.
EP02744892A 2001-02-28 2002-01-28 Procedes et agents pour modifier l'angiogenese humaine Withdrawn EP1366155A1 (fr)

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DE2001109466 DE10109466B4 (de) 2001-02-28 2001-02-28 Verfahren und Mittel zur Modifikation humaner Angiogenese
PCT/EP2002/000859 WO2002068636A1 (fr) 2001-02-28 2002-01-28 Procedes et agents pour modifier l'angiogenese humaine

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DE19628895A1 (de) * 1995-07-17 1997-01-23 Fraunhofer Ges Forschung Metallhaltige Ribonukleotidpolypeptide
DE19810998C1 (de) * 1998-03-13 1999-08-26 Fraunhofer Ges Forschung Metallhaltige Ribonukleotidpolypeptide

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DE19628895A1 (de) * 1995-07-17 1997-01-23 Fraunhofer Ges Forschung Metallhaltige Ribonukleotidpolypeptide
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