EP2179054A1 - Utilisation de polynucléotides pour la détection d'activités géniques afin de différencier une infection locale d'une infection systémique - Google Patents

Utilisation de polynucléotides pour la détection d'activités géniques afin de différencier une infection locale d'une infection systémique

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Publication number
EP2179054A1
EP2179054A1 EP08785273A EP08785273A EP2179054A1 EP 2179054 A1 EP2179054 A1 EP 2179054A1 EP 08785273 A EP08785273 A EP 08785273A EP 08785273 A EP08785273 A EP 08785273A EP 2179054 A1 EP2179054 A1 EP 2179054A1
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European Patent Office
Prior art keywords
gene
seq
infection
patient
nucleic acids
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EP08785273A
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German (de)
English (en)
Inventor
Stefan Russwurm
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Analytik Jena AG
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SIRS Lab GmbH
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/178—Oligonucleotides characterized by their use miRNA, siRNA or ncRNA

Definitions

  • the present invention relates to the use of polynucleotides and / or their gene loci and / or their transcripts for detecting gene activities for distinguishing a state associated with a local infection from a condition of a patient associated with a systemic infection according to claim 1 and the use of Patient samples obtained gene activities for the distinction of a condition associated with a local infection from a condition of a patient associated with a systemic infection according to claim 3 and claim 15.
  • the invention further relates to a method for in vitro measurement of gene activities according to claim 17 and a kit according to claim 25th
  • the present invention relates in particular to marker genes and / or fragments thereof and their use for differentiating local from systemic infections by means of gene expression analyzes.
  • the invention further relates to PCR primers and probes derived from the marker genes.
  • Bacterial cell wall components such as lipopolysaccharides cause changes in cytokine levels in the blood (Mathiak 2003), and individual protein markers such as procalcitonin (PCT) and mannan-binding lectin (MBL) are used to indicate postoperative infections (Siassi 2005).
  • PCT procalcitonin
  • MBL mannan-binding lectin
  • the immune response of blood to bacterial pathogens can also be characterized by functional genomics (Feezor and Moldawer 2003, M. Foti et al, 2006) and bacterial proteins cause transcriptional and translational changes in host cells (FIo 2004).
  • Gene expression profiles can therefore also be used to determine the mechanisms of antibacterial substances (Hutter 2004).
  • a transcriptional analysis of genes in a blood sample was used to characterize differences in surviving and non-surviving sepsis patients (Pachot 2006).
  • transcripts including mRNA and small RNA, especially microRNA and other RNAs
  • quantification of transcripts with variable concentration of blood, blood cells and cells from organs and peripheral tissues, which are located in whole blood, provide a prerequisite for blood-based diagnostic tools represents.
  • the starting point for the invention disclosed in the present patent application is the recognition that gene activities of various genes present in blood cells in samples of an individual in local and systemic infection differ from the gene activities of the affected genes of individuals in which no infection, no local or systemic infection were diagnosed and can be used together or individually as marker genes with disease-related change in concentration of transcripts of blood. Normalization or relative quantification of the activities of these genes can be used as tools for diagnosis, prognosis, therapy and follow-up.
  • a kit according to claim 25 solves the problem as well.
  • the subclaims represent preferred embodiments of the invention.
  • a local infection is an infection in which the pathogens remain at the site of infection and cause symptoms only at this site without redistributing in the body.
  • a local infection may be respiratory infection, diarrhea, or boil on the skin.
  • a systemic infection is an infection in which the pathogens spread through an entire organ system or the entire organism.
  • Biological fluids within the meaning of the invention are understood to be all body fluids of mammals, including humans.
  • a gene is a section of the deoxyribonucleic acid (DNA) that contains basic information for the production of a biologically active ribonucleic acid (RNA).
  • genes are also understood as meaning all derived DNA sequences, partial sequences and synthetic analogs (for example peptido-nucleic acids (PNA)).
  • PNA peptido-nucleic acids
  • Genlocus is the position of a gene in the genome. If the genome consists of several chromosomes, the position within the chromosome on which the gene is located is meant. Different manifestations or variants of this gene are referred to as alleles, all located at the same site on the chromosome, namely the gene locus. Thus, the term "gene locus” includes, on the one hand, the pure genetic information for a specific gene product and, on the other hand, all regulatory DNA segments as well as any additional DNA sequences which are in any functional or non-functional context with the gene at the gene locus. Gene activity: Genetic activity is the measure of the ability of a gene to be transcribed and / or to form translation products.
  • Gene Expression The process of forming a gene product and / or expression of a genotype into a phenotype.
  • a marker gene is a gene that displays a disease-related change in its expression and transcription across different RNA samples and whose variable gene activity is used to diagnose, monitor and control the therapy of various samples.
  • Hybridization Conditions Physical and chemical parameters well known to those skilled in the art that may affect the establishment of a thermodynamic equilibrium of free and bound molecules. In the interest of optimal hybridization conditions, the duration of contact of the probe and sample molecules, cation concentration in the hybridization buffer, temperature, volume, and concentrations and ratios of the hybridizing molecules must be matched.
  • Amplification conditions cyclically changing reaction conditions which allow the amplification of the starting material in the form of nucleic acids.
  • the reaction mixture are the individual components (deoxynucleotides) for the resulting nucleic acids, as well as short oligonucleotides, which can attach to complementary regions in the starting material, as well as a nucleic acid synthesis enzyme, called polymerase.
  • the cation concentrations, pH, volume and the duration and temperature of the individual cyclically repeating reaction steps which are well known to the person skilled in the art are of importance for the course of the amplification.
  • Primer is an oligonucleotide which serves as a starting point for DNA-replicating enzymes such as DNA polymerase. Primers may consist of both DNA and RNA (primer 3, see, e.g., http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi of MIT).
  • a probe is a
  • Nucleic acid fragment (DNA or RNA) (with a molecular marker such as fluorescent markers, in particular molecular beacons, TaqMan ® probes, Isotopic labeling, etc.) and used for sequence-specific detection of target DNA and / or target RNA molecules.
  • a molecular marker such as fluorescent markers, in particular molecular beacons, TaqMan ® probes, Isotopic labeling, etc.
  • PCR is the abbreviation for the term "polymerase chain reaction.”
  • the polymerase chain reaction is a method to amplify DNA in vitro outside a living organism using a DNA-dependent DNA polymerase according to the present invention, to amplify short parts - up to about 3,000 base pairs - of a DNA strand of interest, which may be a gene or just a part of a gene or non-coding DNA sequences.
  • PCR a number of PCR methods are known in the art, all of which are encompassed by the term "PCR”. This applies in particular to the "real-time PCR”.
  • PCR primers typically, PCR requires two primers to set the starting point of DNA synthesis on each of the two strands of DNA, thereby limiting the range of duplication on both sides.
  • primers are well known to those skilled in the art, for example from the website "Primer3", see for example http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi of MIT.
  • RNA transcript For the purposes of the present application, a transcript is understood to mean any RNA product which is produced on the basis of a DNA template.
  • small RNA Small RNAs in general. Representatives of this group are in particular, but not limited to: a) small cytoplasmic RNA (scRNA), which is one of several small RNA molecules in the cytoplasm of a eukaryote. b) snRNA (small nuclear RNA), one of the many small RNA forms found only in the nucleus. Some of the snRNAs play a role in splicing or in other RNA-processing reactions.
  • scRNA small cytoplasmic RNA
  • snRNA small nuclear RNA
  • RNAs small non-protein-coding RNAs, which include the so-called small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs) and small double-stranded RNAs (dsRNAs), which increase gene expression on many levels, including chromatin architecture, RNA editing, RNA stability, translation, and possibly also transcription and splicing.
  • small nucleolar RNAs small nucleolar RNAs
  • miRNAs microRNAs
  • siRNAs short interfering RNAs
  • dsRNAs small double-stranded RNAs
  • these RNAs are processed in multiple ways from the introns and exons of longer primary transcripts, including protein coding transcripts. Although only 1, 2% of the human genome encodes proteins, a large part is nevertheless transcribed.
  • ncRNA non-protein-coding RNAs
  • snoRNAs Small nucleolar RNAs regulate the sequence-specific modification of nucleotides in target RNAs.
  • modifications There are two types of modifications, namely 2'-O-ribosemethylation and pseudouridylation, which are regulated by two large snoRNA families, called box C / D snoRNAs on the one hand, and box H / ACA snoRNAs on the other.
  • box C / D snoRNAs two large snoRNA families
  • box H / ACA snoRNAs Such snoRNAs have a length of about 60 to 300 nucleotides.
  • miRNAs are even smaller RNAs with generally 21 to 25 nucleotides. miRNAs are derived from endogenous short hairpin precursor structures and usually use other loci with similar - but not identical - sequences as the target of translational repression.
  • siRNAs arise from longer double-stranded RNAs or long hairpins, often of exogenous origin. They usually target homologous sequences at the same locus or elsewhere in the genome, where they participate in so-called gene silencing, a phenomenon also called RNAi.
  • miRNAs and siRNAs are fluid.
  • small RNA may also include so-called transposable elements (TEs) and in particular retroelements, which are also understood for the purposes of the present invention by the term “small RNA”.
  • TEs transposable elements
  • retroelements which are also understood for the purposes of the present invention by the term “small RNA”.
  • the invention describes the identification of novel marker genes from blood, suitable microarray probes and their use, also in combination, for the determination of normalized and quantitative expression data from blood and blood cells in microarrays, real-time PCR assays and other measurement systems with or without amplification and with different quantification and Visualization possibilities for the determination of local inflammations and for the differentiation of generalized systemic inflammations, infections and resulting immune reactions in the systemic reaction to it.
  • the method according to the invention is characterized in that, in a sample of a biological fluid, in particular a blood sample or a tissue sample of an individual, the activity of one or more marker genes is normalized by determining the presence and amount of the gene product also relative to the amounts of the gene products of control genes or quantified by further methods known to those skilled in the art, to distinguish between local and systemic infection.
  • sequences from blood and blood cells as well as probes derived therefrom which can be used for the determination, visualization, normalization and quantification of gene activities and transcripts, are disclosed.
  • the sequences of the oligonucleotide probes in a preferred embodiment are set forth in Tables 1 to 5 and correspond to those in the attached sequence listing SEQ ID No 1 to SEQ ID No 69.
  • the sequences of the oligonucleotide probes may also have further sequences, in a preferred embodiment of a length of 50 -100 nucleotides which specifically bind transcripts of the genes set forth in Table 1 to 5 with sequences SEQ ID No 1 to SEQ ID No 69.
  • the length and combination of sequences used in amplification methods such as PCR may be arbitrary as far as they support the desired enzymatic manipulation and amplification.
  • the marker genes can be used singly or in combination of several. Typically, marker gene activity may be as described herein with hybridization probes for microarrays or PCR primers and real-time PCR be determined. However, the marker genes and their expression products can also be determined by other methods known to those skilled in the art, such as, for example, NASBA (Nucleic Acid Sequence-based Amplification) and in various combinations. They can also be determined using a number of other methods or visualization options, for example by means of beach displacement or with the aid of monoclonal antibodies. Probes can be used for the gene, the expression product (mRNA), fragments or expression intermediates that are not completely processed into mRNA.
  • mRNA the expression product
  • the probes bind a specific region of the marker genes or their transcripts disclosed herein.
  • the probes may interact with any region of the gene sequences disclosed herein or sequences transcribed therefrom.
  • the primers and probes may interact via continuous base pairing but may not continuously interact with the complete complementary sequence, the buffer compositions, salt concentrations, washes and temperatures may be chosen to be variable.
  • these changes of the marker genes can be compared with the expression values (or data derived therefrom, such as average values) of one or more reference samples, which need not be determined simultaneously with the target sample but are stored in a database, for example, in the manner of a calibration or as values or tables become.
  • An embodiment of the invention is characterized in that expression values using marker genes of Table 1 to 5 and nucleic acids and transcripts of these marker genes from blood and blood cells by comparing the expression levels with one or more control genes (as in the non-prepublished patent application DE 10 2007 010 252.8) and quantification of marker gene expression values relative to marker nucleic acid.
  • a further embodiment of the invention is characterized in that nucleic acids and DNA probes with the sequences according to Tables 1 to 5 and their binding of RNA including small RNA, in particular microRNA, siRNA and / or repeats and / or of transcripts (RNA or mRNA) in Blood or from blood cells of genes according to Tables 1 to 5 in any combination in solution or immobilized on surfaces or particles or beads and the use of the bound transcripts of these genes for normalization by comparison of the bound amounts (expression levels) of the nucleic acids with one or more probe nucleic acid-bound nucleic acids and for quantification in relation to the bound marker nucleic acid.
  • RNA including small RNA, in particular microRNA, siRNA and / or repeats and / or of transcripts (RNA or mRNA) in Blood or from blood cells of genes according to Tables 1 to 5 in any combination in solution or immobilized on surfaces or particles or beads and the use of the bound transcripts of these genes for normalization by comparison of the bound amounts (expression levels) of the nucleic acids
  • An embodiment of the invention is characterized in that the method for the ex vivo, in vitro differentiation of local and systemic infection based on reference of the RNA amounts from control gene and marker gene comprises the following steps:
  • a further embodiment of the invention is characterized in that the marker gene RNA is hybridized with the DNA before the marker gene RNA is measured and the marker signals of the control RNA / DNA complex are detected, optionally further transformed and optionally in the form of a calibration curve or table.
  • a further embodiment of the invention is characterized in that RNA of the marker genes or parts thereof are identified and quantified by sequencing or partial sequencing, for example via pyrosequencing.
  • a further embodiment of the invention is characterized in that mRNA or microRNA is used as the marker gene RNA.
  • a further embodiment of the invention is characterized in that the DNA for specific binding of the marker gene RNA or its in vitro transcripts at predetermined areas on a support in the form of a microarray arranged, in particular immobilized, is.
  • Another embodiment of the invention is characterized in that the biological sample is that of a human.
  • a further embodiment of the invention is characterized in that the immobilized or free probes derived from sequences corresponding to Tables 1 to 5 are labeled.
  • immobilized or free probes derived from sequences corresponding to Tables 1 to 5 are labeled.
  • self-complementary oligonucleotides so-called molecular beacons
  • They carry a fluorophore / quencher pair at their ends, so that in the absence of a complementary sequence they are present in a folded hairpin structure and only deliver a fluorescence signal with a corresponding sample sequence.
  • the hairpin structure of the Molecular Beacons is stable until the probe hybridizes to the specific capture sequence, resulting in a conformational change and concomitant release of reporter fluorescence.
  • RNA sequences within the meaning of the invention, all derived DNA sequences, partial sequences and synthetic analogs (for example peptidonucleic acids, PNA) are understood as aptamers.
  • PNA peptidonucleic acids
  • RNA of the marker genes is isolated from the whole blood of corresponding patients.
  • the RNA is then labeled, for example radioactively with 32 P or with dye molecules (fluorescence).
  • labeling molecules it is possible to use all molecules and / or detection signals known for this purpose in the prior art.
  • Corresponding molecules and / or labeling methods are also known to the person skilled in the art.
  • the thus-labeled RNA is then hybridized with immobilized on a microarray DNA molecules.
  • the DNA molecules immobilized on the microarray represent a specific selection of the genes according to the present invention for distinguishing between local and systemic infection.
  • the intensity signals of the hybridized molecules are subsequently measured by suitable measuring devices (phosporimagers, microarray scanners) which are well known in the prior art and analyzed by further software-supported evaluations.
  • suitable measuring devices phosporimagers, microarray scanners
  • the expression ratios between the marker genes of the patient sample and the control genes are determined from the measured signal intensities. From the expression ratios of the under- and / or overregulated genes can be, as shown in the below Draw conclusions about the distinction between local and systemic infection.
  • a further application of the method according to the invention consists in the measurement of the differential gene expression for the therapy-accompanying determination of the probability that patients will respond to the planned therapy, and / or for the determination of the response to a specialized therapy and / or on the determination of the Therapy end in the sense of a "drug monitoring" in patients with proven systemic infection and their severity grades.
  • the RNA test RNA and control RNA
  • the RNA samples are marked together and hybridized with selected marker genes as well as control genes immobilized on a microarray, thus allowing the assessment of the likelihood that patients will respond to the planned therapy by assessing the expression relationships between one or more control genes and marker genes and / or whether the initiated therapy is effective and / or how long the patients still need to be treated accordingly and / or whether the maximum therapeutic effect has already been achieved with the dose and duration used.
  • RNA of the genes according to the invention for obtaining quantitative information by hybridization-independent methods, in particular enzymatic or chemical hydrolysis, surface plasmon resonance method (SPR method), subsequent quantification of the nucleic acids and / or derivatives and / or fragments thereof
  • the transcripts of control genes amplified and quantified by means of PCR constitute a further embodiment according to the present invention for normalizing gene expression data in the differentiation of local and systemic infection and their degrees of severity.
  • the intensity signals of the amplified transcripts are subsequently measured by suitable measuring devices (PCR fluorescence detector) and analyzed by further software-supported evaluations.
  • the expression ratios between the marker genes of the patient sample and the control genes are determined from the measured signal intensities. From the expression ratios of the under- and / or overregulated genes can be, as shown in the below Experiments, conclusions on the distinction between local and systemic infection, if necessary, draw their severity.
  • a further application of the method according to the invention consists in the normalization of an optionally amplified mRNA quantity in several samples comprising a) a comparison of the expression values of one or more nucleic acids selected from SEQ ID 1 to SEQ ID 69 via different samples; b) deriving a value for normalizing expression values of one or more nucleic acids selected from SEQ ID 1 to SEQ ID 69 over several samples and c) normalizing the expression of other nucleic acids isolated from several samples based on step b)
  • the invention may further relate to a kit comprising a selection of polynucleotides having the sequences of SEQ ID 1 to SEQ ID 69 and / or gene fragments thereof of at least 1-100, more preferably 1-5 and 1-10 nucleotides for determination of gene expression profiles in vitro in a patient sample, for use as marker genes.
  • the invention may also relate to a kit comprising a selection of hybridization probes according to SEQ ID No.1 to SEQ ID NO. 69 and / or gene fragments thereof containing at least 50 nucleotides for determining gene expression profiles in vitro in a patient sample for use as marker genes.
  • An alternative embodiment of the present invention is a use of gene activities obtained in vitro from at least one patient sample for distinguishing a condition associated with a local infection from a condition of a patient associated with a systemic infection, wherein the gene activities are obtained from a method comprising steps:
  • Amplification conditions wherein the length of the amplified portion comprises 50 to 3000 nucleotides and the markers are those gene products which are present in varying amounts in patients with local and systemic infection;
  • Another application of the via microarray analysis or other quantification methods such as e.g. Genetic activities and gene expression data determined in real-time PCR are used to distinguish local and systemic infection for electronic further processing for the purpose of producing software for diagnostic purposes (eg to assess the severity of an individual immune response, especially in the case of bacterial infection, also in the context of patient data management systems) or expert systems) or for modeling cellular signal transduction pathways.
  • a further embodiment of the invention is characterized in that at least one of the polynucleotides according to SEQ ID No 1 to SEQ ID No 69, in particular nucleic acid probes or their complements are used for binding the transcripts or their complements of the marker genes.
  • a further embodiment of the invention is characterized in that the synthetic analogs of the marker genes or the synthetic oligonucleotides which bind the transcripts of the control genes, in particular comprise about 60 base pairs.
  • a further embodiment of the invention is characterized in that a radioactive marker, in particular 32 P, 14 C, 125 1, 33 P or 3 H, is used as the detectable marker.
  • the detectable marker is a non-radioactive marker, in particular a staining or fluorescence marker, an enzyme marker or immune marker, and / or quantum dots or an electrically measurable signal, in particular potential and / or conductivity and / or capacity change, eg. In hybridizations.
  • a further embodiment of the invention is characterized in that the marker gene RNA and control gene RNA and / or enzymatic or chemical derivatives thereof carry the same label.
  • a further embodiment of the invention is characterized in that the marker gene RNA and control gene RNA and / or enzymatic or chemical derivatives carry different labels.
  • nucleic acid probes are immobilized on a support material, e.g. Glass or plastic, to be immobilized.
  • Another embodiment of the invention is characterized in that the individual DNA molecules are immobilized via a covalent bond to the carrier material.
  • a further embodiment of the invention is characterized in that the individual DNA molecules are immobilized on the carrier material by means of electrostatic and / or dipole-dipole and / or hydrophobic interactions and / or hydrogen bonds.
  • a kit according to claim 25 and clusters of polynucleotides also solve the problem.
  • the present invention is for companion clinical evaluation of patients with local or systemic infection.
  • the present invention may also be used to prepare "in silico" expert systems and / or for "in silico” modeling of more cellular signal transduction pathways.
  • a plurality of specific genes and / or gene fragments are used, which are selected from the group in Tables 1 to 5 consisting of SEQ ID NO. 1 to SEQ ID NO. 69 and gene fragments thereof having at least 5-2000, preferably 20-200, more preferably 20-80 nucleotides.
  • the present invention relates to the use of gene expression profiles obtained in vitro from a patient sample and / or of the probes used therefor, which are selected from the group consisting of SEQ ID no. 1 to SEQ ID NO. 69 and gene fragments thereof with at least 5-2,000, preferably 20-200, more preferably 20-80 nucleotides, for switching off or switching on and / or for activity modification of target genes and / or for determining the gene activity for monitoring local or systemic infection and course a local Infection to a systemic infection of a patient.
  • a further embodiment of the invention is characterized in that at least one specific marker gene and / or gene fragment is selected from the group consisting of SEQ ID NO. 1 to SEQ ID NO. 69 and gene fragments thereof having at least 5-2000, preferably 20-200, more preferably 20-80 nucleotides.
  • Another embodiment of the invention is characterized in that at least 2 to 69 different cDNAs are used.
  • a further embodiment of the invention is characterized in that the SEQ ID NO. 1 - SEQ ID NO. 69 listed genes or gene fragments and / or derived from their RNA sequences are replaced by corresponding synthetic analogues, aptamers and Peptidonukleinklaren.
  • a further embodiment of the invention is characterized in that the synthetic analogs of the genes comprise 5-100, in particular about 70 base pairs.
  • a further embodiment of the invention is characterized in that the gene activities are determined by means of hybridization methods.
  • a further embodiment of the invention is characterized in that the gene activity is determined by means of microarrays.
  • a further embodiment of the invention is characterized in that the gene activity is determined by hybridization-independent methods, in particular enzymatic and / or amplification methods, preferably PCR, subsequent quantification of the nucleic acids and / or of derivatives and / or fragments thereof.
  • a further embodiment of the invention is characterized in that cell samples are optionally subjected to a lytic treatment in order to release their cell contents.
  • Fig. 1 is a heat map showing a random selection from the relevant genes for the distinction between local and systemic infection.
  • the technical replicates (multiple spots of the same sample) on the microarray are filtered out of the corrected and transformed signal intensities depending on their spot quality. For each spot, the highest-labeled replicas are selected and the associated signal intensity is averaged. The expression of spots with only unmeasurable replicates are marked with "NA" (not available).
  • the total RNA from cell lines SIG-M5 served as reference samples.
  • the patients' whole blood in the intensive care unit was approved by the patients using the PAXGene kit according to the manufacturer's instructions (Qiagen). After collection of the whole blood, the total RNA of the samples was isolated using the PAXGene Blood RNA Kit according to the manufacturer's instructions (Qiagen).
  • This mixture was centrifuged at 1000 xg for 5 minutes at room temperature and suspended in 10 ml of the above-mentioned medium.
  • the subsequent cell count gave the following result: 1, 5 x 10 7 cells per ml, 10 ml total volume, total number of cells: 1, 5 x 10 8 . Since the cell count was still insufficient, 2.5 ml of the above-mentioned cell suspension in 30 ml of the above medium was added to a 250 ml (75 cm 2 ) flask (total of 4 flasks). After 72 hours of incubation, 20 ml each of fresh medium was added to the flasks.
  • the cell count was as described above, giving a total cell number of 3.8 x 10 8 cells.
  • the cells were resuspended in 47.5 ml of the above medium in 4 flasks. After an incubation period of 24 hours, the cells were centrifuged and washed twice with phosphate buffer without Ca 2+ and Mg 2+ (Biochrom AG).
  • RNA isolation is carried out using the NucleoSpin RNA L kit (Machery & Nagel) according to the manufacturer's instructions. The procedure described above was repeated until the required number of cells was reached. This was required to achieve the required amount of 6 mg total RNA, which corresponds approximately to an efficiency of 600 ⁇ g RNA per 10 8 cells.
  • RNA of the samples was isolated and tested for quality using the PAXGene Blood RNA Kit (PreAnalytiX) according to the manufacturer's instructions. From each sample, 10 ⁇ g total RNA were aliquoted and, together with 10 ⁇ g total RNA from SIGM5 cells as reference RNA to complementary DNA (cDNA) with the reverse transcriptase Superscript II (Invitrogen) rewritten and the RNA then by alkaline hydrolysis removed the approach. In the reaction mixture, a portion of the dTTP was replaced by aminoallyl-dUTP (AA-dUTP), to allow later the coupling of the fluorescent dye to the cDNA.
  • AA-dUTP aminoallyl-dUTP
  • the cDNA of the samples and controls were covalently labeled with the fluorescent dyes Alexa 647 and Alexa 555 and hybridized on a microarray from SIRS-Lab.
  • the microarray used are 5308 immobilized polynucleotides with a length of 55-70 base pairs, each representing a human gene and control spots for quality assurance.
  • An exemplary microarray is divided into 28 subarrays with a grid of 15x15 spots.
  • the hybridization and subsequent washing or drying was carried out in the hybridization station HS 400 (Tecan) according to the manufacturer for 10.5 hours at 42 0 C.
  • the hybridization solution used consists of the respective labeled cDNA samples, 3.5 ⁇ SSC (1x SSC contains 150 mM sodium chloride and 15 mM sodium citrate), 0.3% sodium dodecyl sulfate (v / v) 25% formamide (V ⁇ /) and 0 each, 8 ⁇ g ⁇ l-1 cot-1 DNA, yeast t-RNA and poly-A RNA.
  • the subsequent washing of the microarrays was carried out with the following program at room temperature: rinse for 90 seconds with washing buffer 1 (2 ⁇ SSC, 0.03% sodium dodecyl sulfate), with Wash Buffer 2 (1x SSC) and finally with Wash Buffer 3 (0.2x SSC). Thereafter, the microarrays were dried under a stream of nitrogen at a pressure of 2.5 bar at 30 0 C for 150 seconds.
  • hybridization signals of the microarrays were read using a GenePix 4000B scanner (Axon) and the expression ratios of the differentially expressed genes were determined using the GenePix Pro 4.0 or 5.0 (Axon) software.
  • the mean intensity of a spot was determined as the median value of the associated spot pixels.
  • each array was normalized one at a time using Box-Cox potency transformations (Box and Cox 1964, median and MAD (median absolute deviations from median).
  • the minimum of the classification error estimated by cross-validation was obtained for 69 gene samples.
  • the determination of the 69 gene samples indicated in Table 1-5 was carried out using the bootstrap method. Thus, the selection of the best 69 gene samples was repeated 5000 times using bootstrap samples and then sorting the gene samples according to the frequency of their selection.
  • the classification error estimated using 100 repetitions of a 10-fold cross-validation for the 69 gene samples given in Table 1-5 is 15.7% overall. In the group of patients with local infection the error is 26,7%, in the group of patients with systemic infection 11,1%. The patients with systemic infection are thus recognized with a safety of approximately 90%.
  • Gene expression was measured from a randomly selected ITS patient whose infection changes from systemic to local. The analysis took into account 8 consecutive ITS days of the patient.
  • the total RNA from cell lines SIG-M5 served as reference samples.
  • A Use according to A, characterized in that the gene activities by means of hybridization methods, in particular those on microarrays and / or enzymatic methods, in particular amplification method, preferably PCR, preferably real-time PCR, are created.
  • probe nucleic acids represent genes and / or gene loci or their transcripts that distinguish a condition associated with a local infection from that associated with a systemic infection Permit the condition of a patient and wherein the probe nucleic acid comprises at least one polynucleotide according to SEQ ID No. 1 to SEQ ID No. 69 or at least one such polynucleotide with a length of 2 to 100% of the number of nucleotides of the individual sequences according to SEQ ID No. 1 to SEQ ID NO.
  • step d) comparing the marker signals obtained in step d) with at least one reference value in order to make a statement as to whether a patient has a condition associated with a local infection or a condition associated with a systemic infection.
  • sample nucleic acid is RNA, in particular total RNA
  • probe nucleic acid is DNA, in particular cDNA, or mRNA.
  • the amplification mixture contains further constituents, in particular deoxynucleotides, polymerases, salts, buffers and fluorescence dyes attaching to nucleic acids.
  • a method of measuring gene activities in vitro for distinguishing a condition associated with a local infection from a condition of a patient associated with a systemic infection comprising the steps of:
  • probe nucleic acids represent genes and / or gene loci and / or their transcripts, which distinguishes a condition associated with a local infection from a with a systemic Infection associated condition of a patient and wherein the probe nucleic acid at least one polynucleotide according to SEQ ID NO. 1 to SEQ ID No. 69 or such a polynucleotide having a length of 2 to 100% of the number of nucleotides of the individual sequences according to SEQ ID No. 1 to SEQ ID No. 69;
  • step d Comparing the marker signals obtained in step d) with at least one reference value to determine whether a patient is experiencing a condition associated with a local infection or a condition associated with a systemic infection.
  • Method according to Q characterized in that the gene fragments comprise 5 to 1000, preferably 20-200, preferably 20-80 nucleotides.
  • Method according to Q to R characterized in that the polynucleotides according to Seq-ID No 1 to SEQ ID No 69 and / or sequences derived therefrom are replaced by: synthetic analogs, aptamers, spiegelmers as well as peptido- and morpholinonucleic acids.
  • Method according to Q to U characterized in that the sample is selected from: tissue, body fluids, in particular blood, serum, plasma, urine, saliva or cells or cell components; or a mixture of them.
  • Method according to Q to V characterized in that samples, in particular cell samples, are subjected to a lytic treatment to release their cell contents.
  • Method according to Q to W characterized in that the sample nucleic acid is RNA, in particular total RNA, the probe nucleic acid is DNA, in particular cDNA, or mRNA.
  • Kit containing at least one of the polynucleotide sequences SEQ ID NO. 1 - SEQ ID NO. 69 and / or primers and / or probes and / or antisense nucleotides therefor or which are specific for detecting the condition of an infection (local or systemic) of a patient, and / or polynucleotides of 2 to 100% of the number of nucleotides in length individual sequences according to SEQ ID No. 1 to SEQ ID No. 69 for the determination of gene activities in vitro in a patient sample, and / or for determining the course of a patient's infection from local to systemic or from systemic to local.
  • Kit according to Y characterized in that the polynucleotide sequences also include gene loci, mRNA, small RNA, in particular scRNA, snoRNA, microRNA, siRNA, dsRNA, ncRNA or transposable elements.
  • FIG. 1 shows a so-called heat map for the representation of a random selection from the relevant genes for the distinction between local and systemic infection.
  • Fig. 1 Shown in Fig. 1 in the rows are the normalized expression data of the individual transcripts centered on the mean (i.e., for each gene, the mean is determined across all patients). The columns represent the different patients. Light gray / white stands for Expression higher than the mean and dark gray / black for Expression lower than the mean.
  • the light gray bar above the heatmap stands for the patients with local infection, the dark gray bar for the patients with systemic infection.
  • a hierarchical clustering was also performed using the "complete" method known to those skilled in the art, using as distance Pearson's correlation distance.
  • the dendrogram on the left shows that the transcripts can essentially be divided into 4 clusters.

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Abstract

La présente invention concerne l'utilisation de polynucléotides ayant une longueur comprise entre 2 et 100% du nombre des nucléotides des différentes séquences représentées par les SEQ ID N° 1 à SEQ ID N° 69 et/ou leurs locus géniques et/ou leurs produits de transcription pour la détection d'activités géniques afin de différencier un état associé à une infection locale d'un état associé à une infection systémique chez un patient, en utilisant toutes les séquences représentées par les SEQ ID N° 1 à SEQ ID N° 69. L'invention concerne également un procédé ainsi qu'un kit de mise en oevre de ce procédé.
EP08785273A 2007-08-03 2008-07-31 Utilisation de polynucléotides pour la détection d'activités géniques afin de différencier une infection locale d'une infection systémique Withdrawn EP2179054A1 (fr)

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DE102007036678.9A DE102007036678B4 (de) 2007-08-03 2007-08-03 Verwendung von Polynukleotiden zur Erfassung von Genaktivitäten für die Unterscheidung zwischen lokaler und systemischer Infektion
PCT/EP2008/006332 WO2009018962A1 (fr) 2007-08-03 2008-07-31 Utilisation de polynucléotides pour la détection d'activités géniques afin de différencier une infection locale d'une infection systémique

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EP2309001A1 (fr) 2009-09-23 2011-04-13 SIRS-Lab GmbH Procédé de détermination et de différenciation in vitro d'états physiopathologiques
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DE102011005235B4 (de) 2011-03-08 2017-05-24 Sirs-Lab Gmbh Verfahren zum Identifizieren einer Teilmenge von Polynucleotiden aus einer dem Humangenom entsprechenden Ausgangsmenge von Polynucleotiden zur in vitro Bestimmung eines Schweregrads der Wirtsantwort eines Patienten
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