(19)
(11)EP 1 347 063 A1

(12)EUROPEAN PATENT APPLICATION

(43)Date of publication:
24.09.2003 Bulletin 2003/39

(21)Application number: 02006257.6

(22)Date of filing:  20.03.2002
(51)Int. Cl.7C12Q 1/68, A61K 31/00, A61K 38/00, C12N 15/63, C12N 5/10, C07K 16/18, A01K 67/027
(84)Designated Contracting States:
AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR
Designated Extension States:
AL LT LV MK RO SI

(71)Applicant: Biofrontera Pharmaceuticals AG
51377 Leverkusen (DE)

(72)Inventors:
  • Lübbert, Hermann, Prof.Dr.
    51381 Leverkusen (DE)
  • Engels, Peter, Dr.
    51429 Bergisch Gladbach (DE)
  • Zwilling, Stefan, Dr.
    51375 Leverkusen (DE)

(74)Representative: Sternagel, Fleischer, Godemeyer & Partner Patentanwälte 
Braunsberger Feld 29
51429 Bergisch Gladbach
51429 Bergisch Gladbach (DE)

  


(54)Multiple genes relevant for the characterisation, diagnosis and manipulation of stroke


(57) The present invention relates to the use of differentially expressed polynucleotide sequences or polypeptides for the characterisation of stroke or progression thereof or progression of neurodegenerative processes as consequence thereof, a method for characterising stroke, a method for identifying therapeutic agents for stroke and the use of such sequences for the development of a medicament.


Description


[0001] The present invention relates to the use of differentially expressed polynucleotide sequences or polypeptides for the characterisation of stroke or progression thereof or progression of neurodegenerative processes as consequence thereof, a method for characterising stroke, a method for identifying therapeutic agents for stroke and the use of such sequences for the development of a medicament.

[0002] Stroke is a rather common and potentially harmful event that often leaves patients severely disabled for the rest of their lives. It is the result of either an interruption of blood and therefore oxygen supply to the brain, or bleeding in the brain that occurs when a blood vessel bursts. The reduced oxygen supply leads to shortage of energy in the affected brain regions and results in the death of neurons around the lesion. The degeneration spreads from the site that is affected by the reduced blood supply into regions which have at all times obtained sufficient oxygen.

[0003] Stroke results from a loss of blood flow to the brain caused by thrombosis or haemorrhage. With an incidence of 250-400 in 100.000 and a mortality rate of around 30%, stroke is a major public health problem. About one-half of the stroke survivors suffer from significant persisting neurological impairment and/or physical disability. Thus, the economic costs of stroke amount to many billions of dollars worldwide.

[0004] A stroke occurs when the blood supply to part of the brain is suddenly interrupted or when a blood vessel in the brain bursts. As a consequence, brain cells die when they no longer receive oxygen and nutrients from the blood or when they are damaged by sudden bleeding into the brain. Some brain cells die immediately after interruption of the blood flow into the brain, while others remain at risk for death and stay in a compromised state for hours. These damaged cells make up the so-called "ischemic penumbra", and with timely treatment these cells could be saved. Stroke ultimately leads to infarction, the death of huge numbers of brain cells, which are eventually replaced by a fluid-filled cavity (or infarct) in the injured brain.

[0005] There are two major forms of stroke: ischemic - blockage of a blood vessel supplying the brain, and hemorrhagic - bleeding into or around the brain. An ischemic stroke can be caused by a blood clot (embolus or thrombus), which is blocking a vessel. It can also be caused by the narrowing of an artery due to the build-up of plaque (a mixture of fatty substances, including cholesterol and other lipids). The underlying pathological process called stenosis is often observed in arteriosclerosis, the most common blood vessel disease. About 80% of all strokes are ischemic strokes. A hemorrhagic stroke is caused by the bursting of an artery in the brain. Subsequently, blood spews out into the surrounding tissue and upsets not only the blood supply but also the delicate chemical balance neurons require to function. Hemorrhagic strokes account for approximately 20% of all strokes.

[0006] A transient ischemic attack (TIA), sometimes called a mini-stroke, starts just like a stroke but then resolves leaving no noticeable symptoms or deficits. The occurrence of a TIA is a warning that the person is at risk for a more serious and debilitating stroke. About one-third of patients who have a TIA will have an acute stroke sometime in the future. The addition of other risk factors compounds a person's risk for a recurrent stroke. The average duration of a TIA is a few minutes. For almost all TIAs, the symptoms go away within an hour. There is no possibility to distinguish whether symptoms will be just a TIA or persist and lead to death or disability.

[0007] Recurrent stroke is frequent; about 25 percent of people who recover from their first stroke will have another stroke within 5 years. Recurrent stroke is a major contributor to stroke disability and death, with the risk of severe disability or death from stroke increasing with each stroke recurrence. The risk of a recurrent stroke is greatest right after a stroke, with the risk decreasing with time. About 3 percent of stroke patients will have another stroke within 30 days of their first stroke and one-third of recurrent strokes take place within 2 years of the first stroke.

[0008] The most important risk factors for stroke are hypertension, arteriosclerosis, heart disease, diabetes, and cigarette smoking. Others include heavy alcohol consumption, high blood cholesterol levels, illicit drug use, and genetic or congenital conditions, particularly vascular abnormalities. People with multiple risk factors compound the destructive effects of these risk factors and create an overall risk greater than the simple cumulative effect of the individual risk factors.

[0009] Although stroke is a disease of the brain, it can affect the entire body. Depending on the affected brain region and the severity of the attack, post-stroke patients suffer from a variety of different symptoms. Some of the disabilities that can result from stroke include paralysis, cognitive deficits, speech problems, emotional difficulties, daily living problems, and pain. Stroke disability is devastating to the stroke patient and family, but therapies are available to help rehabilitate post-stroke patients. The mortality rate observed in ischemic stroke is around 30%. The time window for a medical treatment is narrow and limited to anticoagulants and thrombolytic agents, which must be given immediately (at latest 3 hours) after having a stroke. Unfortunately, there is no effective neuroprotective medication available, which is able to stop the delayed degeneration of neurons following the initial stroke attack.

[0010] Stroke symptoms appear suddenly. The following acute symptoms can be observed: Sudden numbness of the face, arm or leg, difficulties in talking or understanding speech, trouble seeing in one or both eyes, sudden trouble in walking, loss of balance and coordination. Severe headache with no known cause does also occur. Even more importantly, there is a variety of severe disabilities occurring and persisting in post-stroke patients. Paralysis is a frequent disability resulting from stroke. Cognitive deficits (problems with thinking, awareness, attention and learning) are also commonly observed. Post-stroke patients exhibit language deficits and also emotional deficits (like post-stroke depression). Furthermore, an uncommon type of pain, called central pain syndrome (CPS), can occur after having a stroke.

[0011] Currently the only effective treatment for thrombotic stroke is the use of anticoagulants (e.g. heparin), and thromobolytics (recombinant tissue plasminogen activator). Neuroprotective agents, which are effective in animal models, have generally proved ineffective in the clinic, and none are yet registered for use in stroke.

[0012] A number of experimental models have been developed for global ischemia and focal ischemia. The availability of these different models provides an opportunity to investigate mechanisms of stroke. Finding common features in different models or over several time points within one model should provide better insight into the mechanisms critical for stroke, and comparison of the models should help to understand development, progression and consequences of stroke.

[0013] Most common global ischemia models are

a) the two vessel model



[0014] Models of transient global ischemia resulting in patterns of selective neuronal vulnerability are models that attempt to mimic the pathophysiology of cardiac arrest or hemodynamic conditions that result from severe systemic hypotension. Reversible high-grade forebrain ischemia is generated by bilateral common carotid artery (CCA) occlusion. Together with systemic hypotension conditions it reduces the blood flow to severe ischemic levels (Smith et al. 1984, Acta Neuropathol. 64; p319-332). This model of transient global ischemia has the advantage of a one stage surgical preparation, the production of a high-grade forebrain ischemia and the possibility to conduct chronic survival studies in order to assess the potential of neuroprotective drugs.

b) the four vessel model



[0015] This model results in a high-grade forebrain ischemia but is produced in two stages, one to manipulate each of the CCA, and the second stage 24h later to produce the forebrain ischemia. The advantage is that the second step can be produced in awake freely moving animals (Pulsinelli et al. 1982, Ann.Neurol. 11; 491-498). Similar pathohistological results could be obtained for both vessel models.

c) the cardiac arrest model



[0016] Forebrain ischemia models are of value to study cerebral ischemia but these models do not exactly mimic the hemodynamic consequences of a cardiac arrest, which results in a complete ischemia of the brain, spinal cord and extracerebral organs (Katz et al. 1995; J. Cereb. Blood Flow Metab. 15; 1032-1039). The initial cardiac arrest models from Safar et al. (1982, Protection of tissue against hypoxia; Elsevier Biomedical Press; 147-170 ) or Korpaczew et al. (1982, Partol Fizjol Eksp Ter 3; 78-80), were developed further by Katz et al. (1989, Resuscitation 17; 39-53) and Pluta et al. (1991, Acta Neuropathologica 83; 1-11) to models with controllable insult. Katz and colleagues (1995) have reported a reproducible outcome model of cardiac arrest with apneic asphyxia of 8 min, leading to the cessation of circulation at 3-4 min of apnea and resulting in cardiac arrest of 4-5 min. At 72 hr after injury, widespread patterns of ischemic neurons were found in many brain regions, including cerebral cortex, caudate putamen CA1 and CA3 regions of hippocampus, thalamus, cerebellum and brain stem.
Pluta et al. described a primary mechanical cardiac arrest model whereby global ischemia was induced by cardiac arrest for 3 to 10 min with survival periods of the animals from 3 min to 7 days.
Although these models have several limitations, they provide a method for studying the mechanisms of neuronal injury resulting from the clinically
realistic cerebral insult and screening potential cerebral resuscitation therapies.

Focal Ischemia models



[0017] Models of permanent or transient focal ischemia typically giving rise to localised brain infarction have routinely been used to investigate the pathophysiology of stroke. For example, models of middle cerebral artery (MCA) occlusion in a variety of species have gained increased acceptance due to their relevance to the human clinical setting.

a) the permanent MCA Occlusion models



[0018] Tamura and colleagues (1981, J. Cereb. Blood Flow Metab. 1; 53-60) developed a subtemporal approach as standard model of proximal MCA occlusion. In models of permanent MCA occlusion, electrocauterisation of the MCA proximal to the origin of the lateral lenticulostriate arteries is utilised routinely. In these models, severe reductions in blood flow are seen within the ischemic core, with milder reductions in blood flow within the border or penumbral regions. The addition of moderate arterial hypotension has the effect of enlarging infarct volume.

b) the transient MCA Occlusion models



[0019] In human ischemic stroke, recirculation frequently occurs after focal ischemia. Thus, models of transient MCA occlusions have also been developed, mainly in rats or mice, whereby surgical clip or sutures are introduced to induce a transient ischemic insult.

[0020] Some further animal models for stroke are considered in several reviews like the articles of W.D. Dietrich (1998; Int. Review of Neurobiology, Vol.42; 55-101) , Wiebers et al. (1990 Stroke 21; 1-3) or Zivin and Grotta (1990 Stroke 21; 981-983).

[0021] Object of the present invention is to identify and characterise development, conditions (status which elicits), progression and consequences of stroke on a molecular basis.

[0022] This object is met by the use of polynucleotide sequences selected from the group of sequences SEQ ID NO: 1 to 88 or homologues or fragments thereof or the according polypeptides for the characterisation of a) development and /or occurrence of stroke, b) the progression of the pathology of stroke and / or b) the consequences of the pathology of stroke, whereby the characterisation is carried out outside of a living body.

[0023] Polynucleotide sequences SEQ ID NO.1 to 88 are expressed sequence tags (ESTs) representing genes, which are differentially expressed under stroke, particularly under global ischemia in the cardiac arrest model (Pluta et al, 1991 Acta Neuropathol. 83; 1-11).

[0024] The model is explained in more detail in the literature and in the examples below.

[0025] The term "polynucleotide sequence" or "nucleic acid sequence" designates in the present application any DNA or RNA sequence, independent of the length. Thus this term can describe short sequences like PCR primers or probes for hybridisation, as well as whole genes or cDNA of these genes.

[0026] The term "polypeptide" or "amino acid sequence" designates a chain of amino acids, independent of their length, however, in any case more than one amino acid.

[0027] As "homologues" of polynucleotide sequences such polynucleotide sequences are designated which encode the same type of protein as one of the polynucleotide sequences described herein. Accordingly as "homologues" of a polypeptide the polypeptides are designated, which have an amino acid sequence, wherein at least 70 %, preferably 80 %, more preferably 90 % of the amino acids are identical to one of the proteins of the present invention and wherein the replaced amino acids preferably are replaced by homologous amino acids. As "homologous" amino acids are designated those, which have similar features concerning hydrophobicity, charge, steric features etc.. Most preferred are amino acid sequences, containing the species- or family-dependent differences of the amino acid sequence. Particularly as "homologues" sequences are designated those, which correspond to one of the cited sequences in another species or individual. For example if in the present invention a rat model is used and the cited polynucleotide sequence encodes the rat protein, the according polynucleotide sequence and protein of a mouse or of a human is designated as "homologue". Further splice variants and members of gene families are designated as homologues.

[0028] "Fragments" of a polynucleotide sequence are all polynucleotide sequences, which have at least 10 identical base pairs compared to one of the polynucleotide sequences shown in the present application or by the genes represented by these polynucleotide sequences. The term "fragment" encloses therefore such fragments as primers for PCR, probes for hybridisation, DNA fragments included in DNA vectors like plasmids, cosmids, BACs or viral constructs, as well as shortened splice variants of the genes identified herein. As a fragment of a protein (polypeptide) amino acid sequences are designated which have at least three amino acids, preferably at least 10 amino acids. Therefore fragments serving as antigens or epitopes are enclosed in this designation.

[0029] In the present application the term "sequence" is used when either a polynucleotide sequence (= nucleic acid sequence) or a polypeptide (= amino acid sequence) or a protein is meant. That means, when it is irrelevant which type of sequence is used the type is not designated particularly, but with the more common term "sequence".

[0030] In the present application the term "stroke" means the development, occurrence, progression and consequences of the disease state. Several features of the development, occurence and consequences of this disease are described herein above.

[0031] The basis of the models and methods described in the present application is the examination and determination of the expression of genes, which are differentially expressed during development, conditions, progression and consequences of stroke. Therefore for the examination each sequence can be used which allows the determination of the expression rate of the considered gene. Such a sequence can be at least one of the polynucleotide sequences SEQ ID NO. 1 to 88 or homologues or fragments thereof, as well as the polypeptides encoded thereby, however, just as well polynucleotide sequences and the according polypeptides can be used which are (parts of) the genes represented by the polynucleotide sequences SEQ ID NO. 1 to 88.

[0032] According to the invention it has been found, that the genes represented by the polynucleotide sequences SEQ ID NO. 1 to 88 are differentially expressed in the cardiac arrest model of stroke.

[0033] Therefore the present invention provides sequences, which represent genes, which are differentially expressed under stroke. Such polynucleotide sequences and the according polypeptides allow the determination and examination of stroke. Most of these sequences have not yet been regarded in relation to stroke. Sequences which are known to be differentially expressed in connection with stroke conditions are Apolipoprotein E, herein referred to as SEQ ID NO: 40, (J. Cereb. Blood Flow Metab 2001 Oct; 21(10): 1199-1207), β-amyloid precursor protein (APP), herein referred to as SEQ ID NO: 77 (Neuroreport 1996 Nov 4; 7(15-17): 2727-2731), Preproenkephalin, herein referred to as SEQ ID NO: 35 (Brain Res. 1997 Jan 2; 744(1): 185-187), Cathepsin B, herein referred to as SEQ ID NO: 86 (J. Neurosurg. 1997 Nov; 87(5): 716-723).

[0034] For these examinations animal models can be used. As such a model any animal can be used wherein the necessary preparations can be carried out, however mammalian models are preferred, even more preferred are rodents. Most preferred animal models of the present invention are rat and mouse models.

[0035] The sequences of the present invention further can be used for diagnosing stroke of a human outside of the living body by determining the expression levels of at least one of the cited sequences in comparison to the non-disease status. During treatment period of a patient the expression of the presently shown sequences can also be used outside of the body for assessing the efficacy of stroke treatment. In this case blood, cerebrospinal fluid (CSF) or tissue is removed from the patient and expression is determined in the samples.

[0036] For determination and comparison of the expression levels of at least one of the genes identified in the present invention any of the commonly known methods can be used, either on RNA/cDNA level or on protein level. For example PCR, hybridisation, micro array based methods, western blot or 2-D protein gel analysis are suitable methods. One preferred method is the digital expression pattern display method (DEPD method), explained in detail in WO99/42610. The method used for determination of expression levels is not restrictive, as long as expressed amounts can be quantified.

[0037] The sequences of the present invention can further be used to develop new animal models for stroke. By examination of the expression levels of at least one of the shown sequences, a procedure might be determined, which is useful for generating a suitable animal model for different interesting conditions. In particular, useful animal models might be transgenic, knock out, or knock in models.

[0038] In such a newly generated animal model as well as in one of the known models the efficacy of compounds can be tested, using techniques known in the art. As well assay systems can be used that are based on the shown sequences. Such assay systems may be in vivo, ex vivo or in vitro assays. In any case the models or assay systems are contacted with the compound(s) to be tested and samples are obtained from these models / systems, wherein expression levels of the sequences are determined and compared to the non-treated model / system.

[0039] Dependent of the model used the samples can be derived from whole blood, CSF or whole tissue, from cell populations isolated from tissue or blood or from single cell populations (i.e. cell lines).

[0040] In one embodiment of the invention cellular assays can be used. Preferred cells for cellular assays are eukaryotic cells, more preferably mammalian cells. Most preferred are neuronal-like cells, like SHSY5Y (neuroblastoma cell line), hippocampal murine HT-22 cells, primary cultures from astrocytes, cerebral cortical neuronal-astrocytic co-cultures, mixed neuronal/glial hippocampal cultures, cerebellar granular neuronal cell cultures, primary neuronal cultures derived from rat cortex (E15-17), or COS cells (African green monkey, kidney cells); CHO cells (Chinese hamster ovary), or HEK-293 cells (human embryonic kidney).

[0041] Whereas the comparison of the expression levels (disease / non-disease status) of at least one of the provided sequences might give information about the examined disease status, it is preferred to determine the expression levels of more than one of the sequences simultaneously. Thus several combinations of the sequences can be used at different time points. By combination of several sequences a specific expression pattern can be determined indicating and / or identifying the conditions of the disease. The more expression rates are determined simultaneously, the more specific the result of the examination might be. However, good results also can be obtained by combination of only a few sequences. Therefore for the present invention it is preferred to compare the expression rates of at least two of the sequences provided herein, more preferred of at least 4, further more preferred of at least 6 of the sequences.

[0042] Since the presently provided sequences represent genes, which are differentially expressed, the expression rates of the single genes can be increased or decreased independently from each other. "Independently" in this context means that the expression rate of each of the genes can but need not be influenced by each other. In any case expression levels different from the non-disease status might be a hint to the disease status, which is examined.

[0043] The disease status, which is considered in the present invention, is stroke. The preferred types of stroke are ischemic and hemorrhagic stroke. Consequences, which might be related to stroke, are among others severe headache, paralysis, cognitive deficits, speech problems, emotional difficulties, daily living problems, and central pain syndrome.

[0044] Independent whether stroke is diagnosed or characterised, a model for stroke is characterised, the efficacy of stroke treatment or the efficiency of a compound in a model shall be examined, the determination of the expression levels of at least one of the sequences is carried out outside of a living body. A method to obtain such results comprises

a) providing a sample comprising cells or body fluids expressing one or more genes represented by polynucleotide sequences selected from the group of SEQ ID NO. 1 to 88 or homologues or fragments thereof

b) detecting expression of one or more of the genes in said cells;

c) comparing the expression of the genes in the test cells to the expression of the same genes in reference cells whose expression stage is known, and

d) identifying a difference in expression levels of the considered sequences, if present, in the test cell population and the reference cell population.



[0045] As mentioned above, detection of the expression of the genes can be carried out by any method known in the art. The method of detection is not limiting the invention.

[0046] Expression levels can be detected either on basis of the polynucleotide sequences or by detecting the according polypeptide, encoded by said polynucleotide sequence.

[0047] Preferred methods for detection and determination of the gene expression levels are PCR of cDNA, generated by reverse transcription of expressed mRNA, hybridisation of polynucleotides (Northern, Southern Blot systems, In situ hybridisation), DNA-microarray based technologies, detection of the according peptides or proteins via e.g. Western Blot systems, 2-dimensional gel analysis, protein micro-array based technologies or quantitative assays like e.g. ELISA tests.

[0048] The most preferred method for quantitative analysis of the expression levels is the digital expression pattern display method (DEPD), described in detail in WO99/42610.

[0049] The sequences of the present invention can further be used for identifying therapeutic agents and their efficacy for treating stroke. For example a method can be used comprising:

a) providing a test cell population comprising cells capable of expressing one or more genes represented by nucleic acid sequences selected from the group consisting of SEQ ID NO: 1 to 88 or homologues or fragments thereof

b) contacting said test cell population with the test therapeutic agent;

c) detecting the expression of one or more of the genes in said test cell population,

d) comparing the expression of the gene(s) in the test cell population to the expression of the gene(s) in a reference cell population whose disease stage is known; and

e) identifying a difference in expression levels of the considered sequences, if present, in the test cell population and the reference cell population, thereby identifying a therapeutic agent for treating stroke.



[0050] Test cells can be obtained from a subject, an animal model or cell cultures of fresh cells or cell lines. Further in vitro assays may be used.

[0051] A method examining the different expression patterns of the differentially expressed gene(s) therefore can be used for testing agents and compounds for their efficiency for treatment of stroke. Which model is used is not relevant, as long as the model allows the determination of differences in expression amounts.

[0052] In such a model cells are contacted with the interesting agent or compound and expression of at least one of the genes considered in the present invention is determined in comparison to the expression of the same gene in cells which never have been contacted to the according agent / compound. Contacting the cells either can be affected by administering the agent /compound to an animal or by contacting isolated cells of tissue, CSF, or blood or cells of cell lines in culture with the agent / compound.

[0053] By examination of the influence the considered agent(s) / compound(s) have on the expression of at least one of the genes, the efficacy of the agent(s) / compound(s) can be estimated. This allows the decision whether it is worthwhile to develop a medicament containing such an agent or compound.

[0054] Whether the expression is determined on basis of mRNA generation or on basis of protein generation is not relevant, as long as the difference of the expression rate can be determined. Therefore both, the polynucleotide sequences, and the polypeptides or proteins shown in the present application can be used for the development or the identification of a medicament.

[0055] The development of a medicament can be desirable for example if the considered compound / agent has any influence on the regulation of the expression rate or on the activity of any polynucleotide sequence or polypeptide /protein of the present invention. Said influence can be for example acceleration, promotion, increase, decrease or inhibition of the expression or activity.

[0056] Said influence of a compound or agent can be examined by a method comprising contacting a sample comprising one of the nucleic acid sequences or of the polypeptides of the present invention with a compound that binds to said sequence in an amount sufficient to determine whether said compound modulates the activity of the polynucleotide or polypeptide / protein sequence.

[0057] By such a method a compound or agent modulating the activity of any of the nucleic acid sequences or any polypeptides of the present invention can be determined.

[0058] Furthermore the sequences itself can be used as a medicament.

[0059] An example for such a use is the use of a polynucleotide sequence as an antisense agent. Antisense agents, including but not limited to ribonucleotide or desoxyribonucleotide oligomers, or base-modified oligomers like phosphothioates, methylated nucleotides, or PNAs (peptide nucleic acids), can hybridise to DNA or mRNA, inhibiting or decreasing transcription or translation, respectively. Thus, polynucleotide sequences of a gene, which is increased in expression rate under stroke, can be used as antisense agents to decrease the expression rates of said gene. Further such polynucleotide sequences can be used for gene therapy.

[0060] Another example for such a use is the use of a polypeptide or a protein as a medicament. In case that the expression of a gene is decreased under stroke and therefore not "enough" protein is provided by the body to maintain natural (healthy) conditions, said protein can be administered as a medicament. In case a gene is increased under stroke, representing a protective beneficial or adaptive response of the brain, this effect can be further strengthened by adding even more of the corresponding protein as medicament.

[0061] A pharmaceutical composition comprising a polynucleotide sequence or a polypeptide according to the present invention can be any composition, which can serve as a pharmaceutical one. Salts or aids for stabilising the sequences in the composition preferably are present.

[0062] For the determination of the expression of the relevant genes the generated sequences have to be detected. Therefore several reagents can be used, which are for example specific radioactive or non-radioactive (e.g. biotinylated or fluorescent) probes to detect nucleic acid sequences by hybridisation e.g. on DNA microarrays, primer sets for the detection of one or several of the nucleic acid sequences by PCR, antibodies against one of the polypeptides, or epitopes, or antibody- or protein-microarrays. Such reagents can be combined in a kit, which can be sold for carrying out any of the described methods. The isolated polynucleotide or polypeptide sequences therefore can be provided as microarrays ("DNA chips" and "protein chips").

[0063] Further the sequences defined in the present invention can be used to "design" new transgenic animals as model for stroke. Therefore the animals are "created" by manipulating the genes considered in the present application in a way that their expression in the transgenic animal differs from the expression of the same gene in the wild type animal. In which direction the gene expression has to be manipulated (up- or down-regulation) depends on the gene expression shown in the present application. Methods of gene manipulation and methods for the preparation of transgenic animals are commonly known to those skilled in the art.

[0064] For further examinations or experiments it might be desirable to include any of the nucleic acids of the present invention into a vector or a host cell. By including the sequences in a host cell for example cellular assays can be developed, wherein the genes, polynucleotide sequences and the according proteins / polypeptides further can be used or examined. Such vectors, host cells and cellular assays therefore shall be considered as to fall under the scope of the present invention.

Figures:



[0065] Figures 1 to 7 show results of several transcripts differentially expressed in the cardiac arrest model over several time points. Each sequence is examined in their expression levels over a time period of 0.5 hrs after cardiac arrest to 2 years after surgery and compared to sham operated controls. Genes are described as differentially expressed when the sequence is up- or down-regulated at one or more time points with a certain statistical relevant significance value. Over time the expression pattern can be determined as up-regulated in one to seven time points; as down regulated in one to seven time points or as mixed regulated if the type of regulation changes between up- and down-regulation at different time points.
X-axis describes the time points analysed by DEPD, 1= 0.5hrs post operation, 2= 3hrs post operation, 3= 6hrs post operation, 4=day 3 post operation, 5=day 7 post operation, 6= 2 years post operation.

[0066] The Y-axis shows delta h, which represents the normalised difference of expression (peak height) of a certain transcript between a control group and a treated group. x-fold difference in gene expression is calculated by:

0= no change to control, + = up regulation, - = down regulation; (0,2 = 1,5 fold; 0,3 = 1,86 fold; 0,4 = 2,33 fold; 0,5 = 3 fold).

[0067] The following examples are provided for illustration and are not intended to limit the invention to the specific example provided.

Examples:


Example 1: Preparation of rat cardiac arrest model



[0068] Cardiac arrest was performed in female Lewis-rats at 3 months of age (150-220g), resulting in total cessation of blood flow leading to global cerebral ischemia. After 10 min of ischemia, the animals were resuscitated by external heart massage and ventilation. The group size was 2 x 3 animals. For induction of cardiac arrest, a special blunt-end, hook-like probing device was inserted into the right parasternal line across the third "intracostal spaces into the chest cavity. Next, the probe was gently pushed down the vertrebral column until a slight resistance from the presence of the right pulmonary veins was detected. The probe then was tilted 10-20° caudally. Then, the probe was rotated in a counter-clockwise direction about 135-140° under the inferior vena cava. At this position, the occluding part of the device was positioned right under the heart vessle bundle (inferior vena cava, superior right and left vena cava, ascending aorta, and pulmonary trunk). The pulmonary veins (left and right) were closed by the rotation of the occluding part of the hook. In the last step, the probe was pulled up with concomitant compression of the heart vessel bundle against the sternum. The end of the occluding portion of the hook was then positioned in the left parasternal line in the second intercostal space. To prevent upward movement of the chest and to insure complete ligation of the vessels, simple finger pressure was applied downward the sternum, producing total hemostasis and subsequent ventricular arrest. The effect of the whole procedure is total cessation of both arterial and venous blood flow, it essentially represents the onset of clinical death. After 2,5-3,5 min, the probe was released and removed from the chest by reverse procedural succession, and the animals remained in this position until the beginning of resuscitation.
The resuscitation procedure consisted of external heart massage until spontaneous heart function was recovered and controlled respiration occured. During this time, air was pumped through a polyethylene tube inserted intratracheally that was connected to a respirator. External heart massage was produced by the the index and middle fingers rapidly striking against the chest (sternum) for 1-2 min at the level of the fourth intracostal area with a frequency of 150-240/min in continuous succession. The ratio of strikes to frequency of ventilation was 6:1 or 8:1.

[0069] An electrocardiogramm (lead II-EEG) was recorded continuously during the course of the experiment. Moreover, heart activity was monitored using a loud speaker connected to the output lead of the electroencephalograph. Additionally, the cranial bones were exposed at the sagittal and coronal sutures, where silver-needle electrodes were attached for recording on an electrocorticogramm (ECoG). All measurements were registered on a ten-channel electroencephalogramm (Accutrace-100A, Beckmann).

[0070] 2 x 3 sham operated animals served as controls. These animals were treated similarly to the experimental group with one major exception. Under anesthesia, the probe was inserted through the chest wall into the plural cavity as has already been described above but without further manipulation and torsion of the probe. The probe remained in the chest for essentially the same time period (3,5 min) as in the experimental group. The control animals were then returned to their cages for recovery.

[0071] Tissue preparation occurred 0.5hr, 1hr, 6hrs, 3 days, 7 days and 2 years after surgery. Tissues were frozen on liquid nitrogen prior to RNA preparation.

Example 2: Determination of expression levels



[0072] Gene expression profiling by DEPD-analysis starts with the isolation of 5-10µg total RNA. In a second step, double-stranded cDNA is synthesized. Through an enzymatic digest of the cDNA with three different type IIS restriction enzymes, three pools with short DNA-fragments containing single-stranded overhangs are generated. Afterwards, specific DNA-adaptor-molecules are ligated and in two subsequent steps 3.072 PCR reactions are performed by using 1024 different unlabelled 5'primer and a common FAM-fluorescent- labelled 3'-primer in the last PCR step. Subsequently, the 3072 PCR pools are analysed on an automatic capillary electrophoresis sequencer.

[0073] Differential gene expression pattern of single fragments are determined by comparison of normalized chromatogram peaks from the control groups and corresponding operated animals.

Example 3: Sequencing and Databank analysis of the obtained sequences



[0074] Differentially expressed peaks are confirmed on polyacrylamid gels by using radioactive labelled 3' primer instead of the FAM fluorescent primer. Differentially expressed bands are cut from the gel. After an elution step of up to 2 hrs in 60µl 10mM Tris pH8, fragments are re-amplified by PCR using the same primer as used in the DEPD analysis. Resulting PCR products are treated with a mixture of ExonucleaseI and shrimp alkaline phosphatase prior to direct sequencing. Sequencing reactions are performed by using DY-Enamic-ET-dye terminator sequencing kit (Amersham) and subsequently analysed by capillary electrophoresis (Megabace 1000, Amersham).

[0075] Prior to a BLAST sequence analysis (Altschul et al. 1997, Nucleic Acids Res. 25:3389-3402) against Genbank (Release No. 126), all sequences are quality verified and redundant sequences or repetitive motifs are masked.

[0076] Results are shown in Table 1.
Seq-IDascession numbernamefragment length [bp]
1 Z99755 Human DNA sequence from clone CTA-714B7 on chromosome 22q12.2-13.2 Contains pseudo-gene similar to part of COX7B (cytochrome c oxidase subunit VIIb) 323
2   no hit found in DB 323
3 BF418899 UI-R-BJ2-bqk-c-03-0-UI.s1 UI-R-BJ2 Rattus norvegicus cDNA clone UI-R-BJ2-bqk-c-03-0-UI 3', mRNA sequence 323
4   no hit found in DB 138
5 AB023781 Rattus norvegicus mRNA for cathepsin Y, partial cds 301
6 BF420410 UI-R-BJ2-bqb-g-04-0-UI.s1 UI-R-BJ2 Rattus norvegicus cDNA clone UI-R-BJ2-bqb-g-04-0-UI 3', mRNA sequence 258
7 M35826 Rat mitochondrial NADH-dehydrogenase (NDI) gene, complete cds 375
8 AK019199 Mus musculus 11 days embryo cDNA, RIKEN full-length enriched library, clone:2700005I17, full insert sequence 221
9 BF413204 -UI-R-BT1-bny-a-04-0-UI.s1 UI-R-BT1 Rattus norvegicus cDNA clone UI-R-BT1-bny-a-04-0-UI 3', mRNA sequence 251
10 X16555 Rat PRPS2 mRNA for phosphoribosylpyrophosphate synthetase subunit II 208
11 AK017685 Mus musculus 8 days embryo cDNA, RIKEN full-length enriched library, clone:5730466L18, full insert sequence 238
12   no hit found in DB 201
13 Y00964 M musculus mRNA for beta-hexosaminidase 217
14   no hit found in DB 145
15 L17127 Rattus norvegicus proteasome RN3 subunit mRNA, complete cds 311
16   no hit found in DB 171
17 BG380139 UI-R-CS0-btp-e-04-0-UI.s1 UI-R-CS0 Rattus norvegicus cDNA clone UI-R-CSO-btp-e-04-0-UI 3', mRNA sequence 171
18 D86215 Rattus norvegicus mRNA for NADH:ubiquinone oxidoreductase, complete cds 325
19 AK005320 Mus musculus adult male cere-bellum cDNA, RIKEN full-length enriched library, clone:1500031J01, full insert sequence 171
20   no hit found in DB 171
21   no hit found in DB 199
22   no hit found in DB 171
23   no hit found in DB 139
24   no hit found in DB 139
25 BI287855 UI-R-CWOs-ccm-a-07-0-UI.s1 UI-R-CW0s Rattus norvegicus cDNA clone UI-R-CWOs-ccm-a-07-0-UI 3', mRNA sequence 269
26   no hit found in DB 166
27   no hit found in DB 227
28 AC016673 Homo sapiens BAC clone RP11-17N4 from 2, complete sequence 261
29 AB071989 Mus musculus mRNA for Spop. partial cds 365
30   no hit found in DB 248
31 AF178845 Rattus norvegicus calmodulin mRNA, complete cds 482
32 J02701 Rat Na+, K+ -ATPase beta sub-unit protein mRNA, complete cds 418
33 X12553 Rat mRNA for liver cytochrome c oxidase subunit VIa 444
34   no hit found in DB 140
35 Y07503 Rat mRNA for preproenkephalin (A) 244
36 X14876 Rat mRNA for transthyretin 245
37   no hit found in DB 270
38 AI547530 UI-R-C3-sr-b-11-0-UI.s1 UI-R-C3 Rattus norvegicus cDNA clone UI-R-C3-sr-b-11-0-UI 3', mRNA sequence 207
39 M27315 Rattus norvegicus cytochrome c oxidase subunit II (Co II) gene 188
40 J02582 Rat apolipoprotein E gene, complete cds 180
41   no hit found in DB 250
42 U65579 Human mitochondrial NADH de-hydrogenase-ubiquinone Fe-S protein 8, 23 kDa subunit precursor (NDUFS8) nuclear mRNA encoding mitochondrial protein, complete cds 337
43 AF173082 Mus musculus LIN-7 homolog 2 (MALS-2) mRNA, complete cds 158
44   no hit found in DB 250
45 U70268 Rattus norvegicus mud-7 mRNA, 3' UTR 241
46 X96997 O.aries SOX-2 gene 187
47 AK020957 Mus musculus adult male corpora quadrigemina cDNA, RIKEN full-length enriched library, clone:B230104P22, full insert sequence 221
48   no hit found in DB 490
49 BC012314 Mus musculus, Similar to ferritin heavy chain, clone MGC:19422 IMAGE:3488821, mRNA, complete cds 116
50 BB452941 BB452941 RIKEN full-length enriched, 12 days embryo spinal ganglion Mus musculus cDNA clone D130020J05 3', mRNA sequence 159
51 AK019418 Mus musculus 13 days embryo head cDNA, RIKEN full-length enriched library, clone:3110018K01, full insert sequence 377
52 J01435 Rattus norvegicus mitochondrial ATPase subunit 6 gene 224
53 BF387893 UI-R-CA1-bbw-a-03-0-UI.s1 UI-R-CA1 Rattus norvegicus cDNA clone UI-R-CA1-bbw-a-03-0-UI 3', mRNA sequence 200
54 AB033713 Rattus norvegicus mitochondrial gene for cytochrome b, partial cds 149
55   no hit found in DB 154
56 U53513 Rattus norvegicus glycine-, glutamate-, thienylcyclohexylpiperidine-binding protein mRNA, complete cds 162
57 AK004546 Mus musculus adult male lung cDNA, RIKEN full-length enriched library, clone:1200002H13, full insert sequence 301
58   no hit found in DB 304
59 AY004290 Rattus norvegicus scg10-like-protein mRNA, complete cds 321
60   no hit found in DB 306
61   no hit found in DB 155
62 BF544005 UI-R-E0-ce-c-04-0-UI.r1 UI-R-E0 Rattus norvegicus cDNA clone UI-R-E0-ce-c-04-0-UI 5', mRNA sequence 155
63   no hit found in DB 276
64   no hit found in DB 306
65   no hit found in DB 186
66 BC011132 Mus musculus. Similar to special AT-rich sequence binding protein 1, clone MGC:18461 IMAGE:4164993, mRNA, complete cds 239
67 AJ278701 Rattus norvegicus mRNA for cytosolic branched chain aminotransferase (Bcatc gene) 233
68 M14512 Rat Na+,K+-ATPase alpha(+) isoform catalytic subunit mRNA, complete cds 246
69 U42975 Rattus norvegicus Shal-related potassium channel Kv4.3 mRNA, complete cds 309
70 BC004706 BC004706 Mus musculus, heterogeneous nuclear ribonucleoprotein C, clone MGC:5715 IMAGE:3499283, mRNA, complete cds 336
71 BE101398 UI-R-BJ1-aud-e-10-0-UI.s1 UI-R-BJ1 Rattus norvegicus cDNA clone UI-R-BJ1-aud-e-10-0-UI 3', mRNA sequence 190
72 AF073297 Mus musculus small EDRK-rich factor 2 (Serf2) mRNA, complete cds 180
73 D32249 Rattus norvegicus mRNA for neurodegeneration associated protein 1, complete cds 302
74 BF391228 UI-R-CA1-bcq-g-07-0-UI.sl UI-R-CA1 Rattus norvegicus cDNA clone UI-R-CA1-bcq-g-07-0-UI 3', mRNA sequence 166
75 BE109851 UI-R-CAO-axi-b-04-0-UI.sl UI-R-CA0 Rattus norvegicus cDNA clone UI-R-CA0-axi-b-04-0-UI 3', mRNA sequence 205
76   no hit found in DB 163
77 AY011335 Rattus norvegicus amyloid beta precursor protein (App) gene, partial cds 282
78 BC013540 Mus musculus, Similar to retinal short-chain dehydrogenase/reductase 1, clone MGC:19224 IMAGE:4241608, mRNA, complete cds 246
79 X52311 Rat unr mRNA for unr protein with unknown function 335
80 M29358 Rat ribosomal protein S6 mRNA, complete cds 232
81 AC068987 UI-R-CAO-bkh-f-06-0-UI.sl UI-R-CA0 Rattus norvegicus cDNA clone UI-R-CA0-bkh-f-06-0-UI 3', mRNA sequence 369
82 AC068987 UI-R-B01-aqb-b-02-0-UI.s1 UI-R-B01 Rattus norvegicus cDNA clone UI-R-B01-aqb-b-02-0-UI 3', mRNA sequence 381
83   no hit found in DB 219
84 M23953 UI-R-BJ0p-aio-h-09-0-UI.s1 UI-R-BJ0p Rattus norvegicus cDNA clone UI-R-BJ0p-aio-h-09-0-UI 3', mRNA sequence 360
85 AK018721 Mus musculus adult male kidney cDNA, RIKEN full-length enriched library, clone:0610007M20, full insert sequence 488
86 X82396 R. norvegicus mRNA for cathepsin B 457
87   no hit found in DB 215
88 X82550 R.norvegicus mRNA for ribosomal protein L41 249
DB = data base

Example 4: Comparison of differentially expressed sequences over several time points in the cardiac arrest model.



[0077] 0.5hr, 1hr, 3hrs, 6hrs, 3days, 7 days, and 2 years survival time of the animals were chosen as time points for gene expression profiling of the cardiac arrest model. After DEPD analysis peaks obtained as differentially expressed at least at one time point were compared over time to control within the cardiac arrest stroke model. Results are shown in Table 2.
Table 2
Seq-ID No.Regulation
1 down 7d
2 down 7d
3 down 7d
4 down 7d
5 up 3d / up 7d
6 up 3d
7 up 3d
8 down 3d
9 down 3d
10 up 3d
11 down 3d
12 up 3d
13 up 3d
14 up 7d
15 up 7d
16 up 3d
17 down 7d
18 up 7d
19 down 7d
20 down 7d
21 up 7d
22 up 3d / down 7d
23 up 3d
24 down 3d
25 down 7d
26 up 3d
27 down 3d
28 up 3d
29 down 3d
30 down 3d
31 down 7d
32 up 3d
33 up 7d
34 up 3d
35 up 3d / up 7d
36 up 7d
37 up 7d
38 down 7d
39 up 7d
40 up 7d
41 down 7d
42 up 7d
43 up 7d
44 down 7d
45 down 7d
46 up 7d
47 up 7d
48 down 7d
49 up 3d
50 down 7d
51 down 7d
52 up 7d
53 up 3d
54 up 3d
55 up 3d
56 up 3d
57 down 3d
58 down 3d
59 down 3d
60 down 3d
61 up 3d
62 down 3d
63 up 3d
64 up 3d
65 up 3d
66 up 3d / down 7d
67 up 3d
68 up 3d
69 down 3d
70 down 3d / down 7d
71 up 3d
72 up 3d
73 up 3d
74 down 3d
75 down 2y
76 up 2y
77 up 2y
78 down 2y
79 down 2y
80 down 2y
81 up 2y
82 up 2y
83 up 2y
84 up 2y
85 up 2y
86 down 2y
87 up 2y
88 up 2y


[0078] For each DNA fragment, gene expression patterns are obtained in the stroke model compared over several time points. "Up", "down", and "mixed" is defined as time dependent expression at one or more time points in the cardiac arrest model compared to the non-disease model.






































Claims

1. Use of polynucleotide sequences selected from the group of sequences SEQ ID NO: 1 to 34, 36 to 39, 41 to 76, 78 to 85 and 87, 88 or homologues or fragments thereof or the according polypeptides for the characterisation of the development and/or occurrence of stroke and / or the progression of the pathology of stroke and/or consequences of stroke, whereby the characterisation is carried out outside of a living body.
 
2. Use of a combination of at least two of polynucleotide sequences selected from the group of sequences SEQ ID NO: 1 to 88 or homologues or fragments thereof or the according polypeptides for the characterisation of the development and/or occurrence of stroke and / or the progression of the pathology of stroke and/or consequences of stroke, whereby the characterisation is carried out outside of a living body.
 
3. Use of polynucleotide sequences according to claim 1 or 2, characterised in that the sequences are mammalian sequences.
 
4. Method for characterising a stroke status, comprising the comparison of the expression of genes represented by at least one of the sequences selected of the group of SEQ ID NO. 1 to 34, 36 to 39, 41 to 76, 78 to 85 and 87, 88 or homologues or fragments thereof to non-disease status.
 
5. Method for characterising a stroke status, comprising the comparison of the expression of genes represented by at least two of the sequences selected of the group of SEQ ID NO.1 to 88 or homologues or fragments thereof to non-disease status.
 
6. Method according to claim 4 or 5, for diagnosing stroke outside of the living body.
 
7. Method according to claim 4 or 5, for assessing the efficacy of stroke treatment outside of a living body.
 
8. Method according to claim 4 or 5, for characterisation of animal models for stroke.
 
9. Method according to claim 4 or 5, for determining the efficiency of compounds in a model for stroke.
 
10. Method according to any of claims 4 to 9, whereby the expression is determined from in vivo samples, in vitro samples, or ex vivo samples.
 
11. Method according to claim 10, whereby the samples are derived from whole tissues, cerebrospinal fluid (CSF) or blood, or from cell populations isolated from tissues, CSF, or blood, or from cell lines.
 
12. Method according to any of claims 4 to 11, whereby at least a combination of 4 sequences are compared to non-disease status.
 
13. Method according to any of claims 4 to 11, whereby at least a combination of 6 sequences are compared to non-disease status.
 
14. Method according to any of claims 4 to 13, whereby the considered gene expression is increased compared to non-disease status.
 
15. Method according to any of claims 4 to 13, whereby the considered gene expression is decreased compared to non-disease status.
 
16. Method according to any of claims 4 to 13, whereby the considered gene expression for at least one sequence is increased and for at least one sequence is decreased compared to non-disease status.
 
17. The method according to any of claims 4 to 16 comprising:

a) providing a sample comprising cells expressing one or more genes represented by polynucleotide sequences selected from the group of SEQ ID NO. 1 to 88 or homologues or fragments thereof

b) detecting expression of one or more of the genes in said cells;

c) comparing the expression of the genes in the test cells to the expression of the same genes in reference cells whose expression stage is known, and

d) identifying a difference in expression levels of the considered sequences, if present, in the test cell population and the reference cell population.


 
18. A method of identifying a test therapeutic agent for treating stroke in a subject, the method comprising:

a) providing a test cell population comprising cells capable of expressing one or more genes represented by nucleic acid sequences selected from the group consisting of SEQ ID NO: 1 to 88 or homologues or fragments thereof

b) contacting said test cell population with the test therapeutic agent;

c) detecting the expression of one or more of the genes in said test cell population,

d) comparing the expression of the gene(s) in the test cell population to the expression of the gene(s) in a reference cell population whose disease stage is known; and

e) identifying a difference in expression levels of the considered sequences, if present, in the test cell population and the reference cell population, thereby identifying a therapeutic agent for treating stroke.


 
19. Method according to claims 4 to 18, whereby the gene expression is considered or determined by PCR of cDNA, hybridisation of a sample DNA or by detecting the according protein.
 
20. Use of at least one of the polynucleotide sequences of genes represented by polynucleotide sequences selected from SEQ ID NO. 1 to 88 or homologues or fragments thereof or of a protein encoded by any gene represented by any of the polynucleotide sequences selected from SEQ ID NO. 1 to 88 or homologues or fragments thereof for the development or identification of a medicament.
 
21. Use according to claim 20, whereby the medicament changes the expression or the activity of a protein encoded by a gene represented by one of said polynucleotide sequences.
 
22. Use according to claim 20 or 21 for the development of a medicament for the treatment of stroke.
 
23. Use or method according to any of claims 1 to 18 or 22, whereby the stroke is ischemic or hemorrhagic stroke.
 
24. Combination of isolated nucleic acid sequences comprising at least one nucleic acid sequence of a gene that is one of the genes represented by the polynucleotide sequences selected of SEQ ID NO. 1 to 88, or the complement of the nucleic acid sequence as an microarray.
 
25. Combination of isolated polypeptides comprising at least one amino acid sequence encoded by one of the genes represented by the nucleic acid sequences of SEQ ID NO. 1 to 88.
 
26. Use of an isolated nucleic acid sequence of a gene that is one of the genes represented by the polynucleotide sequences selected of SEQ ID NO. 1 to 88 or the according isolated polypeptide as a medicament.
 
27. A pharmaceutical composition comprising any of the nucleic acid sequences of a gene that is one of the genes represented by the polynucleotide sequences selected of SEQ ID NO. 1 to 88 or any of the according polypeptides.
 
28. A kit comprising one or more reagents for detecting one or more genes represented by nucleic acid sequences selected from the group consisting of SEQ ID NO. 1 to 88 or homologues or fragments thereof.
 
29. A vector comprising any nucleic acid sequence of of a gene that is one of the genes represented by the polynucleotide sequences selected of SEQ ID NO. 1 to 88.
 
30. A host cell comprising any isolated nucleic acid sequence of a gene that is one of the genes represented by the polynucleotide sequences selected of SEQ ID NO. 1 to 88 or a vector of claim 29.
 
31. An antibody that selectively binds to a polypeptide encoded by a of a gene that is one of the genes represented by the polynucleotide sequences selected of SEQ ID NO. 1 to 88, and fragments, homologues, analogues and derivatives of the antibody.
 
32. A transgenic animal wherein at least one of the sequences corresponding to the sequences 1 to 88 is manipulated in the animal in comparison to the wild type.
 




Drawing