CN1671734A - CFTR modifier genes and expressed polypeptides useful in treating cystic fibrosis and methods and products for detecting and/or identifying same - Google Patents

Abstract

The discovery that CFTR modifier genes, in particular the Kir4.2 gene, the expressed polypeptide(s), as well as genetic and polypeptide regulators thereof, can be used to treat cystic fibrosis (CF), or at least the conditions that cause CF. Methods and products for detecting and/or identifying CFTR modifier genes, their respective expressed polypeptides, the genetic regulators of such CFTR modifier genes, and the regulators of their respective expressed polypeptides are disclosed. Also disclosed are compositions and methods using these CFTR modifier genes, their respective expressed polypeptides, genetic regulators of these CFTR modifier genes, and/or CFTR modifier polypeptide regulators for the purpose of treating CF, or at least the conditions that cause CF, are disclosed.

Description

Be used for the treatment of CFTR modifying factor and the polypeptide expressed and the detection of cystic fibrosis and/or identify their method and product

Technical field

The application relates to and is used for the treatment of cystic fibrosis (CF), or causes the CFTR modifying factor of the symptom of CF at least, and polypeptide expressed.The application also relates to the hereditary instrumentality that uses this CFTR modifying factor of adjusting, and uses the polypeptides for modulating thing to influence the function and/or the activity of each express polypeptide.The application also relates to and detects and/or identify this CFTR modifying factor, the method for each express polypeptide and product, regulate this modifying factor expression hereditary instrumentality and influence the function and/or the active polypeptides for modulating thing of each express polypeptide.

Background technology

Cystic fibrosis (CF) is human modal mortality genetic diseases.Referring to Boat etc., TheMetabolic Basis of Inherited Diseases (Scriver, volumes such as C.R., McGraw-Hill, NewYork (1989)).At present, in the U.S. thousands of patients CF are arranged.Although standard treatment is arranged now, the mean age of survival has only 26 years old.The pulmonary respiration tract disease is the major cause of morbidity and causes 95% mortality ratio.Although many organs are affected in CF, the M ﹠ M of this disease mucus main and that lung is interior is gathered, and recurrent infection is with too much inflammation is relevant.

The protein product of CF dependency gene is called cystic fibrosis and strides film conduction instrumentality (CFTR).Referring to Riordan etc., Science (1989) 245: 1066-1073.About 1480 amino acid whose protein that CFTR is made up of two repeat element, wherein each element comprises 6 transmembrane segments and a Nucleotide binding domains.Two repeat element are separated by the so-called R-structural domain that a large-scale polarity contains a plurality of potential phosphorylation sites.According to the structural domain structure of its expection, CFTR comprises multi-medicine resistance (MDR) or P-glycoprotein, ox adenosine cyclase, yeast STE6 albumen, and a member in the class related protein of several bacterium amino acid transporters.Referring to Riordan etc., Science (1989) 245: 1066-1073; Hyde etc., Nature (1990) 346: 362-365.This histone matter is characterised in that pump into or pump cell with molecule relevant.

Inferred CFTR and flowed out epithelial cell by the phosphorylation response regulation negatively charged ion that encircles AMP (cAMP)-dependent protein kinase or protein kinase C.Referring to Riordan etc., Science (1989) 245: 1066-1073; Frizzell etc., Science (1986) 233: 558-560; Welsh etc., Nature (1986) 322: 467; Li etc., Nature (1988) 331: 358-360; Hwang etc., Science (1989) 244: 1351-1353.Although the pathogeny of CF is not exclusively understood, but it is believed that epithelial ring AMP dependent form the chloride secretion unusual and too much sodium relevant with the CFTR defective absorbs the liquid bulk inner equilibrium that has changed respiratory tract surface liquid (ASL) again, cause its dehydration, the impaired and infection of mucociliary clearance.Referring to Tarran etc., Molecular Cell, (2001) 8: 149-58.Owing to illustrated the primary structure of CFTR, with the interaction of many functions and many and other cell protein owing to CFTR.Therefore, except the effect of CFTR in regulating the transportation of cAMP-dependent form chlorine, this protein by with cytoskeleton, protein called membrane transporters, and acceptor, albumen approach are selected and degradation mechanism interacts brings into play the multiple-effect effect in many cell processes.Referring to Welsh etc., " Cystic Fibrosis, " Metabolic and Molecular Bases of Inherited Disease (8 ^ThEd.2001), 5121-88 page or leaf.

Many researchs also are supported in the idea that too much inflammatory reaction takes place in the CF lung, but these mechanism that comprise unusually it be unclear that.Other protein of IL-8 and transmitting inflammation signal comprises that the change of NF κ B and iNOS level is relevant with CF, no matter whether there is infection, but causes the approach of the unusual composing type change transmitting inflammation in CFTR probably.Referring to Khan etc., Am J.Respir.Crit.Care Med., (1995) 151: 1075-82; DiMango etc., J.Clin.Invest., (1998) 101: 2598-2605; Elmer etc., Am.J.Physiol., (1999) 276: L466-73.

The chromosomal cftr gene of CF is carried out sequential analysis disclosed the various sudden changes that cause disease.Referring to Cutting etc., Nature (1990) 346: 366-369; Dean etc., Cell (1990) 61: 863:870; With Kerem etc., Science (1989) 245: 1073-1080; Kerem etc., Proc.Natl.Acad.Sci.USA (1990) 87: 8447-8451.In fact, the different sudden change above 800 is relevant with the clinical disease feature of CF in cftr gene.Referring to Welsh etc., " Cystic Fibrosis, " Metabolic andMolecular Bases of Inherited Disease (8 ^ThEd.2001), 5121-88 page or leaf.Colony studies show that modal CF sudden change, and 3 nucleotide deletions (Δ F508) of the 508th phenylalanine of CFTR aminoacid sequence of promptly encoding are relevant with about 70% cystic fibrosis case.This sudden change causes losing efficacy with the epithelial cell chloride channel of cAMP reaction.Referring to Frizzell etc., Science (1986) 233: 558-560; Welsh Science (1986) 232: 1648-1650; Li etc., Nature (1988) 331: 358-360; Quinton, Clin.Chem. (1989) 35: 726-730.In the respiratory tract cell, this causes ion and transport of liquid imbalance.Generally believe that this causes that mucus secretion is unusual, and finally cause pulmonary infection and epithelial cell damage.

In lung, CFTR mainly is distributed in respiratory tract apex zone and the submucosa body of gland epithelial cell.Referring to Engelhardt etc., J.Clin.Invest., (1994) 93: 737-49.Abundance and cell site that CFTR expresses are grown, the strong influence of space and humoral factor, supported the expression of CFTR and function transcribe with post-transcriptional level on the viewpoint of all being regulated.Although carried out extensive studies, the accurate effect in the CF disease pathogenesis is known little to CFTR.On clinical level, even the CF severity of disease also is height change in having the individuality of identical sudden change, this has supported the E﹠H factor can influence the viewpoint of this disease seriousness.Referring to Welsh etc., " Cystic Fibrosis, " Metabolic and Molecular Bases of Inherited Disease (8 ^ThEd.2001), 5121-88 page or leaf.These clinical observation result, with the observations that confirms to exist after in mouse cftr gene target or the sudden change strain difference of CF phenotype seriousness (referring to Rozmahel etc., Nat.Genet., (1996), 12:280-87), support the expression of CFTR and the function in cell processes thereof to be subjected to increasing the weight of in the various organs or alleviated many genes of CF disease or the viewpoint of the influence of approach.

According to existing knowledge, adopt three kinds of general correction solution (with respect to the therapy of being devoted to improve this symptom) to reverse the unusual chlorine (Cl that reduces in the CF respiratory tract at present to the CF gene ^-) secretion and the sodium (Na that increases ⁺) absorb.The ionogen transhipment defective that it is believed that airway epithelial can change respiratory secretions and mucous composition.Referring to Boat etc., The Metabolic Basis of Inherited Diseases (Scriver, volumes such as C.R., McGraw-Hill, New York (1989)); Quinton, FASEB J. (1990) 4: 2709-2717.Therefore, adopt the pharmacology treatment to be devoted to correct the unusual of ionogen transhipment.With the medicine amiloride of atomised form, promptly a kind of inhibition sodium (Na ⁺) passage, thereby suppressing the diuretic(s) that sodium absorbs, the test of carrying out gets along with.PRELIMINARY RESULTS shows that this medicine is safe and shows that the progression of disease speed of measuring by lung function slightly changes.Referring to Knowles etc., N.Eng.J.Med (1990) 322: 1189-1194; App, Am.Rev.Respir.Dis. (1990) 141-605.Can (purinergic) acceptor such as the purine in the Nucleotide stimulation airway epithelial of ATP or UTP.Therefore, they are opened and are different from CFTR chlorine (Cl ^-) a class chlorine (Cl of passage ^-) passage.In vitro tests shows that ATP and UTP can stimulate chlorine (Cl ^-) secretion.Referring to Knowles etc., N.Eng.J.Med (1991) 325-533.Test oligonucleotide stimulates the body internal secretion, thereby the tentative experiment of correcting the unusual ability of ionogen transhipment is just beginning to carry out.

For all pharmacology reagent, in the suitable drug reagent of exploitation treatment CF, be important such as the problem of drug toxicity and administration.The more basic consideration of pharmacology scheme for the CF treatment is the chlorine (Cl relevant with CFTR ^-) whether channel activity be the decisive characteristic that causes this morbid state.Existing infer have another still unidentified CFTR system composition, it is a kind of key instrumentality, and if like this, may be based on chlorine (Cl ^-) the pharmacology scheme of transhipment can successfully regulate ionic equilibrium, but still can not alleviate basic physiological knowledge topic.Referring to U.S. Patent number 5,639,661 (Welsh etc.), on June 17th, 1997 published.If Such is the fact, so may be based on chlorine (Cl ^-) the pharmacology scheme of transhipment can successfully regulate ionic equilibrium, but still can not alleviate basic physiological knowledge topic.

Second scheme of treatment cystic fibrosis, the i.e. direct CFTR activity that lacks of increase of " protein replacements " recombinant C FTR of seeking to send function to the CF mutant cell.It is simple that the protein of CF replaces the therapy notion: be about to merge the high purity recombinant C FTR goods of preparing in liposome or the recombinant viral vector at some and be administered into respiratory tract by instillation or aerosol.Yet the trial that preparation CF albumen replaces therapeutical agent has run into many difficulties.For example, CFTR is used for the soluble protein type that former protein replaces therapy or other therepic use.Also restricted for the membranin amount that can in reconstitution cell, express with biochemical activity.For example, report 10 is arranged in the document ⁵-10 ⁶Individual molecule/cell represents its upper limit (referring to Wang etc., J.Biol.Chem (1989) 264: 14424), by contrast, such as EPO, Regular Insulin, the secretory protein of tethelin and tPA are reported to 2000 molecule/second/cells.

Except ability to express is limited, CFTR, promptly a kind of purifying of purification ratio soluble protein of embrane-associated protein is more difficult.Membranin need dissolve in stain remover.Yet, in the presence of stain remover purifying CFTR represent be one than the required purge process of the soluble proteins process of poor efficiency more.Other potential obstacle that protein replaces scheme comprises: (1) is difficult to approaching by the airway epithelial that the harmful characteristic of the environment in mucus plugging and the CF respiratory tract causes; (2) potential immunogenicity; (3) CFTR may be invalid with the fusion of accepting cell.

The 3rd scheme of cystic fibrosis treatment is the gene therapy scheme, and the DNA of the CFTR that wherein will encode transfers to CF deficient cell (for example, the cell of respiratory tract).Yet, generally be invalid with the method for DNA transfered cell.Since virus in very effective mode with its nucleic acid transfered cell, the defective virus that therefore many gene therapy scheme utilizations are transformed.Yet virus vector admits the space of foreign gene limited.For example, although adeno associated virus (AAV) is an attractive gene therapy vector in many aspects, it only has 4.5Kb to can be used for admitting exogenous DNA.The DNA of coding total length cftr gene has represented its upper limit.In addition, the gene therapy scheme of CF is the same with protein therapeutic can face many identical clinical challenges.

Although to the gene of coding CFTR, the knowledge of expressed proteins product and mechanism of action has had impressive progress in the therapy of exploitation treatment CF based at present, each scheme for the treatment of CF all still has obstacle.Patient's CF M ﹠ M and mucus are gathered, and inflammation is relevant strongly with the tuberculosis that infection causes.Yet disappearance CFTR does not cause tangible tuberculosis in mouse, and showing has replaceable channel or other additional genetic expression to keep lung's homeostasis of mouse.In fact, found that the tuberculosis in the CF mouse model is alterable height.In general, tuberculosis can be slight or non-existent in CFTR mutant mouse.This shows that other gene of expressing influences the function of CFTR strongly in lung.

Be used to study CFTR although developed many external and body inner models, analyze genome reaction that whether CFTR exists owing to the heterogeneity of cell model is complicated with being independent of that CFTR influences the culture condition of cell function and genetic expression.Direct RNA to the human lung tissue of suffering from CF analyzes because almost ubiquitous serious pulmonary infection is and complicated, and this infection can be revised cell response and genetic expression once more, makes that the reaction of identifying CFTR in the body is complicated.

Therefore, still need to disclose and identify and to modify or to strengthen ion transport, chlorine (Cl ^-) transhipment or other co-transport ion, and other active gene or reagent of CFTR influence.The treatment target of identifying CF can be provided like this, and the new gene of potential, the ability of the therapy of reagent and treatment CF.Specifically, need to disclose or identify and more effectively in treatment CF influence ion transport, particularly chlorine (Cl ^-) transhipment alternative route.

The invention summary

The present invention relates to find the CFTR modifying factor, and Kir4.2 gene particularly, and polypeptide expressed, can be used for ability, or expression provides cell (CF-affected cell) function that affords redress and/or the activity of the CF-influence of invalid or poor efficiency CFTR function and/or active sudden change CFTR to treat cystic fibrosis (CF) disease by expressing CFTR for shortage.The invention still further relates to the hereditary instrumentality of having found to regulate this CFTR the modification of gene expression, and the function and/or the active polypeptides for modulating thing that influence each express polypeptide.

Therefore, the invention provides and detect and/or identify the CFTR modifying factor, the CFTR modified polypeptide of Biao Daing is regulated the hereditary instrumentality of this CFTR the modification of gene expression and is influenced the function of the CFTR modified polypeptide of expressing separately and/or the method and the product of active instrumentality separately.Some embodiments of detection/authentication method of the present invention and product comprise: (1) Kir4.2 gene and other CFTR modifying factor and polypeptide expressed thereof can be regulated genetic expression (promptly as screening or mensuration, the generegulation thing), maybe can influence the purposes of potential reagent (for example, medicine) of the functional performance (being the polypeptides for modulating thing) of express polypeptide; (2) CFTR-defective type transgenic nonhuman mammal (for example, mouse) is used to identify potential CFTR modifying factor, and the purposes of the CFTR modified polypeptide of expressing separately; (3) suitable carrier that will contain CFTR modifying factor and/or gene promoter imports Mammals (people and inhuman), yeast, or insect cell or clone are so that can use genetic expression or polypeptide function and/or active change to detect and/or identify potential heredity and/or polypeptides for modulating thing in the mensuration system.

The present invention also provides and has used these CFTR modifying factors, it is polypeptide expressed separately, and other reagent is used for the treatment of composition and the method for CF, comprise: containing or do not having under the condition of other treatment or reagent, the CFTR the modification of gene expression in the cell of the hereditary instrumentality adjusting of (1) use CF influence; (2) function and/or the activity of CFTR modified polypeptide in the cell of use polypeptides for modulating thing influence (for example, strengthening) CF influence; (3) the CFTR modified polypeptide is delivered in the cell of CF influence; And/or (4) are delivered to the CFTR modifying factor in the cell of CF influence.

Surprisingly find Kir4.2, and other CFTR modifying factor unexpectedly can compensate CFTR and lack, or have poor efficiency or invalid sudden change CFTRs (for example CFTR Δ F508) so that treatment CF, or cause the symptom of CF at least.Specifically, find that the Kir4.2 gene is by providing potassium (K ⁺) passage approach and unexpectedly influence and strengthen chlorine (Cl as an alternative ^-) ion transport.In addition, be surprised to find these potassium (K is provided ⁺) passage Kir4.2 genetic expression polypeptide can with such as stimulate chlorine (Cl by CFTR-dependent form passage ^-) all ingredients reaction of ring AMP (cAMP) stimulant (for example, forskolin and IBMX) of ion transport activated and/or regulate.Therefore, because activating, Kir4.2 also strengthens chlorine (Cl ^-) ion transport, therefore regulate Kir4.2 and express (when transcribing or after transcribing), or the function and/or the active pharmacology reagent of the Kir4.2 polypeptide of influence expression also can influence potassium (K valuably ⁺) ion transport, thereby influence chlorine (Cl ^-) ion transport.In fact, advantage of the present invention is to treat the ability of CF, and it passes through alternative route (for example, for Kir4.2, by potassium (K by the expression of regulating endogenous CFTR modifying factor and/or the function/activity that influences endogenous CFTR modified polypeptide ⁺) passage) influence ion transport valuably.

Description of drawings

Fig. 1 is that (relative intensity is at the y-axle with respect to differentially expressed screening (p-value＜0.05) mode chart of wild-type CFTR (+/+) mouse from the mRNAs of FABP-hCFTR/mCFTR (/-) CFTR-defective type intestines-rectification mouse lung results, the right age of mouse is increased in the x-axle and draws), show 27 genes, comprise the Kir4.2 gene, they are potential CFTR modifying factors.

Fig. 2 is a column diagram, shows that CFTR defective type intestines-rectification mouse increases with respect to the Kir4.2mRNA in the wild-type control mice lung.

Fig. 3 is the cloning potassium (K with respect to contrast ⁺) channel activity figure, described activity is that expressed (pA/pF is at the y-axle, the V that represents with mV for Kir4.2 gene pairs cAMP stimulant (combination of forskolin and IBMX) reaction _mAt the x-axle).

Fig. 4 shows CFTR (/-) and CFTR (+/+) each RNAs distribution plan of mouse, the left side is the histogram of log ratio and gene frequency for one group, the right side is the outlier skeleton diagram for one group, and wherein the dotted line end of representing with x and y mark is the outlier of identifying from its quartile (quartile) separately.

Fig. 5 is at existing or not having CFTR and the two dimension level of 315 gene/expressed sequence tag (ESTs) of obviously changing is cluster (dimensional hierarchial clustering) figure.

The expression spectrogram of Fig. 6 stable 54 selected RNAs that change at there not being CFTR.

Fig. 7 is that the level separately of 54 selected RNAs of Fig. 6 is a dendrogram.

Fig. 8,9,10 and 11 represent demonstration to Grind2d (Fig. 8) respectively, and Kir4.2 (Fig. 9), the mRNAs of CEBP δ (Figure 10) and TNFAIP3 (Figure 11) carry out the column diagram that PCR in real time is analyzed.

Figure 12,13,14 and 15 is respectively the iFABP-hCFTR from three monthly ages, CFTR (/-) mouse (Figure 13,15) and iFABP-hCFTR, CFTR (+/+) the fixing photo of the lung tissue of acquisition afterwards of brood mouse (Figure 12,14).

The concise and to the point description of sequence table

SEQ ID NO:1 shows the nucleotide sequence of mouse Kir4.2 gene cDNA.

SEQ ID NO:2 shows the Kir4.2 polypeptide of mouse Kir4.2 genetic expression.

SEQ ID NO:3 shows the nucleotide sequence of people Kir4.2 gene cDNA.

SEQ ID NO:4 shows the nucleotide sequence variation body of people Kir4.2 gene cDNA.

SEQ ID NO:5 shows another varient of the nucleotide sequence of people Kir4.2 gene cDNA.

SEQ ID NO:6 shows the Kir4.2 polypeptide of the people Kir4.2 genetic expression of SEQ ID NOs.3-5.

SEQ ID NO:7 shows another varient of the nucleotide sequence of people Kir4.2 gene cDNA.

SEQ ID NO:8 shows the Kir4.2 polypeptide of the people Kir4.2 genetic expression of SEQ ID NO.7.

Detailed Description Of The Invention

1. Definition

Term used herein " gene " is meant the sequence (for example, DNA and RNA) of the genetic material of the information of carrying coded polypeptide (for example, protein).

Term used herein " RNA " refers to RNA interchangeably, mRNA or tRNA.

Unless this paper has explanation in addition, term " polypeptide " is meant protein, polypeptide or peptide.

Term used herein " carrier " is meant and contains and the nucleotide sequence transfered cell can be caused expressing the DNA or the RNA of this nucleotide sequence in this cell, forms by it basically, or by its medium of forming.

Term used herein " expression vector " is meant and carries the modification plasmid or the virus that can import the suitable host cell and instruct encoded polypeptide expression or synthetic gene or cDNA therein.

Term used herein " plasmid " and " cloning vector " are used interchangeably, be meant can be in cell self-replicating, annular, general less extrachromosomal dna molecule.

Term used herein " cystic fibrosis is striden the film conduction and regulated polypeptide (Cystic FibrosisTransmembrane Conductance Regulator polypeptide) " or " CFTR polypeptide " are meant and contain two membrane spaning domains (MSDs), two Nucleotide is in conjunction with about 1480 amino acid whose protein of territory (NBDs) and single R structural domain, and it serves as the chlorine (Cl that regulated by phosphorylation and ribonucleoside triphosphote ^-) passage.Term " CFTR polypeptide " also can refer to keep the part of those cftr genes of cftr gene functional domain.

Term used herein " CFTR Δ 508 " and " CFTR Δ F508 " are used interchangeably, and are meant the CFTR mutant polypeptide that the phenylalanine of the 508th of the CFTR aminoacid sequence of can not encoding produces.

Term used herein " film conduction instrumentality (CFTR) function or activity are striden in cystic fibrosis " is meant the function of generally being finished by wild-type CFTR.This function can comprise mediation, regulates or the control ion (for example, chlorine (Cl ^-) ion) transport and pass cytolemma.

Term used herein " cell of cystic fibrosis (CF)-defective type or influence " is meant owing to lack CFTR, perhaps because the CFTR mutant polypeptide can not provide CFTR function and/or activity, CFTR function that perhaps provides and/or active poor efficiency and lack the cell that film conduction regulatory function is striden in cystic fibrosis.The example of this cell comprises the CFTR mutant (for example, CFTR Δ F508) of 1300 different variants identifying so far.Referring to, for example, Kunzelmann etc., " Pharmacotherapy of the IonTransport Defect in Cystic Fibrosis, " Clin.Exper.Pharm.Phys. (2001) 28: 857-67; Welsh etc., " Molecular Mechanisms of CFTR Chloride ChannelDysfunction in Cystic Fibrosis, " Cell (1993) 73: 1251-54.

Term used herein " CFTR modifying factor " and " CFTR compensates (compensateory) gene " are used interchangeably, be meant CFTR activity or function in the cell that can compensate CF-influence, comprise that expression can compensate the function of CFTR in the cell of CF-influence and/or the gene of active polypeptide.The example of CFTR modifying factor comprises following positive regulator gene: EST Affymetrix ID#92319 (but ubiquitin family member of mediating protein transportation); Guanine-nucleotide-binding protein α subunit (the receptor pathway albumen of G albumen coupling); Repetin (GenBank accession number X99251); Membrane glycoprotein (GenBank accession number Z22552); Ras-dependency induced by dexamethasone type albumen (DEXRAS1) mRNA; SWAP-70mRNA; Vq96e09.41 cDNA; Zinc finger protein (Peg3) mRNA; Uo89c05.x1 cDNA; Potassium channel 14 (ATP-sensitiveinward rectifier potassium channel 14, GenBank accession number AI314692) is corrected in input with ATP-susceptibility.The example of CFTR modifying factor also comprises following negative regulator gene: Preproapelin (GenBank accession number AB023494); Caspase-12 (GenBank accession number Y13090); Islet cell autoantigen 1 (GenBank accession number U37186); Natriuretic peptide precursor A type (Natriuretic peptide precursortype A, GenBank accession number K02781); MSox7 (GenBank accession number AB023419); Secretion frizzled related protein (secreted frizzled related protein) sFRP-2 (Sfrp2) mRNA (GenBank accession number U88567); U[-M-BH1-ang-b-04-0-U] .s1 cDNA (GenBank accession number AW050325); C88243 cDNA (GenBank accession number C88243); U[-M-BH0-ajq-h-03-0-U] .s1 (GenBank accession number AI853682); IB3 (GenBank accession number X79131); Butyrylcholine esterase (Butyrylcholinesterase) mRNA (GenBank accession number M99492); Homology frame A5 (homeo box A5, GenBank accession number Y00208); Connect protein 37 gap junctional membrane channel protein α 4 (Connexin 37 Gap junction membrane Channel protein α 4, GenBank accession number X57971); Wnt10a mRNA (GenBank accession number U61969); Calnexin (Calnexin, GenBank accession number L18888); Regulator homologue (GenBank accession number M60493); With mRNA (GenBank accession number X84797) similar in appearance to people's hematopoiesis specific proteins 1.Compensation exists gene this paper of the CF morbid state of inefficacy and/or poor efficiency CFTR mutant to be also referred to as the CFTR modifying factor.

Following table is to find to be subjected to up-regulated other gene with respect to mCFTR (+/+) mouse in mCFTR (/-) mouse:

Table 1 is subjected to up-regulated gene

Gene	Code name	Ratio	The P-value	Classification	Accession number
						Envelope protein (env) gene, 3 ends	?Env	?4.02	?1.19E-03	Antigen	M90535
The 3b that the lymphocyte that X-connects is regulated	?Xlr3b	?2.25	?1.48E-02	Antigen	NM_011727
						Polymyositis/scleroderma autoantigen 2	?Pmsc12	?2.53	?2.59E-02	Antigen	NM_016699
Calcium binding protein-D9K	?Calb3	?1.77	?3.33E-02	The calcium combination	NM_009789
						Phosphoglycerate phosphomutase 2	?Pgam2	?2.09	?9.10E-04	The glycolysis-metabolism	NM_018870
Natriuretic peptide acceptor 3	?Npr3	?2.37	?4.85E-03	Inflammatory reaction	NM_008728
						Chitinase, acidity	Chia-is unsettled	?1.50	?8.75E-03	Inflammatory reaction	NM_023186
G CFS 3 acceptors (granulocyte)	?Csf3r	?1.84	?2.86E-02	Inflammatory reaction	NM_07782
						Tumour necrosis factor, α-inducible protein 3	?Tnfaip3	?5.07	?2.96E-02	Inflammatory reaction	NM_009397
Mouse (Mus musculus) interleukin 4 (I1-4) mRNA, complete coding region	?I14	?1.32	?3.85E-02	Inflammatory reaction	U01310

Interleukin-11 β	?I11b	?1.67	?5.38E-02	Inflammatory reaction	BC013644
						S100 calcium binding protein A8 (calgranulin A)	?S100a8	?1.92	?9.48E-03	Inflammatory reaction/calcium combination	NM_013650
S100 calcium binding protein A9 (calgranulin B)	?S100a9	?2.39	?1.93E-02	Inflammatory reaction/calcium combination	NM_009114
						Passage is corrected in the potassium input, subfamily J, and the member 15	?Kcnj15	?3.28	?1.59E-02	Ion transport	NM_019664
The adaptin of inferring is in conjunction with the monomer GTPases of Rho subfamily	Mig-6/ gene 33	?2.12	?2.16E-02	RHO GTPase activator/inflammatory reaction	NM_133753
						3 of male parent expression	?Peg3	?1.74	?3.22E-02	Signal transduction	AB003040
Secretory granule neuroendocrine albumen 1,7B2 albumen	?Sgne1	?6.76	?4.33E-02	Signal transduction	NM_009162
						claudin?8	?Cldn8	?2.86	?4.16E-02	Closely connection/transhipment	BC003868
Mouse c-fos oncogene	?c-Fos	?1.75	?3.87E-03	Transcriptional regulatory	V00727
						Homologue in period (fruit bat)	?Per	?1.76	?1.90E-02	Transcriptional regulatory	NM_011065
Kruppel-like factor 1 (redness)	?K1f1	?2.63	?3.84E-02	Transcriptional regulatory	NM_010635
						RARG-1: but the vitamin A acid arrestin	?RARG-1	?1.50	?3.86E-02	Transcriptional regulatory	NM_023554
CCAAT/ enhancer binding protein C/EBP), δ	?Cebpd	?1.61	?4.15E-02	Transcriptional regulatory	NM_007679
						Solute carrier family 38, the member 4	?Slc38a4	?2.03	?1.95E-04	Transhipment	NM_027052
(ATPase 3 for precursor, macropain) 26S subunit for proteasome	?Psmc3	?1.54	?2.45E-02	Transhipment	NM_008948
						Express in the EST, lung		?3.92	?2.98E-02	Unknown	BG869733
Glutamate receptor, ionization, NMDA2D (ε 4)	?Grin2d	?3.50	?4.46E-02	Transhipment/acceptor	NM_008172
						Mouse membrane glycoprotein gene		?1.62	?5.73E-03	Unknown	Z22552
RIKEN?cDNA?1100001G20	?1100001G ?20Rik	?1.56	?1.74E-02	Unknown	AV006463

Following table is to find to be subjected to negative other gene of transferring with respect to mCFTR (+/+) mouse in mCFTR (/-) mouse:

Table 2 is subjected to the negative gene of regulating

Gene	Code name	Ratio	The P-value	Classification	Accession number
						SA rat hypertension dependency homologue	Sah	-4.44	?8.69E-04	Blood pressure regulation	NM_016870
Adenylate cyclase 4	Adcy4	-1.34	?3.72E-02	The CAMP biosynthesizing	NM_080435
						The formin sample	Fmnl	-2.21	?1.04E-03	The cell growth	NM_019679
Insulin-like growth factor binding protein 2	Igfbp2	-1.23	?6.99E-04	The cell growth	NM_008342
						Insulin-like growth factor binding protein 7	Igfbp7	-1.38	?4.23E-02	The cell growth	NM_008048
Precollagen, the XV type	Coll5a1	-2.07	?4.30E-02	Collagen	NM_009928
						RAD51-sample 1 (Saccharomyces cerevisiae)	Rad5111	-1.56	?1.32E-02	The DNA reorganization/	NM_009014

				The cell growth
						Neurofilament, heavy polypeptide	?Nfh	-1.92	?4.69E-05	Middle filament	M35131
Adaptin mixture AP-2, α 1 subunit	?Ap2a1	-1.52	?3.72E-02	The intracellular protein transportation	NM_007458
						Kinesin family member 3a	?Kif3a	-1.48	?3.72E-02	The intracellular protein transportation	NM_008443
ADP-ribosylation factor 5	?Arf5	-1.34	?1.06E-02	The intracellular protein transportation	NM_007480
Lipase, hormone-sensitive	?Lipe	-2.34	?1.82E-02	Lipid metabolism/cell growth	NM-010719
						Ki antigen; The Psme3 gene of PA28 γ subunit	?Psme3	-1.57	?5.44E-06	Protein degradation	NM_011192
Metallocarboxypeptidase 1	?CPX-1	-3.60	?4.44E-03	Protein degradation	NM_019696
						Yolk sac gene 2	?Ysg2	-3.21	?1.38E-02	Protein degradation	NM_011734
Calcemic factor-A (Pth) gene	?Pth	-2.13	?2.68E-03	Signal transduction	NM_020623
						Tryptophane 2, the 3-dioxygenase	?Tdo2	-2.01	?2.15E-02	Signal transduction	NM_019911
β-3-adrenergic receptor	?Adrb-3	-2.01	?1.31E-02	Signal transduction	NM_013462
						Janus kinases 3	?Jak3	-1.49	?1.24E-02	Signal transduction	NM_010589
Nuclear receptor subunit family 2, the F group, the member 1	?Nr2f1	-1.46	?3.28E-03	Signal transduction	NM_010151
						Interferon regulatory factor 1	?Irf1	-1.19	?8.51E-03	Transcriptional regulatory	NM_008390
Cystic fibrosis is striden the film conduction and is regulated homologue	?Cftr	-2.80	?3.63E-07	Transhipment	NM_021050
						Mouse gap junction protein gene connects protein 37	?GJA4	-1.81	?2.12E-02	Transhipment/cell-cell communication	NM_008120
Dna fragmentation, Chr 4, ERATO Doi 13	?D4Ertd13e	-2.07	?4.45E-02	Unknown	AI842362
						Riken cDNA 2700022J23 gene	?2700022j2 ?3Rik	-1.45	?3.72E-02	Unknown	AI853682

Term used herein " treatment cystic fibrosis (CF) ", " cystic fibrosis (CF) treatment " and similar terms comprise that the cell to the CF influence provides any detectable reducing, alleviate, improve, be benefited or the treatment of other positive effect (directly or indirectly), and for causing CF or any symptom relevant with CF, comprise the defective of the cell aspect of ion transport turnover CF influence, any detectable reducing is provided, alleviate, improve, be benefited or other positive improved any treatment.

Phrase used herein " treatment of CF modifying factor " with coding compensation CFTR function and/or active genetic material (for example is meant, DNA or RNA) shift the cell of CF-influence to reduce, alleviate, or improve the symptom of cystic fibrosis (CF), perhaps treat cystic fibrosis for certain.

Term used herein " CFTR (+/+) " is meant wild-type CFTR mouse.

Term used herein " CFTR (/-) " is meant the CFTR deficient mice.

Term used herein " FABP-hCFTR " is meant the people CFTR that expresses at the transgenic mice enteron aisle.

Term used herein " FABP-hCFTR/mCFTR (/-) " is meant the corrigent transgenic mice of CFTR defective type intestines.

Term used herein " CHO " is meant Chinese hamster ovary.

Term used herein " cAMP " is meant the ring adenosine monophosphate.

Term used herein " SP-C " is meant tensio-active agent albumen-C, is positioned at the major ingredient of the lung surface active liquid in Mammals and other breathing animal breath road.

Term used herein " SPC-h Δ 508 " is meant under the SP-C promoter sequence is regulated the transgenic mice of expressing human CFTR Δ 508 in pulmonary epithelial cells.

Term used herein " Kir4.2 gene " is meant input rectification potassium (K ⁺) channel gene (inwardrectirying potassium (K ⁺) channel), it is expressed and serves as potassium (K ⁺) passage polypeptide and found in the past kidney between the growth period and lung in and in comprising some adult's tissues of kidney and brain, express.The Kir4.2 gene is also referred to as KIR4.2 or KCNJ15, and is positioned on No. 21 karyomit(e) of people.Referring to Gosset etc., " ANew Inward Rectifier Potassium Channel Gene (KCNJ15) Localized onChromosome 21 in the Down Syndrome Chromosome Region 1 (DCR 1), " Genomics (1997) 44: 237-41, quote the document for your guidance.The same with many genes, the allelic variation body can produce similar basically polypeptide, and this paper provides the example from the mankind.The cDNA of mouse Kir4.2 gene has the nucleotide sequence shown in the SEQ IDNO:1 (GenBank accession number AF 085696), and the cDNA of people Kir4.2 gene has SEQ ID NO:3 (GenBank accession number NM_170737), SEQ ID NO:4 (GenBank accession number NM_170736), the nucleotide sequence shown in SEQ ID NO:5 (GenBank accession number NM_002243) or the SEQ ID NO:7 (GenBank accession number Y10745).There is not normal CFTR and having CFTR Δ 508 and do not having that Kir4.2 expression of gene level raises under the condition of normal CFTR.Found that Kir4.2 can express at the domain of dependence of pulmonary respiration road epithelium, thereby the activity that increases the expression of each Kir4.2 polypeptide (mouse is shown in SEQ ID NO:2, and the people is shown in SEQ ID NOs:6 and 8) or strengthen this polypeptide is to walk around chlorine (Cl ^-) transhipment and the CFTR-dependent form defective of cell function.The control region of analyzing Kir4.2 and cftr gene has demonstrated many similaritys, its potential functional cohesion is got up and has confirmed that Kir4.2 is that a kind of accident of CFTR modifying factor is found.For with other gene family member of Kir4.2 gene-correlation referring to Durell etc., " A Family of Putative Kir Potassium Channels in Prokaryotes, " BMCEvolutionary Biology (2001), 1-9 page or leaf; Fakler etc., " Heterooligomeric Assemblyof Inward-Rectifier K+Channels from Subunits of Different Subfamilies:Kir2.1 (IRK1) and Kir 4.1 (BIR10), " Pflugers Arch-Eur.J.Physiol. (1996) 433:77-83 (this paper quotes for your guidance).

Term used herein " gene promoter " is meant adjusting, the partial nucleotide sequence of this gene that control or adjustment (for example, stimulation or inhibition) specific gene is expressed.For example, gene promoter can strengthen this gene transcription and/or translation, thereby increases the mRNA level from this genetic transcription.

Term used herein " genetic expression " and " genetic transcription " are used interchangeably, and are meant the initial step that causes producing the gene polypeptide product in the dna molecular level.Therefore, it is genetic expression that genetic transcription is interpreted as the result in this article, perhaps produce by the polypeptide of the genes encoding of transcribing or expressing.

Term used herein " reporter gene " is meant the genetic material in the transfered cell, DNA normally, and this report gene is expressed in this cell under favourable condition, so that indication has reached the standard that satisfies these favourable conditions.These favourable conditions can include, but not limited to pH, and there is suitable transcription factor storehouse in ionic current, perhaps the molecule or the factor in other cell.

Term used herein " transcription factor " is meant in the genetic transcription first being processed, during, perhaps after in conjunction with DNA or cause other polypeptide in conjunction with polypeptide in the extensive cell of DNA.

Term used herein " hereditary instrumentality " is meant the material that regulatory gene is expressed.In the present invention, hereditary instrumentality typically refers to the material of the interior level of cell of at least a cell polypeptide (for example, protein) that particularly increases or reduce the CFTR the modification of gene expression in the cell of CF influence.The heredity instrumentality can comprise the endogenous cell polypeptide, pharmaceutical agent or other biologically active molecules.

The cell function and/or the active material of term used herein " polypeptides for modulating thing " is meant in the cell of CF influence can be influenced (for example, improve or reduce) at least a CFTR modified polypeptide.The polypeptides for modulating thing can stimulate, and strengthens, or increases the function and/or the activity of this polypeptide, and suppress or reduce this function or activity.Used herein in the cell of CF influence the function and/or the activity of CFTR modified polypeptide influenced by the polypeptides for modulating thing so that directly or indirectly regulate valuably, control or adjust ion transport (for example, chlorine (Cl ^-), potassium (K ⁺), etc.) or other CFTR function and/or activity, so that alleviate or treat CF for certain.

Term used herein " pharmaceutically useful salt " is meant the non-toxic salts (general by free acid and suitable organic or inorganic alkali reaction are prepared) of compound and includes, but not limited to acetate, benzene sulfonate, benzoate, supercarbonate, bisulfate, bitartrate, borate, bromide, calcium, camsylate, carbonate, muriate, clavulanate, Citrate trianion, dihydrochloride, edetate, edisylate, estolate, esylate, fumarate, gluceptate, gluconate, glutaminate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isothionate, lactic acid salt, Lactobionate, lauroleate, malate, maleate, mandlate, mesylate, MB, methyl nitrate, metilsulfate, mucate, napsylate, nitrate, oleate, oxalate, pamaote, palmitate, panthothenate, phosphoric acid salt, diphospate, Polygalacturonate, salicylate, stearate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, and valerate, and the mixture of these salt.

Term used herein " reagent ", " medicine " and " medicine " is used interchangeably, and is meant pharmaceutical compositions, and preparation or compound comprise the material as heredity and/or polypeptides for modulating thing.

Term used herein " Mammals " is meant people and non-human mammal, comprises Primates (for example, people, monkey, baboon, macaque), dog, cat, rabbit, rat, pallasiomy, hamster, mouse, horse, milk cow, goat and be referred to as mammiferous other kind.

Term used herein " experimenter " is meant the Mammals that comprises the CF susceptible.Transgenic nonhuman mammal also attempted to comprise in this term " experimenter "." experimenter " is also referred to as this paper interchangeable " patient ".

Term used herein " comprises " and is meant all ingredients that can be used in combination in the present invention, composition, compound, gene, polypeptide, composition, step etc.Therefore, term " comprises " and comprises that more restrictive term " is made up of it " basically and " being made up of it ".

All amounts used herein, part, ratio and percentage number average by weight, except as otherwise noted.

2. The CFTR modifying factor, the detection and the evaluation of hereditary instrumentality and polypeptides for modulating thing

Although the present invention relates to find lack cftr gene expresses, perhaps expressing can not provide or poor efficiency provides CFTR function or active sudden change CFTR, situation about in the experimenter who suffers from the CF disease, finding for example, but (for example at non-human mammal, the expression that can change the many genes CFTR modifying factor of Kir4.2 gene (that is, such as) mouse) is to provide the surface normal pulmonary function.The expression change of having found these CFTR modifying factors is a kind of compensatory adaptation.Because forfeiture CFTR function and/or active generally in human and other non-human mammal (for example, mouse) produce disease, so this CFTR modifying factor may compensate the effect of CFTR, particularly compensates the forfeiture that CFTR expresses.As a result, these compensatory CFTR modifying factors, and polypeptide expressed can be recovered the homeostasis of lung and can be used for treating CF.

Therefore, the present invention relates to be used for detect and/or identify the CFTR modifying factor, comprise such as the listed positive regulator gene of table 1, and such as the listed negative regulator gene of table 2, the CFTR modified polypeptide of its expression, regulate the hereditary instrumentality of this modification of gene expression and influence its function of polypeptide expressed and/or the method and product of active instrumentality separately.Can use various biologic materials and method to detect and/or identify potential CFTR modifying factor and polypeptide expressed separately, the potential hereditary instrumentality of CFTR modifying factor, and influence the function and/or the active potential polypeptides for modulating thing of polypeptide expressed separately, comprise drug screening; Test; The array and/or the probe of biological molecule (for example, Nucleotide or polypeptide); And mouse and other non-human mammal model.These detections and authentication method and product generally comprise and will contain potential CFTR modifying factor, the CFTR modified polypeptide, the sample of the hereditary instrumentality of CFTR modifying factor and/or the instrumentality of CFTR modified polypeptide contacts with a kind of indicator, in sample, there is potential CFTR modifying factor, the CFTR modified polypeptide, this indicator can identify when the hereditary instrumentality of CFTR modifying factor and/or the instrumentality of CFTR modified polypeptide.

Be used for detecting and/or identifying the CFTR modifying factor, the CFTR modified polypeptide, the suitable product of the hereditary instrumentality of CFTR modifying factor and the instrumentality of CFTR modified polypeptide, test kit and test can be the forms of array; Probe; Cross experiment; Sandwich test; Use dna sequencing and identify (comprising the analysis of using discontinuous a plurality of probes) by hybridization; Use the order-checking of Nucleotide and polypeptide, fingerprinting and mapping; And polypeptide (for example, protein) capture agent array.Referring to, for example, the U.S. Patent number 5,445,934 (Fodor etc.) of issue on August 29 nineteen ninety-five; The U.S. Patent number 6,027,800 (Cronin etc.) of issue on February 22nd, 2000; The U.S. Patent number 6,045,996 (Cronin etc.) of issue on April 4th, 2000; U.S. Patent number 6,077,673 (Chenchik etc.); The U.S. Patent number 6,268,210 (Baier etc.) of issue on July 31 calendar year 2001; The U.S. Patent number 6,270,961 (Drmanac) of issue on August 7 calendar year 2001; The U.S. Patent number 6,355,432 (Fodor etc.) of issue on March 12nd, 2002; And quote all these documents for your guidance.For example, for array, the matrix that the surface is comprised majority (or more typical be various) biological active agents is attached to the surface of different zones, this biological agents can be identified potential CFTR modifying factor, CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor and/or the instrumentality of CFTR modified polypeptide.So this array can be used for screening potential CFTR modifying factor, CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor and/or the instrumentality of CFTR modified polypeptide.For example, this array can with contain potential CFTR modifying factor, the CFTR modified polypeptide, the sample contact of the hereditary instrumentality of CFTR modifying factor and/or the instrumentality of CFTR modified polypeptide, this array also has the indicator that links with it, be used for identifying whether sample exists potential CFTR modifying factor, CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor and/or the instrumentality of CFTR modified polypeptide.

A. CFTR modifying factor and polypeptide

The method of detection and/or evaluation CFTR modifying factor generally is to seek the gene of compensation CFTR expression or activity change.For example, the change of genetic expression can disclose those genes that compensate CFTR expression or loss of activity (or minimizing) in the non-human mammal (for example, mouse) with transgeneic procedure CFTR expression.Being used for detecting and/or identifying that the method for CFTR modifying factor and product comprise uses transgenic nonhuman mammal as measuring the RNA source that the genetic expression of CF disease changes.These methods and product generally comprise and will contain the sample of potential CFTR modifying factor mixture, and the isolate of preferred mRNA or total cell RNA contacts with the indicator that can discern when having potential CFTR modifier mRNA in the sample.These methods and product include, but are not limited to RT-PCR, the Northern trace, and microarray, PCR in real time, or such as the biochip technology of Affymetrix.For example, can separate the mRNA sample that contains unknown mRNAs mixture, measure the change of the genetic expression that can improve the CF sickness influence then from transgenic mice with the sudden change of general or lung specificity cftr gene.This transgenic mice can comprise that having the specific expressed CFTR of transgenosis intestines makes CFTR-null mutation type mouse live through the CFTR-null mutation mouse of Adulthood.Also comprise and use CFTR null mutation type mouse with lung specificity transgene expression people Δ 508-mutant cftr gene.Express or activity change comprising to use CFTR-defective type transgenic mice to identify to compensate CFTR, in the method for those changes that for example in the CF disease, occur or the CFTR modifying factor of sudden change, from suffering from any organ or tissue of CF disease, include but not limited to, the upper respiratory tract epithelium of CFTR-deficient mice, lung, pancreas and intestines results RNA.

An embodiment of this detection and/or authentication method comprises the steps: that (1) provides CFTR mutant mouse or do not have the mouse (for example, by transgene expression CFTR mutator gene or the gene orthomutation by cftr gene) of CFTR; (2) separate the genetic material (normally RNA) of coding CFTR mutant polypeptides or separate the genetic material of the CFTR that do not encode from mouse from CFTR mutant mouse; The change of the genetic expression when (3) using this isolating genetic material evaluation can compensate CFTR sudden change or CFTR shortage.

Preferably this sudden change cftr gene is expressed lacking endogenous CFTR or have in the non-human mammal (for example, mouse) of CFTR-defective type, so that this mutator gene is interested tissue, and the unique CFTR source in the lung for example.Using Affymetrix or other gene chip can analyze from the mRNA expression level in the sample of the transgenic mice with sudden change CFTR and with mRNA expression level (that is contrast) in this sample of wild-type mice compares to measure those genes and has and potentially compensate impaired CFTR function and/or active expression level changes.Further analyze these potential CFTR modifying factor then to confirm that they are by compensating the homeostasis that CFTR function and/or active forfeiture (or impaired) change lung.This checking is usually included in expresses this gene in the low or undetectable cell type of this expression of gene, and measures the function and/or the activity of its encoded polypeptides.Checking also can comprise producing to have disappearance, false demonstration, or the transgenic mice of the described gene of overexpression is to confirm its effect of inferring as the CFTR modifying factor.

Also can use any arrays of immobilized protein known to those skilled in the art (proteomic) to measure the qualitative and quantitative change of systems measurement genetic expression.Referring to, for example, can be used for polypeptide (for example, protein) the capture agent array of this mensuration system in the United States Patent (USP) 6,365,418 (Wagner etc.) (this paper quotes for your guidance) of issue on April 2nd, 2002.Can be from people as herein described or non-human mammal results tissue or organ, fractional separation is with the isolated protein mixture, and is used to measure polypeptide function and/or active qualitative and/or quantitative change.Transgenic nonhuman mammal, for example, the CFTR-deficient mice of CFTR-defective type intestines-rectification mouse or expression sudden change CFTR, and has other sudden change to cftr gene, the mouse that comprises the gene orthomutation can be used for using the arrays of immobilized protein method to detect and/or evaluation CFTR modifying factor.An example that is used for the arrays of immobilized protein technology of this analysis is the Cyphergen system.But homogenate from such as the tissue of the mouse results of FABP-hCFTR/mCFTR (/-) mouse or organ and fractional separation with the isolated polypeptide mixture; The chip that will have the polypeptide mating surface then contacts with polypeptide mixture, uses analysis of biological information to measure and chip surface bonded polypeptide characteristic then.

B. The instrumentality of CFTR modifying factor and polypeptide

The gene region that this area is called gene promoter can be used in the mensuration system to detect and/or to identify the expression which reagent can the specific CFTR modifying factor of potential adjusting.In general, gene promoter comprises the trans-acting factor identification that produced in the cell and one group of cis element of bonded subsequently.The dna binding activity of these trans-acting factors is regulated all expression of gene in any cell.In general, trans-acting factor is the polypeptide that is called transcription factor.Some transcription factors are stimulable types and improve the level of genetic expression, and other are inhibition types and reduce expression level.Each cell type is compared specific one group of peculiar or eclipsed factor of express cell with other cell type.Each gene promoter needs specific one group of transcription factor.Therefore, the phenotype of cell depends on one group of transcription factor of this cell inner expression greatly.

Available reporter gene construct begins the promoter element in the analyzing gene promotor, and wherein gene promoter links to each other with reporter gene in the expression vector.The reporter gene construct can be imported in the homology colony of culturing cell, for example in the colony of immortalization mammal cell line or other suitable clone or type.If the required transcription factor of promotor in the reporter gene construct of this cell expressing q.s, this promotor will cause the reporter gene expression so.Can measure reporter gene polypeptide expressed amount then to measure by the amount or the activity of (1) transcription factor; (2) there is required cis element in the promoter sequence, the promoter activity level of generation.

Because transcription factor is a polypeptide, its activity can be regulated or influence by the polypeptides for modulating agent.Therefore, gene promoter can be placed in the reporter gene construct, suitable cell type is advanced in transfection, and is used to identify by at first influencing the reagent that the indirect regulation reporter gene is expressed in conjunction with the transcription factor activity of testing gene promotor cis element.Provide exploitation to be used to detect like this and/or identified the method for the potential hereditary instrumentality that can regulate the CFTR the modification of gene expression and the ability of product.

In order to make up reporter gene, can use plasmid cloning vector to make up the dna molecular that comprises the testing gene promotor that is operably connected with the reporter gene encoding sequence.Also comprise Mammals intron and SV-40 Poly A sequence in this carrier.Reporter gene can comprise CAT, LacZ, green fluorescent protein (GFP), luciferase, or any other suitable reporter gene.Use comprises calcium phosphate or calcium chloride co-precipitation, the transfection of deae dextran mediation, and the fat transfection, or the various technology arbitrary well-known to those having ordinary skill in the art of electroporation can be with in this construct transfered cell.Cell can be handled with single agents, or amplification and be seeded on 96-or the 384-hole flat board and be used for extensive screening.Allow screening of medicaments library or combinatorial library detecting and/or to identify the reagent of potential as hereditary instrumentality like this, this reagent is used for other cell processes that activation of endogenous transcription factor or regulatory gene are transcribed.But also sequenced genes promoter sequence and use the potential cis element of bioinformatic analysis to determine to encode in the promoter sequence.

In one embodiment, with promoter region, for example the promoter region of the promoter region of Kir4.2 gene and CFTR and pulmonary surfactant protein-D compares.Comprise cAMP response element binding protein (CREBP) with the example of Kir4.2 in the mouse and the total element of CFTR, C-Ets-1, cut-iMe homeodomain protein, liver nf 1, slow virus Poly a-signal is conjugated protein, the nf (NFAT) of activation T-cell, ocatmer-binding factor 1, Pax-3, PU.1, retrovirus Poly A downstream components is conjugated protein, STAT, and RFX1.Therefore, these transcription factors are to regulate the target of the reagent of Kir4.2 expression.

In another embodiment, also can will comprise such as the reporter gene construct transfection of the CFTR modifying factor promoter region of Kir4.2 to advance in the cultured cells so that the assay method of identifying CFTR modifying factor heredity instrumentality to be provided.Can be from mouse as herein described results pulmonary epithelial cells or from the primary culture of the cell of other organ and be used for a kind of assay method, this assay method is used to identify hereditary instrumentality that can serve as the CFTR modifying factor and/or the reagent that serves as the instrumentality of CFTR modified polypeptide.Can use to comprise calcium phosphate or calcium chloride co-precipitation, the transfection of deae dextran mediation, the fat transfection, or the various technology arbitrary well-known to those having ordinary skill in the art of electroporation are with in this construct transfered cell.Cells transfected can include but not limited to former generation, conversion, or the cell of immortalization.The source of cells transfected can include, but are not limited to Mammals (people and inhuman), yeast, or insect cell.Cell can be handled with single agents, or amplification and be seeded on 96-or the 384-hole flat board and be used for extensive screening.Allow screening of medicaments library or combinatorial library to detect and/or to identify the reagent that potential is used to activate the endogenous transcription factor or promotes other cell processes of CFTR modification of gene transcription like this.

In the mensuration system, can use the polypeptid coding area of CFTR modifying factor, or polypeptide itself is to detect and/or to identify the potential reagent that serves as the polypeptides for modulating thing.Use the detection of CFTR modifying factor (for example, Kir4.2 gene) and polypeptide expressed thereof and/or identify that the method and the product of polypeptides for modulating thing comprise: (1) is used to screen the cell culture assays detection system of potential reagent; (2) experimentize with potential reagent and handle the arrays of immobilized protein assay method of the CFTR modified polypeptide of expressing the back.

In the cell culture assays detection system, the CFTR modifying factor can be imported in any cell type widely and therein and express.The cell screening combinatorial library of expressing the CFTR modifying factor be can use then or the function and/or the active material (that is, being the polypeptides for modulating thing) of this express polypeptide independently can be influenced in compound or the medicine.Cells transfected can include but not limited to former generation, conversion, or the cell of immortalization.The source of cells transfected can include but not limited to Mammals (people and non-human mammal), yeast, or insect cell.Transfection method can be to comprise calcium phosphate or calcium chloride co-precipitation, the transfection of deae dextran mediation, fat transfection, or the various technology arbitrary well-known to those having ordinary skill in the art of electroporation.Cells transfected can be handled with single agents, or amplification and be seeded on 96-or the 384-hole flat board and be used for extensive screening.Can be by the use indicator dye, fluorescence, chemoluminescence, or the activity of CFTR modified polypeptide is measured or measured to other indicator method of reflection ionic concn change.Indicator dye, fluorescence, chemoluminescence, the use that is used for other indicator method of this purpose is well-known to those having ordinary skill in the art.

In an embodiment of this detection and/or authentication method, to be placed on such as the CFTR modifying factor cDNA nucleotide sequence of mouse Kir4.2 cDNA nucleotide sequence and contain the human cytomegalic inclusion disease virus promotor, in the expression vector of Mammals intron and Sv-40poly-A sequence.The expression vector stable transfection that will contain Kir4.2 advances in the Chinese hamster ovary celI.Kir4.2 mRNA can detect by RT-PCR, and the Kir4.2 polypeptide can detect by the Western trace, thereby confirms to have expressed Kir4.2 cDNA.Use such as the cAMP-stimulant of forskolin and/or IBMX and handle this cell.After experiment is handled, compare by cell and can measure potassium (K with untransfected ⁺) increase of ionic current.

Use arbitrary arrays of immobilized protein known to those skilled in the art to measure the qualitative and quantitative change that system also can measure CFTR modified polypeptide expression level.The reagent of identifying in above-mentioned cell culture assays detection system can be used for handling in further external or the body.Cell can be from any cell culture assays as herein described system, perhaps from any animal as herein described.But harvested cell or tissue, fractional separation are with the isolated protein mixture, and the CFTR modified polypeptide function and/or the active change of mensuration reflection experiment processing.Detect and/or identify that the method for potential polypeptides for modulating thing comprises the use transgenic nonhuman mammal, for example, intestines-corrigent CFTR-deficient mice, FABP-hCFTR/mCFTR (/-) or express the CFTR-deficient mice of sudden change CFTR, the SPC-h Δ 508/FABP-hCFTR/mCFTR (/-) that is used for arrays of immobilized protein test, and having other sudden change to cftr gene, the mouse that comprises the gene orthomutation is as the model system that experimentizes and handle by the inventive method.In addition, wild-type mice and other non-human mammal species also can be used as experimental animal to measure the effectiveness that influences CFTR modified polypeptide function and/or active reagent.This reagent can be gathered in the crops organ and the tissue of these animals and measure the change that the CFTR modified polypeptide is expressed after the experiment administration.

3. The CFTR modifying factor, the purposes of polypeptide and instrumentality

The invention still further relates to and use CFTR modifier gene, comprise the positive regulator gene that table 1 is listed, and the listed negative regulator gene of table 2, it is polypeptide expressed separately, the hereditary instrumentality of CFTR modifying factor, and/or the polypeptides for modulating thing of CFTR modified polypeptide treatment CF, or the symptom that causes of CF at least, comprise adjusting, the ion transport of the cell of control or adjustment CF-influence.The CFTR modifying factor, polypeptide expressed separately, hereditary instrumentality and polypeptides for modulating thing can be used for the treatment of CF separately, perhaps can be in conjunction with being used for the treatment of CF.For example, can use heredity and polypeptides for modulating thing combination therapy CF.

A. The hereditary instrumentality of CFTR the modification of gene expression

Be used for CFTR modifying factor of the present invention, and Kir4.2 gene particularly, suitable hereditary instrumentality comprise transcription factor (for example, AP1, PU.1); Strengthen the proto-oncogene of transcribing; Interferon-gamma and its analogue; Barbiturate and analogue thereof; NF-κ B and analogue thereof; The nf of active cells and calcium channel activators; PU.1, for example ets factor reagent and GM-CSF; IL-6, IL-1 α, IL-1 β, INF-γ and its analogue; The cAMP analogue, adenylate cyclase activating agent and cAMP phosphodiesterase inhibitor; Retinoid-like and orphan receptor (orphan recaptor) activator, retinoic acid receptor (RAR) agonist, Vogan-Neu, vitamin A acid and its analogue; Steroid generates the factor (steriodogenic factor), glucocorticosteroid and glucocorticoid analogue, mineralocorticoid, oestrogenic hormon, progesterone and its analogue; Betamethasone Valerate (betamethasone), Decadron; With its mixture.

Have been found that the CFTR modifying factor, and Kir4.2 gene particularly, can directly or indirectly influence cell, the cell of CF influence particularly, the ion transport turnover, specifically, can influence the chlorine (Cl of the cell of CF influence ^-) ion transports.Specifically, have been found that and influence substrate outside potassium (K ⁺) the Kir4.2 gene of ion transportation also can strengthen (directly or indirectly) top chlorine (Cl ^-) ion transportation.This means the stimulation of Kir4.2 gene or express to increase and to strengthen normally and the chlorine (Cl in the CFTR-damaged cells particularly ^-Therefore) ion transportation, and can walk around and it is believed that the chlorine (Cl that causes CF ^-) transhipment and the CFTR-dependent form defective of cell function.

CFTR modifying factor instrumentality can be mixed with therapeutic composition or the packing medicine is used for the treatment of the experimenter who suffers from CF.Therapeutic composition comprises at least a above-mentioned hereditary instrumentality for the treatment of significant quantity and the pharmaceutically useful carrier of choosing any one kind of them.The packing medicine comprises at least a above-mentioned hereditary instrumentality, the pharmaceutically useful carrier and use the specification sheets that the treatment of this heredity instrumentality suffers from the experimenter of CF of choosing any one kind of them.Specification sheets can be write or be printed on the scraps of paper, can with the packing drug packaging together, can be to load, install (directly or by remote download, for example by LAN, WAN or internet), perhaps can pass through computer, the electronic media that personal digital assistant (PDA) or other electronic installation read or the form of software (for example, floppy disk or CD ROM disk), or provide how to use any other method that this heredity instrumentality is treated the experimenter's who suffers from CF explanation.

Although above-mentioned hereditary instrumentality can be individually dosed, they are preferably as a part of administration of pharmaceutical formulation.This preparation can comprise pharmaceutically useful carrier known to those skilled in the art, and other therapeutical agent.Can expect that also hereditary instrumentality of the present invention can various pharmaceutically useful forms, for example, as its pharmaceutically useful salt administration.

The suitable dose of the hereditary instrumentality of administration and preparation depends on the type of CFTR sudden change or defective and the seriousness of the symptom of being treated according to the present invention, and also can change according to different experimenters.Measure acceptable or best dosage and generally comprise the level of balance therapy benefit and any danger or the harmful side effect of dosage of the present invention and treatment.Dosage for " treatment effectively " must have required effect, that is, regulate the expression of the CFTR modified polypeptide that this paper limits, thereby cause experimenter's useful improvement, for example transports (for example, Cl with the cell plasma of the CF-influence of this dosage treatment ^-Secretion) improves.Optimal dose can be when giving when suffering from experimenter's administration of CF, for example causing ion transport (for example, chlorine (Cl ^-) secretion) be improved to or near the dosage of wild-type CFTR level.

Except this heredity instrumentality, pharmaceutical formulation of the present invention also can comprise other compound and/or the composition that also helps to alleviate the CF symptom, comprises CFTR modified polypeptide instrumentality hereinafter described.The invention provides the pharmaceutical formulation of treatment CF, it comprises the above-mentioned suitable hereditary instrumentality of safety and treatment significant quantity, the following CFTR modified polypeptide instrumentality of pharmaceutically useful carrier and safety and treatment significant quantity.This heredity instrumentality can make up with more than one polypeptides for modulating thing.The ratio of heredity instrumentality and polypeptides for modulating thing depends on the required dosage of each individualized compound.Preferably, this polypeptides for modulating thing can be used as pharmaceutically useful aqueous solution administration, and wherein this pharmaceutical formulation comprises: the hereditary instrumentality of (1) from about 0.001% to about 10%; (2) from about 10% to about 99% pharmaceutically useful carrier; (3) from about 0.001% to about 10% polypeptides for modulating thing hereinafter described.

The administration that contains or do not contain the hereditary instrumentality of pharmaceutically useful carrier and/or other polypeptides for modulating thing can be by any suitable way, comprises mouth, nose, local (comprising cheek and hypogloeeis), and parenteral (comprise subcutaneous, intramuscular, intravenously and intradermal), vagina or rectum, preferred port and nasal administration.Therefore this preparation comprises those preparations that are adapted to pass through this administration.Can reckon with that optimization approach can be with for example, experimenter's situation and age and change.This preparation is suitable for providing with unit dosage form, and for example, tablet and the lasting capsule that discharges, and any method preparation and the administration that can know by the technician of pharmaceutical field comprise the liposome transfer system.

The preparation of the present invention that is suitable for oral administration can be isolating unit, capsule for example, and cachet or tablet are powder or particle, perhaps are solution, suspension or emulsion.Tablet form can comprise one or more lactose, N.F,USP MANNITOL, W-Gum, yam starch, Microcrystalline Cellulose, gum arabic, gelatin, silicon dioxide colloid, croscarmellose sodium, talcum powder, Magnesium Stearate, stearic acid and other vehicle, tinting material, thinner, buffer reagent, moistening agent, sanitas, seasonings and medicinal compatible carrier.The preparation that is suitable for oral cavity local medication also is included in the lozenge of administration in suitable matrix or the liquid vehicle, lozenge, mouth wash shua and the agent of suction mist.Lozenge form can be included in such as the active ingredient in sucrose and gum arabic or the tragacanth, and contain such as gelatin and glycerine or sucrose and gum arabic emulsion the lozenge of the active ingredient in the inert base of gel and contain analogue except active ingredient such as carrier known in the art.The preparation that is suitable for being locally applied to skin can be used as and contains the ointment that remains to compound and pharmaceutically useful carrier, emulsifiable paste, and gel and paste perhaps provide with endermic plaster.

If to be suitable for the preparation carrier of nasal administration be solid then comprise that granular size is, for example about 20 to 500 microns, can be by the powder of the rapid inhalation of nasal passage.If being liquid then suitable preparation, carrier can be used as, for example, and nasal spray or drops administration.The preparation that is adapted to pass through inhalation comprise put into pressurization such as Refrigerant 12, trichlorofluoromethane, propane, the aerosol preparation in the acceptable propelling agent of nitrogen etc.This promoting agent can be with suitable vehicle aerosolization.For inhalation, this preparation preferably is dissolved in or is scattered in such as in water or the brinish liquid form with such concentration, but promptly fully dissolves and administration proper dosage in the volume that can suck in said composition under this concentration.Proper dosage should be and every day every liter of about composition of 0.001 to about 5.0mmol is placed on about 4 times of respiratory tract surface.Administration can repeat several times in one day, depended on the speed that the composition of the given dose of selection and selection is removed from respiratory tract, and purpose is to keep the chlorine permeability of airway epithelial cell.Can or indicate the sucker administration of dosage by atomizer.The appropriate method of aerosol administration that is used for hereditary instrumentality is also at the United States Patent (USP) 5,543,399 (Riordan etc.) of on August 6th, 1996 issue; The United States Patent (USP) 5,641,662 (Debs etc.) of issue on June 24th, 1997; The United States Patent (USP) 5,827,703 (Debs etc.) of issue on October 27th, 1998; The United States Patent (USP) 5,756,353 (Debs) of issue on May 26th, 1998; The United States Patent (USP) 5,858,784 (Debs etc.) of issue on January 12nd, 1999; The United States Patent (USP) 5,948,681 (Scanlin etc.) of issue on September 7th, 1999; With open in the United States Patent (USP) of issuing on December 14th, 1,999 6,001,644 (Debs etc.), quote all these documents for your guidance.

The preparation that is suitable for administered parenterally comprises can contain antioxidant, buffer reagent, bacteriostatic agent and the moisture and anhydrous aseptic injectable solution that causes this preparation and the isoosmotic solute of purpose recipient blood; With the moisture and anhydrous sterile suspension that can comprise suspension agent and thickening material.The preparation that is suitable for intravenously and intraperitoneal administration can comprise, for example, can contain antioxidant, buffer reagent, bacteriostatic agent and cause the moisture and anhydrous of this preparation and the isoosmotic solute of purpose recipient blood, isotonic sterile injection solution with can comprise suspension agent, solubilizing agent, thickening material, the moisture and anhydrous sterile suspension of stablizer and sanitas.This preparation can be in unit container or multi-dose container, for example, in the ampere and bottle of sealing, and can be freeze dried, only needs the sterile liquid carrier of interim before use adding such as injection water.Can be from the sterilized powder of type noted earlier, interim injection solution of particle and tablet preparation and suspension.

The preparation that is suitable for vagina administration can be used as hysterophore, embolism, emulsifiable paste, gel, paste, foaming agent or sprays administration.The preparation of rectal administration can be used as the suppository that contains suitable matrix and provides.

B. The function of CFTR modified polypeptide or active instrumentality

The CFTR modified polypeptide, and Kir4.2 polypeptide particularly, function and/or active suitable adjustable thing comprise the reagent for adenylate cyclase in the activation target cell of alleviating the useful dosage of CF symptom known to those skilled in the art, for example, adrenergic reagent, catacholamines, cAMP agonist and cAMP additive, forskolin for example, Racemic isoproterenol and salbutamol (albuterol), cAMP and analogue thereof; Various polypeptide hormones for example, stimulate the beta-hypophamine of cAMP; The cAMP phosphodiesterase inhibitor that suppresses the cAMP degraded, alkyl-yellow purine for example, theophylline and aminophylline; The cAMP-specific inhibitor, Rolipram (Shearing AG) for example, glucocorticosteroid, TGF-β (SMAD ₃); Potassium K _ATPPassage is opened agent, cromakalim for example, Pinacidil (pinacidil), Nicoril (nicorandil), minoxidil sulfate (nimoxidil sulphate), aprikalim, diazoxide (diazoxide); Potassium BK _CaPassage is opened agent, NS004 for example, benzimidazolone, 1-ethyl-2-benzimidazolone (1-EBIO) for example, fenamates, dehydroxoyasaponin-I (DHS-I), maxikdiol, cromakalim, nirendipine, and Phloretin; UTP, the 8-oxsoralen (Methoxsalen, 8-MOP), and genistein; The calcium ion agonist, ionomycin for example, A23187, carbachol, bradykinin, Moli 1901 and thapsigargin; People DNase 1; Sodium channel inhibitor, for example guanamprazine (amiloride) and triamterene (triamterene); The pancreatin additive; And composition thereof.

Be used for suitable alkyl-yellow purine of the present invention and comprise methyl xanthine, 3-isobutyl-1-methylxanthine (IBMX) and 1 for example, 3-dimethyl xanthine (theophylline) and other xanthine, for example Papaverine, Pentoxifylline and trimethyl-xanthine.Also can be referring to the United States Patent (USP) 5 of issue on November 22nd, 1994,366,977 (Pollard etc.) (this paper quotes for your guidance), antagonism A1-adenosine cell receptor that this paper is suitable for is wherein disclosed and the compound of antagonism A2-adenosine cell receptor and comprise 8-cyclopentyl-1 not, 3-dipropyl xanthine (CPX), the amino homologue of xanthine (8-[4-[2-aminoethyl aminocarboxyl methoxyl group]-phenyl]-1,3-dipropyl xanthine, XAC), or its treatment go up effective derivative.

Be used for suitable benzoglyoxaline of the present invention or benzimidizole derivatives United States Patent (USP) 6 in issue on December 12nd, 2000,159, those disclosed material in 968 (Cuppoletti) (this paper quotes for your guidance), 2-[(pyridyl particularly)-and methylsulfinyl or methylthio group] benzimidizole derivatives and its salt, omeprazole (omeprazole) for example, lansoprazole (lansoprazole), thimoprazole and pantoprazole, and following illustrative compound: sulfydryl 4-trifluoromethyl-2-[(4-methoxyl group-2-picolyl)]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-3-methyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-5-methyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-3,5-dimethyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-3-methyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-5-methyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-3,5-dimethyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-2-picolyl)-sulfinyl]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-3-methyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-5-methyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-3,5-dimethyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-3-methyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-5-methyl-2-picolyl) sulfinyl]-(1H)-and benzoglyoxaline and 5-trifluoromethyl-2-[(4-methoxyl group-3,5-dimethyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 2-[2-(4-methoxyl group)-methyl sulfinyl]-(5-ethanoyl-6-methyl)-benzoglyoxaline; 2-[2-(4-methoxyl group)-methyl sulfinyl]-(4, the 6-dimethyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-ethanoyl-6-methyl)-benzoglyoxaline; 2-[2-(4-methoxyl group)-methyl sulfinyl]-(5-methoxycarbonyl-6-methyl)-benzoglyoxaline; 2-[2-(4-oxyethyl group)-methyl sulfinyl]-(5-methoxycarbonyl-6-methyl)-benzoglyoxaline; 2-[2-(3-methyl-4-methoxyl group)-methyl sulfinyl]-(5-methoxycarbonyl-6-methyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-methoxycarbonyl-6-methyl)-benzoglyoxaline; 2-[2-(4-methoxyl group-5-methyl)-methyl sulfinyl]-(5-methoxycarbonyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-the 5-methoxycarbonyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-ethanoyl)-benzoglyoxaline; 2-[2-(4-methoxyl group-5-methoxyl group)-methyl sulfinyl]-(5-methoxyl group)-benzoglyoxaline; 2-[2-(3,5-methyl-4-methoxyl group)-methyl sulfinyl]-(5-methoxyl group)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-methyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-chlorine)-benzoglyoxaline; 2-[2-[3-methyl-4-(2,2, the 2-trifluoro ethoxy) pyridyl] methylsulfinyl] benzoglyoxaline (lansoprazole); 2-[2-[3-methyl-4-(2,2,3,3-tetrafluoro propoxy-) pyridyl] methyl sulphur] benzoglyoxaline; The 2-[(2-pyridyl) methylsulfinyl] benzoglyoxaline (thimoprazole); 2-[2-(3,5-dimethyl-4-methoxypyridine base) methylsulfinyl]-5-methoxyl group-1H-benzoglyoxaline (omeprazole); 2-[2-[4-(3-methoxy propoxy)-3-picolyl] methylsulfinyl]-the 1H-benzoglyoxaline; 2-[2-(3,4-dimethoxy-pyridine base) methylsulfinyl]-5-difluoro-methoxy-1H-benzoglyoxaline (pantoprazole); 4-methyl-3-(2,2, the 2-trifluoro ethoxy)-5H-pyrido [1 ', 2 ': 4,5] [1,2,4] thiaziano[2,3-a] benzoglyoxaline-13-a tetrafluoro borate or its pharmaceutically useful salt.

The same with CFTR heredity instrumentality, CFTR modified polypeptide instrumentality can be mixed with therapeutic composition or the packing medicine is used for the treatment of the experimenter who suffers from CF.This therapeutic composition comprises at least a aforementioned polypeptides instrumentality and a kind of pharmaceutically useful carrier for the treatment of significant quantity.This packing medicine comprises at least a aforementioned polypeptides instrumentality and uses the specification sheets that this polypeptides for modulating thing treatment suffers from the experimenter of CF.Specification sheets can be write or be printed on the scraps of paper, can with the packing drug packaging together, can be to load, install (directly or by remote download, for example by LAN, WAN or internet), perhaps can pass through computer, the electronic media that personal digital assistant (PDA) or other electronic installation read or the form of software (for example, floppy disk or CD ROM disk), or any other the suitable method that this heredity instrumentality is treated the experimenter's who suffers from CF explanation of how using is provided.

The suitable dose of the CFTR modified polypeptide instrumentality of administration and preparation depends on the type of CFTR sudden change or defective and the seriousness of the symptom of being treated according to the present invention, and also can change according to experimenter's difference.Measure acceptable or best dosage and generally comprise the level of balance therapy benefit and any danger or the harmful side effect of dosage of the present invention and treatment.Dosage for " treatment effectively " must have required effect, promptly, influence the function and/or the activity of the CFTR modified polypeptide of this paper qualification, thereby cause experimenter's useful improvement, for example, cell plasma transhipment (for example, chlorine (Cl with the CF-of this dosage treatment influence ^-) secretion) improve.Optimal dose can be when giving when suffering from experimenter's administration of CF, for example causing ion transport (for example, chlorine (Cl ^-) secretion) be improved to or near the dosage of wild-type CFTR level.

The suitable medication that is used for these CFTR modified polypeptide instrumentalities, pharmaceutical formulation, pharmaceutically useful carrier, wait can with used same or similar of foregoing hereditary instrumentality.The appropriate method of aerosol administration that is used for CFTR modified polypeptide instrumentality is also at the United States Patent (USP) 5,543,399 (Riordan etc.) of on August 6th, 1996 issue; The United States Patent (USP) 5,641,662 (Debs etc.) of issue on June 24th, 1997; The United States Patent (USP) 5,827,703 (Debs etc.) of issue on October 27th, 1998; The United States Patent (USP) 5,756,353 (Debs) of issue on May 26th, 1998; The United States Patent (USP) 5,858,784 (Debs etc.) of issue on January 12nd, 1999; The United States Patent (USP) 5,948,681 (Scanlin etc.) of issue on September 7th, 1999; With open in the United States Patent (USP) of issuing on December 14th, 1,999 6,001,644 (Debs etc.), quote all these documents for your guidance.

C. The treatment of CFTR modified polypeptide

Provide CFTR modifier function and/or active any method can realize the polypeptide treatment for these cells by the cytolemma that the CFTR modified polypeptide is effectively imported the CF-influence.The significant quantity of CFTR modified polypeptide (that is, is enough to reduce, alleviates, improve, the reagent Combined Preparation that or the amount of the improvement symptom relevant with CF) can pass (for example, through merging or endocytosis) cytolemma separately or with help is to CF experimenter (that is the experimenter who, has the CF deficient cell).Those skilled in the art is according to type and seriousness such as the symptom of being treated, experimenter's body weight and/or age, and the case history before the experimenter, the factor of the route of administration that this reagent is selected can determine what is " significant quantity ".

Reorganization or natural CFTR modified polypeptide can use such as ion-exchange chromatography gel filtration chromatography, the currently known methods of electrophoresis and affinity chromatography purifying from host cell.Referring to Tilly etc., J.Biol.Chem., (1992) 267(14): 9470-73; Anderson etc., Science (1991) 251: 679-682).An embodiment of purification process is included in non-sex change stain remover existence and at first dissolves this protein down.

In order to be used for protein therapeutic, generally with CFTR modified polypeptide and lipid such as stain remover or other amphipathic molecule group, the film vesicle, liposome, virosome, or microsome links.Preferred especially natural fusion or can be transformed into the lipid composite of fusion (for example, by fusion rotein is mixed in the lipid).Fusion rotein can be from virus, parainfluenza virus 1-3 for example, and respiratory syncytial virus (RSV), influenza A, the fusion rotein of Sendai virus and togavirus obtains.Non-virus amalgamation protein comprises the normal cell protein of mediated cell-cytogamy.Other non-virus amalgamation protein comprises sperm protein PH-30, and it is the integral protein that is positioned at the spermoblast surface, it is believed that the fusion between its mediation sperm and the ovum.Referring to Blobel etc., Nature (1992) 356: 248-251.Also have other non-virus amalgamation protein to comprise the chimeric PH-30 albumen that combines composition and PH-30 and decomposition of protein (disintegrin) (for example, bitistatin, barbourin, kistrin, and echiststin) such as PH-30 with influenza virus hemagglutinin.In addition, can use traditional chemical fusogen to merge adipose membrane such as polyoxyethylene glycol (PEG).

The same with CFTR heredity instrumentality, the CFTR modified polypeptide can be mixed with therapeutic composition or the packing medicine is used for the treatment of the experimenter who suffers from CF.This therapeutic composition comprises at least a above-mentioned CFTR modified polypeptide and a kind of pharmaceutically useful carrier for the treatment of significant quantity.This packing medicine comprises at least a above-mentioned CFTR modified polypeptide and uses the specification sheets that this polypeptides for modulating thing treatment suffers from the experimenter of CF.Specification sheets can be write or be printed on the scraps of paper, can with the packing drug packaging together, can be to load, install (directly or by remote download, for example by LAN, WAN or internet), perhaps can pass through computer, the electronic media that personal digital assistant (PDA) or other electronic installation read or the form of software (for example, floppy disk or CD ROM disk), or any other the suitable method that this heredity instrumentality is treated the experimenter's who suffers from CF explanation of how using is provided.

The suitable dose of the CFTR modified polypeptide of administration and preparation depends on the type of CFTR sudden change or defective and the seriousness of the symptom of being treated according to the present invention, and also can change according to experimenter's difference.Measure acceptable or best dosage and generally comprise the level of balance therapy benefit and any danger or the harmful side effect of dosage of the present invention and treatment.Dosage for " treatment effectively " must have required effect,, provides useful improvement to the experimenter that is, for example, transports (for example, chlorine (Cl with the cell plasma of the CF-influence of this dosage treatment ^-) secretion) improve.Optimal dose can be when giving when suffering from experimenter's administration of CF, for example causing ion transport (for example, chlorine (Cl ^-) secretion) be improved to or near the dosage of wild-type CFTR level.

The suitable medication that is used for these CFTR modified polypeptides, pharmaceutical formulation, pharmaceutically useful carrier, wait can with used same or similar of foregoing heredity and polypeptides for modulating thing.The appropriate method of aerosol administration that is used for the CFTR modified polypeptide is also at the United States Patent (USP) 5,543,399 (Riordan etc.) of on August 6th, 1996 issue; The United States Patent (USP) 5,641,662 (Debs etc.) of issue on June 24th, 1997; The United States Patent (USP) 5,827,703 (Debs etc.) of issue on October 27th, 1998; The United States Patent (USP) 5,756,353 (Debs) of issue on May 26th, 1998; The United States Patent (USP) 5,858,784 (Debs etc.) of issue on January 12nd, 1999; The United States Patent (USP) 5,948,681 (Scanlin etc.) of issue on September 7th, 1999; With open in the United States Patent (USP) of issuing on December 14th, 1,999 6,001,644 (Debs etc.), quote all these documents for your guidance.

D. The treatment of CFTR modifying factor

The invention still further relates to the administration of the CFTR modifying factor that contains or do not contain other reagent or treatment, as the therapy of treatment CF.Referring to, for example, the United States Patent (USP) 5 of issue on August 31st, 1993, the United States Patent (USP) 5 of 240,846 (Collins etc.) and issue on September 28th, 1999,958,893 (Welsh etc.) (this paper quotes for your guidance) wherein described the appropriate method of administration according to CFTR modifying factor of the present invention.Recombinant retroviral vector and other cftr gene transfer scheme can be used in the practice of the present invention.Can use the CF epithelial cell and carry the clone of transduceing or shifting CFTR modifying factor wherein.

The CFTR modifying factor that uses in the therapy of treatment CF can pass through such as dna clone, and ordinary method artificial constructed or alternate manner obtains.The transgenosis that is used for this therapy can realize by the whole bag of tricks well known in the art, comprise the transfection of using coprecipitation of calcium phosphate, target cell and the liposome that carries this CFTR modifying factor, the fusion of erythrocyte ghost or spheroplast, the transfer of plasmid and virus vector mediation and the transgenosis of DNA albumen composition mediation.

As selection, the wip gene of coding CFTR modified polypeptide can be mixed and be used in the suitable carriers this gene is transmitted the into cell of CF experimenter's CF influence.(for example can use standard technique, through calcium superphosphate or calcium chloride co-precipitation, the transfection of deae dextran mediation, fat transfection, or electroporation) will the encode CFTR modifying factor (for example, in the described hereinafter suitable expressed sequence box) of suitable polypeptide imports in the cultured cells.Then to allow to express the mode vitro culture reconstitution cell of CFTR modified polypeptide.The preferred host cell that produces the CFTR modified polypeptide for example comprises Mammals (people and non-human mammal) cell, yeast cell and insect cell.

At least a portion that the recombinant viral vector that is used for this therapy comprises reverse transcription virus gene group that can target cell infection with the DNA of its CFTR modifying factor that operationally links to each other." infection " is meant that generally virus is with the process of transfer of genetic material to its host or target cell.Preferably the retrovirus that uses in vector construction of the present invention causes that also replication defective is to remove the influence of virus replication to target cell.In this case, can pack this replication-defective virus genome with helper virus according to routine techniques.In general in practice of the present invention, can adopt any retrovirus of the condition that satisfies above-mentioned infectivity and cftr gene transfer ability.Can reckon with that it also is expected using virus attenuation or strong when the virus vector scheme is used for the transfer of CFTR modifying factor.If in practice of the present invention, be suitable for, also can use the amplification of CFTR modifying factor to strengthen the normal expression level.

The cell of transduction or transgenosis target comprises that needs transmit any cell of CFTR modifying factor according to the present invention.In general, this cell is to have cftr gene defective or insufficient those cells, for example cell of CF influence.The cell of the CF of target influence is epithelial cell preferably, comprises pancreas, sweat gland, liver, intestines, nephrocyte, and even more preferably airway epithelial cell, for example pneumonocyte.

Can be in vivo according to the present invention or external treatment cell or cell mass.For example, in the treatment, can use CFTR modifier carrier of the present invention in vivo to the experimenter, preferably in the solution of biocompatible or pharmaceutically useful drug administration carrier by eating, injection sucks perhaps many other method administrations.Dosage changes with experimenter's difference and level that can be by balance CFTR increased functionality and any danger or harmful side effect are measured.Monitoring transduction level, CFTR modifier are expressed and/or the appearance or the level of CFTR modified polypeptide can help to select and adjust dosage.The present invention also inclusion body transduces outward.Can take out or provide the cell colony with defective type cftr gene from the experimenter, principle according to the present invention is transduceed with the CFTR modifying factor, and (again) imports the experimenter then.

Although the epithelial cell of any CF-influence of available gene transfer method of the present invention and carrier target such as pancreas and sweat gland cells, but because the severe complications of CF is relevant with lung usually, so airway epithelial cell is the most desirable target cell of gene therapy of the present invention.In addition, in view of having been found that airway epithelial cell is infected by recombinant retrovirus easily, it is fully feasible therefore these cells being carried out transgenosis according to the present invention.

The appropriate method that is used for the aerosol administration of CFTR modifying factor, and the method for transfectional cell is also at the United States Patent (USP) 5,543,399 (Riordan etc.) of on August 6th, 1996 issue; The United States Patent (USP) 5,641,662 (Debs etc.) of issue on June 24th, 1997; The United States Patent (USP) 5,827,703 (Debs etc.) of issue on October 27th, 1998; The United States Patent (USP) 5,756,353 (Debs) of issue on May 26th, 1998; The United States Patent (USP) 5,858,784 (Debs etc.) of issue on January 12nd, 1999; The United States Patent (USP) 5,948,681 (Scanlin etc.) of issue on September 7th, 1999; With open in the United States Patent (USP) of issuing on December 14th, 1,999 6,001,644 (Debs etc.), quote all these documents for your guidance.

Can make up comprise with transcribe with the translational control element (for example, promotor, ribosome bind site, operon, or enhanser) operationally link to each other or " the expressed sequence box " of the CFTR modifying factor of the suitable polypeptide of coding under its regulation and control and be used for external or expression in vivo CFTR modified polypeptide.The selection of used controlling element can basis, for example the host cell of transfection and required expression level and change.Some gene promoters that are used for mammalian cell are known in the art and comprise, for example, surfactant protein-C (SP-C) promotor that lung specificity is expressed, phosphorylglyceric acid (PGK) promotor, simian virus 40 (SV 40) early promoter, Rous sarcoma virus (RSV) promotor, adenovirus major late promoter (MLP) and early stage immediately 1 promotor of human cytomegalic inclusion disease virus (CMV).Yet, can use any gene promoter that produces suitable expression level in the present invention.Inducible genes promotor, (for example, from heat shock gene, metallothionein gene, interferon-gene, or the promotor that obtains of steroid hormone responsiveness gene) can be used for regulating according to outside stimulus transcribes.

4. Evaluation comprises the CFTR modifying factor of Kir4.2 gene

The invention still further relates to and detect and identify the CFTR modifying factor; comprise and identify the RNAs that is subjected to existing or not existing in the body influence of CFTR; comprise the gene that is similar to those listed positive regulator genes of table 1 and is similar to those listed negative regulator genes of table 2 with evaluation, and evaluation and CFTR interact or compensation CFTR to keep or the approach of normalizing pulmonary function.In the lung tissue of not having infection or disease, observe lacking the regularization genome reaction of CFTR.

In one embodiment, use wild-type and the CFTR-deficient mice of Affymetrix mouse gene array detection from age-matched, the differential expression of CFTR (+/+) and FABP-hCFTR/mCFTR (/-) or CFTR (/-) the isolating lung mRNAs of mouse (relative intensity is indicated in the y-axle, and mouse is increased in the x-axle to the age) particularly.Also analyzed the CFTR deficient mice of expressing sudden change CFTR in an identical manner, i.e. SPC-h Δ 508/FABP-hCFTR/mCFTR (/-), and have other sudden change to cftr gene, comprise the mouse of doxycycline (doxycyline)-inductive sudden change.Bioinformation screening is analyzed (that is, use p-value＜0.02) and has been disclosed statistically 341 genes with CFTR-defective type phenotypic correlation.Being expressed in CFTR (/-) mouse of CFTR itself significantly descends, and confirmed this mouse model.Use further screening, 27 coincidence statistics checks (p-value＜0.05) are arranged in these 341 genes, confirmation is compared them with wild-type and be there are differences in the CFTR-deficient mice, show that these genes can modify CFTR-dependent form approach potentially, and therefore modify CF lysis (seeing figure l).One of these 27 genes, promptly the active basis of the compensation of Kir4.2 is, all increases in all CFTR-deficient mices of test.Compare with the wild-type mice lung by LightCycler PCR evaluation that Kir4.2 mRNA also increases in the CFTR-deficient mice, confirmed this gene array data (see figure 2) as a result.Referring to Gosset etc., " A New Inward Rectifier Potassium Channel Gene (KCNJ15) Localized on Chromosome 21 in the Down Syndrome ChromosomeRegion 1 (DCR 1), " Genomics (1997) 44: 237-41.Express mouse Kir4.2 cDNA in Chinese hamster ovary celI, use the cAMP-stimulant then, for example the combination of forskolin and IBMX is handled, and can increase potassium (K like this ⁺) the ionic current (see figure 3).Because substrate outside potassium (K ⁺) transhipment reinforcement top chlorine (Cl ^-) transhipment, this shows the stimulation of Kir4.2 or expresses increases the chlorine (Cl that may strengthen in normal or CFTR-damaged cells or the lung ^-) ion transport.Because Kir4.2 expresses in epithelium relevant range, pulmonary respiration road, so the expression increase of Kir4.2 can be used for walking around chlorine (Cl ^-) transhipment and the CFTR-dependent form defective of cell function.

Relate to and detect and identify that another embodiment of CFTR modifying factor is as follows:, can correct the small intestine pathology fully and support the normal postpartum of CFTR (/-) transgenic mice to survive at tripe tallow fat acid binding protein gene promoter (iFABP) control expressing human CFTR cDNA in enteric epithelium down.IFABP-hCFTR, CFTR (/-) mouse can be at blended FVB/N, keeps in the C57BL/6 background and does not have the sign of GI or tuberculosis.Histology has been identified with the contrast of the nest type coupling of expressing CFTR with Biochemical Research and has been compared no obvious pathology in the lung tissue of these mouse.Referring to Zhou etc., Science, (1994), 266: 1705-8; Chroneos, J.Immunol., (2000) 165: 3941-50.Mouse is lived in the small-sized separation cage.The iFABP-hCFTR that grows up, the CFTR (/-) and the lung of control mice are evaluated no bacterial pathogens or are built the group by quantitative culture lung homogenate tissue on the blood agar plate.

With FABP-hCFTR (+/+)/mCFTR (/-) mouse and wild-type FVB/N-mCFTR (+/+) mouse mating, be used to produce F1 FABP-hCFTR (+/-)/mCFTR (/+) mouse.Hybridize these mouse to produce F2 offspring nest type coupling mouse, determine its genotype then.Determine that genotype uses following primer to carry out: the primer that is used for mCFTR PCR is forward primer (intron 9): 5 '-AGG GGC TCGCTC TTC TTT GTG AAC,-3 ', reverse primer (introne 1 0): 5 '-TGG CTG TCT GCTTCC TGA CTA TGG,-3 ', the forward primer that is used for neomycin resistance gene PCR is: 5 '-CACAAC AGA CAA TCG GCT GCT,-3 ', reverse primer is: 5 '-ACA GTT CGG CTGGCG CGA G,-3 ', the forward primer (exon 9) that is used for hCFTR PCR is: 5 '-AAA CTTCTA ATG GTG ATG ACA G-3 '.Reverse primer (exons 1 1) is: 5 '-AGA AAT TCTTGC TCG TTG AC-3 '.Evaluation FABP-hCFTR (+/+)/mCFTR (/-) and hCFTR (+/+)/mCFTR (+/+) mouse.The target mCFTR gene of all CFTR (+/+) mouse all is a heterozygous.

Below among the embodiment, carry out in pairs that cDNA is synthetic to separate the technology variability relevant with hybridization conditions with microarray analysis to minimize with RNA.Mate mouse careful separation lung and remove the transporting tracheae and the mediastinum structure from the nest type of gender matched.In TRIzol reagent (Life Technologies), use the method homogenate lung of manufacturer's suggestion.In order to be minimized in the potential impact of breed difference in the mouse colony, can be from 3,6, and separate lung RNA in the nest type coupling mouse of the gender matched in 11 ages in week.Also from the survival CFTR-in 3 ages in week/-)/hCFTR-/-) and CFTR (+/+) nest type coupling mouse lung separated RNA and be used for and carry the genetically modified mouse of iFABP-hCFTR comparing.

Embodiment 1:

The oligomerization dT that use has the T7 promoter sequence carries out reverse transcription to total RNA, then carries out the second chain cDNA and synthesizes.Use amplification of T7 RNA polymerase and biotin labeling antiserum(antisera) cRNA then, use the method and the Affymetrix gene chip mouse U74aV2 hybridization of Affymetrix suggestion afterwards.Affymetrix MicroArray Suite 5.0 editions is used to use default scan that scanning and quantitate gene chip are set.Collect intensity data from each chip, be scaled to 1500 target strength and use MicroArray Suite and GeneSpring 5.0 (Silicon Genetics, Inc., Redwood City, CA) analytical results.CDNAs and U74aV2 chip (Affymetrix Inc.) hybridization.Normalization was to remove or to minimize the system difference source of chip and gene level during hybridization data was processed in the 2-step.Specifically, in each chip of normalization the distribution of all genes on chip with control sample room difference.Each RNA sample of mCFTR (/-) mouse is with respect to its specific contrast (that is the mCFTR of sex and age-matched (+/+) nest type coupling mouse) normalization.Data are further changed into analysis and the symmetry that the logarithm ratio is used to distribute.By in conjunction with distributional analysis (JMP4, SAS Institute, Inc.) and Welch ANOVA identify the change of rna level.Use outlier frame and quartile frame to draw and identify the outlier that meets superelevation outlier＞=higher level's quartile+1.5 (middle quartile scope) and ultralow outlier＜=subordinate's quartile-1.5 (middle quartile scope) definition.Calculate significantly change by Welch t-check with p value＜=0.05.The P-value of calculating adjustment by Westfall and Young displacement (permutation) is to proofread and correct false positive (GeneSpring 4.2.1, Silicon Genetics).Use unidirectional ANOVA to carry out comparison between each genotype and the age group.In order to identify the gene because of cftr gene type differential expression of all ages, use classification and k-mode cluster to identify and change with the consistent of genetic expression that CFTR lacks reaction at all three time points.Further screen candidate rna s according to reproducibility and absolute strength.Calculate the mean value of each replicate(determination), standard error and difference of coefficients.The replicate(determination) of from analyze, deleting CV＞=50%.The gene that is expressed under the detection level is excluded as experimental noise.

The result:

In order to identify and the gene of CFTR reaction, compared the iFABP-hCFTR in 3,6 or 11 ages in week, mCFTR (/-), iFABP-hCFTR, mCFTR (+/+); The lung RNAs of mCFTR (/-) and mCFTR (+/+) nest type coupling mouse.3 and 6 the week ages isolating RNA to carry out microarray analysis in duplicate.The data of 10 Affymetrix musculus cdnas of normalization group U74Av2 chip and the evaluation significant difference between CFTR defective type (CFTR-) and contrast (CFTR+) mouse.Identify the difference relevant by outlier analysis and/or unpaired t-check with the age.After the normalization, the intensity data of observing the lung tissue that obtains from the institute has age is normal distribution.Similar to the DATA DISTRIBUTION of carrying the FABP-hCFTR gene and so in analysis, comprise from the lung RNA data of 3 week CFTR in age (+/+) and CFTR (/-) mouse (no iFABP-hCFTR transgenosis).

The RNAs of differential expression in order to identify of all ages and CFTR reaction is divided into two groups with CFTR (/-) and CFTR (+/+) data.Fig. 4 represents that the logarithm ratio distributes and the outlier of data splitting group is drawn.Totally 1977 outliers have been identified from 12442 gene/ESTs that analyze.The abundance of 848 RNAs increases; 1129 minimizings.Welch t-check further narrows down to 315 with the progressively reduction of Westfall and Young displacement (step-down permutation) with the RNAs number of differential expression.Use hierarchical clustering to observe and this data set of classifying.Referring to Fig. 5.Have the gene group of similar expression pattern with evaluation and show the obvious fractionated gene expression pattern of 315 selected genes with 2D matrix representation data.The RNAs that (dendrogram top) influenced by CFTR on the chip level forms two different groups.In every group, recently more approaching from the mouse of age-matched from the sample of different ages to the sample of collecting, show that the age also influences genetic expression.On rna level (dendrogram in left side), gene obviously is divided into two main groups: the group that mRNAs increases or reduces in mCFTR (/-) mouse.To surpass further screening-gene of 243 (this data set of gene＜=243 by Affymetrix software 90% are called feminine gender) in all time point expression level difference unanimities (CV＜=50%) and its absolute strength.Further screening is RNAs reduced number to 54, and wherein 29 mouse that mate with its mCFTR (+/+) nest type are compared consistent increasing and 25 minimizings in mCFTR (/-).See Table 1 (positive regulator gene) and table 2 (negative regulator gene).These 54 expression of gene patterns represent in Fig. 6 and 7, have confirmed that the expression pattern of the reactive RNAs of CFTR is of all ages to have a consistence.

The gene of differential expression function known according to it or that infer is further classified.Evaluate and specify the function type of each gene.In order to simplify calculating, suppose that the gene in each type satisfies binominal distribution.Use whole U74Av2 to calculate the binomial probability of each type as the reference data set." inflammatory reaction " is the most representative type of gene that RNAs increases in mCFTR (/-) mouse.At CFTR for want of and increase among the RNAs of its abundance, the most representative is that it influences inflammation, transcribe and transport and by with mCFTR (/-) mouse in the group of the diverse function type of those genes of its expression decreased form.See Table 1 (positive regulator gene) and table 2 (negative regulator gene).Use Welch t-check also to assess the potential impact of FABP-hCFTR transgenosis three age groups to rna expression.Correct RNAs that the difference identified in the analysis of mouse regulates at GI-and in mCFTR (/-) mouse, be subjected to same influence, show that iFABP-hCFTR does not have influence to this gene subgroup.It is expressed the gene do not rely on the iFABP-hCFTR transgenosis and to change and comprises 7 minimizings and RNAs 11 increases.The difference of their expression level not significantly (is not no more than 1.5-doubly), and is as shown in the table:

The influence that table 3 fabp-hCFTR (+/+) expresses lung cdna

Gene	The P-value	Ratio	Total	GeneBank	Bioprocess
						Stride film 9 superfamily members 2	0.0381	-1.13	Tm9sf2	NM_080556	Transportation
Mitochondrial ribosomal protein S24	0.03333	-1.19	Mrps24	AA543858	Unknown
						Oncostatin (oncostatin) acceptor	0.02857	-1.26	Osmr	NM_011019	Signal transduction
RIKEN cDNA 4432409D24 gene	0.02857	-1.17	4432409D24Ri k	AK014481	Unknown
						RIKEN cDNA 5730533P17 gene	0.02857	-1.32	5730533P17Rik	NM_027492	Unknown
The sequence A I323512 that expresses	0.02857	-1.22	AI323512	AI323512	Unknown
						Nf I/B	0.02857	-1.17	Nfib	NM_008687	Transcriptional control
Cold shock territory albumin A	0.02381	-1.25	Csda	NM_139117	Transcriptional control
						Aldehyde dehydrogenase 9, subfamily A1	0.02857	-1.20	Aldh9a1	NM_019993	Oxydo-reductase
Heat shock protein(HSP), 105kDa	0.04762	-1.40	Hsp105	NM_013559	Heat shock response
						RIKEN cDNA 2610318I15 gene	0.02381	-1.14	2610318I15Rik	AK012045	Serine/threonine kinase
Caspase 3, apoptosis dependency L-Cysteine HCL Anhydrous	0.04762	1.13	Casp3	NM_009810	Apoptosis
						Ubiquitin conjugate enzyme 8	0.02857	1.68	Ubce8	NM_019949	Protein modified
T-cell receptors α chain	0.02857	1.49	Tcra	U07662	The cytophylaxis reaction
						Protein tyrosine kinase 2 β	0.02857	1.31	Ptk2b	BF579309	Kinases
The thymine DNA glycosylase	0.02857	1.17	Tdg	NM_011561	DNA repairs
						caspase?7	0.02381	1.23	Casp7	NM_007611	Apoptosis
Chemokine (C-C) acceptor 7	0.02381	1.47	Cmkbr7	NM_007719	Chemotaxis

Compare iFABP-hCFTR, CFTR (/-) (that is, intestines are corrected, lung RNA sample GC) and CFTR (+/+) (no iFARP-hCFTR transgenosis, promptly intestines are not corrigent, sample NGC)

Ratio is defined as: and R=GC/NGC if (GC＞NGC); If (GC＜NGC) then R=-NGC/GC.

Embodiment 2:

For the reactive RNAs that verifies that microarray analysis is identified, use Light cycler_ or timing thermal cycler to carry out real-time RT-PCR, then carry out gel electrophoresis.By above-mentioned separation lung RNAs.By reverse transcription produce cDNAs and use following primer carry out pcr analysis: Kir 4.2 forward primers be 5 '-CTTTGA GTT TGT GCC TGT GGT TTC ,-3 ', reverse primer is 5 '-GCT GTG TGA TTTGGT AGT GCG G ,-3 '; People CFTR is the same; Mouse CFTR forward primer is 5 '-TGC TTC CCTACA GAG TCA TCA ACG G ,-3 ', reverse primer is 5 '-CAC AGG ATT TCC CACAAC GCA GAG-3 '; Beta-actin stdn forward primer is 5 '-TGG AAT CCT GCGGCA TCC ATG AAC; Reverse primer is 5 '-TAA AAC GCA GCT CAG TAA CAGTCC G ,-3 '; The GAPDH forward primer is 5 '-CTT CAC CAC CAT GGA GAA GGC ,-3 ', reverse primer is 5 '-GGC ATG GAC TGT GGT CAT GAG ,-3 '; CEBP δ forward primer is 5 '-CGC AAC AAC ATC GCT GTG ,-3 ', reverse primer is 5 '-GGG CTG GGC AGTTTT TTG;-3 ', the TNF-AIP-3 forward primer is 5 '-GCA CGA ATA CAA GAA ATG GCAGG ,-3 ', reverse primer is 5 '-GGC ATA AAG GCT GAG TGT TCA ,-3 ' CG; Grin 2d forward primer is 5 '-CCT TCT TTG CCG TCA, TCT TTC TTG C ,-3 ', reverse primer is 5 '-AAA CTT CAG GGG TGG GTA TTG CTC C ,-3 '.

The result:

Confirmed the change of the selected rna level in microarray analysis, identified by RT-PCR.Use beta-actin or GAPDH normalization mRNA level.For Kir 4.2 (Kcnj15), CEBP δ, TNF-AIP-3 and Grin2d, CFTR (/-) mouse is compared mRNA with contrast nest type coupling mouse obviously to be increased.See Fig. 8,9,10 and 11.As expection, in mCFTR (/-) mouse, do not detect mouse CFTR with RT-PCR, in carrying the mouse lung of iFABP-hCFTR, do not detect hCFTRmRNA yet.

Embodiment 3:

Selected gene is carried out deep retrieval with characterization of biological function and relevant adjusting approach.Make up U74Av2 note (annotation) database of the band system identifier of all array elements and relevant Genbank accession number thereof.Indicate the gene description, function type, biological procedures, molecular function, cell composition, protein structure domain and documentation ﹠ info.Information resources comprise NetAffy (http://www.affymetrix.com), source retrieval (http://genomewww5.stanford.edu/cgi-bin/SMD/source/), BLAST NCBI, LocusLink, mouse-the retrieval of people's homology (http://www.ncbi.nlm.nih.gov) and gene ontology database (http://www.godatabase.org/chi-bin/go.cgi).The gene of differential expression advances in the function type according to gene ontology opinion defining classification.Represent the selected genes table generally for definite any function type, use whole U74Av2 (containing 12488 little musculus cdnas) as calculating all types of binomial probabilities with reference to data set.Binomial probability is defined by following equation:

$P(k,n,p)=\underset{k=0}{\overset{n}{\mathrm{\Σ}}}\left(\begin{array}{c}n\\ k\end{array}\right)p^k{(1-p)}^{n-k}$

If probability is that p then it are created in n the test and obtain probability of successful k time in given colony (U74Av2).Use disclosed documentation ﹠ info to determine that potential protein/protein or protein/DNA interact.

Prepare array analysis from paired mCFTR (/-) mouse and mCFTR (+/+) nest type coupling mouse (lacking the genetically modified mouse of iFABP-hCFTR), confirmed the discovery of microarray, shown that the change that lacks mCFTR mRNA and this RNA in mCFTR (/-) lung does not all rely on the iFABP-hCFTR transgenosis.

Embodiment 4:

Postpartum, the lung of animal was fixed with 25cm H2O pressure inflatable with 4% paraformaldehyde by trachea cannula.Lung tissue is processed and is embedded in the paraffin according to standard method.The program that is used for immunostaining is at Whitsett etc., and J.Biol.Chem. describes among (2002) 277:22743-49.The antigenic rabbit monoclonal antibodies of anti-110kDa Mac-3 is with 1: 40,000 be used to identify pulmonary alveolar macrophage (Pharmingen, SanDiego, CA).

The result:

Adult iFABP-hCFTR, CFTR (/-); IFABP-hCFTR, CFTR (+/+), and the lung tissue indifference of CFTR (+/+) control mice.See Figure 11,12,13 and 14.There is not lung inflammation, the sign that infects or retrofit.Use Mac-3 dyeing to identify pulmonary alveolar macrophage.The number of pulmonary alveolar macrophage and histology do not change because of mCFTR.Histologic analysis has confirmed that non-structure is unusual in the lung of these mouse, infects or inflammation, the change of having supported genetic expression relevant with CFTR and with age or the incoherent idea of tuberculosis.

Homeostatic the keeping of lung replied relevant with the complicated adaptability of genetic expression in mCFTR (/-) mouse.CFTR influences the encoding transcription factor, ionic channel, the RNAs of transport protein in the membrane receptor, cytokine and cell.Finally, CFTR changes expression with the interactional numerous protein of CFTR by representing the protein-protein interaction that the function of CFTR (/-) albumen composition mediation is carried out transcription response.Its diversity of expressing the gene that changes because of CFTR has been supported such viewpoint, and promptly except regulating the Cl-transhipment, CFTR plays a part different in the various kinds of cell function.In CFTR (/-) mouse the lung homeostasis by the complicated genome that lacks CFTR is replied keep rather than by single substituting Cl-passage be used for keep.At last, genes identified and approach provide CFTR and can influence newly getting in touch between the cell processes of CF tuberculosis morbidity in the present invention.

Although described specific embodiments of the present invention, it is evident that to those skilled in the art and can make various modifications and not depart from the spirit and scope of the invention that appended claims limits it.

Sequence table

＜110〉Childrens Hosp Medical Center (Children ' s Hospital Medical Center)

＜120〉be used for the treatment of the CFTR modifying factor of cystic fibrosis and through polypeptide expressed and detection and/or identify their method and product

<130>CHM-001PAT

<150>60/384,856

<151>2002-05-31

<150>60/384,855

<151>2003-05-31

<160>8

<170>PatentIn?version?3.2

<210>1

<211>1443

<212>DNA

＜213〉mouse

<400>1

gccctctaca?tgcatgctcg?agcggccgcc?agtgtgatgg?atatctgcag?aattcggctt??????60

ggtaagccag?gtgggagaag?gggagcgagg?acgcgccact?taccctgcag?aagattcctg?????120

acctacagag?tgagtgacca?ggtggtccaa?agatggatgc?cattcacctt?ggcatgtcca?????180

gtgccccact?ggtgaagcat?accaacgggg?ttggactcaa?ggcccacaga?ccccgagtca?????240

tgtcaaaggg?tgggcacagt?aatgtgagaa?tcgataaggt?agacggaatc?tatttactct?????300

acctccagga?cttgtggaca?accgtcatcg?acatgaagtg?gcgatacaag?ctcaccctat?????360

ttgctgccac?ctttgtgatg?acctggtttc?tgtttggagt?ggtctactat?gccatagcct?????420

ttattcatgg?tgacttacaa?cttggggaat?ctaattccaa?ccacacaccc?tgcattatga?????480

aagtggactc?tctcacggga?gcattcctct?tttccttgga?atctcagaca?accattggct?????540

acggggtccg?ttccatcaca?gaggagtgtc?cccatgctat?cttcctctta?gtcgcccaac?????600

tggtcatcac?cacattgatt?gagatcttca?ttacggggac?ctttctggct?aaaattgcaa?????660

gacccaaaaa?gcgagccgag?accattaagt?tcagccactg?tgctgtcatc?agcaagcaga?????720

atggaaagct?atgcctggtc?atcagggtgg?ccgacatgag?gaagagtctc?ctgattcagt?????780

gccagctctc?tggaaaactc?ctgcagacac?acgtcaccaa?agagggagaa?cgcattctcg?????840

tcaaccaggc?cactgtcaaa?ttccacgtgg?actcctcttc?cgagagtccc?ttcctcatcc?????900

tgcccatgac?cttctaccac?gtgttggatg?agacaagccc?cctgcgggac?ctcacacccc?????960

aaaacgtaaa?ggagaaggag?tttgagctgg?tggtacttct?caacgccacg?gtggagtcta????1020

ccagcgccgt?ctgccagagc?cgaacgtctt?acatcccgga?ggagatctac?tggggctttg????1080

agtttgtgcc?tgtggtttct?ctctccaaaa?atggaaagta?tgtggctgat?ttcagtcaat????1140

ttgagcagat?caggaagagc?ccgggttgta?ccttctactg?tgccgattct?gagaagcaga????1200

agcttgaaga?acagtacagg?caagaggacc?agagggagcg?ggagctgagg?agcctcctgc????1260

tacagcagag?caatgtctga?ctccaccggc?ctctgggcgt?ctcctaacca?gggctccttt????1320

caaaagcaat?ggctgccatg?aacgtgggat?gggtggcccc?acgtaccaaa?tcacacagac????1380

gttaagccga?attccagcac?actggcggcc?gttactagtg?gatccgagct?cggtaccaag????1440

cca??????????????????????????????????????????????????????????????????1443

<210>2

<211>375

<212>PRT

＜213〉mouse

<400>2

Met?Asp?Ala?Ile?His?Leu?Gly?Met?Ser?Ser?Ala?Pro?Leu?Val?Lys?His

1???????????????5???????????????????10??????????????????15

Thr?Asn?Gly?Val?Gly?Leu?Lys?Ala?His?Arg?Pro?Arg?Val?Met?Ser?Lys

20??????????????????25??????????????????30

Gly?Gly?His?Ser?Asn?Val?Arg?Ile?Asp?Lys?Val?Asp?Gly?Ile?Tyr?Leu

35??????????????????40??????????????????45

Leu?Tyr?Leu?Gln?Asp?Leu?Trp?Thr?Thr?Val?Ile?Asp?Met?Lys?Trp?Arg

50??????????????????55??????????????????60

Tyr?Lys?Leu?Thr?Leu?Phe?Ala?Ala?Thr?Phe?Val?Met?Thr?Trp?Phe?Leu

65??????????????????70??????????????????75??????????????????80

Phe?Gly?Val?Val?Tyr?Tyr?Ala?Ile?Ala?Phe?Ile?His?Gly?Asp?Leu?Gln

85???????????????????90?????????????????95

Leu?Gly?Glu?Ser?Ash?Ser?Asn?His?Thr?Pro?Cys?Ile?Met?Lys?Val?Asp

100?????????????????105?????????????????110

Ser?Leu?Thr?Gly?Ala?Phe?Leu?Phe?Ser?Leu?Glu?Ser?Gln?Thr?Thr?Ile

115?????????????????120?????????????????125

Gly?Tyr?Gly?Val?Arg?Ser?Ile?Thr?Glu?Glu?Cys?Pro?His?Ala?Ile?Phe

130?????????????????135?????????????????140

Leu?Leu?Val?Ala?Gln?Leu?Val?Ile?Thr?Thr?Leu?Ile?Glu?Ile?Phe?Ile

145?????????????????150?????????????????155?????????????????160

Thr?Gly?Thr?Phe?Leu?Ala?Lys?Ile?Ala?Arg?Pro?Lys?Lys?Arg?Ala?Glu

165?????????????????170?????????????????175

Thr?Ile?Lys?Phe?Ser?His?Cys?Ala?Val?Ile?Ser?Lys?Gln?Asn?Gly?Lys

180?????????????????185?????????????????190

Leu?Cys?Leu?Val?Ile?Arg?Val?Ala?Asp?Met?Arg?Lys?Ser?Leu?Leu?Ile

195?????????????????200?????????????????205

Gln?Cys?Gln?Leu?Ser?Gly?Lys?Leu?Leu?Gln?Thr?His?Val?Thr?Lys?Glu

210?????????????????215?????????????????220

Gly?Glu?Arg?Ile?Leu?Val?Asn?Gln?Ala?Thr?Val?Lys?Phe?His?Val?Asp

225?????????????????230?????????????????235?????????????????240

Ser?Ser?Ser?Glu?Ser?Pro?Phe?Leu?Ile?Leu?Pro?Met?Thr?Phe?Tyr?His

245?????????????????250?????????????????255

Val?Leu?Asp?Glu?Thr?Ser?Pro?Leu?Arg?Asp?Leu?Thr?Pro?Gln?Asn?Val

260?????????????????265?????????????????270

Lys?Glu?Lys?Glu?Phe?Glu?Leu?Val?Val?Leu?Leu?Asn?Ala?Thr?Val?Glu

275?????????????????280?????????????????285

Ser?Thr?Ser?Ala?Val?Cys?Gln?Ser?Arg?Thr?Ser?Tyr?Ile?Pro?Glu?Glu

290?????????????????295?????????????????300

Ile?Tyr?Trp?Gly?Phe?Glu?Phe?Val?Pro?Val?Val?Ser?Leu?Ser?Lys?Asn

305?????????????????310?????????????????315?????????????????320

Gly?Lys?Tyr?Val?Ala?Asp?Phe?Ser?Gln?Phe?Glu?Gln?Ile?Arg?Lys?Ser

325?????????????????330?????????????????335

Pro?Gly?Cys?Thr?Phe?Tyr?Cys?Ala?Asp?Ser?Glu?Lys?Gln?Lys?Leu?Glu

340?????????????????345?????????????????350

Glu?Gln?Tyr?Arg?Gln?Glu?Asp?Gln?Arg?Glu?Arg?Glu?Leu?Arg?Ser?Leu

355?????????????????360?????????????????365

Leu?Leu?Gln?Gln?Ser?Asn?Val

370?????????????????375

<210>3

<211>2732

<212>DNA

＜213〉people

<400>3

cagatctctg?ggatcttcag?ctgagattgg?gccccagcac?ctggactcaa?ggcagtagca??????60

gaatcccatg?gtagccaggt?gggtgaaggg?gagcgaggac?gttctacctg?ccttgaagaa?????120

gacacctgac?ctgcggagtg?agtgaccagt?gtttccagag?cctggcaatg?gatgccattc?????180

acatcggcat?gtccagcacc?cccctggtga?agcacactgc?tggggctggg?ctcaaggcca?????240

acagaccccg?cgtcatgtcc?aagagtgggc?acagcaacgt?gagaattgac?aaagtggatg?????300

gcatatacct?actctacctg?caagacctgt?ggaccacagt?tatcgacatg?aagtggagat?????360

acaaactcac?cctgttcgct?gccacttttg?tgatgacctg?gttccttttt?ggagtcatct?????420

actatgccat?cgcgtttatt?catggggact?tagaacccgg?tgagcccatt?tcaaatcata?????480

ccccctgcat?catgaaagtg?gactctctca?ctggggcgtt?tctcttttcc?ctggaatccc?????540

agacaaccat?tggctatgga?gtccgttcca?tcacagagga?atgtcctcat?gccatcttcc?????600

tgttggttgc?tcagttggtc?atcacgacct?tgattgagat?cttcatcacc?ggaaccttcc?????660

tggccaaaat?cgccagaccc?aaaaagcggg?ctgagaccat?caagttcagc?cactgtgcag?????720

tcatcaccaa?gcagaatggg?aagctgtgct?tggtgattca?ggtagccaat?atgaggaaga?????780

gcctcttgat?tcagtgccag?ctctctggca?agctcctgca?gacccacgtc?accaaggagg?????840

gggagcggat?tctcctcaac?caagccactg?tcaaattcca?cgtggactcc?tcctctgaga?????900

gccccttcct?cattctgccc?atgacattct?accatgtgct?ggatgagacg?agccccctga?????960

gagacctcac?accccaaaac?ctaaaggaga?aggagtttga?gcttgtggtc?ctcctcaatg????1020

ccactgtgga?atccaccagc?gctgtctgcc?agagccgaac?atcttatatc?ccagaggaaa????1080

tctactgggg?ttttgagttt?gtgcctgtgg?tatctctctc?caaaaatgga?aaatatgtgg????1140

ctgatttcag?tcagtttgaa?cagattcgga?aaagcccaga?ttgcacattt?tactgtgcag????1200

attctgagaa?acagcaactc?gaggagaagt?acaggcagga?ggatcagagg?gaaagagaac????1260

tgaggacact?tttattacaa?cagagcaatg?tctgatcaca?ggggcgccat?ccaggtttaa????1320

ccctgcaagc?tgtttccaca?tcagaactcc?cttcaaacac?aaagattgct?gtgaaaacga????1380

aaatgtgtag?acgcactctc?aaaaactgca?cggacataca?aaatcaatct?tttcctttga????1440

tcttgtggct?aaaccagcat?ttctgtgttt?gagagatttc?ctgttaggtg?cttcgtctga????1500

aagtgaactc?tcataattca?aattgtataa?aataaagcta?catttctaag?agcttggtgt????1560

agggcaattg?gaataatgtc?ctgttagata?aacagacatt?tagcaatcct?gacattaaaa????1620

ggaaatgtat?ttctatacaa?gattattagc?tgtaatacaa?gatatttatt?taaccaatga????1680

ccttatggct?gagagttgaa?ttgtggttca?gtattcattg?atctcactgc?tttaaaacat????1740

gctctttttg?ttcaagcagg?aaaaatatta?tatctaatta?tgtctatgtg?gtaataccta????1800

aaaagaggta?gagagactct?atgttcacaa?aatatagtaa?tttcattttt?acctttcttc????1860

agtggactga?attgtatgga?aggaaattga?tcagatctgt?aattcacaac?tgtggaaacc????1920

tacacaaatg?gggctgatgc?cattgtttcc?tctattttat?tttatttatt?tttgaatcca????1980

ctatctcttt?ccattcaaaa?tatttccatt?caaaatgtgg?ttcacagacc?cgccacatca????2040

gcaccatccg?agagcttatt?agtaatgtgt?agattctcat?gtcccagcca?gacttacaga????2100

attggggtct?gtatcttaac?aagaacccca?gatgatttgt?gtacaaatga?aagttcgaaa????2160

gctgtgctct?ggacagtgtt?ctcaaagcaa?ctttctgaag?ccgtgtggaa?aactgactct????2220

gtgtgggtcc?cgtatccctg?tggcatccag?acctggcctc?tcttcatttc?attattattg????2280

gccaatattt?ctttcagcca?tttcatttct?tatctattaa?aatggcttgc?ccataaagga????2340

actgcatagg?attatcccct?gacattccaa?gagcaaaata?aagctcttga?gagtaattgt????2400

tgtctcagtc?atgcttgctg?attacagtta?gcacaaaaga?aaaattcagc?tgcctgacaa????2460

tacaggaaat?gttctcagat?gctgatgttt?gtaaggtccg?gtggggccat?gaggaagaag????2520

aggagctgaa?ggtaagaaac?tcataaacaa?gatgactctt?tgatgcatga?acaagatttg????2580

aaaatctcaa?gcctgtaaag?aatacccctg?ctatttaaat?aaagctcata?ccaagaggta????2640

acattttgcc?ccgggccaaa?ttcaggggtc?tagtgccctg?cattcctttg?aggcaaaaaa????2700

taaatgggct?atgactggtt?aaatgtccaa?aa??????????????????????????????????2732

<210>4

<211>2919

<212>DNA

＜213〉people

<400>4

ctcctctgcc?caatgtctcc?caatctcttt?cctttctctc?ttcagttcct?ccaggtaatt??????60

cttactcaaa?cttgtaccaa?cttgtttttg?actgacagtg?aacagtgaga?gagttttctt?????120

cattttgagg?aaccctaaac?acctatcttt?cccaaggcaa?cctgtctgga?ctgagcattt?????180

ctctgacttg?acataacttc?ccatccagcc?aggagtctgc?actcttcagt?ctttgcaggc?????240

agtagcagaa?tcccatggta?gccaggtggg?tgaaggggag?cgaggacgtt?ctacctgcct?????300

tgaagaagac?acctgacctg?cggagtgagt?gaccagtgtt?tccagagcct?ggcaatggat?????360

gccattcaca?tcggcatgtc?cagcaccccc?ctggtgaagc?acactgctgg?ggctgggctc?????420

aaggccaaca?gaccccgcgt?catgtccaag?agtgggcaca?gcaacgtgag?aattgacaaa?????480

gtggatggca?tatacctact?ctacctgcaa?gacctgtgga?ccacagttat?cgacatgaag?????540

tggagataca?aactcaccct?gttcgctgcc?acttttgtga?tgacctggtt?cctttttgga?????600

gtcatctact?atgccatcgc?gtttattcat?ggggacttag?aacccggtga?gcccatttca?????660

aatcataccc?cctgcatcat?gaaagtggac?tctctcactg?gggcgtttct?cttttccctg?????720

gaatcccaga?caaccattgg?ctatggagtc?cgttccatca?cagaggaatg?tcctcatgcc?????780

atcttcctgt?tggttgctca?gttggtcatc?acgaccttga?ttgagatctt?catcaccgga?????840

accttcctgg?ccaaaatcgc?cagacccaaa?aagcgggctg?agaccatcaa?gttcagccac?????900

tgtgcagtca?tcaccaagca?gaatgggaag?ctgtgcttgg?tgattcaggt?agccaatatg?????960

aggaagagcc?tcttgattca?gtgccagctc?tctggcaagc?tcctgcagac?ccacgtcacc????1020

aaggaggggg?agcggattct?cctcaaccaa?gccactgtca?aattccacgt?ggactcctcc????1080

tctgagagcc?ccttcctcat?tctgcccatg?acattctacc?atgtgctgga?tgagacgagc????1140

cccctgagag?acctcacacc?ccaaaaccta?aaggagaagg?agtttgagct?tgtggtcctc????1200

ctcaatgcca?ctgtggaatc?caccagcgct?gtctgccaga?gccgaacatc?ttatatccca????1260

gaggaaatct?actggggttt?tgagtttgtg?cctgtggtat?ctctctccaa?aaatggaaaa????1320

tatgtggctg?atttcagtca?gtttgaacag?attcggaaaa?gcccagattg?cacattttac????1380

tgtgcagatt?ctgagaaaca?gcaactcgag?gagaagtaca?ggcaggagga?tcagagggaa????1440

agagaactga?ggacactttt?attacaacag?agcaatgtct?gatcacaggg?gcgccatcca????1500

ggtttaaccc?tgcaagctgt?ttccacatca?gaactccctt?caaacacaaa?gattgctgtg????1560

aaaacgaaaa?tgtgtagacg?cactctcaaa?aactgcacgg?acatacaaaa?tcaatctttt????1620

cctttgatct?tgtggctaaa?ccagcatttc?tgtgtttgag?agatttcctg?ttaggtgctt????1680

cgtctgaaag?tgaactctca?taattcaaat?tgtataaaat?aaagctacat?ttctaagagc????1740

ttggtgtagg?gcaattggaa?taatgtcctg?ttagataaac?agacatttag?caatcctgac????1800

attaaaagga?aatgtatttc?tatacaagat?tattagctgt?aatacaagat?atttatttaa????1860

ccaatgacct?tatggctgag?agttgaattg?tggttcagta?ttcattgatc?tcactgcttt????1920

aaaacatgct?ctttttgttc?aagcaggaaa?aatattatat?ctaattatgt?ctatgtggta????1980

atacctaaaa?agaggtagag?agactctatg?ttcacaaaat?atagtaattt?catttttacc????2040

tttcttcagt?ggactgaatt?gtatggaagg?aaattgatca?gatctgtaat?tcacaactgt????2100

ggaaacctac?acaaatgggg?ctgatgccat?tgtttcctct?attttatttt?atttattttt????2160

gaatccacta?tctctttcca?ttcaaaatat?ttccattcaa?aatgtggttc?acagacccgc????2220

cacatcagca?ccatccgaga?gcttattagt?aatgtgtaga?ttctcatgtc?ccagccagac????2280

ttacagaatt?ggggtctgta?tcttaacaag?aaccccagat?gatttgtgta?caaatgaaag????2340

ttcgaaagct?gtgctctgga?cagtgttctc?aaagcaactt?tctgaagccg?tgtggaaaac????2400

tgactctgtg?tgggtcccgt?atccctgtgg?catccagacc?tggcctctct?tcatttcatt????2460

attattggcc?aatatttctt?tcagccattt?catttcttat?ctattaaaat?ggcttgccca????2520

taaaggaact?gcataggatt?atcccctgac?attccaagag?caaaataaag?ctcttgagag????2580

taattgttgt?ctcagtcatg?cttgctgatt?acagttagca?caaaagaaaa?attcagctgc????2640

ctgacaatac?aggaaatgtt?ctcagatgct?gatgtttgta?aggtccggtg?gggccatgag????2700

gaagaagagg?agctgaaggt?aagaaactca?taaacaagat?gactctttga?tgcatgaaca????2760

agatttgaaa?atctcaagcc?tgtaaagaat?acccctgcta?tttaaataaa?gctcatacca????2820

agaggtaaca?ttttgccccg?ggccaaattc?aggggtctag?tgccctgcat?tcctttgagg????2880

caaaaaataa?atgggctatg?actggttaaa?tgtccaaaa???????????????????????????2919

<210>5

<211>2867

<212>DNA

＜213〉people

<400>5

ggtgtttgga?gagcccggac?tggatgccca?ctagaatcct?gtgctgtgaa?cagtgagaga??????60

gttttcttca?ttttgaggaa?ccctaaacac?ctatctttcc?caaggcaacc?tgtctggact?????120

gagcatttct?ctgacttgac?ataacttccc?atccagccag?gagtctgcac?tcttcagtct?????180

ttgcaggcag?tagcagaatc?ccatggtagc?caggtgggtg?aaggggagcg?aggacgttct?????240

acctgccttg?aagaagacac?ctgacctgcg?gagtgagtga?ccagtgtttc?cagagcctgg?????300

caatggatgc?cattcacatc?ggcatgtcca?gcacccccct?ggtgaagcac?actgctgggg?????360

ctgggctcaa?ggccaacaga?ccccgcgtca?tgtccaagag?tgggcacagc?aacgtgagaa?????420

ttgacaaagt?ggatggcata?tacctactct?acctgcaaga?cctgtggacc?acagttatcg?????480

acatgaagtg?gagatacaaa?ctcaccctgt?tcgctgccac?ttttgtgatg?acctggttcc?????540

tttttggagt?catctactat?gccatcgcgt?ttattcatgg?ggacttagaa?cccggtgagc?????600

ccatttcaaa?tcataccccc?tgcatcatga?aagtggactc?tctcactggg?gcgtttctct?????660

tttccctgga?atcccagaca?accattggct?atggagtccg?ttccatcaca?gaggaatgtc?????720

ctcatgccat?cttcctgttg?gttgctcagt?tggtcatcac?gaccttgatt?gagatcttca?????780

tcaccggaac?cttcctggcc?aaaatcgcca?gacccaaaaa?gcgggctgag?accatcaagt?????840

tcagccactg?tgcagtcatc?accaagcaga?atgggaagct?gtgcttggtg?attcaggtag?????900

ccaatatgag?gaagagcctc?ttgattcagt?gccagctctc?tggcaagctc?ctgcagaccc?????960

acgtcaccaa?ggagggggag?cggattctcc?tcaaccaagc?cactgtcaaa?ttccacgtgg????1020

actcctcctc?tgagagcccc?ttcctcattc?tgcccatgac?attctaccat?gtgctggatg????1080

agacgagccc?cctgagagac?ctcacacccc?aaaacctaaa?ggagaaggag?tttgagcttg????1140

tggtcctcct?caatgccact?gtggaatcca?ccagcgctgt?ctgccagagc?cgaacatctt????1200

atatcccaga?ggaaatctac?tggggttttg?agtttgtgcc?tgtggtatct?ctctccaaaa????1260

atggaaaata?tgtggctgat?ttcagtcagt?ttgaacagat?tcggaaaagc?ccagattgca????1320

cattttactg?tgcagattct?gagaaacagc?aactcgagga?gaagtacagg?caggaggatc????1380

agagggaaag?agaactgagg?acacttttat?tacaacagag?caatgtctga?tcacaggggc????1440

gccatccagg?tttaaccctg?caagctgttt?ccacatcaga?actcccttca?aacacaaaga????1500

ttgctgtgaa?aacgaaaatg?tgtagacgca?ctctcaaaaa?ctgcacggac?atacaaaatc????1560

aatcttttcc?tttgatcttg?tggctaaacc?agcatttctg?tgtttgagag?atttcctgtt????1620

aggtgcttcg?tctgaaagtg?aactctcata?attcaaattg?tataaaataa?agctacattt????1680

ctaagagctt?ggtgtagggc?aattggaata?atgtcctgtt?agataaacag?acatttagca????1740

atcctgacat?taaaaggaaa?tgtatttcta?tacaagatta?ttagctgtaa?tacaagatat????1800

ttatttaacc?aatgacctta?tggctgagag?ttgaattgtg?gttcagtatt?cattgatctc????1860

actgctttaa?aacatgctct?ttttgttcaa?gcaggaaaaa?tattatatct?aattatgtct????1920

atgtggtaat?acctaaaaag?aggtagagag?actctatgtt?cacaaaatat?agtaatttca????1980

tttttacctt?tcttcagtgg?actgaattgt?atggaaggaa?attgatcaga?tctgtaattc????2040

acaactgtgg?aaacctacac?aaatggggct?gatgccattg?tttcctctat?tttattttat????2100

ttatttttga?atccactatc?tctttccatt?caaaatattt?ccattcaaaa?tgtggttcac????2160

agacccgcca?catcagcacc?atccgagagc?ttattagtaa?tgtgtagatt?ctcatgtccc????2220

agccagactt?acagaattgg?ggtctgtatc?ttaacaagaa?ccccagatga?tttgtgtaca????2280

aatgaaagtt?cgaaagctgt?gctctggaca?gtgttctcaa?agcaactttc?tgaagccgtg????2340

tggaaaactg?actctgtgtg?ggtcccgtat?ccctgtggca?tccagacctg?gcctctcttc????2400

atttcattat?tattggccaa?tatttctttc?agccatttca?tttcttatct?attaaaatgg????2460

cttgcccata?aaggaactgc?ataggattat?cccctgacat?tccaagagca?aaataaagct????2520

cttgagagta?attgttgtct?cagtcatgct?tgctgattac?agttagcaca?aaagaaaaat????2580

tcagctgcct?gacaatacag?gaaatgttct?cagatgctga?tgtttgtaag?gtccggtggg????2640

gccatgagga?agaagaggag?ctgaaggtaa?gaaactcata?aacaagatga?ctctttgatg????2700

catgaacaag?atttgaaaat?ctcaagcctg?taaagaatac?ccctgctatt?taaataaagc????2760

tcataccaag?aggtaacatt?ttgccccggg?ccaaattcag?gggtctagtg?ccctgcattc????2820

ctttgaggca?aaaaataaat?gggctatgac?tggttaaatg?tccaaaa??????????????????2867

<210>6

<211>375

<212>PRT

＜213〉people

<400>6

Met?Asp?Ala?I1e?His?Ile?Gly?Met?Ser?Ser?Thr?Pro?Leu?Val?Lys?His

1???????????????5???????????????????10??????????????????15

Thr?Ala?Gly?Ala?Gly?Leu?Lys?Ala?Asn?Arg?Pro?Arg?Val?Met?Ser?Lys

20??????????????????25??????????????????30

Ser?Gly?His?Ser?Asn?Val?Arg?Ile?Asp?Lys?Val?Asp?Gly?Ile?Tyr?Leu

35??????????????????40??????????????????45

Leu?Tyr?Leu?Gln?Asp?Leu?Trp?Thr?Thr?Val?Ile?Asp?Met?Lys?Trp?Arg

50??????????????????55??????????????????60

Tyr?Lys?Leu?Thr?Leu?Phe?Ala?Ala?Thr?Phe?Val?Met?Thr?Trp?Phe?Leu

65??????????????????70??????????????????75??????????????????80

Phe?Gly?Val?Ile?Tyr?Tyr?Ala?Ile?Ala?Phe?Ile?His?Gly?Asp?Leu?Glu

85??????????????????90??????????????????95

Pro?Gly?Glu?Pro?Ile?Ser?Asn?His?Thr?Pro?Cys?Ile?Met?Lys?Val?Asp

100?????????????????105?????????????????110

Ser?Leu?Thr?Gly?Ala?Phe?Leu?Phe?Ser?Leu?Glu?Ser?Gln?Thr?Thr?Ile

115?????????????????120?????????????????125

Gly?Tyr?Gly?Val?Arg?Ser?Ile?Thr?Glu?Glu?Cys?Pro?His?Ala?Ile?Phe

130?????????????????135?????????????????140

Leu?Leu?Val?Ala?Gln?Leu?Val?Ile?Thr?Thr?Leu?Ile?Glu?I1e?Phe?Ile

145?????????????????150?????????????????155?????????????????160

Thr?Gly?Thr?Phe?Leu?Ala?Lys?Ile?Ala?Arg?Pro?Lys?Lys?Arg?Ala?Glu

165?????????????????170?????????????????175

Thr?Ile?Lys?Phe?Ser?His?Cys?Ala?Val?Ile?Thr?Lys?Gln?Asn?Gly?Lys

180?????????????????185?????????????????190

Leu?Cys?Leu?Val?Ile?Gln?Val?Ala?Asn?Met?Arg?Lys?Ser?Leu?Leu?Ile

195?????????????????200?????????????????205

Gln?Cys?Gln?Leu?Ser?Gly?Lys?Leu?Leu?Gln?Thr?His?Val?Thr?Lys?Glu

210?????????????????215?????????????????220

Gly?Glu?Arg?Ile?Leu?Leu?Asn?Gln?Ala?Thr?Val?Lys?Phe?His?Val?Asp

225?????????????????230?????????????????235?????????????????240

Ser?Ser?Ser?Glu?Ser?Pro?Phe?Leu?Ile?Leu?Pro?Met?Thr?Phe?Tyr?His

245?????????????????250?????????????????255

Val?Leu?Asp?Glu?Thr?Ser?Pro?Leu?Arg?Asp?Leu?Thr?Pro?Gln?Asn?Leu

260?????????????????265?????????????????270

Lys?Glu?Lys?Glu?Phe?Glu?Leu?Val?Val?Leu?Leu?Asn?Ala?Thr?Val?Glu

275?????????????????280?????????????????285

Ser?Thr?Ser?Ala?Val?Cys?Gln?Ser?Arg?Thr?Ser?Tyr?Ile?Pro?Glu?Glu

290?????????????????295?????????????????300

Ile?Tyr?Trp?Gly?Phe?Glu?Phe?Val?Pro?Val?Val?Ser?Leu?Ser?Lys?Asn

305?????????????????310?????????????????315?????????????????320

Gly?Lys?Tyr?Val?Ala?Asp?Phe?Ser?Gln?Phe?Glu?Gln?Ile?Arg?Lys?Ser

325?????????????????330?????????????????335

Pro?Asp?Cys?Thr?Phe?Tyr?Cys?Ala?Asp?Ser?Glu?Lys?Gln?Gln?Leu?Glu

340?????????????????345?????????????????350

Glu?Lys?Tyr?Arg?Gln?Glu?Asp?Gln?Arg?Glu?Arg?Glu?Leu?Arg?Thr?Leu

355?????????????????360?????????????????365

Leu?Leu?Gln?Gln?Ser?Asn?Val

370?????????????????375

<210>7

<211>1708

<212>DNA

＜213〉people

<400>7

gcagtagcag?aatcccatgg?tagccaggtg?ggtgaagggg?agcgaggacg?ttctacctgc??????60

cttgaagaag?acacctgacc?tgcggagtga?gtgaccagtg?tttccagagc?ctggcaatgg?????120

atgccattca?catcggcatg?tccagcaccc?ccctggtgaa?gcacactgct?ggggctgggc?????180

tcaaggccaa?cagaccccgc?gtcatgtcca?agagtgggca?cagcaacgtg?agaattgaca?????240

aagtggatgg?catataccta?ctctacctgc?aagacctgtg?gaccacagtt?atcgacatga?????300

agtggagata?caaactcacc?ctgttcgctg?ccacttttgt?gatgacctgg?ttcctttttg?????360

gagtcatcta?ctatgccatc?gcgtttattc?atggggactt?agaacccggt?gagcccattt?????420

caaatcatac?cccctgcatc?atgaaagtgg?actctctcac?tggggcgttt?ctcttttccc?????480

tggaatccca?gacaaccatt?ggctatggag?tccgttccat?cacagaggaa?tgtcctcatg?????540

ccatcttcct?gttggttgct?cagttggtca?tcacgacctt?gattgagatc?ttcatcaccg?????600

gaaccttcct?ggccaaaatc?gccagaccca?aaaagcgggc?tgagaccatc?aagttcagcc?????660

actgtgcagt?catcaccaag?cagaatggga?agctgtgctt?ggtgattcag?gtagccaata?????720

tgaggaagag?cctcttgatt?cagtgccagc?tctctggcaa?gctcctgcag?acccacgtca?????780

ccaaggaggg?ggagcggatt?ctcctcaacc?aagccactgc?caaattccac?gtggactcct?????840

cctctgagag?ccccttcctc?attctgccca?tgacattcta?ccatgtgctg?gatgagacga?????900

gccccctgag?agacctcaca?ccccaaaacc?taaaggagaa?ggagtttgag?cttgtggtcc?????960

tcctcaatgc?cactgtggaa?tccaccagcg?ctgtctgcca?gagccgaaca?tcttatatcc????1020

cagaggaaat?ctactggggt?tttgagtttg?tgcctgtggt?atctctctcc?aaaaatggaa????1080

aatatgtggc?tgatttcagt?cagtttgaac?agattcggaa?aagcccagat?tgcacatttt????1140

actgtgcaga?ttctgagaaa?cagcaactcg?aggagaagta?caggcaggag?gatcagaggg????1200

aaagagaact?gaggacactt?ttattacaac?agagcaatgt?ctgatcacag?gggcgccatc????1260

caggtttaac?cctgcaagct?gtttccacat?cagaactccc?ttcaaacaca?aagattgctg????1320

tgaaaacgaa?aatgtgtaga?cgcactctca?aaaactgcac?ggacatacaa?aatcaatctt????1380

ttcctttgat?cttgtggcta?aaccagcatt?tctgtgtttg?agagatttcc?tgttaggtgc????1440

ttcgtctgaa?agtgaactct?cataattcaa?attgtataaa?ataaagctac?atttctaaga????1500

gcttggtgta?gggcaattgg?aataatgtcc?tgttggataa?acagacattt?agcaatgctg????1560

acattaaaag?gaaatgtatt?tctatacaag?attattagct?gtaatacaag?atatttattt????1620

aaccaatgac?cttatggctg?agagttgaat?tgtggttcag?tattcattga?tctcactgct????1680

ttaaaacatg?ctccttttgt?tcaagcag???????????????????????????????????????1708

<210>8

<211>375

<212>PRT

＜213〉people

<400>8

Met?Asp?Ala?Ile?His?Ile?Gly?Met?Ser?Ser?Thr?Pro?Leu?Val?Lys?His

1???????????????5???????????????????10??????????????????15

Thr?Ala?Gly?Ala?Gly?Leu?Lys?Ala?Asn?Arg?Pro?Arg?Val?Met?Ser?Lys

20??????????????????25??????????????????30

Ser?Gly?His?Ser?Asn?Val?Arg?Ile?Asp?Lys?Val?Asp?Gly?Ile?Tyr?Leu

35??????????????????40??????????????????45

Leu?Tyr?Leu?Gln?Asp?Leu?Trp?Thr?Thr?Val?Ile?Asp?Met?Lys?Trp?Arg

50??????????????????55??????????????????60

Tyr?Lys?Leu?Thr?Leu?Phe?Ala?Ala?Thr?Phe?Val?Met?Thr?Trp?Phe?Leu

65??????????????????70??????????????????75??????????????????80

Phe?Gly?Val?Ile?Tyr?Tyr?Ala?Ile?Ala?Phe?Ile?His?Gly?Asp?Leu?Glu

85??????????????????90??????????????????95

Pro?Gly?Glu?Pro?Ile?Ser?Asn?His?Thr?Pro?Cys?Ile?Met?Lys?Val?Asp

100?????????????????105?????????????????110

Ser?Leu?Thr?Gly?Ala?Phe?Leu?Phe?Ser?Leu?Glu?Ser?Gln?Thr?Thr?Ile

115?????????????????120?????????????????125

Gly?Tyr?Gly?Val?Arg?Ser?Ile?Thr?Glu?Glu?Cys?Pro?His?Ala?Ile?Phe

130?????????????????135?????????????????140

Leu?Leu?Val?Ala?Gln?Leu?Val?Ile?Thr?Thr?Leu?Ile?Glu?Ile?Phe?Ile

145?????????????????150?????????????????155?????????????????160

Thr?Gly?Thr?Phe?Leu?Ala?Lys?Ile?Ala?Arg?Pro?Lys?Lys?Arg?Ala?Glu

165?????????????????170?????????????????175

Thr?Ile?Lys?Phe?Ser?His?Cys?Ala?Val?Ile?Thr?Lys?Gln?Asn?Gly?Lys

180?????????????????185?????????????????190

Leu?Cys?Leu?Val?Ile?Gln?Val?Ala?Asn?Met?Arg?Lys?Ser?Leu?Leu?Ile

195?????????????????200?????????????????205

Gln?Cys?Gln?Leu?Ser?Gly?Lys?Leu?Leu?Gln?Thr?His?Val?Thr?Lys?Glu

210?????????????????215?????????????????220

Gly?Glu?Arg?Ile?Leu?Leu?Asn?Gln?Ala?Thr?Ala?Lys?Phe?His?Val?Asp

225?????????????????230?????????????????235?????????????????240

Ser?Ser?Ser?Glu?Ser?Pro?Phe?Leu?Ile?Leu?Pro?Met?Thr?Phe?Tyr?His

245?????????????????250?????????????????255

Val?Leu?Asp?Glu?Thr?Ser?Pro?Leu?Arg?Asp?Leu?Thr?Pro?Gln?Asn?Leu

260?????????????????265?????????????????270

Lys?Glu?Lys?Glu?Phe?Glu?Leu?Val?Val?Leu?Leu?Asn?Ala?Thr?Val?Glu

275?????????????????280?????????????????285

Ser?Thr?Ser?Ala?Val?Cys?Gln?Ser?Arg?Thr?Ser?Tyr?Ile?Pro?Glu?Glu

290?????????????????295?????????????????300

Ile?Tyr?Trp?Gly?Phe?Glu?Phe?Val?Pro?Val?Val?Ser?Leu?Ser?Lys?Asn

305?????????????????310?????????????????315?????????????????320

Gly?Lys?Tyr?Val?Ala?Asp?Phe?Ser?Gln?Phe?Glu?Gln?Ile?Arg?Lys?Ser

325?????????????????330?????????????????335

Pro?Asp?Cys?Thr?Phe?Tyr?Cys?Ala?Asp?Ser?Glu?Lys?Gln?Gln?Leu?Glu

340?????????????????345?????????????????350

Glu?Lys?Tyr?Arg?Gln?Glu?Asp?Gln?Arg?Glu?Arg?Glu?Leu?Arg?Thr?Leu

355?????????????????360?????????????????365

Leu?Leu?Gln?Gln?Ser?Asn?Val

370?????????????????375

Claims (35)

1. treatment has the method for the cystic fibrosis among the experimenter of cell of CF-influence, comprises step: give the cell with CF-influence experimenter's administering therapeutic significant quantity be selected from modified polypeptide by CFTR; The hereditary instrumentality of CFTR modifying factor; The instrumentality of CFTR modified polypeptide; Or combinations thereof group in a member.

2. the method for claim 1, comprise to the experimenter and use subunit by guanine-nucleotide-binding protein α, membrane glycoprotein, ras-dependency induced by dexamethasone type albumen (DEXRAS1), potassium channel 14 is corrected in the input of ATP-susceptibility, zinc finger protein (Peg3), secretion frizzled related protein sFRP-2, CFTR modified polypeptide in the group that connection protein 37 gap junctional membrane channel protein α 4 and people's hematopoiesis specific proteins 1 (GenBank accession number X84797) is formed; Be selected from by Kir4.2, EST AffymetrixID#92319, Repetin, SWAP-70, vq96e09.41, uo89c05.x1, Preproapelin, Caspase-12, islet cell autoantigen 1, natriuretic peptide precursor A type, U[-M-BH1-ang-b-04-0-U] .s1, C88243, U[-M-BH0-ajq-h-03-0-U] .s1, butyrylcholine esterase, homology frame A5, Wnt10a, regulator homologue, and the CFTR modified polypeptide of the CFTR the modification of gene expression in the group of table 1 and 2 listed CFTR modifying factors compositions; Be selected from by expressing guanine-nucleotide-binding protein α subunit, membrane glycoprotein, ras-dependency induced by dexamethasone type albumen (DEXRAS1), potassium channel 14 is corrected in the input of ATP-susceptibility, zinc finger protein (Peg3), secretion frizzled related protein sFRP-2, connect protein 37 gap junctional membrane channel protein α 4, hematopoiesis specific proteins 1 (GenBank accession number X84797) with the people, with those selected free Kir4.2, EST Affymetrix ID#92319, Repetin, SWAP-70, vq96e09.41, uo89c05.x1, Preproapelin, Caspase-12, islet cell autoantigen 1, natriuretic peptide precursor A type, U[-M-BH1-ang-b-04-0-U] .s1, C88243, U[-M-BH0-ajq-h-03-0-U] .s1, butyrylcholine esterase, homology frame A5, Wnt10a, regulator homologue, and the hereditary instrumentality of the CFTR modifying factor in the group of those genomic constitutions of the expressed CFTR modified polypeptide of the CFTR modifying factor in the group formed of table 1 and 2 listed CFTR modifying factors; Be selected from guanine-nucleotide-binding protein α subunit, membrane glycoprotein, ras-dependency induced by dexamethasone type albumen (DEXRAS1), potassium channel 14 is corrected in the input of ATP-susceptibility, zinc finger protein (Peg3), secretion frizzled related protein sFRP-2, connection protein 37 gap junctional membrane channel protein α 4 and people's hematopoiesis specific proteins 1 (GenBank accession number X84797), be selected from by Kir4.2, ESTAffymetrix ID#92319, Repetin, SWAP-70, vq96e09.41, uo89c05.x1, Preproapelin, Caspase-12, islet cell autoantigen 1, natriuretic peptide precursor A type, mSox7, U[-M-BH1-ang-b-04-0-U] .s1, C88243, U[-M-BH0-ajq-h-03-0-U] .s1, butyrylcholine esterase, homology frame A5, Wnt10a, regulator homologue, and the instrumentality of the CFTR modified polypeptide in the expressed CFTR modified polypeptide of the CFTR modifying factor in the group formed of table 1 and 2 listed CFTR modifying factors, or its combination.

3. the method for claim 2 comprises the CFTR modified polypeptide of using the CFTR the modification of gene expression to the experimenter, and described CFTR modifying factor is selected from by Kir4.2; Passage is corrected in the potassium input, and the member 15; Subfamily J, the member 15; Solute carrier family 38, the member 4, and (ATPase 3 for precursor, macropain) 26S subunit for proteasome; And glutamate receptor, ionization, the group that NMDA2D (ε 4) forms.

4. the method for claim 2 comprises the hereditary instrumentality of using the CFTR modifying factor to the experimenter, and described CFTR modifying factor is selected from by Kir4.2; Passage is corrected in the potassium input, and the member 15; Subfamily J, the member 15; Solute carrier family 38, the member 4, and (ATPase 3 for precursor, macropain) 26S subunit for proteasome; And glutamate receptor, ionization, NMDA2D (ε 4), regulator homologue are connected the group that protein 37 is formed with mouse gap junction protein gene.

5. the method for claim 2 comprises the instrumentality of using the CFTR modified polypeptide of CFTR the modification of gene expression to the experimenter, and described CFTR modifying factor is selected from by Kir4.2; Passage is corrected in the potassium input, and the member 15; Subfamily J, the member 15; Solute carrier family 38, the member 4, and (ATPase 3 for precursor, macropain) 26S subunit for proteasome; And glutamate receptor, ionization, NMDA2D (ε 4), regulator homologue are connected the group that protein 37 is formed with mouse gap junction protein gene.

6. the method for claim 2 comprises the CFTR modified polypeptide of using Kir4.2 genetic expression to the experimenter; The hereditary instrumentality of Kir4.2 gene; Or the instrumentality of the CFTR modified polypeptide of Kir4.2 genetic expression.

7. be used for detecting and/or identify and be selected from modifying factor by CFTR, the CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor, the method of a member in the group that the instrumentality of CFTR modified polypeptide and combination thereof are formed, it comprises step: will contain potential CFTR modifying factor, the CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor, the instrumentality of CFTR modified polypeptide or the sample of its combination with have potential CFTR modifying factor in the sample, the CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor, the instrumentality of CFTR modified polypeptide, or the discernible indicator contact of its when combination.

8. the method for claim 7 comprises the sample that will contain potential CFTR modifying factor or CFTR modified polypeptide and contacts with described indicator.

9. be used to detect and identify the method for CFTR modifying factor, comprise step:

A., CFTR mutant mouse is provided or does not have the mouse of CFTR;

B. separate the genetic material of coding CFTR mutant polypeptide or never exist the mouse of CFTR to separate the genetic material of the CFTR that do not encode from CFTR mutant mouse; With

The change of the genetic expression when c. using this isolating genetic material evaluation can compensate CFTR sudden change or CFTR shortage.

10. the method for potential CFTR modifying factor in the human body that has the CFTR sudden change or may not have CFTR is suspected in detection and evaluation, comprises step:

A. separate the genetic material of the CFTR mutant polypeptides of may encoding or separate the genetic material of the CFTR that do not encode from the human body that may not have CFTR from the human body of suspecting; With

B. use in the isolating genetic material identified gene expression and lack relevant any potential change with cystic fibrosis or CFTR.

11. be used to screen potential CFTR modifying factor, the CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor, the instrumentality of CFTR modified polypeptide and the array of its combination, this array comprises the matrix with the most biological agents that are attached to the different zones surface, described biological agents can be identified potential CFTR modifying factor, the CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor, the instrumentality of CFTR modified polypeptide or its combination.

12. the array of claim 11, wherein said biological agents is to be selected from by expressing guanine-nucleotide-binding protein α subunit, membrane glycoprotein, ras-dependency induced by dexamethasone type albumen (DEXRAS1), potassium channel 14 is corrected in the input of ATP-susceptibility, zinc finger protein (Peg3), secretion frizzled related protein sFRP-2, CFTR modifying factor in the group that the CFTR modifying factor of connection protein 37 gap junctional membrane channel protein α 4 and people's hematopoiesis specific proteins 1 (GenBank accession number X84797) is formed; Be selected from by Kir4.2, EST Affymetrix ID#92319, Repetin, SWAP-70, vq96e09.41, uo89c05.x1, Preproapelin, Caspase-12, islet cell autoantigen 1, natriuretic peptide precursor A type, mSox7, U[-M-BH1-ang-b-04-0-U] .s1, C88243, U[-M-BH0-ajq-h-03-0-U] .s1, butyrylcholine esterase, homology frame A5, Wnt10a, the regulator homologue, the CFTR modifying factor in the group that table 1 and 2 listed CFTR modifying factors are formed; Or by the CFTR modified polypeptide of any of these CFTR the modification of gene expression; With its combination.

13. the array of claim 12, wherein this biologic material is the CFTR modifying factor.

14. the array of claim 13, wherein this biologic material is to be selected from by expressing guanine-nucleotide-binding protein α subunit, membrane glycoprotein, ras-dependency induced by dexamethasone type albumen (DEXRAS1), the CFTR modifying factor in the group of those genomic constitutions of ATP-susceptibility input rectification potassium channel 14; Kir4.2, EST Affymetrix ID#92319, Repetin, SWAP-70, vq96e09.41, and uo89c05.x1, the CFTR modifying factor that table 1 is listed and its combination.

15. the array of claim 13, wherein this biologic material is to be selected from by expression-secretion frizzled related protein sFRP-2, CFTR modifying factor in the group of those genomic constitutions of connection protein 37 gap junctional membrane channel protein α 4 and people's hematopoiesis specific proteins 1 (GenBank accession number X84797); Preproapelin, Caspase-12, islet cell autoantigen 1, natriuretic peptide precursor A type, mSox7, U[-M-BH1-ang-b-04-0-U] .s1, C88243, U[-M-BH0-ajq-h-03-0-U] .s1, butyrylcholine esterase, homology frame A5, Wnt10a, the regulator homologue, the CFTR modifying factor that table 2 is listed and its combination.

16. be used to screen potential CFTR modifying factor, the CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor, the instrumentality of CFTR modified polypeptide, or the method for its combination, this method comprises step: with the array of claim 10 with contain potential CFTR modifying factor, the CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor, the instrumentality of CFTR modified polypeptide or the contact of the sample of its combination, this array has the indicator that links with it, this indicator is used for identifying whether sample exists potential CFTR modifying factor, CFTR modified polypeptide, the hereditary instrumentality of CFTR modifying factor, the instrumentality of CFTR modified polypeptide, or its combination.

17. be used to screen the method for the hereditary instrumentality of potential CFTR modifying factor, it comprises step: the sample that will contain the hereditary instrumentality of potential CFTR modifying factor contacts with the reporter gene cells transfected culture of the hereditary instrumentality that can identify the CFTR modifying factor.

18. the method for claim 17, it comprises the step that contacts sample, wherein this report gene can be identified and be selected from by expressing guanine-nucleotide-binding protein α subunit, membrane glycoprotein, ras-dependency induced by dexamethasone type albumen (DEXRAS1), potassium channel 14 is corrected in the input of ATP-susceptibility, zinc finger protein (Peg3), secretion frizzled related protein sFRP-2, the hereditary instrumentality of the CFTR modifying factor in the group that the CFTR modifying factor of connection protein 37 gap junctional membrane channel protein α 4 and people's hematopoiesis specific proteins 1 (GenBank accession number X84797) is formed; Be selected from by Kir4.2, EST AffymetrixID#92319, Repetin, SWAP-70, vq96e09.41, uo89c05.x1, Preproapelin, Caspase-12, islet cell autoantigen 1, natriuretic peptide precursor A type, mSox7, U[-M-BH1-ang-b-04-0-U] .s1, C88243, U[-M-BH0-ajq-h-03-0-U] .s1, butyrylcholine esterase, homology frame A5, Wnt10a, regulator homologue, and the CFTR modifying factor in the group formed of table 1 and 2 listed CFTR modifying factors; Or by the CFTR modified polypeptide of any of these CFTR the modification of gene expression; With its combination.

19. the method for claim 18, it comprises the step that contacts sample, wherein this report gene can be identified and be selected from by expressing guanine-nucleotide-binding protein α subunit, membrane glycoprotein, ras-dependency induced by dexamethasone type albumen (DEXRAS1), the hereditary instrumentality of the CFTR modifying factor in the group that the CFTR modifying factor of ATP-susceptibility input rectification potassium channel 14 is formed; Kir4.2, ESTAffymetrix ID#92319, Repetin, SWAP-70, vq96e09.41, and uo89c05.x1, the CFTR modifying factor that table 1 is listed and its combination.

20. the method for claim 18, it comprises the step that contacts sample, wherein this report gene can be identified and be selected from by expression-secretion frizzled related protein sFRP-2, the hereditary instrumentality of the CFTR modifying factor in the group of those genomic constitutions of connection protein 37 gap junctional membrane channel protein α 4 and people's hematopoiesis specific proteins 1 (GenBank accession number X84797); Preproapelin, Caspase-12, islet cell autoantigen 1, natriuretic peptide precursor A type, mSox7, U[-M-BH1-ang-b-04-0-U] .s1, C88243, U[-M-BH0-ajq-h-03-0-U] .s1, butyrylcholine esterase, homology frame A5, Wnt10a, the regulator homologue, the CFTR modifying factor that table 2 is listed and its combination.

21. the method for screening potential CFTR modified polypeptide instrumentality, comprise step: the sample that will contain potential CFTR modified polypeptide contacts with the expression vector cells transfected culture that contains the CFTR modifying factor that can identify the CFTR modified polypeptide.

22. the method for claim 21, comprise the step that contacts sample, wherein this expression vector contains and is selected from by expressing guanine-nucleotide-binding protein α subunit, membrane glycoprotein, ras-dependency induced by dexamethasone type albumen (DEXRAS1), potassium channel 14 is corrected in the input of ATP-susceptibility, zinc finger protein (Peg3), secretion frizzled related protein sFRP-2, CFTR modifying factor in the group of those genomic constitutions of connection protein 37 gap junctional membrane channel protein α 4 and people's hematopoiesis specific proteins 1 (GenBank accession number X84797); Be selected from by Kir4.2, EST Affymetrix ID#92319, Repetin, SWAP-70, vq96e09.41, uo89c05.x1, Preproapelin, Caspase-12, islet cell autoantigen 1, natriuretic peptide precursor A type, mSox7, U[-M-BH1-ang-b-04-0-U] .s1, C88243, U[-M-BH0-ajq-h-03-0-U] .s1, butyrylcholine esterase, homology frame A5, Wnt10a, the regulator homologue, the CFTR modifying factor in the group that table 1 and 2 listed CFTR modifying factors are formed; Or by the CFTR modified polypeptide of any of these CFTR the modification of gene expression; With its combination.

23. the method for claim 22, it comprises the step that contacts sample, wherein this expression vector contains and is selected from by expressing guanine-nucleotide-binding protein α subunit, membrane glycoprotein, ras-dependency induced by dexamethasone type albumen (DEXRAS1), the CFTR modifying factor in the group of those genomic constitutions of ATP-susceptibility input rectification potassium channel 14; Kir4.2, EST Affymetrix ID#92319, Repetin, SWAP-70, vq96e09.41, and uo89c05.x1, the CFTR modifying factor that table 1 is listed and its combination.

24. the method for claim 22, it comprises the step that contacts sample, wherein this expression vector contains and is selected from by expression-secretion frizzled related protein sFRP-2, CFTR modifying factor in the group of those genomic constitutions of connection protein 37 gap junctional membrane channel protein o4 and people's hematopoiesis specific proteins 1 (GenBank accession number X84797); Preproapelin, Caspase-12, islet cell autoantigen 1, natriuretic peptide precursor A type, mSox7, U[-M-BH1-ang-b-04-0-U] .s1, C88243, U[-M-BH0-ajq-h-03-0-U] .s1, butyrylcholine esterase, homology frame A5, Wnt10a, the regulator homologue, the CFTR modifying factor that table 2 is listed and its combination.

25. treatment has the method for the cystic fibrosis among the experimenter of cell of CF-influence, comprises step: the hereditary instrumentality of CFTR modifying factor of giving experimenter's administering therapeutic significant quantity of cell with CF-influence.

26. the method for claim 25 comprises using being selected from by transcription factor, strengthens the proto-oncogene of transcribing, interferon-gamma and its analogue, NF-κ B and analogue thereof, the nf of active cells, calcium channel activators, ets factor reagent, GM-CSF; IL-6, IL-1 α, IL-1 β, INF-γ and its analogue, cAMP analogue, adenylate cyclase activating agent, cAMP phosphodiesterase inhibitor, retinoid-like, orphan receptor activator; The retinoic acid receptor (RAR) agonist, Vogan-Neu, vitamin A acid and its analogue; Steroid generates the factor, glucocorticosteroid, glucocorticoid analogue, mineralocorticoid, oestrogenic hormon, progesterone and its analogue; Betamethasone Valerate, the hereditary instrumentality in the group that Decadron and its mixture are formed.

27. the method for claim 26 wherein should the heredity instrumentality to administration.

28. the method for claim 27 is wherein used this heredity instrumentality to the people.

29. treatment has the method for the cystic fibrosis among the experimenter of cell of CF-influence, comprises step: the polypeptides for modulating thing of CFTR modified polypeptide of giving experimenter's administering therapeutic significant quantity of cell with CF-influence.

30. the method for claim 29, comprise and use the reagent that is selected from by activating adenylate cyclase in the target cell, the cAMP agonist, the cAMP additive, stimulate the polypeptide hormone of cAMP, suppress the cAMP phosphodiesterase inhibitor of cAMP degraded, the cAMP-specific inhibitor, glucocorticosteroid, TGF-β (SMAD ₃); Potassium K _ATPPassage is opened agent, potassium BK _CaPassage is opened agent, benzimidazolone, UTP, 8-oxsoralen, and genistein, calcium ion agonist, people DNase 1 sodium channel inhibitor, pancreatin additive; And composition thereof the polypeptides for modulating thing in the group formed.

31. the method for claim 30 comprises using being selected from by forskolin Racemic isoproterenol and salbutamol, cAMP and analogue thereof, beta-hypophamine, alkyl-yellow purine, aminophylline; Rolipram, glucocorticosteroid, TGF-β (SMAD ₃), cromakalim, Pinacidil, Nicoril, minoxidil vitriol, aprikalim, diazoxide, NS004,1-ethyl-2-benzimidazolone, fenamates, dehydroxoyasaponin-I, maxikdiol, cromakalim, nirendipine, and Phloretin, UTP, 8-oxsoralen, genistein; Ionomycin, A23187, carbachol, bradykinin, Moli 1901, thapsigargin, people DNase 1, guanamprazine, triamterene, the pancreatin additive, and composition thereof the polypeptides for modulating thing in the group formed.

32. the method for claim 30, comprise to use and be selected from by alkyl-yellow purine, polypeptides for modulating thing in the group that benzoglyoxaline or benzimidizole derivatives are formed, this alkyl-yellow purine is selected from by 3-isobutyl-1-methylxanthine, 1, the 3-dimethyl xanthine, Papaverine, Pentoxifylline, trimethyl-xanthine, in the group of forming with its mixture, and this benzoglyoxaline or benzimidizole derivatives are selected from by omeprazole, lansoprazole, thimoprazole, pantoprazole, 4-trifluoromethyl-2-[(4-methoxyl group-2-picolyl) sulfydryl]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-3-methyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-5-methyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-3,5-dimethyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-3-methyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-5-methyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-3,5-dimethyl-2-picolyl) sulphur]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-2-picolyl)-sulfinyl]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-3-methyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-5-methyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 4-trifluoromethyl-2-[(4-methoxyl group-3,5-dimethyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-3-methyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 5-trifluoromethyl-2-[(4-methoxyl group-5-methyl-2-picolyl) sulfinyl]-(1H)-and benzoglyoxaline and 5-trifluoromethyl-2-[(4-methoxyl group-3,5-dimethyl-2-picolyl) sulfinyl]-(1H)-benzoglyoxaline; 2-[2-(4-methoxyl group)-methyl sulfinyl]-(5-ethanoyl-6-methyl)-benzoglyoxaline; 2-[2-(4-methoxyl group)-methyl sulfinyl]-(4, the 6-dimethyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-ethanoyl-6-methyl)-benzoglyoxaline; 2-[2-(4-methoxyl group)-methyl sulfinyl]-(5-methoxycarbonyl-6-methyl)-benzoglyoxaline; 2-[2-(4-oxyethyl group)-methyl sulfinyl]-(5-methoxycarbonyl-6-methyl)-benzoglyoxaline; 2-[2-(3-methyl-4-methoxyl group)-methyl sulfinyl]-(5-methoxycarbonyl-6-methyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-methoxycarbonyl-6-methyl)-benzoglyoxaline; 2-[2-(4-methoxyl group-5-methyl)-methyl sulfinyl]-(5-methoxycarbonyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-the 5-methoxycarbonyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-ethanoyl)-benzoglyoxaline; 2-[2-(4-methoxyl group-5-methoxyl group)-methyl sulfinyl]-(5-methoxyl group)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-methoxyl group)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-methyl)-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-benzoglyoxaline; 2-[2-(3,5-dimethyl-4-methoxyl group)-methyl sulfinyl]-(5-chlorine)-benzoglyoxaline; 2-[2-[3-methyl-4-(2,2, the 2-trifluoro ethoxy) pyridyl] methylsulfinyl] benzoglyoxaline (lansoprazole); 2-[2-[3-methyl-4-(2,2,3,3-tetrafluoro propoxy-) pyridyl] methylthio group] benzoglyoxaline; The 2-[(2-pyridyl) methylsulfinyl] benzoglyoxaline (thimoprazole); 2-[2-(3,5-dimethyl-4-methoxypyridine base) methylsulfinyl]-5-methoxyl group-1H-benzoglyoxaline (omeprazole); 2-[2-[4-(3-methoxy propoxy)-3-picolyl] methylsulfinyl]-the 1H-benzoglyoxaline; 2-[2-(3,4-dimethoxy-pyridine base) methylsulfinyl]-5-difluoro-methoxy-1H-benzoglyoxaline (pantoprazole); 4-methyl-3-(2,2, the 2-trifluoro ethoxy)-5H-pyrido [1 ', 2 ': 4,5] [1,2,4] thiaziano[2,3-a] benzoglyoxaline-13-a tetrafluoro borate, in the group that its pharmaceutically useful salt and its mixture are formed.

33. the method for claim 32 is wherein to this polypeptides for modulating thing of administration.

34. the method for claim 33 is wherein used this polypeptides for modulating thing to the people.

35. the method for claim 32 comprises and uses the polypeptides for modulating thing that is selected from the group of being made up of forskolin and 3-isobutyl-1-methylxanthine.

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