JP2004505648A5 - - Google Patents

Description

EX20 expression was found to be up-regulated, for example by GM-CSF, after stimulation of cytokines important in regulating the inflammatory process in the above-mentioned diseases. In terms of the various leukocyte subsets (leukocyte subsets) inflammatory diseases related over-expression observed in EX20 in, proteins EX20 and encoded thereby are for chromatic Te diagnosis smell of the inflammatory disease, and is encoded The proteins are useful as therapeutic targets for identifying medicaments suitable for treating those diseases.

The terms used herein have the following meanings:
"Isolated" means material that has been removed from its original environment.
"Hybridization" or "hybridization" refers to any process by which a polynucleotide strand binds to its complementary strand through base pairing.
"Stringent conditions" refers to experimental conditions that allow up to 20% base pair mismatch, typically at 65 ° C. in 0.1 × SSC (15 mM NaCl, 1.5 mM sodium citrate, pH 7.0). Twice for 15 minutes.

As used herein, “variant” refers to an amino acid sequence that has been changed by one or more amino acids with respect to the amino acid sequence. Changes can include amino acid substitutions, deletions or insertions. With reference to a nucleotide sequence, the term "variant" as used herein refers to a nucleotide sequence that has been changed by one or more nucleotides; the alteration may include a nucleotide substitution, deletion or insertion. Preferred functional equivalent variants of amino acid SEQ ID NO: 2 or SEQ ID NO: 16 have at least 80%, more preferably at least 90%, and especially more than 95% amino acid sequence identity with SEQ ID NO: 2 or SEQ ID NO: 16 It is.

The polynucleotide probe is capable of selectively hybridizing under stringent conditions to a polynucleotide fragment having a polynucleotide fragment having the sequence of SEQ ID NO: 1 or SEQ ID NO: 15. The probe has a sequence that hybridizes only to its cognate sequence under such hybridization conditions. The DNA probes described above are useful in many screening applications, including Northern and Southern blot analysis, dot and slot blot analysis, and fluorescence in situ hybridization (FISH).

Polynucleotide hybridization probes and complementary polynucleotide sequences of the present invention, in situ (e.g., FISH) hybridization, Northern or Southern blot analysis can be detected using a dot-blot or slot-blot analysis. Abnormalities can also be detected by, for example, sequence-sensitive gel electrophoresis (CSGE) and DNA sequencing. Genetic abnormalities can result in a change in the amino acid sequence of an individual's EX20 protein compared to the amino acid sequence of a normal EX20 protein, or a deletion of the protein. Alternatively, the alteration does not alter the amino acid sequence, but instead alters the sequence of the control element at either the 5'- or 3'-end of the gene, or the sequence of the control element within the intron sequence region of the gene. Can change EX20 gene expression. Changes can also affect how the transcription of the gene is processed or translated. The invention also includes kits for detecting abnormalities in the polynucleotide sequence of the EX20 gene of an individual or for measuring the level of EX20 expression in cells of an individual. Hybridization kit for such use comprises a detectable, e.g. chemiluminescence, may be modified by the inclusion of labeled radioactive or fluorescent therein, wherein the probe and the labeled reagent, That is, it may include other reagents, such as those that incorporate radioisotopes, chemiluminescent or fluorescent groups into the hybridized product, and buffers. A PCR amplification kit can include a primer pair as described above with a DNA polymerase such as Taq polymerase, and can include additional reagents such as amplification buffers and the like. Certain embodiments of the PCR amplification kit may include additional reagents specific to many techniques for detecting polynucleotide changes, including CSGE and DNA sequencing.

Measurement of the level of expression of the polynucleotide (B) can be used in the diagnosis of an inflammatory disease as described above. Accordingly, the invention includes a method of determining whether a subject has an inflammatory disease, eg, an inflammatory disease associated with elevated GM-CSF levels, wherein the method comprises the steps of: in the measurement of the polynucleotide (B), the expression level of a nucleotide sequence in particular comprising a nucleotide sequence that hybridizes thereto with a polynucleotide (B), or stringent conditions comprising the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 15 And comparing the level to the expression level of the polynucleotide in a cell sample from a healthy subject. Elevated expression levels of polynucleotide (B) are indicative of an inflammatory disease. The measured level is indicative of the nature of the inflammatory disease. Expression levels of the polynucleotide (B), for example, Northern blot analysis, reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, immunoprecipitation, Western blot hybridization or immunohistochemistry (immunohistochemistry ) Can be determined.

For example, in a diagnostic test using quantitative RT-PCR, mRNA is isolated from the desired cells, eg, BAL fluid cells or peripheral blood cells. Two primers, one having a sequence identical to the sequence of the region between nucleotides 765 and 783 of SEQ ID NO: 1 and the other being the reverse complement of nucleotides 815-835 of SEQ ID NO: 1 Design a primer having a nucleotide sequence. An appropriately modified and labeled probe is then generated corresponding to the sequence between nucleotides 787-813 of SEQ ID NO: 1. Using the appropriate control gene, these two primers and probe are used in a TaqMan (PE Applied Biosystems) instrument to measure the EX20 mRNA level in samples taken from patients and healthy subjects under investigation. I do. When using an in-situ hybridization for diagnostic purposes, the probe of about 200~1200bp corresponding to any portion of the sequence shown in SEQ ID NO: 1 can be used. Several overlapping probes of various lengths can be used in such tests, such as those described in Example 3. Samples can be collected from the patient and embedded in paraffin. Such a sample can be, for example, peripheral blood cells, bronchoalveolar lavage (BAL) fluid cells, or various tissue sections. Labeled riboprobes prepared as described in Example 3 can then be used to measure EX20 expression levels in these samples and thereby identify inflammatory conditions.

The efficacy of a drug of the invention in inhibiting or ameliorating an inflammatory condition, e.g., inflammatory airway disease, can be demonstrated in animal models, e.g., mouse or rat models of airway inflammation or other inflammatory conditions, e.g., Szarka et al., J. Immunol. Methods (1997) 202: 49-57; Renzi et al., Am. Rev. Respir. Dis. (1993) 148: 932-939; Tsuyuki et al., J. Clin. Invest. (1995) 96: 2924-2931; Cernadas et al (1999) Am. J. Respir. Cell Mol. Biol. 20: 1-8; Durie et al., Clin. Immunol. Immunopathol. (1994) 73: 11-18; and Natl. Acad. Sci. USA (1992) 89: 9784-9788.

Example 1
Blood (200 ml) is collected in test tubes containing sodium citrate from a normal donor with no history of respiratory disease under sterile conditions. Neutrophils are purified by established methods. PBMCs are separated from peripheral blood cells by Ficoll Hypaque (Pharmacia) centrifugation. The remaining cell population, mainly granulocytes and erythrocytes, is treated with an erythrocyte lysis solution (155 mM NH ₄ Cl, 10 mM KHCO ₃ , 0.1 mM EDTA, PH 7.3). To determine purity, granulocytes are stained with Hansel dye (Difco Laboratories Ltd) and differentiated with a high power light microscope. It can be seen that eosinophil contamination is less than 2%. For stimulation, neutrophils are resuspended in RPMI-1640 + 10% FCS at a concentration of 5 million cells / ml. The cells are cultured for 5 hours in the presence or absence of 50 ng / ml human recombinant GM-CSF (R & D System). Total RNA is extracted using TRIZOL reagent (Gibco / BRL) as described by the manufacturer. 1 ml of TRIZOL is used for resuspension of 5 million pelleted neutrophils each. The mRNA is purified using the MESSAGEMAKER mRNA isolation kit (Gibco / BRL) under conditions recommended by the manufacturer. Using 300 ng of mRNA, cDNA is synthesized using Superscript Choice System (Gibco / BRL) and oligo (dT) for first strand synthesis. The double-stranded cDNA is extracted once with an equal volume of phenol: chloroform: isoamyl alcohol and precipitated with 0.5 volume of 7.5 M ammonium acetate and 2.5 volume of ethanol. After centrifugation, the resulting pellet is washed with 70% ethanol and resuspended in 16 μl of sterile water.

cDNA-RAD is essentially described by Hubank, M., and DG Schatz.Nucleic Acids Res. 22: 5640-5648; and O'Neill, MJ, and AH Sinclair.Nucleic Acids Res. 25: 2681-2682. Run as is. The double-stranded cDNA samples prepared from unstimulated and stimulated neutrophils isolated from mRNA, digested with DpnII restriction enzyme, and 24-mer (SEQ ID NO: 7) and 12-mer (SEQ ID NO: 8) Oligo Ligate to the R-adaptor obtained by annealing to nucleotides. The and both "tester" of (cDNA of stimulated neutrophils) "driver" (cDNA of unstimulated neutrophils), generated using the Expand Long Template PCR System and Expand Buffer 1 (Roche Diagnostics) . Five 100 μl PCRs are performed for each tester and ten for each driver. The R-adapter is removed from both the driver and the tester amplicon using DpnII digestion. The J-adapter obtained by annealing the 24-mer (SEQ ID NO: 9) to the 12-mer (SEQ ID NO: 10) is then ligated to a tester population. The subtractive hybridization is performed in a 5 μl reaction at 67 ° C. for 20 hours. To make a different product 1 (DP1), 250 ng of tester cDNA is mixed with 25 μg of driver cDNA in a 100: 1 ratio. The DP1 cDNA is then digested with DpnII to remove the J-adapter and then ligate the N-adapter, which anneals the 24-mer (SEQ ID NO: 11) to the 12-mer (SEQ ID NO: 12) oligonucleotide. It was obtained by To make a different product 2 (DP2), 31.25 ng of the tester is mixed with 25 μg of the driver cDNA in a ratio of 800: 1. The digested and excess adapter is removed by washing the cDNA on a Microcon30 filter (Amicon). The subtracted library is fractionated by agarose gel electrophoresis.

Example 3
This example describes the analysis of expression and tissue distribution of EX20 genes by in situ hybridization.

To generate labeled riboprobes, a subclone containing a 1161 bp insert between the EcoRI and XbaI sites of the pSPORT1 plasmid (Life Technologies) is constructed. The insert of this subclone is identical to the insert of Example 4 below and is prepared in the same manner. This construct uses the SP6 promoter in the vector to transfer the antisense strand of the insert that can be used as probes in in situ hybridization experiments. The T7 promoter in the vector is used to transcribe the sense strand used as a negative control. Serial tissue sections from paraffin-embedded samples are hybridized with radiolabeled cRNA probes synthesized from the 1.2 kb insert. Riboprobes are transcribed in vitro with ³³ P-uridine 5′-triphosphate in the presence of SP6 (antisense) and T7 (sense) RNA polymerase. The probe is then column purified and then subjected to electrophoresis on a 5% TBE-urea acrylamide gel to confirm size and purity. Tissue sections are digested with proteinase K and then hybridized with the probe at approximately 8.0 × 10 ⁸ dpm / ml at 65 ° C. for 18 hours. Slides are treated with RNAse A and washed at 65 ° C. in 0.1 × SSC under stringent conditions for 2 hours. Slides are then coated with Kodak NTB-2 emulsion, exposed at 4 ° C. for 7 days, and developed using Kodak D-19 developer and fixer. Slides are stained with hematoxylin and eosin and photographed using a Sony Digital Photo Camera connected to a Nikon microscope. In addition to hybridization with an antisense probe, another section is hybridized with two types of control probes. All tissues are first screened with a probe for beta-actin mRNA to ensure that RNA is preserved in the sample. The adjacent contiguous section also hybridizes as an antisense probe with a sense control riboprobe from the same region of the gene.

Preparation Cells crude lysate (1x10 ^9), lysis buffer 100ml (20mM Hepes pH 7.9,100mM NaCl, 5% glycerol, 2 mM E- mercaptoethanol, 0.5 mM imidazole, 0.1% Nonidet P- Resuspend in 40, 40 pg / ml AEBSF, 0.5 pg / ml leupeptin, 1 pg / ml aprotinin and 0.7 pg / ml pepstatin A). The cells are incubated on ice for 15 minutes and then centrifuged at 39,000 xg for 30 minutes at 4 ° C.

Metal chelate affinity chromatography is performed on a column connected to a BioCAD chromatography workstation at room temperature. A 20 ml Poros MC / M (16 mmD × 100 mmL) column is filled with Ni ²⁺ before use and after each injection. The column is washed with 10 column volumes (CV) of 50 mM EDTA pH 8, 1 M NaCl followed by 10 CV of water to fill with Ni ²⁺ . The column was filled with 0.1 M NiSO ₄ pH 4.5-5 of 500 ml, washed with water and 10 CV, then any unbound ^{Ni 2+,} is removed by washing with 5CV of 0.3 M NaCl. All steps are performed at a flow rate of 20 ml / min. The filled MC / M column was equilibrated with 5 CV of buffer B (20 mM Hepes pH 7.9, 100 mM NaCl, 5% glycerol, 2 mM E-mercaptoethanol, 1 mM PMSF, 100 mM imidazole) to saturate the site, And fill with 10 CV of buffer A (same as buffer B, except for 0.5 mM imidazole). 90-95 ml of crude lysate per run is loaded onto the column at a flow rate of 20 ml / min. The following steps are performed at a flow rate of 30 ml / min. Any unbound material is removed by washing with 12 CV of buffer A, and EX20 elutes with a gradient over 0 CV of 0-50% buffer B. Fractions (8 ml) are collected over the gradient. The EX20-containing fractions are combined and the protease inhibitor is added to the final concentration described for the lysis buffer above. DTT is also added to a final concentration of 1 mM. The combined fractions are dialyzed overnight at 4 ° C. against 4 liters of 20 mM Hepes pH 7.9, 1 mM DTT, 0.2 mM PMSF. The protein concentration is measured and, if necessary, the sample is concentrated at 4 ° C. on a Millipore Ultrafree-15 centrifugal device (MW cut-off 50 kDa). Rinse the device with water before use. The buffer used for long-term storage at -80 C is 20 mM Hepes pH 7.9, 1 mM DTT, about 100 mM NaCl, 5% glycerol. Glycerol can be removed from the sample upon storage at 4 ° C.