WO2001019867A1 - Prion-related protein - Google Patents

Prion-related protein Download PDF

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Publication number
WO2001019867A1
WO2001019867A1 PCT/EP2000/008937 EP0008937W WO0119867A1 WO 2001019867 A1 WO2001019867 A1 WO 2001019867A1 EP 0008937 W EP0008937 W EP 0008937W WO 0119867 A1 WO0119867 A1 WO 0119867A1
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prion
related protein
nucleic acid
protein
acid according
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PCT/EP2000/008937
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German (de)
French (fr)
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Kay Hofmann
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Memorec Stoffel Gmbh
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; 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
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • 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 nucleic acids encoding the prion-related protein and their use.
  • the prion protein is a surface protein of cells, especially of neurons, which has an important role in the genesis of neurodegenerative diseases such as scrapie, BSE, Creutzfeld-Jakob disease etc.
  • a common factor in these diseases is the presence of an atypically folded form of PrP.
  • the atypical, pathogenic folding of the PrP protein can be triggered by an inheritable mutation in the PrP gene, e.g. in familial fatal insomnia (FFI) and Gerstmann-St syndrome.
  • FFI familial fatal insomnia
  • Gerstmann-St syndrome e.g. in Gerstmann-St syndrome
  • an atypical folding of the PrP protein can also occur sporadically with a genetically unchanged Prp gene.
  • the PrP protein is bound to the outside of the plasma membrane of eukaryotic cells, especially neurons, by means of a GPI anchor (glycosylphosphatidylinositol).
  • GPI anchor glycosylphosphatidylinositol
  • the spatial structure of the non-anchored protein in solution is known. It has a high proportion of alpha helices and is stabilized by a disulfide bridge.
  • the structure of the pathogenic atypically folded form is not known in detail, but it has a high proportion of beta-sheet elements.
  • Atypically folded PrP protein has a protease-resistant portion and shows a tendency to aggregate. Such aggregated PrP protein is found in the brain of individuals suffering from prion disease. The mechanism of how PrP aggregation leads to the demise of neurons and thus the symptoms of the disease is not known.
  • Protein X plays an important role in the conversion of normally folded PrP into the pathogenic form. Protein X binds to the PrP protein and thus accelerates the change in folding.
  • Protein X is incorporated into the PrP aggregates and thus affects the aggregation. If protein X fulfills a physiologically important function and is in deficit compared to PrP, incorporation into the aggregates would result in the cell no longer having enough free protein and being damaged as a result. In this case, the external administration of protein X or a derivative thereof would alleviate the neurodegenerative phenotype of prion diseases.
  • the present invention for the first time makes nucleic acids available which code for a prion-related protein.
  • the prion-related protein is characterized in that it shows significant similarity to known PrP proteins from the human, mouse, sheep, cattle, and chicken species. The similarity can be shown with the help of the "generalized profile" method described in: P. Bucher, K. Karplus, N. Moeri and K. Hofmann Comput. Chem. 20: 3-23 (1996).
  • the prion-related protein is further characterized by the presence of at least one disulfide bridge, the presence of a signal sequence for protein secretion and the presence of a C-terminal hydrophobic sequence which is typically replaced in eukaryotic cells by a glycosylphosphatidylinositol anchor ,
  • FIG. 1 shows the nucleic acid sequence of the mRNA coding for human prion-related protein
  • FIG. 2 shows the nucleic acid sequence of the mRNA coding for murine prion-related protein
  • Figure 3 shows the peptide sequence of the human prion-related protein
  • Figure 4 shows the peptide sequence of the murine prion-related protein
  • the nucleic acid according to the invention is preferably a nucleic acid which codes for the prion-related protein. It is particularly preferably the human and murine prion-related protein.
  • the corresponding nucleic acid sequences are as Seq. ID. No. 1 and Seq. ID. No. 2 disclosed.
  • the corresponding peptide sequences are as Seq. ID. No. 3 and Seq. ID. No. 4 disclosed.
  • the person skilled in the art can easily find the nucleic acids and proteins from other eukaryotes, taking into account the high homology. For this purpose, he can use cross-reacting antibodies for a specific affinity chromatographic purification, or he can synthesize oligonucleotide primers based on the nucleic acid sequence and amplify the nucleic acids sought using the polymerase chain reaction in a cDNA library of the eukaryote.
  • the corresponding cDNA library can be obtained in a manner known per se by isolating mRNA from a tissue sample and subsequent reverse transcription.
  • the amino acid sequence can be derived from the nucleic acid sequence using the genetic code. Alternatively, it is also possible to search for and combine homologous sequences in EST (Expressed Sequence Tags) databases.
  • nucleic acids according to the invention are suitable for the expression of the prion-related protein in pro- or eukaryotic systems.
  • they are also suitable for the expression of the prion-related protein in vivo in the sense of a gene therapy or in particular in the form of fragments, also in a complementary structure, as antisense nucleotides for reducing the expression of the prion-related protein.
  • nucleic acids according to the invention can be produced by chemical synthesis or by duplication in genetically modified organisms by methods known per se to the person skilled in the art.
  • the invention also relates to the prion-related protein obtainable by the expression of the nucleic acids according to the invention.
  • the prion-related protein according to the invention can be produced by expression in genetically modified organisms.
  • Eukaryotic expression systems are particularly suitable.
  • Corresponding eukaryotic expression systems are known to the person skilled in the art, for example pRc / CMV (Stratagene). Purification from genetically modified organisms, particularly in the case of overexpression, provides easy and direct access to the protein according to the invention and also allows isolation in large amounts. Variants of the prion-related protein are also claimed.
  • variants includes both naturally occurring allelic variations of the prion-related protein and recombinant DNA technology (in particular by in vitro mutagenesis with the help of chemically synthesized oligonucleotides) and subsequent expression of proteins which are biological and / or immunological Activity correspond to the prion-related protein.
  • Amino acids can be deleted, inserted or exchanged conservatively. Conservative exchange means that an amino acid is replaced by an amino acid that has similar physicochemical properties.
  • amino acids are interchangeable: serine for / against alanine, alanine for / against glycine, methionine for / against serine, lysine for / against arginine, lysine for / against serine.
  • variants also encompasses N- and / or C-terminal truncated proteins and acetylated, glycosylated, amidated and / or phosphorylated derivatives.
  • Compounds in which the prion-related protein or its variants are coupled to further molecules such as dyes, radionuclides or affinity components also represent variants according to the invention.
  • nucleic acids which code for the prion-related protein or which are complementary to these nucleic acids.
  • the nucleic acids can be, for example, DNA, RNA, PNA or nuclease-resistant analogs.
  • Nuclease-resistant analogs are, in particular, those compounds in which the phosphodiester bond is modified by hydrolysis-stable compounds, for example phosphothioates, methylphosphonates or the like.
  • Short fragments of the nucleic acids are particularly suitable for antisense nucleotides. For reasons of specificity, these should preferably be more than 6, more preferably more than 8 and most preferably more than 12 nucleotides exhibit. For diffusion and cost reasons, they are typically less than 30 nucleotides in length, preferably 24 or less, and even more preferably 18 or fewer nucleotides in length.
  • the invention also relates to derivatives of nucleic acids which are coupled with other molecules for diagnostic or therapeutic purposes, for example with fluorescent dyes, radioactive markers or affinity components, and fragments of the nucleic acids according to the invention and of the nucleic acids complementary to these nucleic acids, and variants of the nucleic acids.
  • Fragments refer to nucleic acids that are shortened on the 5 'or 3' or on both sides.
  • variants is understood to mean that these nucleic acids hybridize under stringent conditions with the nucleic acid according to the invention or nucleic acids complementary thereto.
  • stringent conditions is understood to mean that the hybridization is carried out under conditions in which the temperature is still up to 10 ° C. below the temperature (under otherwise identical conditions) under which exactly complementary nucleic acids would just just hybridize. For example, if a precisely hybridizing nucleic acid hybridizes under given conditions up to a temperature of approx. 55 ° C, then stringent conditions are temperatures equal to or higher than 45 ° C.
  • the preferred temperature range for stringent conditions is 5 ° C, more preferably 3 ° C.
  • the invention further relates to antibodies which are directed against the prion-related protein according to the invention or the nucleic acids according to the invention. These substances are particularly suitable for use in diagnostics, immunoassays known to those skilled in the art, for histological examination and as a medicament for the treatment of conditions which are associated with overexpression of the prion-related protein.
  • Such antibodies according to the invention can be obtained by methods known per se to those skilled in the art by immunization with prion-related protein, ß nucleic acids or peptide and nucleic acid fragments are obtained in the presence of auxiliary reagents.
  • the invention furthermore relates to cell lines which overexpress the prion-related protein according to the invention.
  • Such cell lines are obtainable by transfection with vectors which contain the nucleic acids according to the invention which code for the prion-related protein.
  • the transfection can be carried out, for example, by electroporation.
  • the cell lines are preferably stably transfected.
  • the prion-related protein according to the invention, the nucleic acids according to the invention and the antibodies according to the invention can optionally be contained in medicaments and diagnostic agents together with further auxiliaries.
  • These medicinal and diagnostic agents are suitable for the diagnosis and treatment of diseases which are based on over- or under-expression and / or an increased or reduced activity of the prion-related protein and / or on disorders of protein aggregation, the copper balance of the cell and / or those associated with increased levels of neuronal cell death.
  • a pharmaceutical screening method according to the invention is based on the observation of protein aggregates in cell lines which overexpress the prion-related protein according to the invention, possibly in combination with PrP, at least one potentially pharmaceutically active substance being added. Such cell lines are therefore particularly suitable for the development and testing of pharmaceutical lead structures.
  • a pharmaceutical screening method according to the invention is based on measuring the cell survivability of the cell lines mentioned under the influence of at least one potentially pharmaceutically active substance.

Abstract

The invention relates to a prion-related protein and to its use.

Description

Prionen-verwandtes Protein Prion-related protein
Die vorliegende Erfindung betrifft Nukleinsäuren, die für das Prionen-verwandte Protein codieren, und ihre Anwendung.The present invention relates to nucleic acids encoding the prion-related protein and their use.
Das Prionen-Protein (PrP) ist ein Oberflächenprotein von Zellen, insbesondere von Neuronen, das eine wichtige Rolle bei der Genese von neurodegenerativen Krankheiten wie Scrapie, BSE, Creutzfeld-Jakob-Krankheit etc. innehat. Ein gemeinsamer Faktor dieser Erkrankungen ist das Vorliegen einer atypisch gefalteten Form des PrP. Die atypische, pathogene Faltung des PrP-Proteins kann durch eine vererbbare Mutation im PrP-Gen ausgelöst werden, wie sie z.B. in den Krankheiten familial fatal insomnia (FFI) und Gerstmann-Sträussler Syn- drom vorliegt. Eine atypische Faltung des PrP-Proteins kann jedoch auch sporadisch bei genetisch unverändertem Prp-Gen auftreten. Über einen nicht vollständig aufgeklärten Mechanismus kann solch atypisch gefaltetes PrP-Protein, wenn es mit der Nahrung aufgenommen wird, normal gefaltetes PrP-Protein in die atypisch gefaltete Form konvertieren. Daraus resultiert ein infektiöse Krankheit, die u.a. bei Schafen unter dem Namen Scrapie, bei Rindern unter dem Namen BSE (bovine spongiforme encephalopathy), und beim Menschen unter dem Namen Creutzfeld-Jakob-Krankheit bekannt ist.The prion protein (PrP) is a surface protein of cells, especially of neurons, which has an important role in the genesis of neurodegenerative diseases such as scrapie, BSE, Creutzfeld-Jakob disease etc. A common factor in these diseases is the presence of an atypically folded form of PrP. The atypical, pathogenic folding of the PrP protein can be triggered by an inheritable mutation in the PrP gene, e.g. in familial fatal insomnia (FFI) and Gerstmann-Sträussler syndrome. However, an atypical folding of the PrP protein can also occur sporadically with a genetically unchanged Prp gene. Through an incompletely elucidated mechanism, such atypically folded PrP protein, when ingested with food, can convert normally folded PrP protein to the atypically folded form. This results in an infectious disease that includes in sheep under the name Scrapie, in cattle under the name BSE (bovine spongiform encephalopathy), and in humans under the name Creutzfeld-Jakob disease.
Das PrP-Protein ist mittels eines GPI-Ankers (Glycosyl-Phosphatidylinositol) an die Außenseite der Plasmamembran von eukaryontischen Zellen, vor allem von Neuronen, gebunden. Die räumliche Struktur des nicht-verankerten Proteins in Lösung ist bekannt. Sie weist einen hohen Anteil an alpha-Helices auf und wird durch eine Disulfid-Brücke stabilisiert. Die Struktur der pathogenen atypisch gefalteten Form ist nicht im Detail bekannt, sie besitzt jedoch einen hohen Anteil an beta-Faltblatt Elementen. Atypisch gefaltetes PrP-Protein besitzt einen Pro- tease-resistenten Anteil und zeigt die Tendenz zur Aggregation. Solcherart ag- gregiertes PrP Protein wird im Gehirn von Individuen gefunden, die an einer Prionen-Erkrankung leiden. Der Mechanismus, wie PrP-Aggregation zum Untergang von Neuronen und damit zu den Symptomen der Krankheit führt ist nicht bekannt. Mäuse mit einem arti- fiziell inaktivierten PrP-Gen zeigen keine Krankheit, neuronalen Zelltod, oder einen anderen deutlichen Phänotyp. Es ist daher nicht anzunehmen, dass der Verlust von aktivem PrP-Protein durch Aggregation eine Rolle bei der Krankheit spielt. Ein unbekanntes Protein, in der Literatur als „Protein X„ bezeichnet, spielt eine wichtige Rolle bei der Umwandlung von normal gefaltetem PrP in die patho- gene Form. Protein X bindet an das PrP Protein und beschleunigt so die Veränderung der Faltung.The PrP protein is bound to the outside of the plasma membrane of eukaryotic cells, especially neurons, by means of a GPI anchor (glycosylphosphatidylinositol). The spatial structure of the non-anchored protein in solution is known. It has a high proportion of alpha helices and is stabilized by a disulfide bridge. The structure of the pathogenic atypically folded form is not known in detail, but it has a high proportion of beta-sheet elements. Atypically folded PrP protein has a protease-resistant portion and shows a tendency to aggregate. Such aggregated PrP protein is found in the brain of individuals suffering from prion disease. The mechanism of how PrP aggregation leads to the demise of neurons and thus the symptoms of the disease is not known. Mice with an artificially inactivated PrP gene show no disease, neuronal cell death, or any other clear phenotype. It is therefore unlikely that the loss of active PrP protein through aggregation plays a role in the disease. An unknown protein, referred to in the literature as "Protein X", plays an important role in the conversion of normally folded PrP into the pathogenic form. Protein X binds to the PrP protein and thus accelerates the change in folding.
Eine weitergehende Beschreibung der Eigenschaften des PrP-Proteins und den damit in Verbindung stehenden Erkrankungen findet sich in: SB Prusiner, Proc. Natl. Acad. Sei. USA 95:13363-13383 (1998)A further description of the properties of the PrP protein and the associated diseases can be found in: SB Prusiner, Proc. Natl. Acad. Be. USA 95: 13363-13383 (1998)
Es ist wahrscheinlich, dass das „Protein X„ in die PrP-Aggregate eingebaut wird, und die Aggregation auf diese Weise beeinflusst. Wenn das Protein X eine physiologisch wichtige Funktion erfüllt und im Unterschuss gegenüber PrP vorliegt, würde der Einbau in die Aggregate dazu führen, dass der Zelle nicht mehr genügend freies Protein zur Verfügung steht und dadurch geschädigt wird. Die externe Verabreichung von Protein X oder einem Derivat davon würde in diesem Fall den neurodegenerativen Phänotyp von Prionen-Erkrankungen lindern.It is likely that the "Protein X" is incorporated into the PrP aggregates and thus affects the aggregation. If protein X fulfills a physiologically important function and is in deficit compared to PrP, incorporation into the aggregates would result in the cell no longer having enough free protein and being damaged as a result. In this case, the external administration of protein X or a derivative thereof would alleviate the neurodegenerative phenotype of prion diseases.
Falls diesem Protein keine essentielle Funktion zukommt, könnte durch eine Depletion des „Protein X„ die Umwandlung von normalem PrP in atypisch gefaltetes PrP verhindert oder verlangsamt werden.If this protein has no essential function, depletion of the “Protein X” could prevent or slow down the conversion of normal PrP into atypically folded PrP.
Durch die vorliegende Erfindung werden erstmals Nukleinsäuren verfügbar gemacht, die für ein Prionen-verwandtes Protein codieren. Das Prionen-verwandte Protein ist dadurch charakterisiert, dass es signifikante Ähnlichkeit zu bekannten PrP-Proteinen aus den Spezies Mensch, Maus, Schaf, Rind, Huhn aufweist. Die Ähnlichkeit kann gezeigt werden mit Hilfe der „generalized Profile,, Methode, wie beschrieben in: P. Bucher, K. Karplus, N. Moeri und K. Hofmann Comput. Chem. 20:3-23 (1996).The present invention for the first time makes nucleic acids available which code for a prion-related protein. The prion-related protein is characterized in that it shows significant similarity to known PrP proteins from the human, mouse, sheep, cattle, and chicken species. The similarity can be shown with the help of the "generalized profile" method described in: P. Bucher, K. Karplus, N. Moeri and K. Hofmann Comput. Chem. 20: 3-23 (1996).
Das Prionen-verwandte Protein ist weiterhin charakterisiert durch das Vorliegen von zumindest einer Disulfid-Brücke, das Vorliegen einer Signal-Sequenz zur Proteinsekretion, sowie das Vorliegen einer C-terminalen hydrophoben Sequenz, die in eukaryontischen Zellen typischerweise durch einen Glycosyl- Phosphatidylinositol Anker ersetzt wird.The prion-related protein is further characterized by the presence of at least one disulfide bridge, the presence of a signal sequence for protein secretion and the presence of a C-terminal hydrophobic sequence which is typically replaced in eukaryotic cells by a glycosylphosphatidylinositol anchor ,
Figur 1 zeigt die Nukleinsäure-Sequenz der mRNA kodierend für humanes Prio- nen- verwandtes ProteinFIG. 1 shows the nucleic acid sequence of the mRNA coding for human prion-related protein
Figur 2 zeigt die Nukleinsäure-Sequenz der mRNA kodierend für murines Prio- nen-verwandten ProteinFIG. 2 shows the nucleic acid sequence of the mRNA coding for murine prion-related protein
Figur 3 zeigt die Peptid-Sequenz des humanen Prionen-verwandten ProteinsFigure 3 shows the peptide sequence of the human prion-related protein
Figur 4 zeigt die Peptid-Sequenz des murinen Prionen-verwandten ProteinsFigure 4 shows the peptide sequence of the murine prion-related protein
Bevorzugt handelt es sich bei der erfindungsgemäßen Nukleinsäure um eine Nukleinsäure, die für das Prionen-verwandte Protein codiert. In besonders bevorzugter Weise handelt es sich dabei um das humane und murine Prionen- verwandte Protein. Die entsprechenden Nukleinsäuresequenzen sind als Seq. ID. Nr. 1 und Seq. ID. Nr. 2 offenbart. Die entsprechenden Peptidsequenzen sind als Seq. ID. Nr. 3 und Seq. ID. Nr. 4 offenbart.The nucleic acid according to the invention is preferably a nucleic acid which codes for the prion-related protein. It is particularly preferably the human and murine prion-related protein. The corresponding nucleic acid sequences are as Seq. ID. No. 1 and Seq. ID. No. 2 disclosed. The corresponding peptide sequences are as Seq. ID. No. 3 and Seq. ID. No. 4 disclosed.
Bei Kenntnis der Aminosäure- und Nukleinsäurestruktur des humanen und murinen Prionen-verwandten Proteins kann der Fachmann unter Berücksichtigung der hohen Homologie die Nukleinsäuren und Proteine aus anderen Eukaryonten leicht auffinden. Dazu kann er zum einen kreuzreagierende Antikörper für eine spezifische affinitätschromatographische Aufreinigung einsetzen, oder er kann auf der Grundlage der Nukleinsäuresequenz Oligonukleotidprimer synthetisieren und die gesuchten Nukleinsäuren mit Hilfe der Polymerasekettenreaktion in einer cDNA-Bank des Eukaryonten amplifizieren. Die entsprechende cDNA-Bank kann durch Isolierung von mRNA aus einer Gewebeprobe und anschließende Reverse-Transkription in an sich bekannter Weise erhalten werden. Aus der Nukleinsäuresequenz kann mit Hilfe des genetischen Codes die Aminosäuresequenz abgeleitet werden. Alternativ ist es hierzu auch möglich, homologe Sequenzen in EST (Expressed Sequence Tags)-Datenbanken zu suchen und zu kombinieren.If the amino acid and nucleic acid structure of the human and murine prion-related protein is known, the person skilled in the art can easily find the nucleic acids and proteins from other eukaryotes, taking into account the high homology. For this purpose, he can use cross-reacting antibodies for a specific affinity chromatographic purification, or he can synthesize oligonucleotide primers based on the nucleic acid sequence and amplify the nucleic acids sought using the polymerase chain reaction in a cDNA library of the eukaryote. The corresponding cDNA library can be obtained in a manner known per se by isolating mRNA from a tissue sample and subsequent reverse transcription. The amino acid sequence can be derived from the nucleic acid sequence using the genetic code. Alternatively, it is also possible to search for and combine homologous sequences in EST (Expressed Sequence Tags) databases.
Die erfindungsgemäßen Nukleinsäuren eignen sich zur Expression des Prionen- verwandten Proteins in pro- oder eukaryontischen Systemen. Darüber hinaus sind sie auch zur Expression des Prionen-verwandten Proteins in vivo im Sinne einer Gentherapie oder insbesondere in Form von Fragmenten auch in komplementärer Struktur als Antisense-Nukleotide zur Verringerung der Expression des Prionen-verwandten Proteins geeignet.The nucleic acids according to the invention are suitable for the expression of the prion-related protein in pro- or eukaryotic systems. In addition, they are also suitable for the expression of the prion-related protein in vivo in the sense of a gene therapy or in particular in the form of fragments, also in a complementary structure, as antisense nucleotides for reducing the expression of the prion-related protein.
Die erfindungsgemäßen Nukleinsäuren können durch chemische Synthese oder durch Vervielfältigung in gentechnisch veränderten Organismen nach dem Fachmann an sich bekannten Verfahren hergestellt werden.The nucleic acids according to the invention can be produced by chemical synthesis or by duplication in genetically modified organisms by methods known per se to the person skilled in the art.
Gegenstand der Erfindung ist auch das durch die Expression der erfindungsgemäßen Nukleinsäuren erhältliche Prionen-verwandte Protein.The invention also relates to the prion-related protein obtainable by the expression of the nucleic acids according to the invention.
Das erfindungsgemäße Prionen-verwandte Protein lässt sich durch Expression in gentechnisch veränderten Organismen herstellen. Insbesondere sind eukaryon- tische Expressionssysteme geeignet. Entsprechende eukaryontische Expressionssysteme sind dem Fachmann bekannt wie beispielsweise pRc/CMV (Firma Stratagene). Die Aufreinigung aus gentechnisch veränderten Organismen bietet, insbesondere im Falle der Überexpression, ein leichten und direkten Zugang zum erfindungsgemäßen Protein und erlaubt darüber hinaus die Isolierung in größeren Mengen. Weiterhin beansprucht werden Varianten des Prionen-verwandten Proteins. Unter den Begriff "Varianten" fallen sowohl natürlich vorkommende allelische Variationen des Prionen-verwandten Proteins sowie durch rekombinante DNA- Technologie (insbesondere durch in vitro Mutagenese mit Hilfe von chemisch synthetisierten Oligonukleotiden) und anschließende Expression erzeugte Proteine, die hinsichtlich ihrer biologischen und/oder immunologischen Aktivität dem Prionen-verwandten Protein entsprechen. Dabei können sowohl Aminosäuren deletiert, eingefügt oder konservativ ausgetauscht werden. Konservativer Austausch bedeutet, dass eine Aminosäure durch eine Aminosäure ersetzt wird, die ähnliche physikalisch-chemische Eigenschaften aufweist.The prion-related protein according to the invention can be produced by expression in genetically modified organisms. Eukaryotic expression systems are particularly suitable. Corresponding eukaryotic expression systems are known to the person skilled in the art, for example pRc / CMV (Stratagene). Purification from genetically modified organisms, particularly in the case of overexpression, provides easy and direct access to the protein according to the invention and also allows isolation in large amounts. Variants of the prion-related protein are also claimed. The term "variants" includes both naturally occurring allelic variations of the prion-related protein and recombinant DNA technology (in particular by in vitro mutagenesis with the help of chemically synthesized oligonucleotides) and subsequent expression of proteins which are biological and / or immunological Activity correspond to the prion-related protein. Amino acids can be deleted, inserted or exchanged conservatively. Conservative exchange means that an amino acid is replaced by an amino acid that has similar physicochemical properties.
So sind beispielsweise folgende Aminosäuren austauschbar: Serin für/gegen Alanin, Alanin für/gegen Glycin, Methionin für/gegen Serin, Lysin für/gegen Arginin, Lysin für/gegen Serin.For example, the following amino acids are interchangeable: serine for / against alanine, alanine for / against glycine, methionine for / against serine, lysine for / against arginine, lysine for / against serine.
Insbesondere umfaßt der Begriff Varianten auch N- und/oder C-terminale verkürzte Proteine sowie acetylierte, glykosylierte, amidierte und/oder phosphory- lierte Derivate. Auch Verbindungen, bei denen das Prionen-verwandte Protein oder dessen Varianten mit weiteren Molekülen wie Farbstoffen, Radionukliden oder Affinitätskomponenten gekoppelt sind, stellen erfindungsgemäße Varianten dar.In particular, the term variants also encompasses N- and / or C-terminal truncated proteins and acetylated, glycosylated, amidated and / or phosphorylated derivatives. Compounds in which the prion-related protein or its variants are coupled to further molecules such as dyes, radionuclides or affinity components also represent variants according to the invention.
Beansprucht werden auch Nukleinsäuren, die für das Prionen-verwandte Protein codieren bzw. komplementär zu diesen Nukleinsäuren sind. Bei den Nukleinsäuren kann es sich beispielsweise um DNA, RNA, PNA oder um nukleaseresis- tente Analoga handeln. Nukleaseresistente Analoga sind insbesondere solche Verbindungen, in denen die Phosphodiesterbindung durch hydrolysestabile Verbindungen modifiziert sind, beispielsweise Phosphothioate, Methylphosphonate o.a.Also claimed are nucleic acids which code for the prion-related protein or which are complementary to these nucleic acids. The nucleic acids can be, for example, DNA, RNA, PNA or nuclease-resistant analogs. Nuclease-resistant analogs are, in particular, those compounds in which the phosphodiester bond is modified by hydrolysis-stable compounds, for example phosphothioates, methylphosphonates or the like.
Für Antisensenukleotide sind insbesondere kurze Fragmente der Nukleinsäuren geeignet. Diese sollten aus Gründen der Spezifität bevorzugt mehr als 6, noch mehr bevorzugt mehr als 8 und am meisten bevorzugt mehr als 12 Nukleotide aufweisen. Aus Gründen der Diffusion und der Kosten haben sie üblicherweise eine Länge von weniger als 30 Nukleotiden, bevorzugt 24 oder weniger und noch mehr bevorzugt 18 oder weniger Nukleotide.Short fragments of the nucleic acids are particularly suitable for antisense nucleotides. For reasons of specificity, these should preferably be more than 6, more preferably more than 8 and most preferably more than 12 nucleotides exhibit. For diffusion and cost reasons, they are typically less than 30 nucleotides in length, preferably 24 or less, and even more preferably 18 or fewer nucleotides in length.
Gegenstand der Erfindung sind auch Derivate von Nukleinsäuren, die für diagnostische oder therapeutische Zwecke mit anderen Molekülen gekoppelt sind, beispielsweise mit Fluoreszenzfarbstoffen, radioaktiven Markern oder Affinitätskomponenten, sowie Fragmente der erfindungsgemäßen Nukleinsäuren und der zu diesen Nukleinsäuren komplementären Nukleinsäuren sowie Varianten der Nukleinsäuren.The invention also relates to derivatives of nucleic acids which are coupled with other molecules for diagnostic or therapeutic purposes, for example with fluorescent dyes, radioactive markers or affinity components, and fragments of the nucleic acids according to the invention and of the nucleic acids complementary to these nucleic acids, and variants of the nucleic acids.
Fragmente bezeichnet dabei Nukleinsäuren, die am 5' oder 3' oder an beiden Seiten verkürzt sind. Unter dem Begriff "Varianten" wird verstanden, dass diese Nukleinsäuren unter stringenten Bedingungen mit der erfindungsgemäßen Nukleinsäure bzw. dazu komplementären Nukleinsäuren hybridisieren. Unter dem Begriff "stringente Bedingungen" wird verstanden, dass die Hybridisierung bei Bedingungen durchgeführt wird, bei der die Temperatur noch bis zu 10°C unter der Temperatur liegt (bei sonst identischen Bedingungen), bei der exakt komplementäre Nukleinsäuren gerade noch hybridisieren würden. Wenn beispielsweise eine exakt hybrisierende Nukleinsäure unter gegebenen Bedingungen bis zu einer Temperatur von ca. 55°C hybridisiert, dann sind stringente Bedingungen Temperaturen gleich oder höher 45°C. Bevorzugt ist der Temperaturbereich für stringente Bedingungen von 5°C, noch mehr bevorzugt von 3°C.Fragments refer to nucleic acids that are shortened on the 5 'or 3' or on both sides. The term “variants” is understood to mean that these nucleic acids hybridize under stringent conditions with the nucleic acid according to the invention or nucleic acids complementary thereto. The term “stringent conditions” is understood to mean that the hybridization is carried out under conditions in which the temperature is still up to 10 ° C. below the temperature (under otherwise identical conditions) under which exactly complementary nucleic acids would just just hybridize. For example, if a precisely hybridizing nucleic acid hybridizes under given conditions up to a temperature of approx. 55 ° C, then stringent conditions are temperatures equal to or higher than 45 ° C. The preferred temperature range for stringent conditions is 5 ° C, more preferably 3 ° C.
Des weiteren betrifft die Erfindung Antikörper, die gegen das erfindungsgemäße Prionen-verwandte Protein oder die erfindungsgemäßen Nukleinsäuren gerichtet sind. Diese Substanzen eignen sich insbesondere zum Einsatz in der Diagnostik, dem Fachmann an sich bekannten Immunoassays, zur histologischen Untersuchung sowie als Arzneimittel zur Behandlung von Zuständen, die mit einer Überexpression des Prionen-verwandten Proteins verbunden sind. Solche erfindungsgemäßen Antikörper können mit dem Fachmann an sich bekannten Verfahren durch Immunisierung mit Prionen-verwandtem Protein, erfindungsgemä- ßen Nukleinsäuren oder Peptid- und Nukleinsäurenfragmenten in Gegenwart von Hilfsreagenzien erhalten werden.The invention further relates to antibodies which are directed against the prion-related protein according to the invention or the nucleic acids according to the invention. These substances are particularly suitable for use in diagnostics, immunoassays known to those skilled in the art, for histological examination and as a medicament for the treatment of conditions which are associated with overexpression of the prion-related protein. Such antibodies according to the invention can be obtained by methods known per se to those skilled in the art by immunization with prion-related protein, ß nucleic acids or peptide and nucleic acid fragments are obtained in the presence of auxiliary reagents.
Weiterhin sind Gegenstand der Erfindung Zellinien, die das erfindungsgemäße Prionen-verwandte Protein überexprimieren. Solche Zellinien sind erhältlich durch Transfektion mit Vektoren, die die erfindungsgemäßen Nukleinsäuren, die für das Prionen-verwandte Protein kodieren, enthalten. Im Falle von eukaryonti- schen Zellinien kann die Transfektion beispielsweise durch Elektroporation erfolgen. Die Zellinien sind dabei vorzugsweise stabil transfiziert.The invention furthermore relates to cell lines which overexpress the prion-related protein according to the invention. Such cell lines are obtainable by transfection with vectors which contain the nucleic acids according to the invention which code for the prion-related protein. In the case of eukaryotic cell lines, the transfection can be carried out, for example, by electroporation. The cell lines are preferably stably transfected.
Das erfindungsgemäße Prionen-verwandte Protein, die erfindungsgemäßen Nukleinsäuren sowie die erfindungsgemäßen Antikörper können in Arzneimitteln und Diagnostikmitteln gegebenenfalls zusammen mit weiteren Hilfsstoffen enthalten sein. Diese Arznei- und Diagnostikmittel eigenen sich zur Diagnose und Behandlung von Erkrankungen, die auf einer Über- oder Unterexpression und/oder einer erhöhten oder verminderten Aktivität des Prionen-verwandten Protein und/oder auf Störungen der Proteinaggregation, der Kupferhaushaltes der Zelle beruhen und/oder solche, die mit einem erhöhtem Maß an neuronalen Zelltod einhergehen. Insbesondere sind dies Erkrankungen bei denen das PrP- Protein oder das Prionen-verwandte Protein selber Aggregate bilden oder die Aggregation anderer Proteine beeinflussen.The prion-related protein according to the invention, the nucleic acids according to the invention and the antibodies according to the invention can optionally be contained in medicaments and diagnostic agents together with further auxiliaries. These medicinal and diagnostic agents are suitable for the diagnosis and treatment of diseases which are based on over- or under-expression and / or an increased or reduced activity of the prion-related protein and / or on disorders of protein aggregation, the copper balance of the cell and / or those associated with increased levels of neuronal cell death. In particular, these are diseases in which the PrP protein or the prion-related protein itself form aggregates or influence the aggregation of other proteins.
Ein erfindungsgemäßes pharmazeutisches Screening-Verfahren beruht auf der Beobachtung von Protein-Agggregaten in Zellinien, die das erfindungsgemäße Prionen-verwandte Protein, ggf. in Kombination mit PrP, überexprimiern, wobei mindestens eine potentiell pharmazeutisch wirksame Substanz zugefügt wird. Solche Zellinien eignen sich somit insbesondere zur Entwicklung und Prüfung von pharmazeutischen Leitstrukturen. Alternativ beruht ein erfindungsgemäßes pharmazeutisches Screening-Verfahren auf der Messung der Zell- Überlebensfähigkeit der erwähnten Zellinien unter Einfiuss mindestens einer potentiell pharmazeutisch wirksamen Substanz. A pharmaceutical screening method according to the invention is based on the observation of protein aggregates in cell lines which overexpress the prion-related protein according to the invention, possibly in combination with PrP, at least one potentially pharmaceutically active substance being added. Such cell lines are therefore particularly suitable for the development and testing of pharmaceutical lead structures. Alternatively, a pharmaceutical screening method according to the invention is based on measuring the cell survivability of the cell lines mentioned under the influence of at least one potentially pharmaceutically active substance.

Claims

Patentansprüche claims
1. Nukleinsäure kodierend für Prionen-verwandtes Protein.1. Nucleic acid coding for prion-related protein.
2. Nukleinsäure gemäß Anspruch 1, dadurch gekennzeichnet, dass das Prionen-verwandte Protein folgende Aminosäure-Motive enthält: G-X!-R-X2- X3, wobei Xi entweder N oder Q repräsentiert und X2 und X3 jeweils entweder F oder Y repräsentieren, sowie C-X4-F-W-L, wobei X4 entweder D oder E repräsentiert.2. Nucleic acid according to claim 1, characterized in that the prion-related protein contains the following amino acid motifs: GX ! -RX 2 - X 3 , where Xi represents either N or Q and X 2 and X 3 each represent either F or Y, and CX 4 -FWL, where X 4 represents either D or E.
3. Nukleinsäure gemäß Ansprüchen 1 bis 2, dadurch gekennzeichnet, dass es sie für Prionen-verwandtes Protein eines Säugetiers, insbesondere für humanes oder murines Prionen-verwandtes Protein kodiert.3. Nucleic acid according to claims 1 to 2, characterized in that it codes for prion-related protein of a mammal, in particular for human or murine prion-related protein.
4. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 3 mit der Sequenz gemäß Seq. ID. Nr. 1 oder Seq. ID. Nr. 2.4. Nucleic acid according to at least one of claims 1 to 3 with the sequence according to Seq. ID. No. 1 or Seq. ID. No. 2.
5. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 4 dadurch gekennzeichnet, dass es sich um DNA, RNA, PNA oder nukleaseresistente Analoga handelt.5. Nucleic acid according to at least one of claims 1 to 4, characterized in that it is DNA, RNA, PNA or nuclease-resistant analogs.
6. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 5 dadurch gekennzeichnet, dass es sich um mRNA, cDNA oder genomische DNA handelt.6. Nucleic acid according to at least one of claims 1 to 5, characterized in that it is mRNA, cDNA or genomic DNA.
7. Nukleinsäure dadurch gekennzeichnet, dass sie komplementär zur Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 6 ist.7. Nucleic acid characterized in that it is complementary to the nucleic acid according to at least one of claims 1 to 6.
8. Eukaryontisches Prionen-verwandtes Protein, erhältlich durch Expression der Nukleinsäure gemäß Anspruch 1 bis 6, insbesondere mit der Sequenz gemäß Seq. ID. Nr. 3 oder Seq. ID. Nr. 4. 8. Eukaryotic prion-related protein, obtainable by expression of the nucleic acid according to claims 1 to 6, in particular with the sequence according to Seq. ID. No. 3 or Seq. ID. No. 4.
9. Antikörper, dadurch gekennzeichnet, dass sie gegen Prionen-verwandtes Protein gemäß Anspruch 8 oder eine Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7 gerichtet sind.9. Antibodies, characterized in that they are directed against prion-related protein according to claim 8 or a nucleic acid according to at least one of claims 1 to 7.
10. Zellinie, dadurch gekennzeichnet, dass sie Prionen-verwandtes Protein gemäß Anspruch 8 überexprimiert.10. cell line, characterized in that it overexpresses prion-related protein according to claim 8.
11. Transgenes Säugetier mit Überexpression (gain of function) oder Gendefi- zienz oder Gendefekt (loss of function) für eukaryontische Prionen- verwandtes Protein11. Transgenic mammal with overexpression (gain of function) or gene deficiency or gene defect (loss of function) for eukaryotic prion-related protein
12. Transgenes Säugetier gemäß Anspruch 11 dadurch gekennzeichnet, dass es ein Nagetier ist.12. Transgenic mammal according to claim 11, characterized in that it is a rodent.
13. Arzneimittel enthaltend Prionen-verwandtes Protein gemäß Anspruch 8, eine Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7 und/oder einen Antikörper gemäß Anspruch 9 zusammen mit weiteren Hilfsstoffen.13. Medicament containing prion-related protein according to claim 8, a nucleic acid according to at least one of claims 1 to 7 and / or an antibody according to claim 9 together with further auxiliaries.
14. Diagnostikmittel enthaltend Prionen-verwandtes Protein gemäß Anspruch 8, eine Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7 und/oder einen Antikörper gemäß Anspruch 9 zusammen mit weiteren Hilfsstoffen.14. Diagnostic agent containing prion-related protein according to claim 8, a nucleic acid according to at least one of claims 1 to 7 and / or an antibody according to claim 9 together with other auxiliaries.
15. Verwendung der Arzneimittel gemäß Anspruch 13 oder der Diagnostikmittel gemäß Anspruch 14 zur Diagnose und Behandlung von Erkrankungen15. Use of the medicament according to claim 13 or the diagnostic agent according to claim 14 for the diagnosis and treatment of diseases
16. Verwendung gemäß Anspruch 15, dadurch gekennzeichnet dass es sich bei den Erkrankungen um neurodegenerative Erkrankungen handelt. 16. Use according to claim 15, characterized in that the diseases are neurodegenerative diseases.
17. Verwendung gemäß Anspruch 15, dadurch gekennzeichnet, dass die Erkrankungen auf einer Veränderung der Aggregation des Prion-Proteins PrP und/oder des Prionen-verwandten Proteins gemäß Anspruch 8 und/oder auf Störungen des Kupferhaushaltes, des Zelltodes oder der Zeilproliferation von Neuronen beruhen.17. Use according to claim 15, characterized in that the diseases are based on a change in the aggregation of the prion protein PrP and / or the prion-related protein according to claim 8 and / or on disorders of the copper balance, cell death or cell proliferation of neurons ,
18. Verfahren zum Screening von Wirkstoffen dadurch gekennzeichnet, dass die Veränderung der Expression oder Aktivität des Prionen-verwandten Proteins in Zellinien gemäß Anspruch 10 bei Zugabe von mindestens einer möglichen pharmazeutisch wirksamen Substanz gemessen wird.18. A method for screening active substances, characterized in that the change in expression or activity of the prion-related protein in cell lines according to claim 10 is measured when at least one possible pharmaceutically active substance is added.
19. Verwendung der Zellinie gemäß Anspruch 10 zur Entwicklung und Prüfung von pharmazeutischen Leitstrukturen.19. Use of the cell line according to claim 10 for the development and testing of pharmaceutical lead structures.
20. Verfahren zur Herstellung des Prionen-verwandten Proteins gemäß Anspruch 8 durch chemische Peptidsynthese oder durch Expression in gentechnisch veränderten Organismen, insbesondere in eukaryontischen Expressionssystemen.20. A method for producing the prion-related protein according to claim 8 by chemical peptide synthesis or by expression in genetically modified organisms, in particular in eukaryotic expression systems.
21. Verfahren zur Herstellung einer Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7 durch chemische Synthese oder durch Vervielfältigung in gentechnisch veränderten Organismen.21. A method for producing a nucleic acid according to at least one of claims 1 to 7 by chemical synthesis or by duplication in genetically modified organisms.
22. Varianten des Prionen-verwandten Proteins gemäß Anspruch 8.22. Variants of the prion-related protein according to claim 8.
23. Nukleinsäure gemäß mindestens einem der Ansprüche 1 bis 7, dadurch gekennzeichnet, dass es sich um Derivate, Fragmente oder Varianten der Nukleinsäuren handelt. 23. Nucleic acid according to at least one of claims 1 to 7, characterized in that it is derivatives, fragments or variants of the nucleic acids.
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