A genetically modified cell and its use in a method for detecting pyrogens in a sample

Abstract

The present invention relates to a genetically modified cell and its use in a method for detecting pyrogens or mycoplasms in a sample. The invention further relates to a kit comprising said genetically modified cell which can be used to detect pyrogens or mycoplasms in a sample.

Description

[0001] A GENETICALLY MODIFIED CELL AND ITS USE IN A METHOD FOR DETECTING PYROGENS IN A SAMPLE

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to a genetically modified cell and its use in a method for detecting pyrogens or mycoplasms in a sample. The invention further relates to a kit comprising said genetically modified cell which can be used to detect pyrogens or mycoplasms in a sample.

[0004] BACKGROUND OF THE INVENTION

[0005] Pyrogens are a broad category of substances, which can activate immune cells, leading to the release of pro-inflammatory cytokines such as IL-1 p, IL-6, and TNF-a and the subsequent induction of fever in a subject. Pyrogens include endotoxins (e.g. lipopolysaccharides (LPS)) and non-endotoxins (e.g. lipoproteins) derived from microorganisms. The detection of pyrogenic contaminants is a critical aspect in the pharmaceutical industry, in laboratories and in health care institutions, since such contaminants can severely compromise the safety, stability, and efficacy of pharmaceutical products, in particular parenterally applied medicinal products, potentially leading to severe health risks when administered to patients. Undetected contaminations can also severely impact experimental research, rendering the obtained data unusable. Sensitive detection methods for the presence of pyrogenic contaminants, ensuring a pyrogen-free manufacturing and filling process for pharmaceutical products, as well as contaminant-free working conditions, are therefore of vital importance.

[0006] Pyrogens can be detected by several tests such as the in vivo rabbit pyrogen test (RPT) based on measuring the rise in body temperature of healthy rabbits after intravenous injection of a sample containing a pyrogen and the limulus amebocyte lysate test (LAL), also known as bacterial endotoxin test (BET), using blood (amebocytes) of the horseshoe crab (Limulus polyphemus) which coagulates in the presence of pyrogenic contaminants. The use of RPT and LAL is ethically questionable because of the need to sacrifice a large number of animals and further poses risks due to inherent differences between distantly related species.

[0007] More recently, an animal-free alternative called monocyte activation test (MAT) was developed. The human cellbased MAT makes use of the intrinsic capability of primary human monocytes or monocytoid cell lines to respond to various microbial molecular signatures with the expression of immune mediators, such as TNF-a or IL-6, which serve as a readout for the presence of pyrogens. Said tests are available and validated by regulatory agencies, but several disadvantages remain. The use of primary cells is not only time-consuming and labor-intensive, but it also leads to a high variability between tests and long incubation times are needed to produce measurable amounts of cytokines. Forthat reason, there have been several approaches to enhance the sensitivity and specificity of MATs as described in WO 2007 / 076411 A1, WO 2024 / 110508 A1 and EP 4286535 A1. To address the problem of MATs based on the isolation of cells from human blood samples, studies by Battin et al. (PLoS ONE (2017) 12(5): e0178220) and He et al. (Sig Transduct Target Ther (2024) 9:33) tried to overcome said limitation by exploring reporter cell lines, but all these cell-based approaches require incubation times of more than 20 hours to reach their endpoint. A study by Seumen et al. (Scientific Reports (2021) 11:24414) describes the generation of a genetically modified immune cell line for use in the detection of non-endotoxin pyrogens.

[0008] Nevertheless, there is still a significant need for an optimized and accelerated cell-based testing system that ensures and controls pyrogen-free production, which will advance pharmaceutical safety, improve patient outcomes, and align with ethical principles by reducing reliance on animal testing. Moreover, it is desirable to provide a method for detection of both endotoxin and non-endotoxin pyrogens.

[0009] In addition to detecting pyrogenic contaminants, the detection of mycoplasma contamination is a further critical aspect in the pharmaceutical industry, in laboratories and in health care institutions. Mycoplasma species are small self-replicating microorganisms lacking a cell wall and are among the most frequent contaminants in cell culture systems. Although mycoplasma species are not classified as classical pyrogens, they can activate immune cells, leading to the release of pro-inflammatory cytokines, and they may compromise product safety, efficacy, and experimental validity. Current mycoplasma detection methods, such as culture-based assays, DNA staining, or PCR-based techniques, are time-consuming and labor-intensive. Therefore, there remains a need for rapid, sensitive, and preferably cell-based detection systems capable of identifying mycoplasms in pharmaceutical products and experimental research processes.SUMMARY OF THE INVENTION

[0010] The present invention is directed to a genetically modified cell comprising a reduced expression of at least two negative regulators of Toll-like receptor (TLR)-signaling compared to a cell which is not genetically modified, and a reporter gene operably linked to a TLR-responsive promoter.

[0011] The present invention further relates to a method for the detection of pyrogens in a sample, comprising the steps of: (a) providing the genetically modified cells according to the invention in a suitable cell culture medium; (b) contacting the cells with said sample and (c) determining expression of the reporter protein.

[0012] The present invention further relates to a method for the detection of mycoplasms or mycoplasma-derived components in a sample, comprising the steps of: (a) providing the genetically modified cells according to the invention in a suitable cell culture medium; (b) contacting the cells with said sample and (c) determining expression of the reporter protein.

[0013] The present invention further relates to a kit comprising the genetically modified cells according to the invention and at least one small molecule inhibitor of intracellular signaling.

[0014] The invention is further directed to the use of the genetically modified cell according to the invention or the kit according to the invention for the detection of pyrogens or mycoplasms or mycoplasma-derived components in a sample.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS FIGURE 1: Downregulation of both PP2ACA and SOCS1 increases NF-KB dependent reporter gene activation

[0016] PP2ACAKDcells and PP2ACAKD / SOCS1KOcells (clones lzCeO77 and lzCeO78) were stimulated with 30 ng / ml NEP-LP (Non-Endotoxin Pyrogenic Lipopeptide), namely the synthetic triacylated lipoprotein Pam3CSK4, or were kept in the presence of pyrogen-free water (unstimulated). Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to unstimulated cells) was determined by flow cytometry 6 h after stimulation. Bar graphs depict the MFI ± standard error of mean obtained from three independent experiments. Each experiment was conducted on a representative count of 10.000 cells.

[0017] FIGURE 2: Treatment of PP2ACAKD / SOCS1KOcells with sephin-1 and guanabenz further increases NF-KB dependent reporter gene activation

[0018] PP2ACAKD / SOCS1K0cells (clone lzCeO78) were stimulated with 30 ng / ml NEP-LP, namely the synthetic triacylated lipoprotein Pam3CSK4, or were kept in the presence of pyrogen-free water (vehicle, unstimulated). Further, sephin-1 (10 pM) and guanabenz (12.5 pM) were added to the cells prior to treatment with NEP-LP or the cells were not treated with sephin-1 and guanabenz. Black circles indicate that the cells were treated with the respective agent, whereas white circles indicate that the cells were not treated with the respective agent. Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to unstimulated cells) was determined by flow cytometry analysis 6 h after stimulation. Bar graphs depict the MFI ± standard error of mean obtained from three independent experiments. Each experiment was conducted on a representative count of 10.000 cells.

[0019] FIGURE 3: Increased expression of CD14 increases NF-KB dependent reporter gene activation in response to endotoxin

[0020] PP2ACAKD(clone [lzCe001]CD14+) cells with an increased CD14 expression and PP2ACAKD / SOCS1KOcells (clone [lzCeO78]CD14+) with an increased CD14 expression were stimulated with 0.5 EU / ml RSE (Reference Standard Endotoxin) or were kept in the presence of pyrogen-free water (vehicle, unstimulated). Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to unstimulated cells) was determined by flow cytometry analysis 6 h after stimulation. Bar graphs depict the MFI ± standard error of mean obtained from three independent experiments. Each experiment was conducted on a representative count of 10.000 cells.

[0021] FIGURE 4: Treatment of cells with increased CD14 expression with sephin-1 and guanabenz further increases NF-KB dependent reporter gene activation in response to endotoxin

[0022] PP2ACAKD / SOCS1KOcells having an increased CD14 expression were stimulated with 0.5 EU / ml RSE (Reference Standard Endotoxin) or were kept in the presence of pyrogen-free water (unstimulated). Further, sephin-1 (10 pM) and guanabenz (12.5 pM) were added to the cells prior to treatment with RSE or the cells were not treated withseph in-1 and guanabenz. Black circles indicate that the cells were treated with the respective agent, whereas white circles indicate that the cells were not treated with the respective agent. Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to unstimulated cells) was determined by flow cytometry analysis 6 h after stimulation. Bar graphs depict the MFI ± standard error of mean obtained from three independent experiments. Each experiment was conducted on a representative count of 10.000 cells. FIGURE 5: Downregulation of negative regulators of TLR signaling — including SOCS1, USP-4, TBK-1, PP1B, PP1G, PP2ACA, PP2BA, PP5C, PPM1D, PPM1G, or PPM1N — enhances NF-KB-dependent reporter gene activation in response to Pam3CSK4 stimulation

[0023] Cells having a knock-out of SOCS-1, USP-4, TBK-1, PP1B, PP1G, PP2BA, PP5C, PPM1D, PPM1G, PPM1N or a knock-down of PP2ACAwere stimulated with 100 ng / ml Pam3CSK4 or were kept in the presence of pyrogen-free water (unstimulated). Mock cells are THP-1 cells transfected with a sgRNA-negative control vector and thus lacking downregulation of any negative regulator of TLR signaling. The dashed line indicates the threshold above which NF-KB-dependent reporter gene activation is higher than in Mock controls. Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to Mock unstimulated cells) was determined by flow cytometry 3 h after stimulation. Bar graphs depict the MFI ± standard error of mean obtained from three independent experiments. Each experiment was conducted on a representative count of 10.000 cells.

[0024] FIGURE 6: Combined downregulation of PP2ACA with either PP1G or PPM1D further enhances NF-KB-dependent reporter gene activation compared to downregulation of PP2ACA alone in response to various pyrogenic stimuli

[0025] PP2ACAKDcells (black bar, indicated as Mock), PP1GK0 / PP2ACAKD(grey bar) and PPM 1 DK0 / PP2ACAKD(light grey bar) cells were stimulated with 1 EU / ml Reference Standard Endotoxin (RSE), 0.25 pg / ml Flagellin, 5 nM Fibroblaststimulating lipopeptide 1 (FSL-1), 10pg / ml Polyinosinic:polycytidylic acid(Poly (l:C)) or 5pg / ml Thiazoquinoline derivative (CL075). Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to stimulated PP2ACAKDcells) was determined by flow cytometry 3 h after stimulation. For each stimulus the activation of the NF-KB dependent reporter gene in PP2ACAKDcells was used as reference and was always set to 1; the activation in PP1GK0 / PP2ACAKDand PPM1DK0 / PP2ACAKDcells is expressed relative to this reference. Bar graphs depict the MFI ± standard error of mean obtained from three independent experiments. Each experiment was conducted on a representative count of 10.000 cells.

[0026] FIGURE 7: Culture of PP2ACAKD / SOCS1KOcells in low serum medium does not impair sensitivity of NF-KB dependent reporter gene activation in response to Pam3CSK4 stimulation

[0027] PP2ACAKD / SOCS1KOcells having an increased CD14 expression were kept either in medium comprising 0.5% FCS (circle and light grey column) or in medium comprising 10% FCS (square and darker grey column) and were stimulated with 30 ng / ml Pam3CSK4 or were kept in the presence of pyrogen-free water (unstimulated). Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to unstimulated cells) was determined by flow cytometry analysis 3 h after stimulation. Bar graphs depict the MFI ± standard error of mean obtained from four independent experiments. Each experiment was conducted on a representative count of 10.000 cells.

[0028] FIGURE 8: PP2ACAKD / SOCS1KOcells show increased NF-KB dependent reporter gene activation in response to various stimuli

[0029] PP2ACAKD / SOCS1KOcells having an increased CD14 expression were stimulated for 6 hours with 1 pg / pl human genomic DNA (hgDNA), 0.3 pg / ml Gardiquimod (Gardi), 0.1 pg / ml lipopolysaccharides (LPS), 0.01 pg / ml Tumor necrosis factor alpha (TNF-a), 0.25 pg / ml Flagellin, 10 nM Fibroblast-stimulating lipopeptide 1 (FSL-1), 100 ng / ml Pam3CSK4, 0.1 pg / ml heat-killed Salmonella typhimurium (HKST), 0.1 pg / ml heat-killed Staphylococcus aureus (HKSA), 100 pg / ml Zymosan, 1 pM N-methyl-4-nitro-2-(4-(4-(trifluoromethyl)phenyl)-1H-imidazol-1-yl)aniline (CU-T219), 5 pg / ml lipoteichoic acid (LTA), 10 pg / ml polyinosinic: polycytidylic acid (Poly (l:C)), and 5 pg / ml peptidoglycan from Staphylococcus aureus (PGN-SA) or were kept in the presence of pyrogen-free water (unstimulated). Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to unstimulated cells) was determined by flow cytometry analysis 6 h after stimulation. Bar graphs depict the MFI ± standard error of mean obtained from four independent experiments. Each experiment was conducted on a representative count of 10.000 cells.FIGURE 9: Increased expression of CD36 in PP2ACAKD / SOCS1KOcells increases NF-KB dependent reporter gene activation in response to mycoplasma-derived components

[0030] PP2ACAKD / SOCS1KOcells with an increased CD14 expression (clone lzCeO81H-3P, light grey circles) and PP2ACAKD / SOCS1KOcells (clone lzCeO81H-3PCD36+, grey squares) with an increased CD36 and CD14 expression were stimulated in assay medium with CHO SN Myco+ (Chinese hamster ovary cell (CHO) supernatant (SN) positive for Mycoplasma after 3 days of culture, confirmed by PCR), CHO SN Myco- (Chinese hamster ovary cell (CHO) supernatant (SN) negative for Mycoplasma after 3 days of culture, confirmed by PCR), or were kept in the presence of assay medium and endotoxin-free water (unstimulated). Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to unstimulated cells) was determined by flow cytometry analysis 3 h after stimulation. Bar graphs depict the MFI ± standard error of mean obtained from three independent experiments. Each experiment was conducted on a representative count of 10.000 cells. FIGURE 10: Increased expression of CD36 of PP2ACAKD / SOCS1KOcells increases NF-KB dependent reporter gene activation in response to mycoplasma-derived components in a time-dependent manner PP2ACAKD / SOCS1KOcells (clone lzCeO81H-3PCD36+, grey circles) with an increased CD36 and CD14 expression were stimulated in assay medium (0.5% FCS + cell culture medium + 12.5 pM Sephin-1 + 12.5 pM Guanabenz) with CHO SN Myco- (Chinese hamster ovary cell (CHO) supernatant (SN) negative for Mycoplasma after 3 days of culture, confirmed by PCR) for 3 hours or with CHO SN Myco+ (Chinese hamster ovary cell (CHO) supernatant (SN) positive for Mycoplasma after 3 days of culture, confirmed by PCR) for 1, 2 or 3 hours, respectively. Unstimulated controls were maintained in assay medium (unstimulated). Activation of the NF-KB dependent reporter gene encoding mCFP (MFI (median fluorescent intensity) ratio relative to unstimulated cells) was determined by flow cytometry analysis after the indicated stimulation periods. Bar graphs depict the MFI ± standard error of mean obtained from three independent experiments. Each experiment was conducted on a representative count of 10.000 cells.

[0031] DETAILED DESCRIPTION OF THE INVENTION GENERAL DEFINITIONS

[0032] Before the invention is described in detail with respect to some of its preferred embodiments, the following general definitions are provided.

[0033] The present invention as illustratively described in the following may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein.

[0034] The present invention will be described with respect to particular embodiments and with reference to certain figures, but the invention is not limited thereto but only by the claims.

[0035] Where the term “comprise” or “comprising” is used in the present description and claims, it does not exclude other elements or steps. For the purpose of the present invention, the term “consisting of” is considered to be an optional embodiment of the term “comprising of”. If hereinafter a group is defined to comprise at least a certain number of embodiments, this is also to be understood to disclose a group which optionally consists only of these embodiments. Where an indefinite or a definite article is used when referring to a singular noun e.g. “a” or “an”, “the”, this includes a plural form of that noun unless specifically stated. Vice versa, when the plural form of a noun is used it refers also to the singular form.

[0036] Furthermore, the terms first, second, third or (a), (b), (c) and the like in the description and in the claims are used for distinguishing between similar elements and not necessarily for describing a sequential or chronological order. It is to be understood that the terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein are capable of operation in other sequences than described or illustrated herein.

[0037] In the context of the present invention any numerical value indicated is typically associated with an interval of accuracy that the person skilled in the art will understand to still ensure the technical effect of the feature in question. As used herein, the deviation from the indicated numerical value is in the range of ± 10%, and preferably of ± 5%. The aforementioned deviation from the indicated numerical interval of ± 10%, and preferably of ± 5% is also indicated by the terms “about” and “approximately” used herein with respect to a numerical value.

[0038] The term "gene" refers to a nucleic acid sequence that comprises coding sequences necessary for the production of an mRNA, a polypeptide or polypeptide precursor. The mRNA or polypeptide can be encoded by a full-lengthcoding sequence or by any portion of the coding sequence as long as the desired activity or functional properties of the full-length polypeptide are retained. The term also encompasses the coding region of a gene and the sequences located adjacent to the coding region on both the 5' and 3' ends, such that the gene corresponds to the length of the full-length mRNA. The term "gene" encompasses both cDNA and genomic forms.

[0039] The term “expression” or “gene expression” as used herein refers to the process of synthesis of a gene product, preferably a functional RNA or protein. Gene expression generally comprises DNA transcription, optionally RNA processing and in the case of protein-expressing genes, RNA translation into protein. According to the invention, “reduced expression” in a genetically modified cell refers to a decrease in the amount of a specific gene product such as an RNA or a protein produced by a genetically modified cell, measured relative to a cell which is not genetically modified. The term “reduced expression” corresponds to a measurable decrease in the amount of a specific gene product such as an RNA or a protein produced by a genetically modified cell of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% measured relative to a cell which is not genetically modified. Reduced expression includes complete loss of expression (e.g., a knockout), partial reduction of expression (e.g., a knockdown), or decreased stability or activity of the gene product. Furthermore, as used herein, “reduced expression” of a specific gene product refers to a decrease in the amount of the gene product to an extent sufficient to cause a measurable impairment or alteration of its associated biological function. In one embodiment, reduced expression of a negative regulator of TLR signaling selected from SOCS1, USP-4, TBK-1, PP1 or a catalytic subunit thereof, PP2B or a catalytic subunit thereof, PP5C, PPM1D, PPM1G, and PPM1N refers to a complete loss of expression of the gene product, i.e. to a knockout. In one embodiment, reduced expression of a negative regulator of TLR signaling selected from SOCS1, USP-4, TBK-1, PP1B, PP1G, PP2BA, PP5C, PPM1D, PPM1G, and PPMIN refers to a complete loss of expression of the gene product, i.e. to a knockout. In another embodiment, reduced expression of the negative regulator PP2ACA refers to a reduction of expression of the gene product, i.e. a knockdown. The expression of PP2ACA is reduced by at least 40%, at least 50%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%. Techniques to genetically modify cells to reduce the expression of a specific gene product are known to the skilled person and described in further detail herein.

[0040] According to the invention, "increased expression" in a genetically modified cell refers to an increase in the amount of a specific gene product such as an RNA or a protein produced by a genetically modified cell, measured relative to a cell which is not genetically modified. The term “increased expression” corresponds to a measurable increase in the amount of the specific gene product produced by a genetically modified cell of at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350% or at least 400% measured relative to a cell which is not genetically modified. In one embodiment, increased expression may also be defined in terms of fold-change, for example as at least 1-fold, at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, or at least 5-fold increase, measured relative to a cell which is not genetically modified. Increased expression may be achieved by any suitable means known to the person skilled in the art, including introduction or transformation of a polynucleotide encoding the gene, for example by introducing additional copies using suitable gene delivery or gene transfer systems, including viral and non-viral vectors, by activating the endogenous gene (e.g., using CRISPR activation, transcriptional activators, or epigenetic modulators), or by stabilizing the RNAor protein (e.g., through modified coding sequences, regulatory elements, or post-translational modifications).

[0041] The increase or decrease in the amount of a specific gene product may be determined using standard quantitative methods, including qPCR, immunoblotting, ELISA, flow cytometry or mass spectrometry.

[0042] The terms “nucleic acid” or “nucleic acid molecule” are used interchangeably herein to refer to a biomolecule composed of nucleotides. The terms “nucleic acid sequence” or “nucleotide sequence” refer to the order of nucleotides in a strand of DNA or RNA. The nucleic acid sequence determines the genetic information that can be transcribed into RNA and translated into a protein. The nucleic acid can be synthesized in vitro or can be naturally occurring, i.e. isolated from nature. The nucleic acid molecule can be comprised within a eukaryotic or prokaryotic organism, a eukaryotic or prokaryotic cell, a cell nucleus or a cell organelle, as part of a genome or as an individual molecule. According to the invention, a synthesized nucleic acid molecule comprising elements not linked in nature is a recombinant nucleic acid molecule and it can be comprised within a plasmid, a vector or an artificial chromosome.The terms “sequence identity”, “% sequence identity”, “% identity”, “% identical” or “sequence alignment” are used interchangeably herein and refer to the result of the comparison of a first nucleic acid sequence to a second nucleic acid sequence, or a comparison of a first amino acid sequence to a second amino acid sequence. The sequence identity is calculated as a percentage based on the comparison. The result of this calculation can be described as “percent identical” or “percent ID.” A sequence identity may be determined by a program, which produces an alignment and calculates identity counting both mismatches at a single position and gaps at a single position as non-identical positions in the final sequence identity calculation. Preferably, the sequence identity is determined over the entire length of the first and second nucleic acid sequence.

[0043] According to this invention, a pairwise global alignment is produced, meaning that two sequences are aligned over their complete length, usually using a mathematical approach, called alignment algorithm.

[0044] According to the invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453). Preferably, the program “NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the current invention, with using the programs default parameter (polynucleotides: gap open=10.0, gap extend=0.5 and matrix=EDNAFULL; polypeptides: gap open=10.0, gap extend=0.5 and matrix=EBLOSUM62). After aligning two sequences, in a second step, an identity value is determined from the alignment produced. For this purpose, the %-identity is calculated by dividing the number of identical residues by the length of the alignment region which shows the respective sequence over its complete length multiplied with 100: %-identity = (identical residues I length of the alignment region which shows the respective sequence over its complete length) *100.

[0045] For calculating the percent identity of two nucleic acid sequences the same applies as for the calculation of percent identity of two amino acid sequences with some specifications. For nucleic acid sequences encoding a protein the pairwise alignment shall be made over the complete length of the coding region of the sequence from start to stop codon excluding introns. Introns present in the other sequence, to which the sequence is compared, shall also be removed for the pairwise alignment. After aligning two sequences, in a second step, an identity value is determined from the alignment produced. Percent identity is calculated by %-identity = (identical residues I length of the alignment region which shows the sequence from start to stop codon excluding introns over its complete length) *100.

[0046] The term “amino acid sequence” herein refers to the specific order in which amino acids are arranged to form a protein or polypeptide. The sequence of amino acids can be defined using different abbreviations for each amino acid. There are 20 standard amino acids commonly found in proteins, and they are represented by their abbreviations, i.e. each amino acid can be denoted by a single letter (one-letter code) or by a three-letter abbreviation (three-letter code).

[0047] The term “polypeptide” is defined herein as a polymer consisting of at least ten amino acids. According to the invention a recombinant polypeptide can be a polypeptide native to a cell in which structural modifications, e.g. disruptions, deletions, substitutions, and / or insertions, have been made by recombinant DNA techniques to alter the native polypeptide, or a polypeptide native to a cell whose expression is quantitatively altered or whose expression is directed from a genomic location different from that in the native cell as a result of manipulation of the DNA of the cell by recombinant DNA techniques, or whose expression is quantitatively altered as a result of manipulation of the regulatory elements of the polynucleotide by recombinant DNA techniques e.g., a stronger promoter; or a polynucleotide native to the cell, but not integrated within its natural genetic environment as a result of genetic manipulation by recombinant DNA techniques.

[0048] The term “disruption of a nucleic acid sequence” refers to introducing insertions or deletions of nucleotides in a nucleic acid sequence, usually within the coding region of a gene. Insertions or deletions can be created by frameshift mutations that disrupt the open reading frame (ORF) of a gene, leading to a nonfunctional protein or the reduced expression of the protein.

[0049] The term “deletion of a nucleic acid sequence” refers to the removal of a larger segment of nucleotides in a nucleic acid sequence, preferably the removal of the complete coding sequence of a gene.

[0050] Variants of amino acid sequences include amino acid sequences comprising insertions, additions, deletions, or substitutions relative to a reference amino acid sequence. The variant preferably has the same function as the wildtype protein.

[0051] The term "fragment of a nucleic acid sequence" as used herein refers to a portion or subsection of a longer nucleic acid sequence (DNA or RNA). The fragment may be naturally occurring (e.g. a cleavage product) or artificially generated (e.g. by PCR, restriction digestion, or chemical synthesis). The fragment of a nucleic acid sequence preferably encodes a fragment of a protein which has the same function as the full-length protein.The terms “introduction of a polynucleotide” or “transformation of a polynucleotide” as used herein encompass the transfer of an exogenous polynucleotide into a cell, irrespective of the method used for the transfer. That is, the term “transformation of a polynucleotide” as used herein is independent of vector, shuttle system, or host cell, and it not only relates to the polynucleotide transfer method of transformation as known in the art (cf. , for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Labora-tory Press, Cold Spring Harbor, NY), but it encompasses any further kind of polynucleotide transfer methods such as, but not limited to, transduction or transfection.

[0052] To express polypeptides recombinantly in a cell an expression cassette comprising one or more nucleotide sequences as described herein is used. Typically, an expression cassette comprises three elements: a promoter sequence, an open reading frame and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. Additional regulatory elements may include transcriptional as well as translational enhancers. The promoter may be either constitutive or inducible. A constitutive promoter drives continuous, unregulated expression of a polypeptide, whereas an inducible promoter controls the expression of a polypeptide in response to specific environmental signals or chemicals. An intron sequence may also be added to the 5' untranslated region (UTR) or to the coding sequence to increase the amount of the mature message that accumulates in the cytosol. The expression cassette may be part of a vector which is present separate from the genome of a cell or may be integrated into the genome of a cell and replicated together with the genome of its host cell. The expression cassette usually is capable of increasing or decreasing expression of a polypeptide.

[0053] Also disclosed herein is an expression vector comprising one or more of the nucleotide sequences as described herein. The term “vector” as used herein comprises any kind of construct suitable to carry foreign polynucleotide sequences for transfer to another cell or for stable or transient expression within a given cell. The polynucleotide encoding the polypeptide used in the method of the present invention may be introduced into a vector by means of standard recombinant DNA techniques. Once introduced into the vector, the polynucleotide comprising a coding sequence may be suitable to be introduced (by transformation, transduction, transfection, etc.) into a cell or cell organelle. An expression vector may be chosen which is suitable for expression of the polynucleotide sequence in a cell or host cell organelles. Suitable vectors for this purpose are available to the person skilled in the art and can be taken, for example, from the brochures of companies including Novagen, Promega, New England Biolabs, Biosearch technologies, Cambio, VWR, Clontech or Gibco BRL.

[0054] An expression vector as used herein may provide additional segments for transcription and translation of a foreign polynucleotide upon transformation into a cell or cell organelles. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication that is required for maintenance and / or replication of the expression vector in a specific cell type, one or more selectable markers, a polyadenylation signal, a suitable site for the insertion of foreign coding sequences such as a multiple cloning site etc.

[0055] PYROGENS

[0056] Pyrogens are substances that can trigger an immune response in a subject by activating a cascade of immunological processes which lead to an increase in the body’s internal temperature beyond normal levels (fever). A biological activity of a pyrogen is its capacity to induce fever in a subject, which is also referred to herein as its pyrogenicity.

[0057] A pyrogen may be present in a pharmaceutical product or composition, a product for cell culture or laboratory use, a cosmetic or personal care product or a parenteral nutrition solution or on a surface of a medical device.

[0058] In one embodiment, the pyrogen is an endotoxin, such as a lipopolysaccharide (LPS). Endotoxins, such as lipopolysaccharides, are cellular components of bacteria such as Gram-negative bacteria which constitute the main component of their outer cell walls. LPS is a large, amphipathic molecule composed of a lipid and polysaccharide moiety, found in the outer membrane of Gram-negative bacteria. The presence of endotoxins in the blood stream of a subject is associated with multiple adverse symptoms, including fever, hypotension, nausea, shivering, and shock, and can lead to complications such as disseminated intravascular coagulation (DIC), endotoxin shock, and acute respiratory distress syndrome (ARDS). In one embodiment, the endotoxin is a cellular component, preferably a lipopolysaccharide (LPS), of a Gram-negative bacterium. The term "gram-negative bacterium” refers to a bacterium that generally does not retain the crystal violet stain used in the standard Gram staining method, as opposed to a "Gram-positive” bacterium, which generally retains the stain. Further, gram-negative bacteria are characterized by the presence of a thin peptidoglycan cell wall between the cytoplasmic membrane and the outer membrane. In one embodiment, the Gram-negative bacterium is a pathogenic or potentially pathogenic bacterium. Examples of pathogenic or potentially pathogenic Gram-negative bacteria are bacteria of the genera Escherichia, Salmonella, Shigella, Pseudomonas, Neisseria, Haemophilus, Bordetella, Vibrio, and the like (Akira et al. Cell (2006) 124(4):783-801), such as Escherichia coll, Neisseria meningitidis, Haemophilus influenzae, Salmonellaenteridis, Pseudomonas aeruginosa and Vibrio cholerae. Synthetic or standardized analogues of endotoxin pyrogens include Reference Standard Endotoxin (RSE) for example commercially available preparations such as Lonza™ USP Reference Standard Endotoxin (RSE) or the PyroMAT® Reference Standard Endotoxin provided by Millipore.

[0059] In one embodiment, the pyrogen is a non-endotoxin pyrogen (NEP). Non-endotoxin pyrogens include microbe-associated molecular patterns (MAMPs) and pathogen-associated molecular patterns (PAMPs), with examples being bacterial cellular components such as bacterial proteins (e.g., flagellins), peptidoglycans (e.g., peptidoglycan from Staphylococcus aureus (PGN-SA)), lipoproteins, lipoteichoic acid (LTA), fibroblast-stimulating lipopeptide 1 (FSL-1), macrophage-activating lipopeptide-2, heat-killed bacteria such as heat-killed Salmonella typhimurium (HKST) or heat-killed Staphylococcus aureus (HKSA), viral pyrogens such as double-stranded RNA, yeast pyrogens, zymosan, fungal pyrogens (e.g. yeast or fungal polysaccharides), single-stranded DNA molecules such as human genomic DNA (hgDNA), and endogenous pyrogens such as tumor necrosis factor alpha (TNF-a). In one embodiment, the pyrogen is a cellular component, preferably a bacterial protein (e.g., flagellin), of a Gram-positive bacterium. In one embodiment, the Gram-positive bacterium is pathogenic or potentially pathogenic. Examples of pathogenic or potentially pathogenic Gram-positive bacteria are bacteria of the genera Streptococcus, Staphylococcus, Corynebacterium, Listeria, Bacillus (e.g., Bacillus subtilis), Clostridium and the like (Henderson B, Wilson M. Cytokine (1996) 8(4):269-82). In some cases, a flagellin may be from a Gram-positive bacterium, for example from Bacillus subtilis. Synthetic analogues of NEPs include, but are not limited to, the triacetylated lipopeptide Pam3CSK4, the synthetic analogue of double-stranded RNA Poly (I :C), the imidazoquinoline compound Gardiquimod, the thiazoquinoline derivative CL075, N-methyl-4-nitro-2-(4-(4-(trifluoromethyl)phenyl)-1H-imidazol-1 -yl)aniline (CU-T219) and the short single-stranded DNA molecules CpG ODN.

[0060] In one embodiment, the pyrogen is selected from the group consisting of Pam3CSK4, RSE, Flagellin, FSL-1, Poly ( I: C), and CL075. In another embodiment, the pyrogen is selected from the group consisting of RSE, Flagellin, FSL-1, Poly (l:C), and CL075. In another embodiment, the pyrogen is selected from the group consisting of RSE, Pam3CSK4, Flagellin, FSL-1, Poly (l:C), CL075, TNF-a, HKST, HKSA, zymosan, CU-T219, LTA and PGN-SA. TLR-SIGNALING

[0061] Within the present invention, the pyrogen is a toll-like receptor (TLR) agonist. TLR agonists are molecules that specifically bind to TLRs and activate downstream signaling pathways. TLRs are a key part of the innate immune system and their activation by a TLR agonist triggers inflammatory responses, including the production of pro-inflammatory cytokines such as TNF-a, IL-1 p, and IL-6, which contribute to the febrile response (fever) and other systemic effects (Kawai and Akira Nature Immunology (2010) 11(5), 373-384). There are 10 TLRs identified in humans (TLR-1 to TLR-10), wherein TLR-1 to TLR-9 are well-characterized and mainly mediate the detection of diverse microbial molecular signatures such as pathogen-associated molecular patterns (PAMPs). Accordingly, TLRs are able to specifically respond to microbial contaminants and each TLR is specialized in detecting specific types of microbial contaminants (Chakraborty S. et al. Front Immunol. (2023) 14:1227833 and Hu DN et al. Exp Eye Res. (2022) 216:108943). TLR-1 agonists include triacylated lipopeptides, derived from bacterial cell membranes, and synthetic analogues thereof such as Pam3CSK4. TLR-2 is able to form heterodimers with TLR-1 or TLR-6 and to bind various microbial products, including lipoproteins and peptidoglycans from both Gram-positive and Gram-negative bacteria. Lipoteichoic acids (LTA) act as agonists of TLR-2 and Fibroblast-stimulating lipopeptide-1 (FSL-1), which is a synthetic analogue of bacterial lipopeptides, is an agonist of TLR-2 / TLR-6. Known agonists of TLR-3 include double-stranded RNA(dsRNA) molecules, being a molecular signature of viral infections. A synthetic analogue of such a TLR-3 agonist is Poly( I : C). LPS or RSE are known as agonists of TLR-4. Flagellin, the protein subunit of bacterial flagella, acts as TLR-5 agonist. TLR-6 forms a heterodimer with TLR-2 and is bound by diacylated lipopeptides like MALP-2 (macrophage-activating lipopeptide-2), derived from the cell wall of mycoplasma species. Known agonists of TLR-7 include single-stranded RNA(ssRNA) molecules, found in viral and bacterial pathogens, and the synthetic analogues imiquimod and gardiquimod. TLR-8 agonists include singlestranded RNA molecules and imidazoquinoline and thiazoquinoline derivatives. The synthetic compound resiquimod is known to be a TLR-8 agonist. TLR-9 agonists include unmethylated CpG motifs, commonly found in bacterial DNA, and CpG oligodeoxynucleotides are synthetic oligonucleotides that mimic these motifs. In one embodiment, the pyrogen is an agonist of TLR-1, TLR-1 / TLR-2, TLR-2, TLR-2 / TLR-6, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8 and TLR-9. In one embodiment, the pyrogen is an agonist of TLR-1 / TLR-2, TLR-2 / TLR-6, TLR-3, TLR-5, TLR-6, TLR-7, TLR-8 and TLR-9. In one embodiment, the pyrogen is a non-endotoxin pyrogen and an agonist of TLR-1 / TLR-2, TLR-2 / TLR-6, TLR-5 and TLR-9. In one embodiment, the pyrogen is an endotoxin and an agonist of TLR-2 / TLR-4 and TLR-4. In one embodiment, the pyrogen is a non-endotoxin pyrogen and an agonist of TLR-1. In one embodiment, the pyrogen is a non-endotoxin pyrogen and an agonist of TLR-2. In one embodiment, the pyrogen is a non-endotoxin pyrogen and an agonist of TLR-1 / TLR-2. In one embodiment, the pyrogen is a nonendotoxin pyrogen and an agonist of TLR-2 / TLR-6. In one embodiment, the pyrogen is a non-endotoxin pyrogenand an agonist of TLR-3. In one embodiment, the pyrogen is an endotoxin and an agonist of TLR-2 / TLR-4. In one embodiment, the pyrogen is an endotoxin and an agonist of TLR-4. In one embodiment, the pyrogen is a nonendotoxin pyrogen and an agonist of TLR-5. In one embodiment, the pyrogen is a non-endotoxin pyrogen and an agonist of TLR-6. In one embodiment, the pyrogen is a non-endotoxin pyrogen and an agonist of TLR-7. In one embodiment, the pyrogen is a non-endotoxin pyrogen and an agonist of TLR-8. In one embodiment, the pyrogen is a non-endotoxin pyrogen and an agonist of TLR-9.

[0062] In one embodiment, the pyrogen is an endotoxin binding to a TLR, preferably the pyrogen is LPS binding to TLR4. In one embodiment, the pyrogen is a non-endotoxin pyrogen (NEP) binding to TLR, preferably the pyrogen is a triacetylated lipopeptide such as Pam3CSK4 binding to TLR-1.

[0063] Within the context of the present invention, mycoplasma species and mycoplasma-derived components are likewise encompassed as TLR agonists. “Mycoplasma species” are cell wall-deficient microorganisms lacking classical endotoxin structures such as LPS, lipoteichoic acid, or murein fragments, but expressing membrane-associated lipoproteins and lipopeptides. As used herein, the term “mycoplasma-derived components” refers to structural or molecular constituents originating from mycoplasma cells, including, but not limited to, membrane fractions, membrane-associated lipoproteins, acylated lipopeptides, lipid-modified proteins, and other surface-exposed molecules capable of immune recognition. An exemplary mycoplasma-derived component is macrophageactivating lipopeptide-2 (MALP-2), which functions as agonists of the TLR-2 / TLR-6 heterodimer and thereby activates downstream signaling pathways, resulting in the production of pro-inflammatory cytokines such as TNF-a, IL-1 p, and IL-6.

[0064] As used herein, the term “detection of mycoplasms or mycoplasma-derived components” refers to the detection of intact mycoplasma organisms and / or mycoplasma-derived components that are capable of acting as TLR agonists by specifically binding to TLRs and activating downstream signaling pathways. The mycoplasms or mycoplasma-derived components to be detected may be present in a pharmaceutical product or composition, a product for cell culture or laboratory use, a cosmetic or personal care product or a parenteral nutrition solution or on a surface of a medical device.

[0065] The binding of TLR agonists to TLRs can be detected using several assays known to the skilled person, including, but not limited to, cell-based reporter assays comprising a TLR-responsive promoter, co-immunoprecipitation (CoIP), Western Blot assays and fluorescence-based binding assays

[0066] Accessory proteins involved in TLR-signaling are molecules that assist TLRs in recognizing ligands, enhancing ligand binding, or stabilizing the receptor-ligand interaction. Said accessory proteins do not necessarily signal directly but contribute to optimal TLR activation and downstream signaling. Said accessory proteins are particularly useful for the detection of endotoxins such as LPS. Examples for accessory proteins involved in TLR-signaling include, but are not limited to, CD14 (Cluster of Differentiation 14), MD-2 (Myeloid Differentiation Factor 2), LBP (LPS-Binding Protein), CD16, CD36 and TRIL (TLR-4 interactor with leucine-rich repeats). Preferably, the accessory protein involved in TLR-signaling used in the present invention is CD14, MD-2 and / or LBP. CD14 exists in soluble and membrane-bound forms which can both bind to microbial components such as LPS, forming a CD14-LPS complex, thereby enhancing the sensitivity of TLR-4 to LPS (Tobias et al. J Biol Chem. (1995) 270(18): 10482-8). MD-2 directly binds to LPS and forms a complex with TLR-4. LBP binds LPS in circulation, forms an LBP-LPS complex, and transfers LPS to CD14 and subsequently to the TLR-4 / MD-2 complex.

[0067] Also preferably, the accessory protein involved in TLR-signaling used in the present invention is CD14 and / or CD36. In a more preferred embodiment, the accessory proteins involved in TLR-signaling used in the present invention are CD14 and CD36. CD36 functions as a co-receptor primarily in TLR-2 / TLR-6 signaling pathways by facilitating the recognition and internalization of diacylated lipopeptides and other microbial components (Lee et al. Nat Rev Immunol. (2012) 12(3): 168-179).

[0068] Upon binding of a pyrogen ligand or a TLR agonist, including mycoplasms or a mycoplasma-derived component acting as a TLR agonist, to a TLR (e.g. binding of LPS to TLR-4, binding of LTAto TLR-2, or binding of flagellin to TLR-5), several signaling pathways are initiated, ultimately leading to the activation of transcription factors such as Nuclear Factor kappa B (NF-KB), Interferon regulatory factors (IRFs), AP-1 (Activator Protein 1) and STAT (Signal Transducer and Activator of Transcription) and the transcription of the genes controlled by these transcription factors. Downstream signaling of TLRs primarily activates MyD88 (Myeloid differentiation factor 88), leading to the activation of NF-KB and further culminates in a pro-inflammatory response. The MyD88-dependent signaling pathway can further activate MAPKs (Mitogen-Activated Protein Kinases), such as JNK, ERK, and p38 MAPK, which then phosphorylate and activate AP-1 components, involved in the regulation of pro-inflammatory cytokines like TNF-a, IL-1 p, and IL-6 (Ahmad et al. Cell Physiol Biochem. (2014) 34(3):929-42). Downstream signaling of TLRs can further activate JAKs (Janus Kinases), which phosphorylate STATs (especially STAT1, STAT3, andSTAT6), leading to their dimerization and nuclear translocation (Ji et al. Cell Biosci (2023) 13:230). In particular downstream of TLR-3 and TLR-4, a TRIF-dependent pathway activates IRF3 / 7 (Ullah et al. J Leukoc Biol. (2016) 100(1 ):27-45). Hence, promoters activated in response to TLR binding are designated as “TLR-responsive promoters”.

[0069] Negative regulators serve to decrease or halt the activation of specific pathways, thus preventing overactivation or unregulated signaling. TLR signaling is tightly controlled by negative regulators of TLR-signaling to prevent excessive inflammation and cell damage. Negative regulators of TLR-signaling include, but are not limited to, SOCS1, PP2ACA, PP1 and the catalytic subunits PP1A, PP1B and PP1G thereof, PPM1D, PP2B and the catalytic subunits PP2BA, PP2BB or PP2BC thereof, USP-4, TBK-1, PP5C, PPM1G, PPM1N, A20, SOCS3, and IRAK-M.

[0070] The suppressor of cytokine signaling (SOCS) proteins, e.g. SOCS1 and SOCS3, are an integral component of a negative feedback loop to dampen TLR responses. SOCS1 acts by inducing the ubiquitination and proteasomal degradation of key TLR associated proteins (Mansell et al. Nat. Immunol (2006) 7(2): 148-55 and Kinjyo et al. Immunity (2002) 17(5):583-91 ).

[0071] The serine / threonine phosphatase PP2ACA has a well-established negative regulatory role in TLR-mediated NF-KB activation (Shanley et al. J. Immunol. (2001) 166(2):966-72 and Sun et al. J. Immunol. (2017) 198(1 ):404-416). The de-ubiquitinating enzyme A20 plays a crucial role in inhibiting NF-KB activation (Pujari et al. Immunol Res. (2013) 57(1-3): 166-71 ).

[0072] IRAK-M is another negative regulator of the TLR / IL-1 receptor (IL-1 R) signaling pathway (Kobayashi et al. Cell (2002) 110(2): 191 -202).

[0073] Ubiquitin-specific protease 4 (USP-4) is a deubiquitinating enzyme that modulates multiple signaling pathways, including the NF-KB signaling pathway, by stabilizing key regulatory proteins and controlling their ubiquitination status, thereby contributing to the negative regulation of excessive pathway activation (Hu et al. Front Cell Dev Biol. (2021) 18;9:595159).

[0074] In addition, the Ca2+ / calmodulin-dependent serine / threonine phosphatase PP2B, also known as calcineurin, can play an indirect but significant role in the negative regulation of TLR signaling. In vertebrates, three genes (PPP3CA, PPP3CB, and PPP3CC) encode the three catalytic subunits PP2BA, PP2BB or PP2BC. Calcineurin is activated by calcium and calmodulin and can dephosphorylate key signaling molecules in the TLR signaling pathway, thereby providing a braking mechanism to prevent overactivation.

[0075] Protein phosphatase-1 (PP1) is a ubiquitously expressed protein serine / threonine phosphatase and has been reported to play both the roles of a negative and a positive regulator in TLR-mediated innate immune responses. In humans, three catalytic subunits PP1A, PP1B and PP1G are encoded (Seumen et al., Commun Signal (2021); 19(1 ):45).

[0076] TANK-binding kinase 1 (TBK-1 ) is an important serine / threonine-protein kinase that mediates phosphorylation and is involved in innate immune responses (Seumen et al., Commun Signal (2021); 19(1 ):45, Ooi et al. Proc Natl Acad Sci USA(2014) 4;111(5): 1909-14).

[0077] Protein phosphatase 5 (PP5), also called PP5C, is a ubiquitously expressed serine / threonine phosphatase that modulates multiple signaling pathways, including stress and MAPK pathways, by dephosphorylating key regulatory proteins (Hinds et al. Int J Biochem Cell Biol. (2008) 40(11 ):2358-62).

[0078] Members of the metal-dependent protein phosphatase (PPM) family participate in diverse cellular processes, including cell proliferation, growth, survival, apoptosis, metabolism, and stress signaling. Emerging evidence also indicates that PPM phosphatases can modulate TLR-initiated signaling cascades. In particular, the wild-type p53-induced phosphatase 1 (Wip1, encoded by PPM1D) has been shown to contribute to negative feedback regulation of TLR-induced NF-KB signaling and PPM1N has been suggested to play a role in the immune response to bacterial stimuli or in the regulation of inflammation (Seumen et al., Commun Signal (2021); 19(1 ):45). Additionally, PPM1G has been reported to restrict innate immune signaling (Yu et al., Sci Adv (2020); 6(47)).

[0079] GENETICALLY MODIFIED CELL

[0080] The term “genetically modified cell” refers to a cell whose genetic material has been altered in a way that does not occur naturally through processes like mutation, recombination, or natural selection, and therefore differs from a cell which is not genetically modified. The term “genetic modification” relates to a modification that alters expression of the genethat is targeted or the functional activity of the gene product. Within the present invention, the geneticallymodified cell has been modified to reduce the expression of at least two negative regulators of Toll-like receptor (TLR)-signaling. In one embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding a negative regulator of Toll-like receptor (TLR)-signaling.

[0081] In one embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding SOCS1. In one embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding SOCS1 according to SEQ ID NO: 8.

[0082] In one embodiment, the genetically modified cell has been modified to reduce the expression of PP2ACA. In one embodiment, the genetically modified cell has been modified to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0083] In one embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding SOCS1 and to reduce the expression of PP2ACA. In one embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding SOCS1 according to SEQ ID NO: 8 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0084] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding USP-4. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding USP-4 according to SEQ ID NO: 15. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding USP-4 and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding USP-4 according to SEQ ID NO: 15 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0085] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding TBK-1. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding TBK-1 according to SEQ ID NO: 16. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding TBK-1 and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding TBK-1 according to SEQ ID NO: 16 and to reduce the expression of PP2ACAencoded by SEQ ID NO: 9.

[0086] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1 ora catalytic subunit thereof. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1 or a catalytic subunit thereof and to reduce the expression of PP2ACA.

[0087] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1A. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1 A according to SEQ ID NO: 34. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1A and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1 A according to SEQ ID NO: 34 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0088] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1B. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1B according to SEQ ID NO: 32. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1B and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1 B according to SEQ ID NO: 32 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0089] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1G. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1G according to SEQ ID NO: 17. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1G and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1G according to SEQ ID NO: 17 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2B or a catalytic subunit thereof. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2B or a catalytic subunit thereof and to reduce the expression of PP2ACA.

[0090] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BA according to SEQ ID NO: 18. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BA and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BA according to SEQ ID NO: 18 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0091] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BB. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BB according to SEQ ID NO: 39. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BB and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BB according to SEQ ID NO: 39 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0092] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BC. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BC according to SEQ ID NO: 37. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BC and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP2BC according to SEQ ID NO: 37 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0093] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP5C. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP5C according to SEQ ID NO: 19. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP5C and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP5C according to SEQ ID NO: 19 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0094] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1D. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1D according to SEQ ID NO: 20. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1D and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1D according to SEQ ID NO: 20 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0095] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1G. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1G according to SEQ ID NO: 21. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1G and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1G according to SEQ ID NO: 21 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.

[0096] In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1N. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1N according to SEQ ID NO: 22. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1N and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1N according to SEQ ID NO: 22 and to reduce the expression of PP2ACA encoded by SEQ ID NO: 9.In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding at least one negative regulator selected from the group consisting of SOCS1, PP1G and PPM1D and to reduce the expression of PP2ACA. In another embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 8, 17 and 20 and to reduce the expression of the nucleic acid sequence according to SEQ ID NO: 9.

[0097] In a preferred embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding SOCS1 and to reduce the expression of PP2ACA. In another preferred embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PP1G and to reduce the expression of PP2ACA. In another preferred embodiment, the genetically modified cell has been modified by disruption of a nucleic acid sequence encoding PPM1D and to reduce the expression of PP2ACA.

[0098] There are multiple techniques to genetically modify cells to reduce the expression of one or more proteins, each targeting different steps of the gene expression process. Said techniques include, but are not limited to, RNA interference (RNAi), CRISPR-Cas, CRISPR interference (CRISPRi), transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), antisense oligonucleotides (ASOs), use of translational repressors and the use of proteasomal degradation techniques (e.g. degron-tagging system), wherein CRISPR-Cas, TALENs and ZNFs can be specifically used for the disruption and / or deletion of nucleic acid sequences.

[0099] The genetically modified cell of the present invention may be a genetically modified mammalian cell, preferably a genetically modified human cell, more preferably a genetically modified human immune cell and most preferably a genetically modified monocyte cell. Human immune cells include, but are not limited to, macrophages, dendritic cells, granulocytes, T cells, B cells, NK cells, lymphocytes, myeloid cells and virally or chemically transformed cells. Human immune cells can be immortalized or non-immortalized cells. Immortalized cells can be cells of an immortalized human cell line. Preferably, the immortalized human cell line is a monocytic human cell line. Immortalized monocytic human cell lines include, but are not limited to, THP-1 cells, U937 cells, Mono Mac 6 (MM6) cells, HL-60 cells and MDM (Monocyte-Derived Macrophages) cells. The THP-1 cell line is a suspension cell line isolated from peripheral blood of an acute monocytic leukemia patient and, depending on culture conditions, these cells can differentiate into macrophages and dendritic cells. THP-1 cells are widely used for MAT assays due to their monocytic origin, TLR responsiveness, ability to produce cytokines and consistency in immune responses. Cell lines as described herein are available from suppliers such as ATCC. In one embodiment, the genetically modified cell of the present invention is a cell of an immortalized human cell line. In one embodiment, the genetically modified cell of the present invention is a cell of a monocytic human cell line. In a preferred embodiment, the genetically modified cell of the present invention is a THP-1 cell.

[0100] CRISPR CAS

[0101] In one embodiment, a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas (CRISPR-associated) method is used for the disruption of a nucleic acid sequence in a cell. The use of this technology in genome editing is well described in the art, for example in Adli et al. (Nat Commun (2018) 9(1):1911). In short, CRISPR is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes encoding an endonuclease as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. There are different CRISPR-Cas systems available, depending on the Cas endonuclease used (e.g. Cas9, Cas12 or Cas13). The terms “guide RNA” or “sense strand of the guide RNA” or“gRNA” are used interchangeably herein and relate to a polynucleotide sequence that can form a complex with a Cas endonuclease and enables the Cas endonuclease to recognize and optionally cleave a DNA target site. The guide RNA comprises a nucleic acid sequence complementary to the nucleic acid sequence encoding the target site. The term “target site” refers to a nucleic acid sequence in the genome of a cell at which a double-strand break is induced in the cell’s genome by a Cas endonuclease. The “target site” comprises the nucleic acid sequence to be disrupted.

[0102] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence in the genetically modified cell of the invention. In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence leading to a reduced expression of the gene product encoded by the nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding a negative regulator of TLR-signaling.

[0103] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding SOCS1 or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%,at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8.

[0104] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding USP-4 or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 15 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 15.

[0105] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding TBK-1 or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 16 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 16.

[0106] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PP1A or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 34 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 34.

[0107] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PP1B or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 32 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 32.

[0108] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PP1G or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17.

[0109] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PP2BA or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 18 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 18.

[0110] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PP2BB or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 39 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 39.

[0111] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PP2BC or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 37 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 37.

[0112] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PP5C or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 19 or a nucleic acid sequence being at least 90%,at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 19.

[0113] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PPM1D or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20.

[0114] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PPM1G or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 21 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 21.

[0115] In one embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence encoding PPM1N or a fragment of said nucleic acid sequence. In a preferred embodiment, the CRISPR-Cas9 method is used for the disruption of a nucleic acid sequence according to SEQ ID NO: 22 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 22.

[0116] In one embodiment, the CRISPR-Cas9 method used in the present invention comprises introducing and coexpressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding a negative regulator of TLR-signaling.

[0117] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding SOCS1 or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1.

[0118] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding USP-4 or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 23 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 23. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 23 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 23.

[0119] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding TBK-1 or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 24 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 24. In one embodiment, the genetically modified cell of the presentinvention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 24 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 24.

[0120] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PP1 A or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 35 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 35. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 35 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 35.

[0121] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PP1 A or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 36 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 36. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 36 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 36.

[0122] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PP1 B or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 33 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 33. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 33 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 33.

[0123] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PP1G or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25.

[0124] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PP2BA or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 26 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 26. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 26 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 26.

[0125] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PP2BB or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 40 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 40. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 40 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 40.

[0126] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PP2BC or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 38 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 38. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 38 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 38.

[0127] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PP5C or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 27 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 27. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 27 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 27.

[0128] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PPM1D or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28.In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PPM1G or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 29 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 29. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 29 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 29.

[0129] In one embodiment, the CRISPR-Cas9 method comprises introducing and co-expressing in a cell Cas9 and a gRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PPM1N or a fragment of said nucleic acid sequence. In one embodiment, the CRISPR-Cas9 method comprises introducing and coexpressing in a cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 30 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 30. In one embodiment, the genetically modified cell of the present invention is produced by the CRISPR-Cas9 method which comprises introducing and co-expressing in the cell Cas9 and the gRNA having the nucleic acid sequence according to SEQ ID NO: 30 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 30.

[0130] In one embodiment, Cas9 and a gRNA are introduced into a cell by any method known in the art, including, but not limited thereto, lentiviral transduction, adenoviral or AAV vectors, electroporation, lipofection, microinjection, nanoparticle-based delivery, and / or topical application. In one embodiment, the Cas9 and a gRNA are introduced into a cell by lentiviral transduction. Lentiviral transfer vectors can be constructed as described in the art.

[0131] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of SOCS1 protein expression. The term “complete loss of SOCS1 protein expression” means that no signal is obtained when a SOCS1 -specific antibody is used to detect SOCS1 expression, for example in a Western blot analysis.

[0132] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of USP-4 protein expression. The term “complete loss of USP-4 protein expression” means that no signal is obtained when a USP-4-specific antibody is used to detect USP-4 expression, for example in a Western blot analysis.

[0133] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of TBK-1 protein expression. The term “complete loss of TBK-1 protein expression” means that no signal is obtained when a TBK-1-specific antibody is used to detect TBK-1 expression, for example in a Western blot analysis.

[0134] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PP1 protein expression or expression of a catalytic subunit thereof. The term “complete loss of PP1 protein expression or expression of a catalytic subunit thereof means that no signal is obtained when a PP1-specific or catalytic subunit-specific antibody is used to detect PP1 expression or expression of a catalytic subunit thereof, for example in a Western blot analysis.

[0135] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PP1 A protein expression. The term “complete loss of PP1 A protein expression” means that no signal is obtained when a PP1A-specific antibody is used to detect PP1A expression, for example in a Western blot analysis.

[0136] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PP1 B protein expression. The term “complete loss of PP1 B protein expression” means that no signal is obtained when a PP1B-specific antibody is used to detect PP1B expression, for example in a Western blot analysis.In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PP1G protein expression. The term “complete loss of PP1G protein expression” means that no signal is obtained when a PP1G-specific antibody is used to detect PP1G expression, for example in a Western blot analysis.

[0137] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PP2B protein expression or expression of a catalytic subunit thereof. The term “complete loss of PP2B protein expression or expression of a catalytic subunit thereof” means that no signal is obtained when a PP2B-specific or catalytic subunit-specific antibody is used to detect PP2B expression or expression of a catalytic subunit thereof, for example in a Western blot analysis.

[0138] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PP2BA protein expression. The term “complete loss of PP2BA protein expression” means that no signal is obtained when a PP2BA-specific antibody is used to detect PP2BA expression, for example in a Western blot analysis.

[0139] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PP2BB protein expression. The term “complete loss of PP2BB protein expression” means that no signal is obtained when a PP2BB-specific antibody is used to detect PP2BB expression, for example in a Western blot analysis.

[0140] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PP2BC protein expression. The term “complete loss of PP2BC protein expression” means that no signal is obtained when a PP2BC-specific antibody is used to detect PP2BC expression, for example in a Western blot analysis.

[0141] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PP5C protein expression. The term “complete loss of PP5C protein expression” means that no signal is obtained when a PP5C-specific antibody is used to detect PP5C expression, for example in a Western blot analysis.

[0142] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PPM1D protein expression. The term “complete loss of PPM1D protein expression” means that no signal is obtained when a PPM ID-specific antibody is used to detect PPM 1D expression, for example in a Western blot analysis.

[0143] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PPM1G protein expression. The term “complete loss of PPM1G protein expression” means that no signal is obtained when a PPM1G-specific antibody is used to detect PPM1G expression, for example in a Western blot analysis.

[0144] In one embodiment, the CRISPR-Cas9 method used to produce the genetically modified cell of the present invention leads to the complete loss of PPM1N protein expression. The term “complete loss of PPM1N protein expression” means that no signal is obtained when a PPM1N-specific antibody is used to detect PPM1N expression, for example in a Western blot analysis.

[0145] RNAi

[0146] In one embodiment, RNAi is used for the reduction of the protein expression in a cell. The use of this technology is well described in the art, for example in Isenmann et al. (Micromachines (2023) 14(7): 1321 ). In short, RNAi refers to the process of sequence-specific post-transcriptional gene silencing in animals mediated by short interfering RNAs (siRNAs). RNAi is triggered by the presence of double stranded RNA(dsRNA) or short hairpin RNA(shRNA), which is introduced into a cell. The term “shRNA” refers to a single-stranded RNA molecule that forms a hairpin loop. Both dsRNA and shRNA molecules comprise a nucleic acid sequence complementary to the nucleic acid sequence encoding the target mRNA. The term “target mRNA” refers to a nucleic acid sequence corresponding to the gene of interest whose expression is intended to be reduced.

[0147] In one embodiment, the RNAi method is used to reduce the expression of a negative regulator of TLR-signaling encoded by the target mRNA. In one embodiment, the target mRNA comprises a nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence. In a preferred embodiment, the target mRNA has the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9.In one embodiment, the RNAi method comprises introducing into a cell a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding a negative regulator of TLR-signaling. In one embodiment, the RNAi method comprises introducing into a cell a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding PP2ACA protein or a fragment of said nucleic acid sequence. In one embodiment, the RNAi method comprises introducing into a cell the shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2. In one embodiment, the genetically modified cell of the invention is obtained by the RNAi method comprising introducing into a cell the shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2.

[0148] In one embodiment, a shRNA is introduced into a cell by any method known in the art, including, but not limited to, lentiviral transduction, adenoviral or AAV vectors, electroporation, lipofection, microinjection, nanoparticle-based delivery and / or topical application. In one embodiment, a shRNA is introduced into a cell by lentiviral transduction. Lentiviral transfer vectors can be constructed as described in the art.

[0149] FURTHER GENETIC MODIFICATIONS

[0150] In one embodiment, the genetically modified cell of the present invention further comprises an increased expression of at least one accessory protein involved in TLR-signaling. In one embodiment, the genetically modified cell of the present invention further comprises an increased expression of at least one accessory protein involved in TLR-signaling selected from the group consisting of CD14 or a variant thereof, MD-2 or a variant thereof LBP or a variant thereof and CD36 or a variant thereof. Variants of CD14, MD-2, LBP or CD36 can comprise truncations or mutations in their amino acid sequences but still retain functional activity. A variant of CD14 can comprise a truncation in the amino acid sequence at the amino acid 152 (see Juan et al. J Biol Chem. (1995) 270(3): 1382-7). Variants of LBP are described in Meng et al. Front Immunol. (2021) 12:681810 and Zeng et al. Ann Surg. (2012) 255(1):147-57). In one embodiment, the genetically modified cell of the invention comprises an increased expression of CD14 or a variant thereof compared to a cell which is not genetically modified. In a preferred embodiment, the CD14 or a variant thereof is encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5.

[0151] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, PP1G protein and / or PPM1D protein, a reduced expression of PP2ACA protein and an increased expression of CD14 or a variant thereof.

[0152] In one embodiment, the genetically modified cell of the invention comprises an increased expression of MD-2 or a variant thereof compared to a cell which is not genetically modified. In a preferred embodiment, the MD-2 or a variant thereof is encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6.

[0153] In one embodiment, the genetically modified cell of the invention comprises an increased expression of LBP or a variant thereof compared to a cell which is not genetically modified. In a preferred embodiment, the LBP or a variant thereof is encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7.

[0154] In one embodiment, the genetically modified cell of the invention comprises an increased expression of CD36 or a variant thereof compared to a cell which is not genetically modified. In a preferred embodiment, the CD36 or a variant thereof is encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31.In one embodiment, the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, PP1G protein and / or PPM1D protein, a reduced expression of PP2ACA protein and an increased expression of CD36 or a variant thereof.

[0155] In one embodiment, the genetically modified cell of the invention comprises an increased expression of CD36 or a variant thereof and CD14 or a variant thereof compared to a cell which is not genetically modified. In a preferred embodiment, the CD36 or a variant thereof is encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and the CD14 or a variant thereof is encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5.

[0156] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, PP1G protein and / or PPM1D protein, a reduced expression of PP2ACA protein, an increased expression of CD36 or a variant thereof and an increased expression of CD14 or a variant thereof.

[0157] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0158] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0159] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0160] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0161] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0162] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0163] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.In one embodiment, the genetically modified cell of the invention comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0164] In one embodiment, the genetically modified cell of the invention comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0165] REPORTER SYSTEM

[0166] The genetically modified cell of the present invention comprises at least one reporter gene operably linked to a TLR-responsive promoter. In one embodiment, the reporter gene is a gene encoding a detectable gene product which is expressed in the genetically modified cell of the invention in response to the induction of TLR signaling. In one embodiment, the reporter gene is a gene encoding a detectable gene product which is expressed in the genetically modified cell of the invention in response to pyrogens which bind to a TLR and activate intracellular signal transduction pathways leading to the activation of a TLR-responsive promoter and consequently to the expression of the detectable gene product. In another embodiment, the reporter gene is a gene encoding a detectable gene product which is expressed in the genetically modified cell of the invention in response to mycoplasma or a mycoplasma-derived component or molecule acting as a TLR agonist which binds to a TLR and activates intracellular signal transduction pathways leading to the activation of a TLR-responsive promoter and consequently to the expression of the detectable gene product.

[0167] The reporter gene is operably linked to a TLR-responsive promoter, i.e. the reporter protein encoded by the reporter gene is expressed under the control of a TLR-responsive promoter. The genetically modified cell of the present invention may also comprise two independent reporter genes, wherein each reporter gene is operably linked to a different TLR-responsive promoter, leading to two independently regulated reporter proteins.

[0168] The reporter gene is used to identify the activity of the TLR signal transduction pathway in the genetically modified cell of the invention and to determine the effects of stimuli on the activity of said TLR signal transduction pathway. Reporter proteins encoded by the reporter gene include, but are not limited to fluorescent reporter proteins, enzymatic reporter proteins, chemiluminescent reporter proteins and bioluminescent reporter proteins. Fluorescent reporter proteins include, but are not limited to, a monomeric cyan fluorescent protein (mCFP), a green fluorescent protein (GFP), a red fluorescent protein (RFP) and a yellow fluorescent protein (YFP). In one embodiment, the reporter protein is a fluorescent reporter protein. In one embodiment, the fluorescent reporter protein is a monomeric cyan fluorescent protein (mCFP). In a preferred embodiment, the fluorescent reporter protein is a monomeric cyan fluorescent protein (mCFP) encoded by the nucleic acid sequence according to SEQ ID NO: 3 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 3.

[0169] Enzymatic reporter proteins include, but are not limited to, luciferase, horseradish peroxidase (HRP), p-galactosidase, alkaline phosphatase and glucuronidase. A chemiluminescent reporter protein includes, but is not limited to, luciferase. A bioluminescent reporter protein includes, but is not limited to, aequorin.

[0170] The detection of the reporter protein can be quantitative (e.g. measurement of fluorescence intensity or enzyme activity) or qualitative (e.g. presence / absence of a color change or luminescence) and depends on the type of reporter gene used. Hence, the expression of the reporter gene can be detected by fluorescence, luminescence, enzyme activity or colorimetric changes. Methods to measure the expression of the reporter gene include, but are not limited to, fluorescence microscopy, flow cytometry, plate reader assays and immunoblotting (e.g. western blot). In one embodiment, the reporter gene encodes a reporter protein and expression of the reporter gene is detected by measuring fluorescence emission at a specific wavelength.

[0171] A "promoter" is a region of DNAthat binds RNA polymerase before initiating the transcription of DNA into RNA. It directs RNA polymerase to bind to DNA, to open the DNA helix, and to begin RNA synthesis. The promoter sequence comprises a transcription initiation site as well as RNA polymerase binding domains. Eukaryotic promoters usually contain "TATA" boxes and "CAT" boxes. Some promoters are "constitutive" and initiate transcription in the absence of regulatory influences. Some initiate transcription exclusively or selectively in one or a few tissue types. Some promoters are "inducible" and initiate gene transcription under the influence of an inducer. Induction can occur, for example, as the result of a physiological response, a response to outside signals, or artificial manipulation.

[0172] Within the present invention, the induction of the promoter within the reporter system results from TLR-signaling which leads to the activation of transcription factors such as NF-KB, IRFS, AP-1 or STATs.

[0173] NF-KB is a family of dimeric transcription factors that regulate gene expression involved in immune and inflammatory responses. In one embodiment the TLR-responsive promoter is recognized by NF-KB transcription factors and comprises kappa binding sites. Kappa binding sites are specific DNA sequences recognized by NF-KB transcription factors and comprise a consensus DNA sequence of 5-GGGRNYYYCC-3' (in which R is a purine, Y is a pyrimidine, and N is any nucleotide) (Wan et al. Cold Spring Harb Perspect Biol. (2009)1(4): a000067). Disjunct kappa binding sites are two or more kappa binding sites which are located apart from each other on the DNA and which are separated by nucleic acid sequences which do not comprise kappa binding sites. Following activation of TLR signaling, kappa binding sites are recognized and bound by NF-KB proteins, which enhance transcription. In one embodiment, the TLR-responsive promoter comprises at least two copies of kappa binding sites. In one embodiment, the TLR-responsive promoter comprises at least 2 to 14, at least 4 to 12, at least 5 to 11, at 6 to 10 or at least 7 to 9 copies of a kappa binding site. In a preferred embodiment, the TLR-responsive promoter comprises 8 copies of a kappa binding site. In one embodiment, the kappa binding site has the nucleic acid sequence according to SEQ ID NO: 4 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 4. In a preferred embodiment, the TLR-responsive promoter comprises 8 kappa binding sites, each kappa binding site having the nucleic acid sequence according to SEQ ID NO: 4 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 4. In one embodiment, the TLR-responsive promoter has the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0174] In one embodiment, the reporter gene comprises a nucleic acid sequence encoding a fluorescent protein which is operably linked to an NF-KB-dependent promoter. In one embodiment, the reporter gene comprises a nucleic acid sequence encoding a monomeric cyan fluorescent protein (mCFP) which is operably linked to a NF-KB-dependent promoter comprising 8 copies of a kappa binding site. In one embodiment, the reporter gene comprises the nucleic acid sequence according to SEQ ID NO: 3 or a nucleic acid sequence being at least 90% identical to the nucleic acid sequence according to SEQ ID NO: 3 which is operably linked to the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10. In one embodiment, the reporter gene operably linked to a TLR-responsive promoter has the nucleic acid sequence according to SEQ ID NO: 11 or a nucleic acidsequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 11.

[0175] Binding of a pyrogen or mycoplasma or a mycoplasma-derived component or molecule acting as a TLR agonist to a TLR also leads to the activation of Interferon regulatory factors (IRFs) being a family of transcription factors playing a key role in linking TLR activation to the production of type I interferons and other cytokines. Downstream of TLR signaling, IRFs such as IRF3 or IRF7 are phosphorylated and further dimerize and translocate to the nucleus, where they bind to interferon-stimulated response elements (ISREs) in the promoters of type I interferon genes (e.g. IFN-p) and other inflammatory genes, initiating their transcription. The ISRE has the consensus sequence GAAANNGAAAG / CT / C, where N denotes any nucleotide (Honda, K et al. Nat Rev Immunol (2006) 6(9):644-58). Hence, in one embodiment the TLR-responsive promoter comprises at least one ISRE.

[0176] The AP-1 binding site to which AP-1 transcription factors bind has the nucleic acid sequence ATGAGTCAT. Hence, in one embodiment the TLR-responsive promoter comprises at least one AP-1 binding site. The STAT binding site to which STAT transcription factors bind has the nucleic acid sequence TTCN3-4GAA. Hence, in one embodiment the TLR-responsive promoter comprises at least one STAT binding site.

[0177] CELL CULTURE

[0178] Culturing cells as used herein means that said cells are contained in a cell culture medium under conditions suitable for maintaining viability and supporting growth of said cells. Said conditions include maintaining cells at a temperature of 36°C to 37°C and a pH of 7.4, optionally adding exogenous CO2 at a concentration of 5% to 7%. The skilled person will be aware of suitable cell culture media for supporting the growth of cells, preferably cells of an immortalized human cell line, more preferably cells of a monocytic immortalized human cell line, most preferably of THP-1 cells.

[0179] The terms "medium", "cell culture medium" and "culture medium" are used interchangeably herein. Cell culture media can be in liquid or solid form, including gelatinous media such as agar, agarose, gelatin and collagen matrices. Preferably, the cell culture medium is in liquid form. Most media generally are composed of four basic chemical groups: amino acids, carbohydrates, inorganic salts, and vitamins. This serves as the basis for a more complex medium, to which supplements such as serum, buffers, growth factors, lipids, and the like are added. Antibiotics such as gentamicin, penicillin and streptomycin can also be included in the cell culture medium to suppress the growth of bacteria in the culture.

[0180] Examples of suitable cell culture media include, but are not limited to, Eagle’s Basal Medium, Minimum Essential Medium, Dulbecco’s Modified Eagle’s Medium (DMEM), Medium 199, Nutrient Mixtures Ham’s F-10 and Ham’s F-12, McCoy’s 5A, Dulbecco’s MEM / F12, alpha modified Minimal Essential Medium (alphaMEM), Roswell Park Memorial Institute Media 1640 (RPM1 1640) and Iscove’s Modified Dulbecco’s Medium (IMDM). In one embodiment, the cell culture medium is RPMI 1640. In one embodiment, the genetically modified cells are THP-1 cells, and the cell culture medium is RPMI 1640. In one embodiment, the cell culture medium is IMDM. In one embodiment, the genetically modified cells are THP-1 cells, and the cell culture medium is IMDM. In one embodiment, the cell culture medium is DMEM. In one embodiment, the genetically modified cells are THP-1 cells, and the cell culture medium is DMEM.

[0181] Conventionally, cells are maintained in cell culture using media comprising at least about 0.25% to 15% (v / v) serum, such as fetal bovine serum (FBS), fetal calf serum (FCS), adult bovine serum (ABS), human serum and serum replacements.

[0182] In the context of cell culture, the term "maintaining" refers to the ongoing care and management of cultured cells to ensure their viability, functionality, and proper growth conditions over time. The duration for which a cell culture can be maintained depends on several factors, including cell type, culture conditions, and passage number. Cell lines can be maintained indefinitely if properly passaged. In one embodiment, the genetically modified cell according to the invention can be maintained in a suitable cell culture medium. In one embodiment, the suitable cell culture medium comprises FCS in a concentration from about 5% (v / v) to about 15% (v / v), about 6% (v / v) to about 14% (v / v), about 7% (v / v) to about 13% (v / v), about 8% (v / v) to about 12% (v / v), about 9% (v / v) to about 11 % (v / v), more preferably, the FCS concentration is 10% (v / v). In one embodiment, the cell culture medium is RPMI 1640 comprising 10% (v / v) FCS. In one embodiment, the genetically modified cells according to the invention are maintained in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS.

[0183] In the context of cell culture, the term “starvation” refers to the deliberate reduction or removal of essential nutrients such as serum, glucose, amino acids, or growth factors from the culture medium for a specific period of time. Serumstarvation means reducing or completely removing serum (e.g. FCS) from the culture medium and cell culture medium comprising a reduced amount of serum or comprising no serum is referred to as “serum starved cell culture medium”. The genetically modified cells of the invention can be kept in a serum starved cell culture medium for a period of time, e.g. from about 0.5 hours to about 3 hours, about 1 hour to about 2.5 hours or about 1.5 hours to about 2 hours. In a preferred embodiment, the genetically modified cells according to the invention are kept in a serum starved cell culture medium for about 1 hour. In one embodiment, the serum starved cell culture medium can comprise FCS in a concentration from about 0.25% (v / v) to about 1.5% (v / v) or from about 0.3% (v / v) to about 1.25% (v / v), more preferably from about 0.4% (v / v) to about 1% (v / v) or from about 0.5% (v / v) to about 0.9% (v / v), most preferably, the FCS concentration is 0.5% (v / v). In one embodiment, the serum starved cell culture medium is RPMI 1640 comprising 0.5% (v / v) FCS. In a preferred embodiment, the genetically modified cells according to the invention are kept in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS for about 1 hour. In one embodiment, the genetically modified cells of the invention can be first maintained in a cell culture medium comprising FCS in a concentration from about 5% (v / v) to about 15% (v / v), preferably in a cell culture medium comprising FCS in a concentration 10% (v / v) and before adding the sample, said cell culture medium can be replaced by a serum starved cell culture medium comprising FCS in a concentration from about 0.25% (v / v) to about 1.5% (v / v), preferably by a serum starved cell culture medium comprising FCS in a concentration of 0.5% (v / v). In a preferred embodiment, the genetically modified cells of the invention are first maintained in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS and before adding the sample, said cell culture medium is replaced by a serum starved cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS.

[0184] METHOD FOR THE DETECTION OF PYROGENS IN A SAMPLE

[0185] The invention further relates to a method for the detection of pyrogens in a sample, comprising the steps of: (a) providing the genetically modified cells according to the invention in a suitable cell culture medium; (b) contacting the cells with said sample and (c) determining expression of the reporter protein.

[0186] GENETICALLY MODIFIED CELL FOR THE DETECTION OF NON- ENDOTOXIN PYROGENS

[0187] In one embodiment, the invention relates to a method for the detection of non-endotoxin pyrogens in a sample, comprising the steps of: (a) providing the genetically modified cell of the invention comprising a reduced expression of at least two negative regulators of Toll-like receptor (TLR)-signaling compared to a cell which is not genetically modified in a suitable cell culture medium; (b) contacting the cells with said sample and (c) determining expression of the reporter protein.

[0188] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein and PP2ACA protein compared to a cell which is not genetically modified, and a reporter gene operably linked to a TLR-responsive promoter.

[0189] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the SOCS1 protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein, achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0190] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein, achieved by using the RNAi method with a shRNA comprising the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 and a reduced expression of PP2ACA protein, achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0191] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein, achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0192] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein and PP2ACA protein compared to a cell which is not genetically modified, and a reporter gene operably linked to a TLR-responsive promoter.

[0193] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP1G protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein, achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0194] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25, and a reduced expression of PP2ACA protein, achieved by using the RNAi method with a shRNA comprising the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0195] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%,at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 and a reduced expression of PP2ACA protein, achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0196] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein, achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0197] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein and PP2ACA protein compared to a cell which is not genetically modified, and a reporter gene operably linked to a TLR-responsive promoter.

[0198] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PPM1D protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein, achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0199] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28, and a reduced expression of PP2ACA protein, achieved by using the RNAi method with a shRNA comprising the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0200] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 and a reduced expression of PP2ACA protein, achieved by degradation of the nucleic acid sequence accordingto SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0201] In one embodiment, the method for the detection of non-endotoxin pyrogens in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein, achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0202] GENETICALLY MODIFIED CELL FOR THE DETECTION OF ENDOTOXIN PYROGENS

[0203] In one embodiment, the invention relates to a method for the detection of endotoxins in a sample, comprising the steps of: (a) providing the genetically modified cell of the invention comprising a reduced expression of at least two negative regulators of Toll-like receptor (TLR)-signaling and an increased expression of at least one accessory protein involved in TLR-signaling compared to a cell which is not genetically modified in a suitable cell culture medium; (b) contacting the cells with said sample and (c) determining expression of the reporter protein.

[0204] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein and PP2ACA protein and an increased expression of at least one accessory protein involved in TLR-signaling selected from the group consisting of CD14 or a variant thereof, MD-2 or a variant thereof and LBP or a variant thereof compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0205] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the SOCS1 protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence and the increased expression of at least one accessory protein involved in TLR-signaling selected from the group consisting of CD14 or a variant thereof, MD-2 or a variant thereof and LBP or a variant thereof compared to a cell which is not genetically modified and a reporter system gene operably linked to a TLR-responsive promoter.

[0206] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and / or an increased expression of MD-2 encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6 and / or an increased expression of LBP encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0207] In a preferred embodiment, the method for the detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0208] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and / or an increased expression of MD-2 encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6 and / or an increased expression of LBP encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0209] In a preferred embodiment, the method for the detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100%identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0210] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and / or an increased expression of MD-2 encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6 and / or an increased expression of LBP encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0211] In a preferred embodiment, the method for the detection of endotoxins in a sample uses the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0212] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein and PP2ACA protein and an increased expression of at least one accessory protein involved in TLR-signaling selected from the group consisting of CD14 or a variant thereof, MD-2 or a variant thereof and LBP or a variant thereof compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0213] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 methodwith a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP1 G protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence and the increased expression of at least one accessory protein involved in TLR-signaling selected from the group consisting of CD14 or a variant thereof, MD-2 or a variant thereof and LBP or a variant thereof compared to a cell which is not genetically modified and a reporter system gene operably linked to a TLR-responsive promoter.

[0214] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and / or an increased expression of MD-2 encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6 and / or an increased expression of LBP encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0215] In a preferred embodiment, the method for the detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0216] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD14 encoded by the nucleicacid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and / or an increased expression of MD-2 encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6 and / or an increased expression of LBP encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0217] In a preferred embodiment, the method for the detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0218] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and / or an increased expression of MD-2 encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6 and / or an increased expression of LBP encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0219] In a preferred embodiment, the method for the detection of endotoxins in a sample uses the genetically modified cell of the invention comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acidsequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0220] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein and PP2ACA protein and an increased expression of at least one accessory protein involved in TLR-signaling selected from the group consisting of CD14 or a variant thereof, MD-2 or a variant thereof and LBP or a variant thereof compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0221] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PPM1D protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2 AC A protein achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence and the increased expression of at least one accessory protein involved in TLR-signaling selected from the group consisting of CD14 or a variant thereof, MD-2 or a variant thereof and LBP or a variant thereof compared to a cell which is not genetically modified and a reporter system gene operably linked to a TLR-responsive promoter.

[0222] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and / or an increased expression of MD-2 encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6 and / or an increased expression of LBP encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0223] In a preferred embodiment, the method for the detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0224] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and / or an increased expression of MD-2 encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6 and / or an increased expression of LBP encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0225] In a preferred embodiment, the method for the detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1 D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0226] In one embodiment, the method forthe detection of endotoxins in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, atleast 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5 and / or an increased expression of MD-2 encoded by the nucleic acid sequence according to SEQ ID NO: 6 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 6 and / or an increased expression of LBP encoded by the nucleic acid sequence according to SEQ ID NO: 7 or a nucleic acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 7, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0227] In a preferred embodiment, the method for the detection of endotoxins in a sample uses the genetically modified cell of the invention comprises a reduced expression of PPM1D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0228] GENETICALLY MODIFIED CELL FOR THE DETECTION OF MYCOPLASMS

[0229] In one embodiment, the invention relates to a method for the detection of mycoplasms in a sample, comprising the steps of: (a) providing the genetically modified cell of the invention comprising a reduced expression of at least two negative regulators of Toll-like receptor (TLR)-signaling and an increased expression of CD36 or a variant thereof compared to a cell which is not genetically modified in a suitable cell culture medium; (b) contacting the cells with said sample and (c) determining expression of the reporter protein.

[0230] In one embodiment, the invention relates to a method for the detection of mycoplasms in a sample, comprising the steps of: (a) providing the genetically modified cell of the invention comprising a reduced expression of at least two negative regulators of Toll-like receptor (TLR)-signaling and an increased expression of CD36 or a variant thereof and an increased expression of CD14 or a variant thereof compared to a cell which is not genetically modified in a suitable cell culture medium; (b) contacting the cells with said sample and (c) determining expression of the reporter protein.

[0231] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein and PP2ACA protein and an increased expression of CD36 or a variant thereof compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0232] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequenceencoding the SOCS1 protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence and the increased expression of CD36 or a variant thereof compared to a cell which is not genetically modified and a reporter system gene operably linked to a TLR-responsive promoter.

[0233] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0234] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0235] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0236] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical tothe nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0237] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0238] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0239] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein and PP2ACA protein and an increased expression of CD36 or a variant thereof and an increased expression of CD14 or a variant thereof, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0240] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the SOCS1 protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence and the increased expression of CD36 or a variant thereof and an increased expression of CD14 or a variant thereof, compared to a cell which is not genetically modified and a reporter system gene operably linked to a TLR-responsive promoter.

[0241] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%,at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0242] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 1 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 1, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0243] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0244] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0245] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded bythe nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0246] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of SOCS1 protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 8 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 8 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded bythe nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded bythe nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0247] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein and PP2ACA protein and an increased expression of CD36 or a variant thereof compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0248] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding thePP1 G protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence and the increased expression of CD36 or a variant thereof compared to a cell which is not genetically modified and a reporter system gene operably linked to a TLR-responsive promoter.

[0249] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0250] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0251] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0252] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical tothe nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0253] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0254] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0255] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein and PP2ACA protein and an increased expression of CD36 or a variant thereof and an increased expression of CD14 or a variant thereof, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0256] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP1 G protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence and the increased expression of CD36 or a variant thereof and an increased expression of CD14 or a variant thereof, compared to a cell which is not genetically modified and a reporter system gene operably linked to a TLR-responsive promoter.

[0257] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequenceaccording to SEQ ID NO: 25, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0258] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 25 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 25, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0259] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0260] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical tothe nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0261] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded bythe nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0262] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of PP1G protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 17 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 17 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded bythe nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded bythe nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0263] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein and PP2ACA protein and an increased expression of CD36 or a variant thereof compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0264] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PPM1D protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACAprotein achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence and the increased expression of CD36 or a variant thereof compared to a cell which is not genetically modified and a reporter system gene operably linked to a TLR-responsive promoter.

[0265] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0266] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0267] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1 D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0268] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1 D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleicacid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0269] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of PPM1D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified.

[0270] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of PPM1D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0271] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein and PP2ACA protein and an increased expression of CD36 or a variant thereof and an increased expression of CD14 or a variant thereof, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter.

[0272] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PPM1D protein or a fragment of said nucleic acid sequence, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA comprising a nucleic acid sequence complementary to the nucleic acid sequence encoding the PP2ACA protein or a fragment of said nucleic acid sequence and the increased expression of CD36 or a variant thereof and an increased expression of CD14 or a variant thereof, compared to a cell which is not genetically modified and a reporter system gene operably linked to a TLR-responsive promoter.

[0273] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequenceaccording to SEQ ID NO: 28, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0274] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1D protein, achieved by using the CRISPR-Cas9 method with a guide RNA having the nucleic acid sequence according to SEQ ID NO: 28 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 28, and a reduced expression of PP2ACA protein achieved by using the RNAi method with a shRNA having the nucleic acid sequence according to SEQ ID NO: 2 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 2 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0275] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1 D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0276] In one embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention which comprises a reduced expression of PPM1 D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 and a reduced expression of PP2 AC A protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical tothe nucleic acid sequence according to SEQ ID NO: 9 and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded by the nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0277] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of PPM1D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded by the nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded bythe nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified.

[0278] In a preferred embodiment, the method for the detection of mycoplasms in a sample uses the genetically modified cell of the invention comprises a reduced expression of PPM1D protein, achieved by disruption of the nucleic acid sequence according to SEQ ID NO: 20 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 20 using the CRISPR-Cas9 method and a reduced expression of PP2ACA protein achieved by degradation of the nucleic acid sequence according to SEQ ID NO: 9 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 9 using the RNAi method and an increased expression of CD36 encoded bythe nucleic acid sequence according to SEQ ID NO: 31 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 31 and an increased expression of CD14 encoded bythe nucleic acid sequence according to SEQ ID NO: 5 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 5, compared to a cell which is not genetically modified and a reporter gene operably linked to a TLR-responsive promoter having the nucleic acid sequence according to SEQ ID NO: 10 or a nucleic acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the nucleic acid sequence according to SEQ ID NO: 10.

[0279] (a) PROVIDING CELLS

[0280] According to the invention, in step (a) cells can be provided in a suitable cell culture medium. In one embodiment, the genetically modified cells according to the invention are provided in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS. In another embodiment, the genetically modified cells according to the invention are provided in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS.(a1) ADDING AT LEAST ONE SMALL MOLECULE INHIBITOR OF INTRACELLULAR SIGNALING The method of the present invention preferably comprises a step (a1) of adding at least one small molecule inhibitor of intracellular signaling to the genetically modified cells provided in step (a).

[0281] A small molecule inhibitor of intracellular signaling can be a low molecular weight compound that specifically interferes with signaling pathways inside cells. These inhibitors block or modulate the activity of proteins or enzymes involved in transmitting signals, thereby affecting cellular responses such as proliferation, differentiation, survival, or apoptosis. Small molecule inhibitors of intracellular signaling act by targeting key components of intracellular signaling pathways and include, but are not limited to, phosphatase inhibitors, serine / threonine phosphatase inhibitors, kinase inhibitors, protease inhibitors, receptor antagonists, transcription factor modulators and second messenger inhibitors. In one embodiment, the small molecule inhibitor of intracellular signaling targets a component of TLR-signaling. In one embodiment, the small molecule inhibitor of intracellular signaling targets a component of TLR-signaling and enhances TLR signaling. In one embodiment, the small molecule inhibitor of intracellular signaling can be a phosphatase inhibitor targeting a phosphatase involved in TLR-signaling. In one embodiment, the small molecule inhibitor of intracellular signaling can be a phosphatase inhibitor targeting a phosphatase involved in TLR-signaling and enhancing TLR signaling.

[0282] Phosphatase inhibitors include, but are not limited to guanabenz, sephin-1, cyclosporin A, FK506 (also known as Tacrolimus), okadaic acid, calyculin acid, tautomycin, microcystin-LR, cantharidin and fostriecin (Takai et al. Physiol Sci. (2018) 68(1 ): 1-17 and Schwaninger et al. Schmiedebergs Arch Pharmacol. (1993) 348(5):541 -5). In one embodiment, a small molecule inhibitor of intracellular signaling can inhibit the protein phosphatase PP1, in particular the PP1 regulatory subunit GADD34, leading to enhanced TLR-signaling (Gu et al. J. Immunol. (2014) 192(6):2846-56). In a preferred embodiment, the small molecule inhibitor of intracellular signaling inhibiting the protein phosphatase PP1 is guanabenz (CsHsChN^ and / or sephin-lfCsHgClN^ (Lin et al. Am. J. Pathol. (2008) 173(5): 1508-17 and Das et al. Science (2015) 348(6231 ):239-42). Guanabenz has the following structure (I):

[0283]

[0284] In one embodiment, the phosphatase inhibitor is cyclosporin A or FK506. In one embodiment, the small molecule inhibitor of intracellular signaling can inhibit calcium signaling. In one embodiment, the small molecule inhibitor of intracellular signaling inhibiting calcium signaling is 2-ABP (2-aminoethoxydiphenyl borate) (Braun et al. Molecular Pharmacology (2003) 63(6): 1304-11). Most preferably, guanabenz and sephin-1 are added to the cells.

[0285] According to the invention, the small molecule inhibitor of intracellular signaling can be added to the cells for a specific period of time before the cells are contacted with the sample. According to the invention, the small molecule inhibitor of intracellular signaling is added to the cells for about 5 minutes to about 45 minutes, preferably for about 10 minutes to about 40 minutes, more preferably for about 12 minutes to about 35 minutes or for about 15 minutes to about 30 minutes, even more preferably for about 17 minutes to about 25 minutes or for about 18 minutes to about 22 minutes and most preferably for 20 minutes before the cells are contacted with the sample.

[0286] In one embodiment, the sephin-1 is added to the genetically modified cells according to the invention at a concentration from about 5 pM to about 27.5 pM, preferably from about 6.5 pM to about 25 pM or from about 7 pM to about 22.5 pM or from about 7.5 pM to about 20 pM, more preferably from about 8 pM to about 18 pM or from about 8.5 pM to about 16 pM or from about 9 pM to about 14 pM, even more preferably from about 9.5 pM to about 12 pM or from about 10 pM to about 11 pM, and most preferably, the sephin-1 is added to the genetically modifiedcells according to the invention at a concentration of 10 pM. In one embodiment, the sephin-1 is added to the genetically modified cells according to the invention at a concentration of 10 pM in a cell culture medium which is RPM1 1640 comprising 10% (v / v) FCS. In a preferred embodiment, the sephin-1 is added to the genetically modified cells according to the invention at a concentration of 10 pM in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS.

[0287] In one embodiment, the guanabenz is added to the genetically modified cells according to the invention in a concentration from about 7.5 pM to about 25 pM, preferably from about 8 pM to about 22.5 pM or from about 8.5 pM to about 20 pM or from about 8.5 pM to about 19 pM, more preferably from about 9 pM to about 18 pM or from about 9.5 pM to about 17 pM or from about 10 pM to about 16 pM, even more preferably from about 10.5 pM to about 15 pM or from about 11 pM to about 14 pM, from about 11.5 pM to about 13.5 pM or from about 12 pM to about 13 pM, and most preferably, the guanabenz is added to the genetically modified cells according to the invention at a concentration of 12.5 pM. In one embodiment, the guanabenz is added to the genetically modified cells according to the invention at a concentration of 12.5 pM in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS. In a preferred embodiment, the guanabenz is added to the genetically modified cells according to the invention at a concentration of 12.5 pM in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS.

[0288] In one embodiment, the guanabenz at a concentration of 12.5 pM and the sephin-1 at a concentration of 10 pM are added to the genetically modified cells according to the invention in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS. In a preferred embodiment, the guanabenz at a concentration of 12.5 pM and the sephin- 1 at a concentration of 10 pM are added to the genetically modified cells according to the invention in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS.

[0289] ADDING AT LEAST ONE ACCESSORY PROTEIN INVOLVED IN TLR-SIGNALING

[0290] For the detection of endotoxin pyrogens in a sample the genetically modified cell of the invention comprises a reduced expression of at least two negative regulators of Toll-like receptor (TLR)-signaling and an increased expression of at least one accessory protein involved in TLR-signaling compared to a cell which is not genetically modified and at least one accessory protein involved in TLR-signaling can be added to the genetically modified cells provided in step (a) or step (a1 ) or can be pre-incubated with the sample, before the genetically modified cells are contacted with the mixture comprising said sample and the at least one accessory protein involved in TLR-signaling in step (b).

[0291] Accessory proteins involved in TLR-signaling have been described in detail above. In one embodiment, the accessory protein involved in TLR-signaling is selected from the group consisting of CD14 or a variant thereof, MD- 2 or a variant thereof and LBP or a variant thereof. These proteins can be purchased from manufacturers such as R&D systems, or they can be produced using cells commonly used for protein expression (e.g. HEK293T cells or Pichia Pastoris cells).

[0292] According to the invention, at least one accessory protein involved in TLR-signaling can be added to the cells for a specific period of time. According to the invention, at least one accessory protein involved in TLR-signaling is added to the cells for about 5 minutes to about 45 minutes, preferably for about 10 minutes to about 40 minutes, more preferably for about 12 minutes to about 35 minutes or for about 15 minutes to about 30 minutes, even more preferably for about 17 minutes to about 25 minutes or for about 19 minutes to about 22 minutes and most preferably for 20 minutes.

[0293] In one embodiment, the at least one accessory protein involved in TLR-signaling is added to the genetically modified cells according to the invention at a concentration from about 2 ng / ml to about 18 ng / ml, preferably from about 4 ng / ml to about 16 ng / ml or from about 5 ng / ml to about 15 ng / ml or from about 6 ng / ml to about 14 ng / ml, more preferably from about 7 ng / ml to about 13 ng / ml, even more preferably from about 8 ng / ml to about 12 ng / ml or from about 9 ng / ml to about 11 ng / ml, and most preferably, the at least one accessory protein involved in TLR-signaling is added to the genetically modified cells according to the invention at a concentration of 10 ng / ml. In one embodiment the at least one accessory protein involved in TLR-signaling is added to the genetically modified cells according to the invention at a concentration of 10 ng / ml in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS. In a preferred embodiment, the at least one accessory protein involved in TLR-signaling is added to the genetically modified cells according to the invention at a concentration of 10 ng / ml in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS.

[0294] In one embodiment, MD-2 having the amino acid sequence according to SEQ ID NO: 13 or an amino acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the amino acid sequenceaccording to SEQ ID NO: 13 is added to the genetically modified cells of the invention. In one embodiment, MD-2 having the amino acid sequence according to SEQ ID NO: 13 or an amino acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the amino acid sequence according to SEQ ID NO: 13 is added to the genetically modified cells of the invention in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS. In one embodiment, MD-2 having the amino acid sequence according to SEQ ID NO: 13 or an amino acid sequence being at least 90%%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the amino acid sequence according to SEQ ID NO: 13 is added to the genetically modified cells of the invention in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS.

[0295] According to the invention, at least one accessory protein involved in TLR-signaling can be incubated with the sample for a specific period of time before the genetically modified cells are contacted with the mixture comprising the sample and the at least one accessory protein involved in TLR-signaling. According to the invention, at least one accessory protein involved in TLR-signaling is pre-incubated with the sample for about 5 minutes to about 45 minutes, preferably for about 10 minutes to about 40 minutes, more preferably for about 12 minutes to about 35 minutes or for about 15 minutes to about 30 minutes, even more preferably for about 17 minutes to about 25 minutes or for about 19 minutes to about 22 minutes and most preferably for 20 minutes.

[0296] In one embodiment, the at least one accessory protein involved in TLR-signaling is pre-incubated with the sample at a concentration from about 2 ng / ml to about 18 ng / ml, preferably from about 4 ng / ml to about 16 ng / ml or from about 5 ng / ml to about 15 ng / ml or from about 6 ng / ml to about 14 ng / ml, more preferably from about 7 ng / ml to about 13 ng / ml, even more preferably from about 8 ng / ml to about 12 ng / ml or from about 9 ng / ml to about 11 ng / ml, and most preferably, the at least one accessory protein involved in TLR-signaling is pre-incubated with the sample at a concentration of 10 ng / ml. In one embodiment, the at least one accessory protein involved in TLR-signaling is incubated with the sample at a concentration of 10 ng / ml for 20 minutes before the genetically modified cells are contacted with the mixture comprising the sample and the at least one accessory protein involved in TLR-signaling in step (b).

[0297] In one embodiment, CD14 having the amino acid sequence according to SEQ ID NO: 12 or an amino acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the amino acid sequence according to SEQ ID NO: 12 and / or LBP having the amino acid sequence according to SEQ ID NO: 14 or an amino acid sequence being at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the amino acid sequence according to SEQ ID NO: 14 are incubated with the sample, before the genetically modified cells are contacted with the mixture comprising the sample and CD14 and / or LBP

[0298] In one embodiment, CD14 having the amino acid sequence according to SEQ ID NO: 12 or an amino acid sequence being at least 90%%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the amino acid sequence according to SEQ ID NO: 12 and LBP having the amino acid sequence according to SEQ ID NO: 14 or an amino acid sequence being at least 90%%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 98.5 %, at least 99%, at least 99.5% or at least 100% identical to the amino acid sequence according to SEQ ID NO: 14 are incubated with the sample before the genetically modified cells are contacted with the mixture comprising the sample, CD14 and LBP.

[0299] (b) CONTACTING CELLS WITH A SAMPLE

[0300] SAMPLES

[0301] The sample to be analyzed by the method of the invention can be taken from a product such as a pharmaceutical product, a product for cell culture or laboratory use, a cosmetic or personal care product or a parenteral nutrition solution to be tested for the presence of a pyrogen or mycoplasms.

[0302] In one embodiment, a sample may be taken from a pharmaceutical product or composition. Examples of such pharmaceutical products or compositions include, but are not limited to, solid dosage forms such as tablets, capsules, powders for oral suspension and granules, liquid dosage forms such as oral solutions and suspensions, injectable solutions, topical creams and eye drops, gels, inhalation products, parenteral formulations such as intravenous solutions, and nasal sprays.

[0303] In one embodiment, a sample may be taken from a product for cell culture or laboratory use. Examples of such products include but are not limited to cell culture media, reagents, biological buffers, plasticware such as culturedishes and tubes, bioprocessing equipment, endotoxin-free water, purified proteins and enzymes, clinical reagents and diagnostic kits and animal derived products.

[0304] In one embodiment, a sample may be taken from a cosmetic or personal care product. Examples of such products include but are not limited to injectable cosmetics, wound healing and skin care products, facial masks, topical pharmaceuticals and medicinal products, nasal sprays, eyecare products, oral care products, deodorants, body lotions and creams and hair care products.

[0305] In one embodiment, a sample may be taken from a parenteral nutrition solution. Examples of such products include but are not limited to total parenteral nutrition (TPN) solutions, partial parenteral nutrition (PPN) solutions, amino acid solutions, fat emulsion solutions, electrolyte solutions and vitamin and trace element solutions.

[0306] In one embodiment, a sample may be taken from a surface to be tested for the presence of a pyrogen or mycoplasms, for example from a surface of a medical device or instrument. Examples of medical devices and instruments include bedpans, cannulas, cardioverters, defibrillators, catheters, dialysers, electrocardiograph machines, enema equipment, endoscopes, gas cylinders, gauze sponges, surgical scissors, hypodermic needles, syringes, infection control equipment such as masks, gowns, face shields, and goggles, instrument sterilizers, kidney dishes, nasogastric tubes, surgical scalpels, nebulizers, ophthalmoscopes, otoscopes, pipettes, proctoscopes, radiographers, sphygmomanometers, thermometers, tongue depressors, transfusion kits, tuning forks, ventilators, watches, and the like. Such a sample may be taken, for example, by rinsing the surface to be tested with a solution (e.g., water or a buffer), collecting the rinsate, and utilizing it for sample preparation.

[0307] INCUBATION

[0308] According to the method of the invention, the genetically modified cells are contacted with a sample in step (b). In one embodiment, the genetically modified cells are contacted with the sample in step (b) and are further incubated with said sample for a period of time. In one embodiment, the genetically modified cells are contacted with the sample in step (b) and further incubated with said sample for a period of time in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS. In one embodiment, the genetically modified cells are contacted with the sample in step (b) and further incubated with said sample for a period of time in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS.

[0309] According to the invention, the genetically modified cells are incubated with the sample for about 1 hour to about 16 hours, for about 1.25 hours to about 14 hours, for about 1.75 hours to about 12 hours, for about 2 hours to about 10 hours, for about 2.5 hours to about 9 hours, for about 3 hours to about 8 hours, for about 3.5 hours to about 7 hours, for about 4 hours to about 6.5 hours, for about 5 hours to about 6 hours or for about 5.5 hours to about 6 hours.

[0310] For the detection of non-endotoxin pyrogens in a sample the genetically modified cell of the invention is incubated with the sample for about 1 hour to about 7 hours, preferably for about 1.5 hours to about 6 hours or for about 2 hours to about 5 hours, more preferably for about 2.5 hours to about 4 hours, even more preferably for about 3 hours to about 3.5 hours and most preferably for 3 hours.

[0311] For the detection of endotoxin pyrogens in a sample the genetically modified cell of the invention is incubated with the sample for about 3 hours to about 16 hours, preferably for about 3.5 hours to about 14 hours or for about 4 hours to about 12 hours, more preferably for about 4.5 hours to about 10 hours or for about 5 hours to about 9 hours, even more preferably for about 5.5 hours to about 8 hours or for about 6 hours to about 7 hours and most preferably for 6 hours.

[0312] For the detection of mycoplasms in a sample the genetically modified cell of the invention is incubated with the sample for about 0.5 hours to about 7 hours, preferably for about 1 hour to about 6 hours or for about 2 hours to about 5 hours, more preferably for about 2.5 hours to about 4 hours, even more preferably for about 3 hours to about 3.5 hours and most preferably for 3 hours.

[0313] In one embodiment, the genetically modified cells of the invention in a cell culture medium are incubated with the sample for 3 hours. In one embodiment, the genetically modified cells of the invention are incubated with the sample for 3 hours in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS. In one embodiment, the genetically modified cells of the invention are incubated with the sample for 3 hours in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS. In one embodiment, the genetically modified cells of the invention in a cell culture medium are incubated with the sample for 4 hours. In one embodiment, the genetically modified cells of the invention are incubated with the sample for 4 hours in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS. In one embodiment, the genetically modified cells of the invention are incubated with the sample for 4 hours in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS. In one embodiment, thegenetically modified cells of the invention in a cell culture medium are incubated with the sample for 5 hours. In one embodiment, the genetically modified cells of the invention are incubated with the sample for 5 hours in a cell culture medium which is RPMI 1640 comprising 10% (v / v) FCS. In one embodiment, the genetically modified cells of the invention are incubated with the sample for 5 hours in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS. In one embodiment, the genetically modified cells of the invention in a cell culture medium are incubated with the sample for 6 hours. In one embodiment, the genetically modified cells of the invention are incubated with the sample for 6 hours in a cell culture medium which is RPM1 1640 comprising 10% (v / v) FCS. In one embodiment, the genetically modified cells of the invention are incubated with the sample for 6 hours in a cell culture medium which is RPMI 1640 comprising 0.5% (v / v) FCS.

[0314] According to the invention, the contacting of the cells with the sample can be the addition of the sample or samples to the cell culture medium comprising the cells. It can also be the addition of the cell culture medium comprising the cells to the sample or samples. The contacting may be done for example in a microwell plate, a cell culture dish, a cell culture flask or a fermenter and the like. The contacting may be done under controlled conditions, such as a temperature of 36°C to 37°C and a pH of 7.4, optionally adding exogenous CO2 at a concentration of 5% to 7%. In one embodiment, contacting is done in a 96-well plate. In one embodiment, contacting is done in a 384-well plate. In cases where a 96-well plate or a 384-well plate is used, a single sample can be tested in each well. Microwell plates are widely available from commercial suppliers. The cells may be present at a specific number in each well during the contacting step. In one embodiment, the cells are present in each well at a number from about 0.1 x 105cells to about 1.5 x 105cells, from about 0.2 x 105cells to about 1.25 x 105cells or from about 0.3 x 105cells to about 1 x 105cells.

[0315] In one embodiment, contacting is done in a 96-well plate and the genetically modified cells according to the invention are present in each well at a number from about 0.5 x 105cells to about 1.5 x105cells, preferably fr...

Claims

CLAIMS1 ) A genetically modified cell comprising:a) a reduced expression of at least two negative regulators of Toll-like receptor (TLR)-signaling compared to a cell which is not genetically modified, andb) a reporter gene operably linked to a TLR-responsive promoter.2) The genetically modified cell according to claim 1, wherein the negative regulators of TLR-signaling are proteins selected from the group consisting of SOCSI, PP2ACA, PP1 and the catalytic subunits PP1A, PP1B and PP1G thereof, PPM1D, PP2B and the catalytic subunits PP2BA, PP2BB and PP2BC thereof, USP-4, TBK-1, PP5C, PPM1G, PPM1N, A20, SOCS3, and IRAK-M, preferably wherein the negative regulators of TLR-signaling are the proteins SOCS1 and PP2ACA.3) The genetically modified cell according to claim 1 or 2, comprising a reduced expression of PP2ACA and at least one negative regulator selected from the group consisting of SOCS1, PP1G and PPM1D.4) The genetically modified cell according to claim 2 or 3, wherein the expression of the SOCS1 protein is reduced by a disruption of the nucleic acid sequence encoding the SOCS1 protein, preferably wherein the nucleic acid sequence encoding the SOCS1 protein is disrupted by using the CRISPR-Cas method.5) The genetically modified cell according to any one of claims 2 to 4, wherein the expression of the PP2ACA protein is reduced by RNA interference (RNAi) targeting the nucleic acid sequence encoding the PP2ACA protein.6) The genetically modified cell according to any one of claims 1 to 5, wherein the TLR-responsive promoter is a NF-KB-dependent promoter.7) The genetically modified cell according to any one of claims 1 to 6, further comprising an increased expression of at least one accessory protein involved in TLR-signaling compared to a cell which is not genetically modified.8) The genetically modified cell according to claim 7, wherein the at least one accessory protein involved in TLR-signaling is CD14.9) The genetically modified cell according to claim 7, wherein the at least one accessory protein involved in TLR-signaling is CD36.10) The genetically modified cell according to any one of claims 1 to 9, wherein the cell is an immune cell, preferably wherein the immune cell is a cultivated human monocytic cell, preferably THP-1.11 ) A method for the detection of pyrogens in a sample, comprising the steps of:(a) providing genetically modified cells according to any one of claims 1 to 8 in a suitable cell culture medium; (b) contacting the genetically modified cells with said sample;(c) determining expression of the reporter gene.12) A method for the detection of mycoplasms or mycoplasma-derived components in a sample, comprising the steps of:(a) providing genetically modified cells according to any one of claims 1 to 7 and 9 in a suitable cell culture medium;(b) contacting the genetically modified cells with said sample;(c) determining expression of the reporter gene.13) The method according to claim 11 or 12,further comprising a step (a1 ) of adding at least one small molecule inhibitor of intracellular signaling to the genetically modified cells, preferably, wherein the small molecule inhibitor of intracellular signaling is selected from the group consisting of sephin-1, guanabenz, FK506, cyclosporin A and 2-ABR14) The method according to any one of claims 11 to 13, wherein step (b) further comprises incubating the genetically modified cells with said sample for a period of time, preferably, wherein the period of time is about 2 hours to 10 hours.15) The method according to any one of claims 11 to 14, wherein the sample is selected from the group consisting of:a) pharmaceutical products;b) medical devices;c) products for cell culture and laboratory use;d) cosmetic and personal care products; ande) parenteral nutrition solutions.16) The method according to any one of claims 11 to 15,wherein in step (b) the genetically modified cells are contacted with said sample in a culture medium comprising serum in a concentration of about 0.25% to 1.5%, preferably of 0.5%.17) The method according to any one of claims 11 and 13 to 16,wherein at least one accessory protein involved in TLR-signaling is added to the cells of step (a) or step (a1 ) or is incubated with the sample before the genetically modified cells are contacted with the mixture comprising the sample and the at least one accessory protein involved in TLR-signaling.18) A kit comprising:a) genetically modified cells according to any one of claims 1 to 8; andb) at least one small molecule inhibitor of intracellular signaling.19) Use of the genetically modified cell according to any one of claims 1 to 8 or the kit according to claim 18 for the detection of pyrogens in a sample.20) A kit comprising:a) genetically modified cells according to any one of claims 1 to 7 and 9; andb) at least one small molecule inhibitor of intracellular signaling.21 ) Use of the genetically modified cell according to any one of claims 1 to 7 and 9 or the kit according to claim 20 for the detection of mycoplasms or mycoplasma-derived components in a sample.

Images