WO2003058021A2 - Nouvelles sequences d'adn induisant l'apoptose - Google Patents

Nouvelles sequences d'adn induisant l'apoptose Download PDF

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Publication number
WO2003058021A2
WO2003058021A2 PCT/EP2003/000270 EP0300270W WO03058021A2 WO 2003058021 A2 WO2003058021 A2 WO 2003058021A2 EP 0300270 W EP0300270 W EP 0300270W WO 03058021 A2 WO03058021 A2 WO 03058021A2
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Prior art keywords
nucleic acid
acid molecule
poly
peptide
apoptosis
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PCT/EP2003/000270
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German (de)
English (en)
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WO2003058021A3 (fr
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Kerstin KÖNIG-HOFFMAN
Michael Kazinski
Rolf Schäfer
Björn KESPER
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Xantos Biomedicine Ag
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Priority to AU2003235789A priority Critical patent/AU2003235789A1/en
Publication of WO2003058021A2 publication Critical patent/WO2003058021A2/fr
Publication of WO2003058021A3 publication Critical patent/WO2003058021A3/fr

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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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/04—Drugs for disorders of the muscular or neuromuscular system for myasthenia gravis
    • 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
    • A61P25/08—Antiepileptics; Anticonvulsants
    • A61P25/12—Antiepileptics; Anticonvulsants for grand-mal
    • 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
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

Definitions

  • the present invention relates to nucleic acid molecules which encode apoptosis-associated (poly) peptides.
  • the (poly) peptides induce or inhibit apoptosis.
  • the invention further relates to (poly) peptides encoded by these nucleic acids, vectors which contain the nucleic acid molecules and hosts transformed with these nucleic acid molecules.
  • the hosts are transgenic non-human mammals.
  • the invention relates to methods for identifying test substances which activate or inhibit the (poly) peptides according to the invention directly or indirectly and for improving such test substances.
  • the invention relates to methods for the production of pharmaceuticals or medical products, in which the identified or improved test substance is formulated with a pharmaceutically acceptable carrier or diluent, as well as pharmaceuticals that can be used to induce or inhibit apoptosis and to treat corresponding diseases.
  • Apoptosis is the genetically encoded suicide program that is induced in eukaryotic cells under certain physiological or pathological conditions.
  • the induction of apoptosis must be regulated extremely precisely because hyperactivity can lead to degenerative diseases.
  • reduced apoptosis induction can contribute to tumor progression.
  • transgenic mice containing multiple transgenes have produced transgenic mice containing multiple transgenes, the functions of which are determined by examining the phenotype (Simonet et al., Cell 89 (1997), 309-319 and Smith et al., Nat. Genet. 16 (1997) , 28-36).
  • a disadvantage of the in vitro methods is that the results obtained do not readily correlate with complex regulated cell biological effects. In turn, studies on transgenic animals are very complex and tedious.
  • Nucleic acid sequences Small plasmid pools corresponding to 20 clones from normalized cDNA expression libraries are transiently introduced into the human kidney cell line 293.
  • the apoptosis-inducing activity of a nucleic acid sequence is determined manually by microscopic inspection for morphological features characteristic of apoptosis.
  • the apoptosis-inducing adenine nucleotide translocase-1 (ANT-1) gene was identified using this method.
  • the ANT-1 gene is considered to be the cause of the degenerative cardiac disease dilatory cardiomyopathy (DCM) (PCT / EP00 / 08812).
  • DCM degenerative cardiac disease dilatory cardiomyopathy
  • ANT-1 gene or the protein encoded thereby can be used for the development of medicaments for combating certain diseases
  • the previously identified apoptosis-associated sequences can only be used to develop drugs against a limited number of diseases which are causally related to apoptosis.
  • certain genes not yet known or not yet associated with apoptosis or their gene products in the induction or inhibition of apoptosis interact. In this case it would be desirable if the molecular relationships could be clarified, since this would lead to particularly promising approaches for combating apoptosis-related diseases.
  • the prerequisite for this goal is that as many genes or gene products associated with apoptosis as possible are identified, or that direct correlations between genes or gene products and apoptosis can be established.
  • the present invention relates to a nucleic acid molecule encoding a (poly) peptide which is apoptosis-associated and (a) which is a nucleic acid molecule with one of the nucleotide acid sequences of SEQ ID NO: 1-119 and SEQ ID NO: 209-398; (b) is a nucleic acid molecule encoding a (poly) peptide having one of the amino acid sequences of SEQ ID NO: 120-208 and SEQ ID NO: 399-579; (c) is a nucleic acid molecule which comprises the nucleic acid molecule according to (a) or (b); (d) is a nucleic acid molecule in which at least one nucleotide is substituted, deleted or inserted compared to the nucleic acid molecule according to (a), (b) or (c); (e) is a nucleic acid molecule that hybridizes under stringent conditions to the complementary strand of the nucleic acid molecule according to (a) and
  • poly peptide includes polypeptides (proteins) as well as peptides.
  • Peptides have a maximum number of 30 amino acids, whereas amino acid chains with more than 30 amino acids are called polypeptides.
  • apoptosis-associated in combination with nucleic acids / (poly) peptides describes those nucleic acids / (poly) peptides that are associated with the occurrence of apoptotic processes in a cell, in particular in a mammalian cell can use the nucleic acids / (poly) peptides to induce apoptosis
  • Nucleic acids / (poly) peptides i.e. Nucleic acids / (poly) peptides that can induce and / or promote apoptotic processes.
  • the nucleic acids according to the invention are particularly preferably dominant apoptosis-inducing nucleic acids which are capable of inducing apoptosis when expressed in a cell and which produce the characteristics characteristic of apoptosis, such as DNA fragmentation, morphological peculiarities etc.
  • the nucleic acids can be in double-stranded or single-stranded form, e.g. as DNA or RNA.
  • the isolated nucleic acids can develop their cellular effect by expression, in particular by overexpression in cells.
  • apoptosis-associated includes that the nucleic acids or (poly) peptides have no direct influence on the induction of apoptosis, but are part of a cascade, for example an enzymatic cascade or a signal transduction cascade, and other members of this cascade are one have a direct influence on the induction of apoptosis.
  • a member located downstream in this cascade is the trigger of apoptosis
  • the activation or inhibition of the nucleic acids or the (poly) peptides identified in connection with this invention can nevertheless have an influence on apoptosis, since in this way the Expression of the downstream member affected, e.g. is prevented.
  • the crucial control levers for apoptosis such as induction, can be located upstream in the cascade.
  • the expression of the nucleic acids or the (poly) peptides identified in connection with this invention is then also given, which in turn ultimately have an effect on apoptosis, for example its induction can.
  • such changes in the activity of different members of the cascade lead to an "apoptosis-associated" behavior of the nucleic acids or the (poly) peptides that were identified in connection with this invention.
  • nucleic acid molecules that encode (poly) peptides that are associated with apoptosis have been identified.
  • the invention further encompasses those sequences which encode the same (poly) peptide as the specifically disclosed sequences, that is to say those sequences which differ from the specific ones distinguish disclosed sequences by the degeneration of the genetic code. Also encompassed by the invention are nucleic acid sequences which comprise the sequences specifically disclosed.
  • sequences extend a maximum of 5 kB, preferably not more than 3 kB and particularly preferably not more than 1 kB, of the sequences described in detail or differing from them by the degeneracy of the genetic code in 5 'and / or 3' Direction.
  • sequences encode fusion proteins, for example.
  • the nucleic acids according to the invention further comprise those molecules which differ from the above-mentioned molecules by at least one substituted, deleted or inserted nucleotide.
  • the possible different combinations of these mutations are also included.
  • the term “at least” clarifies that the differences can also relate to several nucleotides. These different nucleotides can be strung together, for example in the case of a larger deletion, or can be scattered over the sequence, for example as substitutions for individual nucleotides.
  • nucleic acid molecules or the (poly) peptides encoded by them retain the properties associated with apoptosis
  • the molecules differing from the specifically disclosed nucleic acids can be used, for example, with the methods disclosed in the examples be tested for the apoptosis-associated properties.
  • Nucleic acid molecules which hybridize with the complementary strands of the abovementioned nucleic acid molecules under stringent conditions and have the stated function are also encompassed by the invention.
  • Stringent hybridization conditions can be set by the person skilled in the art with knowledge of the specifically disclosed nucleic acid sequences. It is known that the stringency of the hybridization conditions depends on various factors. One of these factors is the salt concentration in the Hybridization solution. This will usually not exceed 750 mM NaCl and 75 mM sodium citrate. The salt concentrations are preferably below 250 mM NaCl and 25 mM sodium citrate.
  • stringent hybridization conditions are hybridization at 65 ° C in 0.2 ⁇ SSC, 0.1% SDS, or 0.5xSSC, 0.1% SDS at 65 C C. After Hybris mecanicsrepress are not specifically binding nucleic acid molecules removed by washing. Suitable washing conditions include those in 1XSSC, 0.1% SDS at 62-68 ° C or in 0.2XSSC, 0.1% SDS at 55-65 ° C.
  • the person skilled in the art can adapt the hybridization conditions to the necessary conditions with the help of his specialist knowledge with the aid of suitable literature.
  • the invention further includes nucleic acid molecules which are at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical to the aforementioned nucleic acid molecules or which encode (poly) peptides which are at least 80%, preferably at least 90%, more preferably at least 95%, even more preferably at least 98% and most preferably at least 99% identical to the (poly) peptides encoded by the above-mentioned nucleic acid molecules, provided that these Nucleic acid molecules have apoptosis-associated properties.
  • the percentage match can be determined electronically using suitable programs. Such programs are the MEGALIGN program (DNASTAR; Inc. Madison, Wis.) Or BLAST (Altschul et al., Nucl. Acids Res. 25 (1997), 3389-3402.
  • nucleic acid molecules mentioned above can be of natural or non-natural origin. If the nucleic acids are of natural origin, for example, they can represent allelic variants of the specifically disclosed nucleic acid molecules. Non-naturally occurring nucleic acids can be produced recombinantly and have certain changes in the sequence which lead to desired changes in the properties of the encoded (poly) peptide. Methods suitable for this include molecular biological techniques such as site-specific mutagenesis, PCR, restriction cleavage and ligation. The definition according to the invention also includes fragments of the abovementioned nucleic acids which retain the properties associated with apoptosis.
  • All of the molecules mentioned can also have improved apoptosis-associated properties compared to the specifically disclosed nucleic acid molecules.
  • An improvement (activation or inhibition) according to the invention would mean an increase by at least 20%, preferably by at least 30%, more preferably by at least 50% and particularly preferably by at least 80%.
  • molecules as mentioned above which still have at least 30%, preferably at least 50%, more preferably at least 70% and most preferably at least 90% of the apoptosis-associated properties of the specifically disclosed nucleic acid molecules or the (poly) peptides encoded thereby , The apoptosis-associated properties can be measured and quantified as shown in the examples.
  • the invention encompasses nucleic acid molecules which are transcribed by one of the nucleic acids shown above.
  • the transcription into an mRNA can take place in a cell or a cell cluster or in an in vitro system.
  • the apoptosis-associated nucleic acids according to the invention can originate from various organisms, eukaryotic organisms such as nematodes, e.g. C. elegans, arthropods such as Drosophila, Cordata and vertebrates are preferred. Sequences from mammals, in particular from the mouse or from humans, are particularly preferred.
  • the nucleic acid according to the invention can be of various chemical nature.
  • it can be a PNA (“peptide nucleic acid”).
  • the nucleic acid molecule is DNA.
  • This preferred embodiment includes cDNA as well as genomic DNA.
  • the nucleic acid molecule is a gene.
  • gene encompasses molecules or sequences which, in addition to the coding sequence, also include intron sequences and the necessary regulatory sequences for expression. These include promoters, polyadenylation sites, enhancers, etc.
  • the nucleic acid molecule is RNA.
  • RNA is mRNA.
  • the (poly) peptide encoded thereby induces apoptosis.
  • drugs or medical products can be developed which can be used in diseases which are characterized by a lack of apoptotic activity.
  • diseases include tumor diseases and autoimmune diseases such as multiple sclerosis, rheumatoid arthritis, viral infections and lupus. This can be achieved, for example, by overexpressing the nucleic acids, but also by activating or possibly inhibiting the above-mentioned cascades elsewhere.
  • the nucleic acids according to the invention or the expression products can be used to develop drugs or medical products which can be used in diseases which are characterized by an excess of apoptotic activity.
  • Corresponding diseases include degenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease or stroke.
  • appropriate drugs can be developed by activating or inhibiting other members of the above cascades.
  • the polypeptide is a fusion protein.
  • the (poly) peptide according to the invention can be covalently linked in the fusion protein to another therapeutically active (poly) peptide, in other preferred embodiments it is linked to a (poly) peptide which is used as a label (“tag”)
  • (Poly) peptides can modulate, for example intensify or mitigate, the action of the (poly) peptide according to the invention by independently initiating or modulating processes which directly or in a synergistic manner with the (poly) peptide according to the invention influence the desired pharmacological activity
  • Examples of such (poly) peptides are interferons or interleukins, known peptides which serve as a label (for example for purification or for simplified detection) are FLAG-tag or HIS-tag, and proteins which can be used for labeling include GFP ("Green Flourescent Protein").
  • nucleic acid molecules with at least 15 nucleotides, which hybridizes specifically to the nucleic acid molecule according to the invention.
  • Nucleic acid molecules of the specified length statistically hybridize only once to the human genome under stringent conditions. They can be used specifically in vivo or in vitro to inhibit the transcription or translation of the nucleic acid molecules according to the invention.
  • These nucleic acid molecules according to the invention are preferably DNAs (“antisense” DNAs).
  • these nucleic acid molecules according to the invention are “antisense” RNAs or preferably RNAi's (Bosher and Labouesse, Nat. Cell. Biol. 2 (2000), E 31-26).
  • the present invention relates to a vector comprising the nucleic acid molecule according to the invention.
  • the nucleic acids according to the invention are in operative linkage with an expression control sequence, so that they are in a suitable host cell can be transcribed and optionally translated.
  • Expression control sequences usually comprise a promoter and optionally regulatory sequences such as operators or enhancers. Translation initiation sequences may also be present.
  • Suitable expression control sequences for prokaryotic or eukaryotic host cells are known to the person skilled in the art (see, for example, Sambrook et al., Supra).
  • the recombinant vector according to the invention can also contain conventional elements such as an origin of replication and a selection marker gene.
  • recombinant vectors for example plasmids, cosmids, phages, viruses etc.
  • plasmids for example plasmids, cosmids, phages, viruses etc.
  • Starting materials for the production of the recombinant vectors according to the invention are commercially available (for example from Stratagene, InVitroGen or Promega).
  • the present invention relates to a host into which the nucleic acid molecule according to the invention or the vector according to the invention has been introduced.
  • the host according to the invention is a recombinant cell which has been transformed or transfected with a nucleic acid according to the invention or a vector according to the invention.
  • the transformation or transfection can be carried out according to known methods, e.g. by calcium phosphate coprecipitation, lipofection, electroporation, particle bombardment or viral infection.
  • the cell according to the invention can contain the recombinant nucleic acid in extrachromosomal or chromosomally integrated form.
  • the recombinant cell is of eukaryotic origin. Suitable eukaryotic cells include CHO cells, HeLa cells and others. Many of these cells are available through depositaries such as the ATCC or the DMSZ.
  • prokaryotic cells also belong to the hosts according to the invention. Bacterial cells of the type E. coli are particularly preferred as prokaryotic cells.
  • the present invention relates to a nucleic acid molecule which is derived from the nucleic acid molecule according to the invention and differs therefrom by mutation.
  • This embodiment of the nucleic acid molecule according to the invention can have different types of mutations or combinations thereof and have an apoptosis-associated property which is modified or gradually changed compared to the specifically disclosed nucleic acids or the (poly) peptides encoded thereby.
  • the nucleic acid molecule encodes a polypeptide which is no longer associated with apoptosis.
  • nucleic acid according to the invention for example knock-out mice or corresponding other non-human animals (preferably other non-human knock-out mammals) can be produced (Hogan et al., "Manipulating the Mouse Embryo", Cold Spring Harbor Lanoratory (1986) These no longer have functional copies of the nucleic acid molecule / gene according to the invention in the genome and allow conclusions to be drawn about the physiological function of the gene or the (poly) peptide encoded thereby.
  • the present invention relates to a vector comprising the nucleic acid molecule according to the invention which is derived from the nucleic acid molecule according to the invention and differs therefrom by mutation and which preferably encodes a polypeptide which is no longer associated with apoptosis.
  • This embodiment of the vector according to the invention can also be used for the production of non-human knock-out animals.
  • the present invention relates to a host that does not have a functional copy of the nucleic acid molecule according to the invention.
  • Such hosts can be created by various genetic manipulations. Examples of knockout mice are shown by Sauer B., Methods 14 (1998), 381-292.
  • the nucleic acid molecule according to the invention or the vector according to the invention may also have been introduced into the host, the nucleic acid molecule according to the invention, which in this embodiment can also be contained in the vector according to the invention, is derived from the apoptosis-associated nucleic acid molecule according to the invention, which is shown in sequence and / or differs from it by mutation and which preferably encodes a polypeptide that is no longer apoptosis-associated. This deficiency can be converted into a homozygous state, for example by crossing.
  • this is a transgenic non-human animal, preferably a mammal.
  • the transgenic non-human animal according to the invention can preferably have different genetic constitutions. It may (i) constitutively or inducibly overexpress the gene of a nucleic acid according to the invention, (ii) contain the endogenous gene of a nucleic acid according to the invention in inactivated form, (iii) contain the endogenous gene of a nucleic acid according to the invention completely or partially replaced by a mutated gene of a nucleic acid according to the invention , (iv) have a conditional and tissue-specific overexpression or underexpression of the gene of a nucleic acid according to the invention or (v) have a conditional and tissue-specific knock-out of the gene of a nucleic acid according to the invention.
  • the transgenic animal preferably additionally contains an exogenous gene of a nucleic acid according to the invention under the control of a promoter which allows overexpression.
  • the endogenous gene of a nucleic acid according to the invention can be overexpressed by activating and / or exchanging its own promoter.
  • the endogenous promoter of the gene of a nucleic acid according to the invention preferably has a genetic change which leads to a changed expression of the gene.
  • the genetic modification of the endogenous promoter includes a mutation of individual bases as well as deletion and insertion mutations.
  • a preferred embodiment thus relates to a transgenic animal that constitutively or inducibly overexpresses the gene of a nucleic acid according to the invention.
  • the introduced gene of a nucleic acid according to the invention can optionally have additional mutations.
  • Another preferred embodiment relates to a transgenic animal which contains the endogenous gene of a nucleic acid according to the invention in inactivated form.
  • the gene of a nucleic acid according to the invention is preferably inactivated by introducing a knock-out mutation by means of homologous recombination or by introducing an antisense construct or an RNAi contract.
  • a third preferred embodiment relates to a transgenic animal in which the endogenous gene of a nucleic acid according to the invention is completely or partially replaced by a mutated gene of a nucleic acid according to the invention.
  • a fourth preferred embodiment relates to a transgenic animal which has a conditional and tissue-specific overexpression or underexpression of the gene of a nucleic acid according to the invention.
  • the transgenic animal has a conditional and tissue-specific knock-out of the gene of a nucleic acid according to the invention.
  • this is a transgenic rodent, preferably a transgenic mouse, a transgenic rabbit, a transgenic rat, or a transgenic sheep, a transgenic cow, a transgenic goat or a transgenic pig.
  • mice have numerous advantages over other animals. They are easy to hold and their physiology is considered a model system for that of humans.
  • the production of such gene-manipulated animals is well known to the person skilled in the art and is carried out by customary methods (see, for example, Hogan, B., Beddington, R., Costantini, F. and Lacy, E. (1994), Manipulating the Mouse Embryo ; A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).
  • Such a transgenic animal according to the invention can be used for the genetic and / or pharmacological examination of diseases which are associated with excessive or reduced or no expression of a gene of a nucleic acid according to the invention or other members of the cascades mentioned.
  • the transgenic animals according to the invention can serve as a model for the diseases associated with the gene of a nucleic acid according to the invention in humans or else in farm animals. For example, the effects of drugs or gene therapies on the course of the disease can be determined.
  • the animals can also be useful for diagnosis or early detection of a disease.
  • cell culture systems in particular human cell culture systems, can also be used for the applications which are described for the non-human transgenic animal according to the invention.
  • the present invention relates to a method for producing a
  • the (poly) peptide can, for example, by conventional methods, for example after disruption of corresponding cells by ion exchange, size selection or
  • Affinity chromatography etc. can be purified.
  • the (poly) peptide is isolated from the culture supernatant.
  • the (poly) peptide is isolated from a body fluid.
  • the body fluid is milk or serum.
  • the present invention relates to a (poly) peptide encoded by the nucleic acid molecule according to the invention or produced by the method according to the invention.
  • apoptosis-associated (poly) peptides according to the invention as well as the other embodiments of the (poly) peptides according to the invention can be obtained by expression of the apoptosis-associated or the nucleic acids derived therefrom, by chemical synthesis or by combinations of both methods.
  • the present invention relates to a receptor which binds specifically to the nucleic acid molecule according to the invention or the (poly) peptide according to the invention.
  • the term “specifically binds” means that the receptor does not, or essentially not, cross-react with other nucleic acid molecules or (poly) peptides, including those with a similar primary sequence or a similar three-dimensional structure.
  • Cross-reactivity can be determined using methods known in the art (see Hariow and Lane “Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988).
  • competitive assays can be used, for example, in which the receptor is incubated together with the labeled (poly) peptide according to the invention and a (poly) peptide competing therewith, the latter being able to be used in different concentrations.
  • the receptor is an antibody or a fragment or derivative thereof or an aptamer.
  • Antibody fragments include Fv, Fab and F (ab 2 ) 'fragments.
  • the derivatives include scFvs (Hariow and Lane, loc. Cit.).
  • Antibodies can be of polyclonal or monoclonal origin.
  • this is a monoclonal antibody.
  • the present invention further relates to a carrier which carries one or more of the nucleic acid molecules according to the invention, one or more vectors according to the invention, one or more hosts according to the invention, one or more (poly) peptides according to the invention or one or more receptors according to the invention.
  • the hosts are excluded from being non-human mammals. However, it is preferred that they are unicellular of prokaryotic or eukaryotic origin.
  • this is a solid carrier.
  • the carrier is a membrane, a chip, a glass surface, a silicon carrier or a mitrotiter plate.
  • the nucleic acid molecules etc. immobilized on the support are applied in an ordered pattern. This will significantly facilitate the assignment of the properties to the nucleic acid etc. in tests for desired properties, for example in a high-throughput screen.
  • the microtiter plate has at least 24 wells, preferably at least 96, 384 or 1536 wells. If, for example, cells are carried by the carrier and this carrier is, for example, a microtiter plate, the term “carry” also includes the fact that the cells in the wells of the microtiter plate are cultivated in solid or liquid nutrient medium or in a physiologically acceptable liquid.
  • nucleic acids, vectors, hosts and (poly) peptides according to the invention can be used to identify new active substances for the therapy and prevention of apoptosis-associated diseases.
  • the use in known cellular or molecular screening assays is advised, which may be carried out in high-throughput format.
  • the present invention also relates to a method for identifying an inhibitor of the (poly) peptide according to the invention, comprising the steps (a) contacting the (poly) peptide with one or more test substances under conditions which affect the binding of the test substance (s) to the (poly) allow peptide; and (b) demonstrating whether the test substance (s) limit or suppress the apoptosis-associated properties of the (poly) peptide.
  • test substance (s) to the (poly) peptide
  • Physiological conditions for example incubation in physiological saline, are suitable for many purposes.
  • the proof can also be designed according to the design of the experiment.
  • the apoptosis-associated properties can be examined as explained in the examples.
  • a reduction in the measurable activity allows conclusions to be drawn that the test substance (s) can be suitable as an inhibitor.
  • the present invention further relates to a method for identifying an activator of the (poly) peptide according to the invention, comprising the steps (a) bringing the (poly) peptide into contact with one or more test substances under conditions which affect the binding of the test substance (s) to the (poly) allow peptide; and (b) demonstrating whether the test substance (s) enhance the apoptosis-associated properties of the (poly) peptide.
  • the present invention relates to a method for identifying an inhibitor of the nucleic acid molecule according to the invention, comprising the steps (a) bringing the nucleic acid molecule into contact with one or more test substances under conditions which allow the test substance (s) to bind to the nucleic acid molecule; and (b) detection of whether the test substance (s) restrict or prevent the apoptosis-associated properties of the nucleic acid or of the (poly) peptide encoded thereby.
  • the present invention further relates to a method for identifying an activator of the nucleic acid molecule according to the invention, comprising the steps (a) bringing the nucleic acid molecule into contact with one or more test substances under conditions which permit the test substance (s) to bind to the nucleic acid molecule; and (b) detection of whether the test substance (s) enhance the apoptosis-associated properties of the nucleic acid molecule or of the (poly) peptide encoded thereby.
  • Inhibition / activation of the nucleic acid means the inhibition / activation of the expression of the nucleic acid on the transcriptional and / or on the translational level or on the basis of post-translational modifications.
  • the present invention relates to a method for identifying an inhibitor or activator of the biological function of the nucleic acid molecule according to the invention or of the (poly) peptide encoded by the nucleic acid in a cellular system or comprising the steps (a) of contacting the nucleic acid according to the invention or that of the nucleic acid encoded (poly) peptide with one or more test substances and (b) detection of whether the test substance (s) restrict or prevent or activate the biological function or the apoptosis-associated properties of the nucleic acid molecule.
  • the test preferably takes place in a cell system or cells transfected with the nucleic acid according to the invention, preferably of mammalian origin.
  • the transfection can be stable or transient.
  • a genetically modified organism can also be used instead of the cellular system. Examples of such genetically modified organisms are C. elegans, Drosophila, or zebrafish.
  • the term “biological function” can mean the apoptosis-associated property itself, for example the apoptosis-inducing property.
  • this term can represent biological properties that do not lead directly to apoptosis, but can be used as a "readouf system to allow conclusions to be drawn about the apoptosis-associated properties.
  • the expression level of the nucleic acid can be regarded as a biological property, regardless of the function of the encoded polypeptide, provided that upstream or downstream (in the latter, for example, by repression mechanisms) influence the level of expression of the nucleic acid according to the invention becomes part of a cascade, it is possible to draw direct or indirect conclusions about the apoptosis-associated properties, for example the probability that apoptosis is induced in this cell.
  • the enzymatic activity of the (poly) peptide can be measured (if this polypeptide has an enzymatic activity), it being possible to draw conclusions about the apoptosis-associated properties from the amount of enzyme expressed or from the conversion of substrate.
  • the method according to the invention relates to a method in which, if a plurality of test substances are used, the following steps are carried out: (a) testing different test substances in different reaction vessels, those test substances which have the apoptosis-associated properties of the nucleic acids or of them coded (poly) peptides or the reaction vessels containing them do not influence are no longer taken into account in the further test procedure; (b) test substances in reaction vessels which influence the apoptosis-associated properties of the nucleic acids or the nucleic acids encoded by them are distributed to new reaction vessels and the test is repeated; and (c) step (b) is repeated until a single test substance is identified, to which the change in the apoptosis-associated properties can be assigned.
  • This embodiment of the method according to the invention allows the desired substance (s) to be isolated from a pool of substances in a simple manner. With this approach, larger numbers of test substances can be screened per unit of time.
  • nucleic acids or the (poly) peptides are coupled to a reporter system or the A reporter system is added to the test approach and the reporter system delivers
  • Binding of the test substance (s) to the (poly) peptide a detectable signal.
  • Reporter systems suitable for the method according to the invention include: SEAP
  • test substances are low molecular weight substances, peptides, aptamers, antibodies or fragments or derivatives thereof.
  • Antibodies, fragments and derivatives thereof have already been defined above.
  • Peptides and low molecular weight substances can be obtained in particular from commercially available substance libraries.
  • the method is carried out in the host according to the invention.
  • the transgenic non-human animals according to the invention and in particular the transgenic mice prove to be particularly suitable for testing the substances. As already explained above, results obtained from these animals particularly allow conclusions to be drawn about the situation in humans. Of course, this is particularly relevant for medical questions.
  • this is a high-throughput method.
  • High-throughput methods are particularly preferred because they accomplish the largest possible number of expression patterns to be examined with minimal expenditure of time.
  • the samples to be analyzed are advantageously transferred to solid supports such as, for example, microchips and fixed there by generally known methods or else transferred to microtiter plates, where in vitro assays for the properties associated with apoptosis can also be carried out.
  • the method is computer-assisted.
  • the present invention further relates to a method for improving the pharmacological properties of the test substances identified by the method according to the invention, wherein (a) the binding site of the test substance to the nucleic acid molecule or the (poly) peptide and optionally the binding site of the nucleic acid molecule or the (poly) peptide identified to the test substance; (b) the binding site of the test substance and optionally the nucleic acid molecule or the (poly) peptide is modified by molecular modeling; and (c) modifying the test substance such that its binding specificity or binding affinity or binding avidity is increased for the nucleic acid molecule or the (poly) peptide.
  • This method as well as the methods for modifying (improving the pharmacological properties, etc.) of the substances described below, preferably also include the steps for identifying the substances, as described above.
  • the identification of the binding site of the drug by means of binding-specific mutagenesis and the analysis of chimeric proteins can take place, for example, by modifications in the primary (poly) peptide sequence which influence the affinity of the drug; this usually enables precise mapping of the binding pocket for the inhibitor / activator or the drug constituted therefrom.
  • step (b) the following protocols can be used: As soon as the effector site for the test substance has been mapped, the exact residues which interact with different parts of the test substance can be identified using a combination of those from the mutagenesis studies and computer simulations of the Structure of the binding site, provided that the exact three-dimensional structure of the test substance / drug is known (if this is not the case, this can be calculated by means of computational simulation). If the test substance itself is a peptide, it can also be mutated to determine which residues interact with other residues, for example in the desired polypeptide.
  • the test substance can be modified to increase its binding affinity or its potency and specificity. For example, if there are electrostatic interactions between a particular residue of the desired polypeptide and a region of the test substance / drug molecule, the total charge in this region can be modified, which increases this special interaction.
  • Computer programs can be helpful in identifying binding sites. Suitable computer programs can be used to identify interactive sites of a putative test substance and the polypeptide by computer-assisted searches for complementary structure motifs (Fassina, Immunomethods 5 (1994), 114-120). Other suitable computer systems for the computer-aided design of proteins and peptides are described in the prior art, for example in Berry, Biochem. Soc. Trans.
  • the three-dimensional and / or crystallographic structure of the activators of the expression of the (poly) peptide according to the invention can be used for the design of peptidomimetic activators, for example in connection with the (poly) peptide identified according to the invention (Rose, Biochemistry 35 (1996), 12933 -12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).
  • the binding sites in step (a) are determined by site-specific mutagenesis.
  • the present invention also relates to a method for improving the pharmacological properties of a test substance which has been identified or improved by the method according to the invention, the test substance being further modified as a lead structure in order to improve a modified active center, a modified activity spectrum, a modified organ specificity Activity, reduced toxicity (an improved therapeutic index), reduced side effects, a delayed start of therapeutic activity or the length of therapeutic activity, changed pharmacokinetic parameters (absorption, distribution, metabolism or excretion), modified physicochemical parameters (solubility, hygroscopic properties, color, taste, smell, stability, condition), improved general specificity, organ / tissue specificity, and / or an optimized administration form and - route has what by the esterification of carboxyl groups, hydroxyl groups with carboxylic acids, hydroxyl groups to, for example, phosphates, pyrophosphates, sulfates, "hemisuccinates" or the formation of pharmaceutically acceptable salts, pharmaceutically acceptable complexes or the synthesis of pharmac
  • the identified, improved or modified test substance is further pharmacologically improved by peptidomimetics.
  • Approaches based on peptidomimetics are known in the prior art and are described, for example, in Rose, Biochemistry 35 (1996), 12933-12944 or Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558.
  • the invention further relates to a process for the manufacture of a medicament or medicinal product, wherein the nucleic acid molecule according to the invention, the vector according to the invention, the host according to the invention, extracts of the host according to the invention, the (poly) peptide according to the invention, the receptor according to the invention and / or the process according to the invention obtained inhibitor or activator formulated with a pharmaceutically acceptable carrier or diluent.
  • Apoptosis-associated diseases can be characterized on the one hand by an abnormally reduced apoptosis and thus by hyperproliferation, for example tumor diseases, autoimmune diseases and viral infections (Thompson, Science 267 (1995), 1456-1462).
  • apoptosis-associated diseases can also be characterized by abnormally increased apoptosis and thus degenerative phenomena, such as Alzheimer's disease, Huntington's disease, Parkinson's disease, reperfusion damage, stroke and alcohol damage to the liver (Thompson (1995), supra ).
  • the therapeutic or preventive application comprises the administration of an active substance to a diseased organism in a sufficient dosage to alleviate or cure the apoptosis-associated disease or to prevent the onset of an apoptosis-associated disease.
  • an apoptosis-associated nucleic acid is administered on a gene therapy vector, for example an adenovirus, a retrovirus, an adeno-associated virus, etc., in order to bring about increased expression of the apoptosis-associated nucleic acid in a diseased target cell.
  • an antisense nucleic acid can also be administered, for example on a gene therapy vector or also directly if a reduction in the expression of the apoptosis-associated nucleic acid is sought.
  • apoptosis-associated polypeptides or modulators of the activity of such apoptosis-associated polypeptides can be administered.
  • the active substances are administered by known methods, for example in gene therapy (Anderson, Nature 392 (1998), 25-30) or protein therapy (Schwarze et al., Science 285 (1999), 1569-1572).
  • Suitable pharmaceutically acceptable carriers and / or diluents are known to the person skilled in the art and include, for example, phosphate-buffered saline solutions, water, emulsions, such as, for example, oil / water emulsions, various types of wetting agents or detergents, sterile solutions, etc.
  • Medicaments which comprise such carriers can be formulated using known conventional methods. These drugs can be administered to an individual in an appropriate dose. Administration can be oral or parenteral, for example intravenous, intraperitoneal, subcutaneous, intramuscular, local, intranasal, intrabronchial, oral or intradermal, or via a catheter at one location in an artery.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic ester compounds such as ethyl oleate, which are suitable for injections.
  • Aqueous carriers include water, alcoholic aqueous solutions, emulsions, suspensions, saline solutions and buffered media.
  • Parenteral carriers include sodium chloride solutions, Ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, and bound oils.
  • Intravenous carriers include, for example, liquid, nutrient, and electrolyte supplements (such as those based on Ringer's dextrose.
  • the drug may also include preservatives and other additives such as antimicrobial compounds, antioxidants, complexing agents, and inert gases.
  • other active ingredients such as interleukins, growth factors, differentiation factors, interferons, chemotactic proteins or a non-specific immunomodulatory agent may be included.
  • the treating doctor determines the type of dosage according to the clinical factors.
  • the type of dosage depends on various factors, such as, for example, the body size or weight, the body surface, the age, gender or general health of the patient, but also on the agent to be administered specifically, the duration and mode of administration, and other medications that may be administered in parallel.
  • a typical dose can be, for example, in a range between 0.001 and 1000 ⁇ g, doses below or above this exemplary range being conceivable, especially taking into account the factors mentioned above.
  • the dose should be in a range between 1 ⁇ g and 10 mg units per day.
  • the active substances in these preparations will usually be present in a concentration of greater than 10 ⁇ g / ml of a physiological buffer.
  • the active ingredient (s) can also be present in solid form in a concentration of 0.1 to 99.5% by weight of the total mixture.
  • the active ingredient (s) in total amounts of about 0.001 to 100 mg / kg, preferably in total amounts of about 0.01 to 10 mg / kg body weight per 24 hours, optionally as a continuous infusion or in the form of several Single doses to be administered to achieve the desired result.
  • the dose should be in the range between 1 ⁇ g and 10 mg units per kilogram of body weight per day.
  • the drug can be administered topically, locally or systemically.
  • the present invention further relates to the use of the nucleic acid molecule according to the invention, a vector or host containing this nucleic acid molecule, the (poly) peptide according to the invention or an activator of this nucleic acid or this (poly) peptide identified or improved according to the method according to the invention
  • Inhibitor of the nucleic acid molecule according to the invention or of the (poly) peptide according to the invention or of an antisense construct for the nucleic acid molecule according to the invention or of a receptor which specifically binds this (poly) peptide for the production of a medicament or medical product Prevention or treatment of a tumor disease, an autoimmune disease and an and yiral disease.
  • the tumor disease is leukemia, a carcinoma or a sarcoma.
  • the autoimmune disease is multiple sclerosis, rheumatoid arthritis, diabetes or lupus.
  • the viral disease is based on an infection with hepatitis or influenza viruses.
  • the present invention relates to a use of the nucleic acid molecule according to the invention, a vector or host containing this nucleic acid molecule, the (poly) peptide according to the invention or an activator of this nucleic acid or this (poly) peptide identified or improved according to the method according to the invention Inhibitor of the nucleic acid molecule according to the invention or of the (poly) peptide according to the invention or of an antisense construct for the nucleic acid molecule according to the invention or of a receptor specifically binding this (poly) peptide for the production of a medicament or medical device for the prevention or treatment of degenerative diseases.
  • the degenerative diseases include Alzheimer's disease, Huntington's disease, Parkinson's disease, reperfusion damage, stroke and alcohol damage to the liver.
  • the present invention further relates to a composition
  • a composition comprising one or more of the nucleic acid molecules according to the invention, one or more vectors according to the invention, one or more hosts according to the invention, one or more (poly) peptides according to the invention, or one or more of the inhibitors or activators that have been identified or improved by the method according to the invention.
  • this is a pharmaceutical composition.
  • the composition contains a pharmaceutically acceptable carrier or diluent as described above.
  • this is a diagnostic composition.
  • the individual components of the diagnostic composition are preferably packaged in one or more containers. These can in turn be packed in packaging intended for sale, provided with appropriate instructions for using the diagnostic composition (the diagnostic kit).
  • the diagnostic application for which the diagnostic composition can be used preferably comprises a qualitative and / or quantitative detection of the apoptosis-associated nucleic acid, e.g. in the form of a transcript, or the polypeptide encoded thereby, in a sample, in particular a sample which was taken from a diseased organism, for example a patient.
  • Proof can be provided in the usual way, e.g. by nucleic acid hybridization or amplification reactions such as PCR or by protein detection using antibodies. Numerous techniques for this are known to the person skilled in the art.
  • the detection can also be done by using the isolated genes on a DNA chip. This allows several, e.g. all genes or meaningful fragments thereof are examined simultaneously in one experiment.
  • this is a kit.
  • the invention relates to a diagnostic method comprising a qualitative and / or quantitative detection of the apoptosis-associated nucleic acid, for example in the form a transcript, or the polypeptide encoded therein, in a sample, in particular a sample which was taken from a diseased organism, for example a patient.
  • the detection can be carried out in the usual way, for example by nucleic acid hybridization or amplification reactions such as PCR or by protein detection using antibodies. Numerous techniques for this are known to the person skilled in the art.
  • the detection can also be done by using the isolated genes on a DNA chip. This allows several, eg all genes to be examined simultaneously in one experiment.
  • the invention thus relates to a method for diagnosing a tumor disease, an autoimmune disease, a viral disease or a degenerative disease, wherein the nucleic acid or the (poly) peptide according to the invention is detected qualitatively or quantitatively and compared with a normalized value from a healthy tissue.
  • the comparison with the healthy tissue in which the nucleic acid or the (poly) peptide was determined qualitatively or quantitatively, can go directly into the test and be carried out as a control, or can be introduced into the test on the basis of data available in silico. Deviations, e.g. Increased values of the expression products can indicate the induction of apoptosis with the corresponding clinical pictures.
  • the above-mentioned diseases are broken down in more detail in the application at further points above.
  • Figure 1 Nucleic acid sequences SEQ ID NO: 1-119.
  • the sequences SEQ ID No 1-84 contain at the 5 'end three nucleotides "AGG" of the vector sequence which do not belong to the sequence according to the invention.
  • the sequences SEQ ID 110 and SEQ ID 114 contain at the 5' end three nucleotides "AAG” of the Vector sequence that does not belong to the sequence according to the invention.
  • the sequences SEQ ID No 85-88, SEQ ID 90-95 contain at the 5 'end four nucleotides “GAGG” of the vector sequence which do not belong to the sequence according to the invention.
  • sequences SEQ ID No 96-109, SEQ ID 111-113, SEQ ID 115-117 contain at the 5 'end four nucleotides "AAAG" of the vector sequence which do not belong to the sequence according to the invention.
  • sequences SEQ ID No 89 contain at the 5' end five nucleotides "GAGCG” of the vector sequence which are not part of the sequence sequence according to the invention belong. Only the sequences according to the invention without vector sequences are specified in the sequence listing.
  • FIG. 1 Amino acid sequences SEQ ID NO: 120-208
  • FIG. 4 further nucleic acid sequences SEQ ID NO: 209-213
  • Example 1 Isolation of apoptosis-inducing genes
  • the apoptosis-inducing genes were found on a genetic screen in the human cell line HEK 293T (Grimm and Leder (1997), supra), which is based on the iterative transfection of small expression plasmid pools from a normalized gene library and the subsequent determination of the programmed cell death CPRG and CDD + assay.
  • the transfection of individual clones from a positive plasmid pool made it possible to determine the apoptosis-inducing gene.
  • the apoptosis-inducing sequences were obtained from a human embryonic cDNA library (SEQ ID 001 - SEQ ID 095, SEQ ID 214-338).
  • the human embryonic cDNA library was created by Scinet, Braunschweig based on the method shown in US 5702898.
  • the starting material for this cDNA library was mRNA from embryonic / fetal tissue (embryos 9, 12 and 16 weeks old).
  • the primary titer of the first cDNA bank was 3 ⁇ 10 5 cfu / ⁇ g DNA and the average insert size was 1.0 kb.
  • This library was subsequently mixed with a second embryonic / fetal cDNA library, in which only cDNA fragments larger than 1.5 kB were used for ligation.
  • the primary titer of the second library was 04 x 10 5 cfu / ⁇ g DNA and the average insert size was 1.8 kB.
  • a multiple normalization was then carried out, so that 200,000 individual clones represented the normalized cDNA library.
  • the sequences SEQ ID 096 - SEQ ID 119, SEQ ID NO: 209-213 and SEQ ID 339-344 were obtained from a liver cDNA library.
  • the normalization and construction of a liver cDNA library was carried out as described by Grimm and Leder (J. Exp. Meth. 185 (1997), 1137-1142) and Sasaki et al. (Nucleic Acids Res. 22 (1994), 987-992).
  • liver cDNA library mRNA from the liver of 10 week old BalbC mice was normalized by association of abundant mRNA species with antisense cDNA molecules covalently coupled to latex beads and subsequent separation by centrifugation. After two rounds of hybridization, 1.5 ⁇ g (from originally 4 ⁇ g) mRNA were obtained and used to prepare a cDNA library using a cDNA synthesis kit (Gibco BRL, Gaithersburg, MD).
  • the cDNA molecules were inserted into a modified pcDNA3 vector (Invitrogen) under the control of the cytomegalovirus (CMV) promoter, in which the neomycin resistance gene had been deleted.
  • CMV cytomegalovirus
  • the DNA was introduced by electroporation into E. coli SURE cells (Stratagene, Corp. La Jolla, CA), which were then immediately frozen.
  • the library contained approximately 4x106 clones. Individual clones were inoculated with the aid of a robot in holes of 96-hole blocks (Qiagen, Hilden, Germany) in LB medium and cultured for 30 hours with shaking at 300 rpm. After identifying a positive clone in the CPRG assay, the DNA was transfected again to confirm the result. The remaining DNA was used to transform bacteria (E.coli SURE cells) for large-scale plasmid isolation and to sequence the inserted DNA. On the basis of the DNA sequence, a sequence comparison with commercial sequence databases was carried out with the aid of the "Blast" computer program.
  • the sequences SEQ ID 345-383 were obtained from an MGC clone collection (IRAK collection ("Mammalian Gene Collection”; RZPD, Berlin). This collection comprises human cDNA clones from human cell lines and tissues and is in Strausberg RL, Feingold EA, Klausner RD, Collins FS, The Mammalian Gene Collection, Science, 1999, 286, 455-457.
  • IRAK collection (“Mammalian Gene Collection”; RZPD, Berlin). This collection comprises human cDNA clones from human cell lines and tissues and is in Strausberg RL, Feingold EA, Klausner RD, Collins FS, The Mammalian Gene Collection, Science, 1999, 286, 455-457.
  • sequences SEQ ID 384-398 were obtained from a clone collection (Human Fill Length Clone Collection) from Origene Technologies Inc., Rockville USA.
  • 96-hole blocks with bacteria were centrifuged for 5 min at 3000 g (Sigma centrifuges, Osterode am Harz, Germany). The supernatant was decanted and the blocks turned over for 2-3 minutes. Then 170 ul buffer P1 (50 mM Tris-HCl / 10mM EDTA pH 8.0) was added and the bacterial pellets were completely Vortex treatment resuspended for 10 to 20 min. After adding 170 ⁇ l of buffer P2 (200 mM NaOH, 1% SDS), the block was sealed with film, mixed by inverting and incubated for 5 min at room temperature. The lysis was terminated by adding 170 ⁇ l of 4 ° C. cold buffer P3 (3 M potassium acetate pH 5.5).
  • the supernatant was transferred to 96-well polyoxymethylene microtiter blocks after centrifugation at 6000 rpm for 10 min.
  • 150 ul silicon oxide suspension was added and incubated for 20 min at room temperature.
  • the plates were centrifuged for 5 minutes at 6000 rpm.
  • the supernatant was carefully decanted and 400 ul acetone (-20 ° C) was added.
  • the plates were again vortexed (30 sec) and centrifuged for 3 min at 6000 rpm. This acetone washing process was repeated once.
  • the plates were first dried at room temperature for 5 minutes and then for 5 minutes in a vacuum chamber.
  • the pellets were resuspended in 75 ul water (60 ° C) and centrifuged at 6000 rpm and 4 ° C for 10 min. The supernatant was stored in a 96-well microtiter plate at -20 ° C.
  • Human HEK 293T cells were grown in DMEM supplemented with 5% fetal calf serum (Sigma, Deisenhofen, Germany) in a humidified 5% CO 2 atmosphere. For transfections, the cells were transferred to 96-well plates and treated with 2 ⁇ g plasmid DNA using the calcium phosphate coprecipitation method as described by Roussel et al. (Mol. Cell. Biol. 4 (1984), 1999-2009).
  • the apoptosis-inducing activity of the transfected nucleic acids was determined photometrically using a CDD + assay and a CPRG assay.
  • the loss of membrane integrity in the CPRG assay was determined as a parameter of apoptosis induction.
  • apoptotic HEK 293 cells the cell membrane becomes permeable to CPRG and CPRG is subsequently converted in the cytoplasm by ⁇ -galactosidase.
  • the product of this enzymatic reaction is a red dye that can be measured photometrically at 570 nm.
  • HEK 293 cells were each co-transfected with 1.5 ⁇ g of the following cDNAs: control vector pcDNA (empty vector without nucleic acid according to the invention), nucleic acid according to the invention and Nedd-2 cDNA - and 0.5 ⁇ g ⁇ -galactosidase reporter plasmid and apoptosis 24 determined after transfection in the CPRG assay.
  • control vector pcDNA empty vector without nucleic acid according to the invention
  • nucleic acid according to the invention and Nedd-2 cDNA - and 0.5 ⁇ g ⁇ -galactosidase reporter plasmid and apoptosis 24 determined after transfection in the CPRG assay.
  • 30 ⁇ l of a 3 mM CPRG solution were added to the culture supernatant, the cells were incubated for 2 h at 37 ° C. and 5% CO 2 and the optical density was then measured at 570 nm.
  • the DNA fragmentation in the CDD + assay (Cell Death Detection ELISAPLUS, Röche Diagnostics GmbH, Mannheim, Germany) was determined as a parameter for the induction of apoptosis.
  • HEK 293 cells were each transfected with 2.0 ⁇ g of the following cDNAs - control vector pcDNA, nucleic acid according to the invention, Nedd-2 cDNA - and the DNA fragmentation was determined photometrically 24 hours after transfection using a CDD + assay according to the manufacturer.
  • the empty DNA vector used the apoptosis-inducing cDNA of Nedd-2 served as positive control.
  • a further determination of the apoptosis-inducing properties of nucleic acids can be carried out by quantitative fragmentation (Nicoletti et al .; Journal of Immunological Methods, 139 (1991) 271-279), caspase activation (CaspaTag - Caspase3 (DEVD) Activity Kit (Intergen), Determination of the mitochondrial potential (Ushmorov et al., Blood, 93/7 (1999) 2342-2352, cytochrome C release (Ushmorov et al., Blood, 93/7 (1999) 2342-2352), rh Annexin / FITC Kit ( Bender Medsystems) or lamin cleavage using cleaved Lamin A antibodies (cell signaling).
  • FIGS. 1 and 4 The apoptosis-inducing genes identified by the genetic screen are listed in FIGS. 1 and 4 and in the sequence listing. If an identity or homology to known genes could be determined by means of a sequence comparison, this is indicated (FIGS. 3 and 5).

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Abstract

L'invention concerne des molécules d'acide nucléique codant pour des (poly)peptides associés à l'apoptose. Dans des modes de réalisation préférés, les (poly)peptides induisent ou inhibent l'apoptose. La présente invention porte également sur des (poly)peptides codés par ces acides nucléiques, des vecteurs comportant ces molécules d'acide nucléique et des hôtes transformés par ces molécules d'acide nucléique. Les hôtes sont de préférence des mammifères transgéniques non humains. Cette invention concerne aussi des procédés pour identifier des substances tests, lesquelles activent ou inhibent directement ou indirectement les (poly)peptides de l'invention, ainsi que des procédés pour améliorer de telles substances tests. Enfin, ladite invention porte sur des procédés pour réaliser des médicaments ou des produits médicaux, dont la formule comprend la substance test identifiée ou améliorée avec un excipent pharmaceutiquement acceptable ou un agent de dilution pharmaceutiquement acceptable, ainsi que des médicaments qui peuvent être utilisés pour induire ou inhiber l'apoptose et pour traiter des maladies associées.
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EP1544617A2 (fr) * 2003-09-22 2005-06-22 Bernd Dr. Hinzmann Utilisation d'une substance liant la Nifie 14 pour la diagnose et le traitement de cancer
EP1544617A3 (fr) * 2003-09-22 2005-06-29 Bernd Dr. Hinzmann Utilisation d'une substance liant la Nifie 14 pour la diagnose et le traitement de cancer
WO2005054507A2 (fr) * 2003-12-04 2005-06-16 University Of Sheffield Depistage genetique
WO2005054507A3 (fr) * 2003-12-04 2005-08-25 Univ Sheffield Depistage genetique
WO2007015113A2 (fr) * 2005-08-04 2007-02-08 Oxford Genome Sciences (Uk) Ltd Nouveaux isoformes de proteines et leurs utilisations
WO2007015113A3 (fr) * 2005-08-04 2007-12-27 Oxford Genome Sciences Uk Ltd Nouveaux isoformes de proteines et leurs utilisations
EP1837404A1 (fr) * 2006-03-24 2007-09-26 Universität Zu Köln Mutant de l'ATP13A2 appartenant à la classe 5 du typ P des ATPases et son utilisation pour le diagnostic et la thérapie des maladies neurodégénératives
US8173434B2 (en) 2006-04-04 2012-05-08 Diadexus, Inc. PCan065 antibody compositions and methods of use
WO2012029061A2 (fr) 2010-08-31 2012-03-08 Uri Wormser Polypeptides dérivés d' α-1 antitrypsine et leurs procédés d'utilisation
WO2012029061A3 (fr) * 2010-08-31 2012-08-16 Uri Wormser Polypeptides dérivés d' α-1 antitrypsine et leurs procédés d'utilisation
US9518107B2 (en) 2010-08-31 2016-12-13 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Pharmaceutical compositions containing polypeptides derived from α-1 antitrypsin and methods of use thereof
CN103030688A (zh) * 2011-09-30 2013-04-10 北京大学 抑制癌细胞生长的短肽及其编码基因与应用
CN103030688B (zh) * 2011-09-30 2014-09-17 北京大学 抑制癌细胞生长的短肽及其编码基因与应用
US20140271818A1 (en) * 2013-03-12 2014-09-18 Michael Anthony James Targeting CLPTM1L By RNA Interference for Treatment and Prevention of Cancer
US9265789B2 (en) * 2013-03-12 2016-02-23 The Medical College Of Wisconsin, Inc. Targeting CLPTM1L by RNA interference for treatment and prevention of cancer
US20160235821A1 (en) * 2013-10-01 2016-08-18 Korea Research Institute Of Bioscience And Biotechnology Pharmaceutical composition for preventing and treating cancer, containing cyb5r3 gene or protein as active ingredient
US10973884B2 (en) * 2013-10-01 2021-04-13 Korea Research Institute Of Bioscience And Biotechnology Pharmaceutical composition for preventing and treating cancer, containing CYB5R3 gene or protein as active ingredient

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