CN1348376A - Method for validating/invalidating target(s) and pathways - Google Patents

Method for validating/invalidating target(s) and pathways Download PDF

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CN1348376A
CN1348376A CN00806759A CN00806759A CN1348376A CN 1348376 A CN1348376 A CN 1348376A CN 00806759 A CN00806759 A CN 00806759A CN 00806759 A CN00806759 A CN 00806759A CN 1348376 A CN1348376 A CN 1348376A
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receptor
oligonucleotide
adenosine
mrna
disease
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乔纳森·W·尼斯
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Epigenesis Pharmaceuticals Inc
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Abstract

A method of determining the existence of a correlation between a function of a disease or condition and a gene or mRNA encoding a target polypeptide suspected of being associated with a disease or condition, comprises obtaining oligonucleotides (oligos) consisting of up to about 15 % adenosine (A), preferably having no adenosine content, and which is anti-sense to a target selected from the group consisting of target genes and their corresponding mRNAs, genomic and mRNA flanking regions selected from the group consisting of 3' and 5' intron-exon borders and the juxta-section between coding and non-coding regions, and all mRNA segments encoding polypeptides associated with a pre-selected disease or condition; selecting amongst the oligos one that significantly inhibits or ablates expression of the polypeptide encoded by the mRNA upon in vitro hybridization to the target mRNA; administering to a subject an amount of the selected oligo effective for in vivo hybridization to the target mRNA; and assessing a subject=s function that is associated with the disease or condition before and after administration of the oligo; wherein a change in the function=s value greater than about 70 % indicates a positive correlation, between about 40 and about 70 % a possible correlation, and below about 30 % a lack of correlation. The present method preferably administers the oligos in situ where the target is located, e.g. into the subject=s respiration when validating targets associated with malignant and other pulmonary and respiratory functions, so that the agent has direct access to the lungs. Alternatively, such desAdenosine oligos may be delivered directly to the CNS or other organs, tissues and organ systems, by means of known delivery formulations.

Description

Be used to confirm target and approach effectively/invalid method
Background of invention
Invention field
The present invention relates to confirm target gene, for example nervus centralis (CNS) gene effective method, this method is used low adenosine content (low A) or is not contained the antisense oligonucleotide (oligos) of adenosine (taking off A (desA)).This method can be produced its product by the inhibition target gene and be screened target gene and function thereof.This method is specially adapted to use in the body, because individual many functions respond to adenosine, the effect of adenosine may be covered the detectable function of particular target gene.
Background technology
The sequence of expectation everyone genoid in following 1-3 (about 100,000) all will become known.This information provides chance for being designed for the novel drugs for the treatment of in the past or tormenting now human nearly all disease.But a large amount of accumulations of this sequence itself can not be used to develop new medicine, unless there is technology can distinguish the function of these genes, particularly weaken the latent effect of its function, i.e. therapeutical effect or toxicology effect.For example, must design new method and come test function quickly and accurately, thereby test newfound gene prod is used for the drug development program as " target " purposes.In order to preserve the drug development resource, must confirm as early as possible that in the process of drug development " target " is effective or invalid.Even like this, the enforcement of described method still needs a large amount of drug development programs is applied to the known but gene of Unknown Function of a series of sequences.Assessing newfound gene prod is to use antisense oligonucleotide to eliminate the function of target gene in external or body as a kind of quite potential method of the value of the focus of drug development program.Though this method is extremely important in theory, one of problem is that antisense oligonucleotide might and discharge its nucleotide in forming at external or vivo degradation.Evidence suggests that one of product of oligonucleotide degraded--adenosine has very high biological activity in some tissue.Adenosine can mediate the multiple-effect effect, comprises inhibition neurotransmission, the maintenance thalamus spindle rhythm and pace of moving things, induced hypnotic, D1 and the antagonism of D2 dopamine receptor, the Autonomous Control that resists-nociception, mediates alcoholic acid various effects (comprising locomotor ataxia), cardiac function, bronchoconstriction, negative chronotropic, weakens myotility and nerve conductivity attenuating, resists-beta-adrenergic effect and kidney sodium retention.Clearly, for example organize at some in the tissues such as CNS, high response asthma lung, heart, kidney, also can locally activate adenosine receptor even only discharge the adenosine of trace.This will cause and can't explain target affirmation data in credible, clear and definite mode.Therefore, the antisense oligonucleotide that contains adenosine can not provide clear and definite target to confirm data best, because their degraded can cause the effect of multiple-effect adenosine mediation.
The several years basic Neuroscience Research of carrying out has been established for example glutamic acid and aspartic acid and the various pathologic state relation between apoplexy and the CNS wound for example of excitatory amino acid (EAA) central nervous system (CNS) mediator in the past.As if for example, the main mechanism of cerebral ischaemia, apoplexy or post-traumatic nervous tissue degeneration relates to the excessive activation of EAA system in the brain, that is, and and the excessive release of glutamate, Glu and aspartate.This process is called the excitatory toxicity of delay, and some neural cell group is optionally to the excitatory toxicity sensitivity.
Have now found that adenosine also has the activity that suppresses presynaptic EAA release, thereby can weaken its excitatory toxicity, therefore, its release will seriously disturb the target of this system to confirm research.Because the effect of adenosine is normally receptor-mediated by the extracellular, therefore, the aggregation that the pharmacology of adenosine is correlated with is extracellular.Adenosine also has nerve-behavior effect, and plays the CNS inhibitor, that is to say and can suppress neural activity.It still is natural anticonvulsant and tranquilizer.New studies show that, under normal circumstances, adenosine can hypnotic, therefore can disturb the approach that relates to the reaction of sleep dependency, for example sleep apnea.There is more and more evidences to show that adenosine is a kind of important " the tired factor ", and can disturbs affirmation research the molecule that supports sleep cycle.For example, in brain, studies show that key receptor is positioned on the neurocyte that brain wakes network up.Some scientists be sure of, adenosine can by targeting in IC wake up network for example cholinergic system examine hypnotic as the cholinergic basal forebrain and the mesopontine cholinergic that stimulate sleep.The effect of all these adenosines all can be disturbed in system that it works and the target in the approach and confirm research.
Therefore, be starved of the method that to screen a large amount of genes and expression product thereof rapidly, effectively,, thereby determine its purposes in being designed for treatment and target gene and/or its expression product diseases associated and treatment of conditions agent with definite its function.In addition, also need to be suitable for to test the function of individual gene and avoid exciting the become method of other gene function of difficulty of the explanation that may make the result simultaneously.
Summary of the invention
The function that the present invention relates to confirm disease or disease and coding infer the gene of target polypeptide that may be relevant or the dependency between the mRNA with disease or disease effectively/invalid or measure the method whether this dependency exists.This method generally includes, obtain and contain the at most oligonucleotide (oligos) of about 15% adenosines (A), this oligonucleotide for be selected from target gene and corresponding mRNA thereof, be selected from 3 ' and 5 ' intron-exon border and coding region and noncoding region between genome and the mRNA flanking region of part arranged side by side and the segmental target of all mRNA of the coding polypeptide relevant with previously selected disease or disease be antisense; From oligonucleotide, select when external and said target mrna hybridization and can obviously suppress or eliminate the oligonucleotide of expressing by the mRNA encoded polypeptides; Use the selected oligonucleotide that is enough in vivo with the amount of said target mrna hybridization to individuality; Be evaluated at the function of using the oligonucleotide front and back individuality relevant then with disease or disease; Wherein, the change of function value is greater than about 70% expression positive correlation, and it is relevant to express possibility between about 40 to about 70%, and it is uncorrelated to be lower than about 30% expression.The suitable application of the inventive method be used to illustrate may with the disease or relevant gene or the gene net of disease that influence lung, brain, heart, kidney, tumor, blood, immune system, skin, eye, nasal passage, scalp, testis, cervix uteri, oral cavity, pharynx, esophagus, intestinal (small intestinal and large intestine), synovial tissue, muscle, ovary and auditory meatus etc., normally contain or derive from the various cells of target site.
Present invention is described hereinafter with reference to concrete accompanying drawing.Those skilled in the art are easy to find out other purpose of the present invention, advantage and characteristics from description.
Summary of drawings
Fig. 1 has shown the experiment of using saline (contrast), adenosine (o) and dAMP (P) respectively to rabbit.Saline is influence not, and adenosine and dAMP have all caused that in the mode of dose dependent similar expansion and contraction reduces.This result shows, nucleoside for example dAMP can be directly or in degraded and/or produce the physiological role of adenosine after being metabolized to adenosine at adenosine receptor.
Fig. 2 of the present invention has confirmed it is to contain adenosine (A) rather than those oligonucleotide (oligos) that does not contain adenosine (taking off A) can discharge bioactive adenosine is arranged.The adenosine that discharges activates adenosine receptor and causes biological response, the signal that this biological response can disturb preparation to observe in target is confirmed to study.Use the antisense oligonucleotide of D2EHDTPA at random (oligos) of two kinds of 21-aggressiveness to the rabbit that suffers from asthma, a kind of adenosine (triangle) that contains, a kind of is to take off A oligonucleotide (circle).The oligonucleotide that contains adenosine causes the obvious reduction of air flue expansion and contraction, has reacted the A receptor active, and takes off the not influence of A random oligonucleotide.
Fig. 3 and 4 has confirmed antisense oligonucleotide, and the affirmation target with lung or airway disorders relevant is effective effectively.Fig. 3 has illustrated adenosine A 1The oligonucleotide of receptor antisense and mispairing contrast antisense oligonucleotide influence to bronchus dynamic retractility rate in rabbit model.Fig. 3 has illustrated adenosine A 1The specificity of the oligonucleotide of receptor antisense is by A 1The A that exists in the airway tissue of adenosine receptor antisense strategy 1And A 2The quantitaes of adenosine receptor.
With reference to the description of following preferred embodiment, with the present invention may be better understood.
The description of preferred embodiment
The inventor is for the new technique of the function that is provided for finding quickly and effectively target gene and product thereof and purposes and proposed the present invention.The inventor infers, can be by using his discovery before own, promptly can using low adenosine anti-sense oligonucleotide (oligos) in individual body and can not cause that the adverse side effect that is mediated by adenosine receptor successfully finishes the present invention.Method of the present invention can be by assessing newfound gene and the gene outcome value as the focus of drug development program at external and/or body internal diabetes except the antisense oligonucleotide of its function.Though use antisense oligonucleotide to eliminate gene expression very important theoretical value arranged in the past, the inventor can degrade and discharge the nucleotide of its composition in vitro and in vivo about antisense oligonucleotide and the discovery of nucleoside has hindered their successful Application and explained their ineffectivity.The inventor evidence suggests, one of catabolite of oligomeric nucleoside, and adenosine monophosphate, the same with adenosine have a very high biological activity in multiple tissue.Known adenosine can mediate the multiple-effect effect, comprises the antagonism of inhibition neurotransmission, induced hypnotic, D1 and D2 dopamine receptor, the Autonomous Control that resists-nociception, mediates alcoholic acid various effects (comprising locomotor ataxia), cardiac function, bronchoconstriction, negative chronotropic, weakens myotility and nerve conductivity attenuating, resists-beta-adrenergic effect and kidney sodium retention.Clearly, for example organize at some in the tissues such as CNS, lung, heart, kidney, also can in local environment, activate adenosine receptor even only discharge the adenosine of trace.This will cause and can't explain target affirmation data in credible, clear and definite mode.Embodiments of the invention 30 and 31 and Fig. 1 and Fig. 2 can the degrade adenosine of delivery of biologically active of the oligonucleotide that contains adenosine has been described.This figure has illustrated it is to contain adenosine (A) rather than those oligonucleotide (oligos) that does not contain adenosine (taking off A) can discharge bioactive adenosine is arranged.In disclosed example, use the antisense oligonucleotide of D2EHDTPA at random (oligos) of two kinds of 21-aggressiveness to the rabbit that suffers from asthma, a kind of adenosine (＞) that contains, a kind of is to take off A oligonucleotide (O).The result shows that the oligonucleotide that contains adenosine causes the obvious reduction of air flue expansion and contraction, has reacted the adenosine receptor activity, takes off the then not influence of A random oligonucleotide.The embodiment 13 that vide infra.In addition, confirm that also adenosine nucleoside (dAMP) has the effect to adenosine receptor similar to adenosine.The embodiment 30 that vide infra.
The result who is discussed among research work described herein and the embodiment clearly illustrates that, by method of the present invention being applied to novel targets (when it is found) and/or known target spot (when it is associated with its function), successfully realized confirming the effective of target.Experimental research also confirms, when adopting selectively targeted non-di-phosphate ester antisense oligonucleotide, by contending with or weaken effect by the target protein mediation, method of the present invention confirm target effectively/be high selectivity and effectively when invalid.For all targeting adenosine A 1The antisense oligonucleotide of receptor mrna, targeting adenosine A 2b1 antisense oligonucleotide of receptor mrna, targeting A 32 antisense oligonucleotides of receptor mrna, 1 antisense oligonucleotide of targeting bradykinin receptor, confirmation can be eliminated the effect that contends with that is mediated by the specificity adenosine receptor by exogenous administration adenosine.In addition, method of the present invention confirm regioselective target effectively/be specific when invalid, can not suppress other target spot, as targeting in adenosine A 1With shown in the antisense oligonucleotide of Kallidin I gene and mRNA.In addition, the result shows, the method for the present invention that adopts low adenosine content or do not contain the oligonucleotide of adenosine only produces low-down or do not have adverse side effect or toxicity fully.This is illustrated in when highly effective and specific affirmation target effective method is provided is 100% success, is for example confirmed in respiratory system.The present invention can be widely used in an identical manner encoding and relate to or the target spot of breathing/pulmonary system proteic all genes relevant with airway disorders and corresponding mRNA and other system relevant with specific disease or disease (described disease or disease may for example CNS is relevant with specific function or terminal point).Also by the oligonucleotide of the same race that phosphorothioate bond has replaced method of the present invention is compared with phosphodiester oligonucleotide and phosphodiester bond wherein.The result who uses the inventive method shows than phosphodiester oligonucleotide beat all superiority is arranged.Therefore, the antisense oligonucleotide of high adenosine content (33%) is unsuitable for obtaining clear and definite target and confirms data, because their degraded can cause the effect of multi-purpose adenosine-mediation.The employed low adenosine oligomer of method of the present invention does not obviously have these side effect.The invention describes a kind of method that can be used for carrying out clear and definite " confirming that target is effective ", the release that this method can be used for respiratory tract for example or pulmonary system, CNS and adenosine can cause other organ or the system of multiple-effect effect.This method relates to be used low A or takes off the A antisense oligonucleotide, promptly have low A content or do not contain the oligonucleotide of adenosine, thereby can be when degraded release have the adenosine of obvious biological activity in a large number.Method of the present invention can be used for all basically genes; but be specially adapted to following gene subclass: g protein coupled receptor, neuro hormone receptor, neuropeptide receptor, neurotransmitter receptor, G albumen, calciphorin, sodium channel protein, potassium channel receptor, chloride channel receptor that is to say normal or all basically genes and the following listed gene of expressing among the CNS, heart, lung, kidney, blood, immune system of disease arranged.Therefore, confirmation method described in the invention not only can be used for gene as herein described and system, but also can be used for other gene genus and subgenus and idiotype network.
Successful drug development program depends on rapidly, assesses exactly the suitable degree of candidate gene product as the drug design target spot.In the past, this process need expended considerable time and human and financial resources.The invention provides rapid, believable various biologys confirm in the system target effectively/invalid method, this method is used proprietary low or take off adenosine anti-sense oligonucleotide (this paper is referred to as and takes off A-ASON).A-ASON is taken off in use, method of the present invention can with can't reach with traditional technology speed and accuracy confirm potential gene target spot effectively/invalid.Take off A-ASON accuracy and the velocity level higher than traditional method is provided.Take off that A-ASON can not degrade and the release meeting is disturbed the adenosine of the biologically active that target confirms by causing the inductive side effect of adenosine.In ASON, there is a subgroup to be used for respiratory administration, be called RASON at this.The inventor has carried out method of the present invention to confirm adenosine A with taking off A-RASON 1, A 2And A 3Target is effective.Method of the present invention also can be after site-specific functional gene be eliminated, be used for the CNS target spot that relates to biochemistry, behavioristics, physiology's assessment in any zone of brain, this method is used and is taken off A-BASON, promptly can not degrade and discharge the antisense oligonucleotide (original position administration in brain) of adenosine, and it is the main regulator of cerebrophysiology.Except confirm the lung target spot experimental effectively, with the relevant target spot of CNS method of the present invention has been described, generally include with taking off the A antisense oligonucleotide and carry out the rapid target of multistep and confirm.
The step of beginning need be discerned and be used for whole body system or the zone that target is confirmed, for example CNS, respiratory system, kidney, heart area etc.Then, utilize common library for example GenBank or other publicly-owned library or private library for example specific company proprietary library.For example, the specific library that includes all g protein coupled receptors (GPCR) that are recorded among the both privately and publicly owned data base.Have about 250 GPCR at present.Therefore, target confirmation method of the present invention be applicable to test one group of target will selecting for example GPCR when taking off the reduction of A antisense oligonucleotide, incidents such as which type of physiology, biophysics, biology, behavioristics can take place with suitable.For this reason, design and synthesize according to following description and take off the target gene that the A antisense oligonucleotide is used for selecting in advance, for example above-mentioned GPCR.To take off the A antisense oligonucleotide then and test, at first be testing in vitro, for example uses suitable cell line, primary cell culture or other cell tissue.Described testing in vitro can be used for determining the designed multiple antisense material that takes off which is in the A antisense oligonucleotide " active the strongest " at specific target.That is to say, anyly can regulate or eliminate gene expression best downwards.This can carry out with the test that the given activity of cell is associated with biophysics, biology, physiological test or other.In other cases, in vitro system, reject the target confirmation that target gene itself just can provide usefulness.Select the strongest active taking off the A antisense oligonucleotide and carry out application in the body then, each zone that for example directly is instilled into brain is carried out CNS research, is administered into the lung target spot relevant with respiratory system etc.This can finish by methods known in the art, for example, for the target spot of brain, implants intubate by ground, solid location; For breathing target spot, pass through inhalation; For the blood target spot, by the general administration; For organ and other local system and parenchyma and blood vessel heart cell, by the original position administration; For the kidney target spot, by sucking or directly instiling and carry out the general administration.Can adopt behavioristics, biophysics, physiology, biochemistry, immunology and other test, and in each animal, obtain data to eliminate one or more target genes by using suitable antisense oligonucleotide.The for example food intake of behavioristics's function or terminal point, anxiety, libido, cognition etc., perhaps for example temperature, electroencephalogram (ECG), electrocardiogram (EKG), glomerular filtration volume and content, ion retention, loss of proteins etc. of physiology's terminal point can be assessed by methods known in the art.These steps illustrate in flow chart as follows with CNS.
Figure A0080675900181
Method of the present invention depends on low A or takes off A-ASON and is applied to breathing (taking off A-RASON), CNS (taking off A-BASON), kidney (taking off A-KASON), heart (taking off A-CASON), blood (taking off A-SASON), immune system (taking off A-IASON), malignant tissue's (taking off A-MASON) and other function and do not discharge the adenosine with notable biological activity amount.By this mode, method of the present invention has prevented that disadvantageous adenosine receptor activates in lung, CNS, kidney, heart, blood, immune system, the malignant cell aggregation.The result who obtains with present known method not too can illustrate problem, because conventional antisense constructs contains the adenosine of 25% normal (height) level of having an appointment and can activate adenosine receptor.This effect makes the result thicken and makes functional explanation is become chaotic.For example, in lung, the adenosine that oligonucleotide discharges will cause the secretion of change (bronchus is dwindled), inflammation and the surfactant of air flue diameter, and all effects in this case are all irrelevant with the affirmation of different targets.These " side effect " make the data that obtain in this case with oligonucleotide or ribozyme to make an explanation.Equally, when the antisense oligonucleotide that will contain adenosine was used for brain, its effect makes manyly can be confirmed that the effective function of target becomes as terminal point and be difficult to explain.For example, adenosine can cause change and many other effects of antagonism, the CNS blood flow of Autonomous Control, D1 and the D2 dopamine receptor of the inhibition, induced hypnotic of neurotransmission, anti--nociception, mediation alcoholic acid various effects (comprising locomotor ataxia), cardiac function.When use antisense oligonucleotide high response lung, CNS and other contain adenosine receptor or system that adenosine is responded in carry out target when confirming research, obviously should not have the adenosine of significant quantity.Owing to these reasons, method of the present invention provides a kind of superior target to confirm instrument.The useful especially application of the inventive method is research and the relevant treatment field of cell and many other systems of respiratory tract, CNS, blood, pernicious and growth out of control, these systems can be respectively as target spot, and measures one or more functions relevant with this target respectively.
Because method of the present invention is to invent in the period of history of the uniqueness of bioscience, it will allow rapider and more effectively utilize from the order-checking information that human genome obtained.The sequence of understanding range gene in the human genome is the thing that modern medicine is dreamed of.Finishing of it can produce complete novel medicament to special genes and their functional cohesion then, and these medicines can be better near ill heart and not existing side effects of pharmaceutical drugs.A large amount of sequences of storage provide important target spot at present.Method of the present invention provides a kind of invaluable instrument, this instrument can be used to identification extremely important for function in the body, that is to say for the very important gene of disease and whether the function that can be used to determine to suppress described gene has the purposes in the treatment.Whether the function of determining suppressor gene has the method for therapeutic use to be called " confirming that target is effective ".It is a very important early stage step in the drug discovery process, helps to determine whether to consume the novel drugs that critical resource exploitation is used to suppress the target gene function.Thus, confirm target effectively and confirm target invalid be no less important, confirm target invalid be to confirm to suppress its function not have functional (therapeutic) effect.Confirming as soon as possible in drug discovery process that target is invalid can prevent the wasting of resources in barren drug development work.This will cause rapider, the more productive target spot of concentrated area research more.It is rapidly of the present invention and confirm the difference that the target effective method can produce successfully and fail accurately the treatment of bulk information, the information that for example obtains from the Human Genome Project is used.The in vivo test of antisense oligonucleotide in the methods of the invention can be at the animal model of external and important diseases, comprise respiratory disorder for example asthma, hormone disease, heredopathia, obesity etc., comprises in the model of disease, hematologic disease etc. of CNS, kidney, heart implementing.Can adopt test known in the art to realize the present invention, for example, in clear-headed, rodent, rabbit or primate, for example the plethysmography technology of the complete health in TruePrimateJ or other species freely.Therefore, method of the present invention helps to determine whether have dependency between gene that the function and the coding of disease or disease are inferred relative target polypeptide or mRNA.This method itself generally includes following steps: obtain and contain the at most oligonucleotide (oligos) of about 15% adenosines (A), this oligonucleotide for be selected from target gene and corresponding mRNA thereof, be selected from 3 ' and 5 ' intron-exon border and coding region and noncoding region between genome and the mRNA flanking region of part arranged side by side and the segmental target of all mRNA of the coding polypeptide relevant with previously selected disease or disease be antisense; From oligonucleotide, select when external and said target mrna hybridization and can obviously suppress or eliminate the oligonucleotide of expressing by the mRNA encoded polypeptides; Use the selected oligonucleotide that is enough in vivo with said target mrna hybridization amount to individuality; Be evaluated at the function of using the oligonucleotide front and back individuality relevant then with disease or disease; Wherein, the change of function value is greater than about 70% expression positive correlation, and it is relevant to express possibility between about 40 to about 70%, and it is uncorrelated to be lower than about 30% expression.
Antisense oligonucleotide can be by selecting to contain at least 4 vicinities the fragment of target of the nucleic acid that is selected from G and C make up, and obtain about 4,6,8,10 to the long content that contain selected fragment and C and G of about 15,25,45,60 nucleotide be about 0%, about 3%, about 5%, about first oligonucleotide of 10%, about 12% to about 15%.Perhaps, can select the target fragment according to its type and/or active scope, described activity can be used for different purposes.If present, the adenosine (to all) that any amount can be arranged for example can be combined with the thymidine base by " general " or alternate base but in adenosine A 1, A 2a, A 2bAnd A 3Receptor has less than the heteroaromatic base of about 0.3 adenosine base agonist activity or in adenosine A 2aReceptor does not have active heteroaromatic base to replace.The heteroaromatic base can be pyrimidine and purine, and it can be substituted, for example by O, halogen, NH 2, SH, SO, SO 2, SO 3COOH and side chain and condensed primary and secondary amino; alkyl; alkenyl; alkynyl group; cycloalkyl; Heterocyclylalkyl; aryl; heteroaryl; alkoxyl; alkenyloxy; acyl group; the ring acyl group; aryl-acyl; chain oxy-acetylene; cycloalkyloxy; aroyl; arylthio; aryl-sulfonyl oxygen (sulfoxyl); halogenated cycloalkyl; alkyl-cycloalkyl; the alkenyl cycloalkyl; the alkynyl group cycloalkyl; halogenated aryl; alkylaryl; the alkenyl aryl; the alkynyl group aryl; aryl alkyl; aromatic yl alkenyl; aryl alkynyl chain; cycloalkyl aryl replaces, and it again can be by O; halogen; NH 2, primary, the second month in a season and tertiary amine, SH, SO, SO 2, SO 3, cycloalkyl, Heterocyclylalkyl and heteroaryl replace.But other chemical compound and other substituent group also are applicable to method of the present invention.Usually, pyrimidine and purine be 1,2,3,4,7 and 8 replacement, but also comprise other replacement.The example of pyrimidine and purine is theophylline, caffeine, diprophylline, etofylline, acefylline piperazine, bamifylline, enprofylline and the xanthine with following chemical structural formula
Figure A0080675900211
R wherein 1And R 2Be H, alkyl, alkenyl or alkynyl group independently of one another, R 3Be H, aryl, bicyclic alkyl, bicycloenyl, two cycloalkynyl radicals, cycloalkyl, cycloalkenyl group, cycloalkynyl radical, O-cycloalkyl, O-cycloalkenyl group, O-cycloalkynyl radical, NH 2-alkyl amino-ketone oxygen base alkoxyl-aryl and single and dialkyl aminoalkyl-N-alkyl amino-SO 2Aryl.General and object lesson alternate base be 3-nitro-pyrrole-2 '-deoxynucleoside, 5-nitro-indole, 2-deoxyribosyl base-(5-nitroindoline), 2-desoxyribofuranose base-(5-nitroindoline), 2 '-deoxyinosine, 2 '-deoxidation nebularine, 6H, 8H-3,4-dihydro-pyrimidin also [4,5-c] oxazine-7-ketone or 2-amino-6-methoxyl group amidopurin, but also can use other base.First-selection does not have activity at adenosine receptor, that is to say at adenosine receptor both do not had agonist properties not have the neplanocin of antagonist properties yet.In another preferred embodiment, if there is the CpG dinucleotide in the oligonucleotide, this method can also with methylated cytosine ( mC) come at least one unmethylated C in the replaced C pG dinucleotide, but also can replace a plurality of or whole replacements.Also can adopt other C-5 on pyrimidine to modify, for example the C-5 propine.In order to realize this method, preferably with the one or more of antisense oligonucleotide or all connect residue and replace or modify: methyl phosphonate with being selected from following residue, phosphotriester, thiophosphate, phosphorodithioate, bora phosphate ester (boranophosphate), formacetal, thioformacetal, thioether, carbonic ester, carbamate, sulfuric ester, sulphonic acid ester, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, 2 '-methylene (methyl-imino), (MMI), 2 '-methoxy (MOM), 2 '-methoxy ethyl (MOE), 2 '-methylene oxygen base (methyl-imino) is (MOMA), 2 '-methoxy (MOM), 2 '-the O-methyl, (for example C-5 propine) residue and combination thereof that phosphoramidate and C-5 replace.The antisense oligonucleotide that is applicable to this method be about 7, about 9, about 11, about 13, about 15, about 18, about 21 to about 25, about 28, about 30, about 35, about 40, about 45, about 50, about 55, about 60 mononucleotides are long, but other length also suits.
Method of the present invention also can merge use with a kind of can be by the material of cell internalizing or picked-up and targeting known in the art in the material of the cell antisense oligonucleotide that is connected of transferrins, asialoglycoprotein and Succ-PEG-DSPE for example.In one embodiment, oligonucleotide links to each other with carrier, described carrier can be protokaryon or eucaryon.The example of carrier is known in the art, need not to further describe in the present invention.The amount that the dosage of antisense oligonucleotide normally can effectively reduce output or the availability of mRNA or increase its degraded maybe can reduce the amount of the amount of existing polypeptide.For example, when gene to be identified is relevant with respiratory function, can be with antisense oligonucleotide directly to the lung administration.When gene and function are relevant with other system, preferably with nucleic acid to for example brain, heart, kidney, bladder, gonad and reproductive system, breathing and pulmonary system, tumor, blood, immune system, lung, skin, eye, nasal passage, scalp, testis, cervix uteri, oral cavity, pharynx, esophagus, small intestinal and large intestine, synovial tissue, muscle, ovary, the auditory meatus etc. under cancerous condition and the various cell in-situ administrations that derive from selected target site of affected zone.In many cases, disease or disease can influence the zone of some system or system, for example above-mentioned those.For example, the allergy that respiratory disorder may mediate with increase, inflammation, the IgE-of bronchoconstriction, production and other symptom of surfactant are for example at asthma, allergic rhinitis, COPD, lung tumor, ARDS etc.When disease or disease were relevant with immunology kakergasia, target spot can be selected from immunoglobulin and antibody receptor, cytokine and cytokine receptor, gene and other gene outcome and corresponding mRNA, gene and mRNA flanking region and the intron and the exon border etc. of encode they and correlation function.When disease or disease were relevant with malignant tumor or cancer, target spot can be selected from the gene outcome relevant with cancer, their gene and mRNA, gene and mRNA, genome and mRNA flanking region and exon and intron border etc. relevant with oncogene encode.
Being used for antisense oligonucleotide of the present invention can produce by the following method: select target spot from the polypeptide relevant with disease that influences lung airway and/or disease, their gene and RNA, genome and mRNA flanking region and gene and mRNA exon and the intron border of encoding, obtain the target gene and mRNA, genome and the mRNA flanking region of mRNA and the mRNA sequence on gene and mRNA exon and intron border that are selected from corresponding to coding target polypeptide then; Select at least a mRNA fragment, synthetic one or more oligonucleotide to selected mRNA fragment antisense; And, as needs, with general or alternate base replace one or more A (s) so that in the oligonucleotide content of A be reduced to the highest 10% of all nucleotide that are about.The object lesson Nf κ B transcription factor of the polypeptide relevant of coding with pulmonary system, interleukin-8 receptor (IL-8R), interleukin-15 receptor (IL-5R), interleukin 4 receptors (IL-4R), interleukin-13 receptor (IL-3R), il-1 β (IL-1 β), interleukin-11 beta receptor (IL-1 β R), the eotaxin, trypsinlike enzyme, main basic protein, (2-adrenoceptor kinases, endothelin-receptor A, endothelin-receptor B, the anterior endothelium angiogenic peptide that contracts is former, bradykinin b 2 receptor, the IgE high-affinity receptor, interleukin-11 (IL-1), interleukin 1 receptor (IL-1R), interleukin 9 (IL-9), interleukin-9 receptor (IL-9R), interleukin 11 (IL-11), interleukin-11 receptor (IL-llR), the inducible nitric oxide synthase, cyclo-oxygenase (COX), adhesion molecule 1 (ICAM-1) vascular cell adhesion molecule (VCAM) in the cell, Rantes, endothelial leucocyte adhesion molecule (ELAM-1), the monocyte activation factor, chemotactic factor for neutrophil, the neutrophil elastoser, sozin 1,2 and 3, the muscarine acetylcholinergic receptor, platelet activating factor, tumor necrosis factor, the 5-lipoxygenase, phosphodiesterase IN, the P material, P material receptor, histamine receptor, chymase, the CCR-1 CC-chemokine receptor, the CCR-2 CC-chemokine receptor, the CCR-3 CC-chemokine receptor, the CCR-4 CC-chemokine receptor, the CCR-5 CC-chemokine receptor, the prostaglandin receptoroid, the GATA-3 transcription factor, the neutrophil adhesion receptor, map kinase, interleukin-9 (IL-9), the NFAT transcription factor, STAT 4, MIP-1 α, MCP-2, MCP-3, MCP-4, cyclophilin, phospholipase A2, basic fibroblast growth factor, metalloproteases, the CSBP/p38 map kinase, Trypsin
Figure A0080675900241
Receptor, PDG2, interleukin-3 (IL-3), il-1 β (IL-1 β), cyclosporin A-conjugated protein, FK5-is conjugated protein, α 4 β 1 select albumen, fibronectin, α 4 β 7 select albumen, Mad CAM-1, LFA-1 (CD11a/CD18), PECAM-1, LFA-1 selects albumen, C3bi, PSGL-1, E-selects albumen, P-selects albumen, CD-34, L-selects albumen, p150,95, Mac-1 (CD11b/CD18), fucosyltransferase, VLA-4, CD-18/CD11a, CD11b/CD18, ICAM2 and ICAM3, C5a, CCR3 (eotaxin's receptor), CCR1, CCR2, CCR4, CCR5, LTB-4, the AP-1 transcription factor, Protein kinase C, cysteinyl leukotriene receptor, Tachychinnen receptor (tach R), I kappa b kinase 1 ﹠amp; 2, STAT 6, c-mas and NF-interleukin-6 (NF-IL-6).With CNS; eye; the polypeptide that cardiovascular is relevant with cardiorespiratory system or the example of gene are that (about 250 kinds is known to g protein coupled receptor; about 750-1; suppose for 000 kind; still need to check order); the neuropeptide gene; the neuropeptide receptor gene; the excitatory amino acid receptor gene; the chloride channel gene; calcium channel gene; the purinoceptor gene; adrenergic receptor gene; the serotonin receptor gene; the serotonin transporter gene; the excitatory amino acid transporter gene; potassium channel gene; tyrosine kinase; phosphorylase; acetylcholinergic receptor; the cholecystokinin receptor; nitricoxide synthase; dopamine receptor; cholinoceptor; angiotensin; angiotensin receptor; the ion channel that comprises potassium channel; structural protein; comprise and myelin formation/demyelination and axle and tree; neurotransmitter release medium and structure; the structural protein that the disease of ophthalmic, particularly retina and dependency structure is relevant; calcitonin and receptor thereof; neurocalcin and receptor thereof; CGRP and receptor thereof; atrial natriuretic peptide and receptor thereof; brain natriuretic factor(peptide) and receptor thereof; Kallidin I and receptor thereof; the Baro receptor; the GABA/GABA receptor; the benzodiazepine receptor; acetylcholine esterase; Fructus Cannabis ester receptor; calmodulin, CaM and receptor thereof; calcium/sodium exchanging pump; carbonic anhydrase; catecholamine and receptor thereof; histamine receptor; muscarinic receptor; opiate receptor; chemotactic factor; CAT; cholecalciferol; inflammatory mediator; comprise cytokine; interleukin; interferon and receptor thereof; the enzyme of lipoxygenase pathway; protease; DP (PGD2) receptor; the enzyme that phosphoinositide is relevant; endothelin and receptor thereof; enkephalinase; enkephalin; the benzodiazepine receptor; the GABA transaminase; galanin and receptor thereof; the gastrin releasing factor; somatomedin; growth factor receptor inhibitors; cyclin; nucleoside kinase; nucleoside monophosphate kinase; the oncogene receptor; 5-hydroxy tryptamine (5HT) receptor; ADH; IGF; Insulin receptor INSR; lactamase; the kainate receptor; kallikrein and receptor thereof; leukotriene-relevant enzyme; lipomodulin; L-NMMA and receptor; melanotropin; steroidal transport protein and metabolic enzyme and synthase; NMDA and receptor; morphine receptor; MPTP; neurokinin and receptor thereof; cigarette alkali receptor; phospholipase; platelet activating factor; signal conductive protein; PDGF; dynorphin; prostacyclin; prolactin antagonist and receptor thereof; prolactin release inhibiting factor; prohormone; prostaglandins s; prostaglandin and receptor thereof; thrombin; thrombinogen; pteroylglutamic acid and receptor thereof; the lysergic acid receptor; Serpentis and scorpion venom receptor; the renin-angiotensin system composition; reverse transcriptase; second message,second messenger and relevant enzyme; the sodium channel; somatostatin and receptor thereof; growth hormone and receptor thereof; the p material; the k material; synaptic transmitter; tachykinin; the Fugu ocellatus toxin receptor; thromboxane and receptor thereof; thyroxin; hormone; thyrotropin and receptor; Protirelin; T4; the T3 topoisomerase; tumor necrosis factor and receptor thereof; TGF and receptor thereof; xanthine oxidase; virus messenger RNA s; antibacterial mRNA; oxytocin and receptor thereof; cholecystokinin (Chlecystokinin) and receptor thereof; angiotensin and receptor thereof; monoamine oxidase, MAO; tyrosine-kinases connects receptor etc.Other specific target gene is, for example, G albumen and g protein coupled receptor, calciphorin and relevant protein receptor, sodium channel protein and relevant protein receptor, potassium channel protein and relevant protein receptor and chloride channel albumen and relevant protein receptor, neurotransmitter and neurotransmitter receptor, neuro hormone and neuro hormone receptor, neuropeptide and neuropeptide receptor etc. comprise those that this patent is cited.Other target gene is, for example, G albumen and g protein coupled receptor, calciphorin and relevant protein receptor, sodium channel protein and relevant protein receptor, potassium channel protein and relevant protein receptor and chloride channel albumen and relevant protein receptor, neurotransmitter and neurotransmitter receptor, neuro hormone and neuro hormone receptor, neuropeptide and neuropeptide receptor etc.
In the method, composition can be used outward, oral, intracavity is used, intranasal administration, anal is used, intravaginal is used, intrauterine is used, intracranial is used, pulmonary administration, use in the kidney, (intranodularly) uses in the joint, intraarticular is used, intraotically uses, use in the lymph, applied dermally, use in the cheek, intravenous is used, subcutaneous administration, intramuscular is used, use in the tumor, use in the gland, ophthalmic is used, intracranial is used, use in the organ, use in the blood vessel, use in the sheath, by implanting, suction is used, intradermal is used, use in the lung, in ear is used, on skin or the scalp or on the cervix uteri (for example local application), use in the heart, pass through slow release, continue to discharge or use by pump etc.Target gene relevant with different system and disease and mRNA are for example transcription factor of coded polypeptide, stimulate and activation factor, cytokine and receptor thereof, interleukin, interleukin-2-receptor, chemotactic factor, chemokine receptors, specificity and non-specific enzyme that endogenous produces, immunoglobulin, antibody receptor, central nervous system (CNS) and peripheral nervous and non-nervous system receptor, CNS and peripheral nervous and non-nervous system peptide mediator, adhesion molecule, sozin, somatomedin, vasoactive peptide, peptide receptor and protein-bonded gene and mRNA and corresponding to gene and the mRNA of oncogene.The administration of oligonucleotide can be undertaken by the oral formulations that contains liquid-carrier, for example solution, suspensoid, oil-in-water and water in oil emulsion, and/or with the form administration of powder, dragee, tablet, capsule, spray, aerosol, solution, suspension and Emulsion.When with the form administration of topical formulations, carrier can be selected from cream, gel, ointment, spray, aerosol, patch, solution, suspension and emulsion.When preparation was injectable preparation, carrier can be selected from moisture and alcoholic solution and suspension, oil solution and suspension, oil-in-water and water-in-oil emulsion etc.When preparation is a rectum when using preparation, it can be the form of suppository, and when preparation was percutaneous preparation, carrier can be selected from moisture and alcoholic solution, oil solution and suspension, oil-in-water and water-in-oil emulsion etc., but also can use other carrier.Percutaneous preparation can be iontophoretic percutaneous preparation, and carrier wherein is selected from moisture and alcoholic solution, oil solution and suspension, oil-in-water and water-in-oil emulsion, and said preparation also can contain transdermal enhancer, and it all is known in the art that many transdermal enhancers are arranged.The preparation that is equally applicable to postpone administration is implantable capsule or the cartridge case that contains preparation.In this case, carrier also can be selected from moisture and alcoholic solution and suspension, oil solution and suspension, oil-in-water and water-in-oil emulsion, hydrophobic carrier, the for example capsule of lipid or granule, for example by N-(1-[2,3-two oily acyloxy] propyl group)-N, N, the liposome and the crystallite of N-trimethyl-ammonium methyl sulphate and/or the preparation of other lipid.Use the preparation that preparation preferably can suck, for example form of aerosol for pulmonary.In order to prolong the time of contact of target region, can be with oligonucleotide by conveyings such as local implantation, suppository, sublingual formulation, all these preparations all are known in the art.
Confirm that the factor that this method is better than other method is to obtain more credible and accurate data.Antisense ribozyme technology is unstable in the environment in vivo, exists adenosine to hinder in ribozyme and other oligonucleotide and obtains believable data, for example, contains in the system of a large amount of adenosine receptors at high response respiratory tract and other.Up to the present, also do not have other method confirm can be clearly at the system that contains adenosine reduction target spot and provide credible simultaneously and dependency accurately in the respiratory tract for example.In addition, method of the present invention can be used for illustrating for example neuron idiotype network of idiotype network, and this is the quantum leap for isolation identification individual gene in wideer scope.This can finish by the following method: select more than one target spots relevant with metabolic pathway, then it is tested respectively and test with other target spot, that is to say, a kind of antisense oligonucleotide of applied once, be two kinds, three kinds etc. then, then to the result compare with determined whether connection, whether they work etc. successively.Method of the present invention can produce the idiotype network data base, this data base is suitable for replenishing a kind of meticulousr drug discovery methods, to satisfy the medical need of the strictness in the field relevant with CNS such as cognition, memory, pain, anxiety, behavior disorder, trophic behavior, hunger and satiety and neurological disease.Method of the present invention comprises four basic fields: be used to distinguish the functioning gene group of developing relevant idiotype network with novel drugs, in situ hybridization is to understand the distribution of these networks in brain, privately owned site specific function gene is eliminated (SSFGA) to determine the function of each gene in the network, be used for qualitative and quantitative analysis gene and idiotype network and analyze, be used for distinguishing that gene and idiotype network are the extrapyramidal system physiology of the effect of cardiovascular and pulmonary system for example in the multifactor behavioristics of the participation situation of various behaviors relevant with medical science, the biophysics, analyses such as biochemistry.
This method can comprise fields such as pain, satiety, anxiety/dysthymic disorder, libido, cognition/cognitive disorder, sleep/sleep disorder for the application of CNS.The ability of distinguishing the functional sense of the new gene discerned and idiotype network depends on the assessment to their spatial imagery.For example, available in situ hybridization is determined selected gene and accurate three-dimensional (3-D) position of idiotype network in brain, and is used for making gene to eliminate studying accurately targeting in its meaning as the drug candidate of drug development program of assessment.The site specific function gene is eliminated (SSFGA) and is provided a kind of optionally in the required system realm method of reduction expression of target gene in the brain for example.Be used to confirm that the effective SSFGA of CNS target can or take off the A antisense oligonucleotide and carry out with the low A that designs according to following description.Use other method of antisense oligonucleotide that indefinite data are provided, because adenosine can be degraded and discharge to used oligonucleotide, and the adenosine the strongest a kind of endocrine that is biological activity, for example at CNS with contain in other target system of adenosine receptor.The adenosine that discharges when oligonucleotide is degraded can suppress or promote neurotransmission (this depends on residing system and specific zone, for example brain zone when it discharges), induced hypnotic, influence nociception, change the rhythm and pace of moving things of thalamus spindle, alleviate or strengthen the effect of multiple medicine, influence the Autonomous Control of cardiovascular function and breathing, the presynaptic function that suppresses Ca+ stream and GABA, suppress neuron discharge spontaneous and that be excited, suppress the release of neurotransmitter, reduce postsynaptic irritability, inhibition is assumed to the long-term reinforcement of the basic incident of learning and memory, and cause pleiotropy by the bronchiectasis that causes a side.Obviously, confirm that at target in the research be incompatible by in brain or laboratory animal, the degrade adenosine that discharges obvious amount of oligonucleotide.Be suitable for use as the CNS target and confirm that the neurological of terminal point and behavioristics's test are known in the art.For example, memory test, three-dimensional or spatial ability, cognition, motor control, to the sensitivity of outside stimulus and reactivity, vision, eye coordinate, dermal sensation ability, taste and smell identification etc.The test of physiologic parameters also is known in the art, and can depend on electric conductivity for example EKG and EEG, or other function such as heart rate and cardiac rhythm, water are drained, the measurement of sleep pattern etc.In many cases, the side effect of side effect, particularly cardiopulmonary, kidney and other side effect have constituted and have made the underproof main cause of potential drug development CNS target spot.Conversely, it is defective that the CNS side effect often makes suitable therapeutic cardiopulmonary, kidney and other medicines again.The evidence of the cardiopulmonary influence that the CNS target spot that obtains as early as possible when therefore, being preferably in developing drugs to weaken is caused.When this influence is found late in development sequence, they can make program evening many stages are cancelled.Method of the present invention adopts the test of operation, electrophysiology and other type of analysis means of the prior art, key to assess various injurious effects, and for example reduction is breathed target spot any deleterious side effect of target spot to CNS or cardiovascular system breathed in cardiovascular influence and reduction.For example, can understand in depth further the potential impact of reduction candidate CNS target spot in the pulmonary function research clear-headed, that carry out in the animal freely and the research of cardiac function.
Target confirm can be by above-mentioned SSFGA with targeting in the low A of the known receptor of the various functions that in human pathology (for example neuropeptide tyrosine (NPY)/leptine receptor) and trophic behavior, have a supposition and the novel targets from CNS gene library, found or take off the A antisense oligonucleotide and carry out.Can carry out the SSFGA targeting of special receptor to animal model, assess the change of various actions then, for example trophic behavior, analgesia, motor behavior, sensorimotor reflection, gating process, property and reproductive behavio(u)r, anxiety, learning and memory etc.The broad-based behavior test of this group provides a kind of balancing method of sensitivity for the influence of the specific CNS target spot that weakens, and provides a kind of conclusive knowledge in the incipient stage of the decision process of drug development.Combine with physiology/biophysics/biochemical analysis, this method provides rapid, enhanced mensuration candidate target spot as the further judgement of the probability of the valuable focus of drug development trial.Method of the present invention is improved existing method in the affirmation of gene and albumen target spot, shows to use targeting in influence and the symptomatology and the variation that adenosine anti-sense oligonucleotide comes the expression of suppressor gene product and tests these methods of taking off of the gene relevant with different system.The present invention is a prerequisite with the inventor's new discovery, that is, when oligonucleotide when metabolism is its mononucleotide in vivo, can discharge has bioactive adenosine metabolism thing.The adenosine metabolism thing can be degraded and discharge to the oligonucleotide that contains adenosine (A), and this metabolite can activate the adenosine receptor that for example is arranged in lung and cause bronchoconstriction, inflammation etc.Technology of the present invention based on the design targeting in gene relevant with pathological system and the antisense oligonucleotide of mRNA with relating to difference in functionality, disease.Oligonucleotide is modified reducing their adenosine content, thereby made when degraded because the appearance of the caused adverse side effect of release of adenosine reduces to minimum.Do the statistical significance that has improved viewed result like this, particularly when adenosine receptor may relate to or opposite effect similar to observed effect, perhaps the change of the dynamic equilibrium of the experimental model by causing observed system improves indirectly.By this mode, the inventor with the special genes be target spot design one or more optionally with the bonded antisense oligonucleotide of corresponding mRNA, as needs, can be by maybe not activating adenosine A with general or alternative base 1, A 2a, A 2bOr A 3The neplanocin of receptor replaces reducing adenosine content wherein.On the empirical basis before it, the inventor thinks, except " regulate downwards " or eliminate the specific gene, can also be by selecting to lack the low RNA protein fragments of thymidine (T) or thymidine (T) content or can combine with thymidine but the one or more adenosines that are present in the designed oligonucleotide of nucleotide base (being called general or alternative base) replacement that can not activate the effect in lung etc. of adenosine receptor and generation adenosine increase result's accuracy with other.Because adenosine (A) is a kind of and the complementary nucleotide of thymidine (T), when T appears among the RNA, antisense oligonucleotide will contain A in same position.In order to reach consistent, in this patent, when reading from left to right, all RNA and oligonucleotide all with 5 ' represent to the strand of 3 ' direction, although their complementary series is also included within the scope of the present invention.In addition, all nucleotide bases and aminoacid are all represented with the mode that the IUPAC-IUB biochemical nomenclature commission is recommended, or are represented with the code (for aminoacid) of known 3 letters.
Method of the present invention can be used for confirming that many target genes are effective or invalid, from the individual gene relevant with a function of individuality to an intrasystem network or the relevant multiple one gene of approach.Efficiency confirmed/invalidly be meant that evidence confirms whether a kind of special genes is relevant with one of functions such as multiple biophysics, biochemistry, physiology, behavioristics by experiment.Even a kind of gene is not effect when beginning, but also it can be tested with another kind of gene target spot, because the expression that may need to eliminate them simultaneously just can be observed effect.The A content that the adenosine content of antisense material of the present invention has a reduction discharges A when preventing this material degradation in vivo.For example,, there are a large amount of genes relevant, comprise following table 1 is listed those with different functions if this system is lung or respiratory system.
1：NfκB -8 ( IL-8R ) -5 ( IL-5R ) -4 ( IL-4R ) -3 ( IL-3R ) -1β ( IL-1β ) -1β ( IL-1βR ) β2- AB B2 ( B2BR ) IgE ( ) -1 ( IL-1 ) 1 ( IL-1R ) -9 ( IL-9 ) -9 ( IL-9R ) -11 ( IL-11 ) -11 ( IL-11R ) ( COX ) 1 ( ICAM-1 ) P ( VCAM ) Rantes ETA ( ELAM-1 ) -2 ( COX-2 ) GM-CSF； EDN1 monocyte activation factor chemotactic factor for neutrophil neutrophil elastoser sozin 1; 2,3 muscarine acetylcholinergic receptor platelet activating factor tumor necrosis factor α 5-LOX phosphodiesterase IN Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 acceptor histamine receptor chymase CCR-1CC chemokine receptors proleulzin ( IL-2 ) interleukin-4 ( IL-4 ) IL-12 ( IL-12 ) IL-5 ( IL-5 ) interleukin-6 ( IL-6 ) IL-7 ( IL-7 ) interleukin-8 ( IL-8 ) IL-12 acceptor ( IL-12R ) IL-7 acceptor ( IL-7R ) il-1 ( IL-1 ) IL-14 acceptor ( IL-14R ) IL-14 CCR-2 CC-chemokine receptor CCR-3 CC-chemokine receptor CCR-4 CC-chemokine receptor CCR-5 CC-chemokine receptor prostaglandin receptoroid GATA-3 transcription factor neutrophil adhesion receptor map kinase interleukin-15 ( IL-15 ) interleukin-15 acceptor ( IL-15R ) interleukin-11 ( IL-11 ) interleukin-11 acceptor ( IL-11R ) NFAT transcription factor STAT4MIP-1 α MCP-2MCP-3 MCP-4 cyclophilin ( A; B etc.) phospholipase A2 basic fibroblast growth factor metalloproteinases CSBP/p38 map kinase trypsinlike enzyme acceptor PDG2 interleukin-3, (IL-3) interleukin-10, (IL-10) cyclosporin A-select protein fiber to connect protein alpha 4 β 7 in conjunction with albumen FK506-bindin alpha 4 β 1 is selected albumen cMad CAM-1 LFA-1, (CD11a/CD18) PECAM-1 LFA-1 selects PROTEIN C 3bi PSGL-1E-to select albumen CD62P CD-34 L-to select albumen p150; 95 Mac-1 (CD11b/CD18) fucosyltransferase VLA-4STAT-1 STAT-2CD-18/CD11a CD11b/CD18ICAM2 and ICAM3 C5aCCR3 (eotaxin CCR1; CCR2; CCR4, the CCR5 acceptor) LTB-4 AP-1 transcription factor protein kinase c cysteinyl leukotriene receptor Tachykinnen acceptor (tach R) I kappa b kinase 1 ﹠ amp; 2 interleukin-2 receptors (IL-2R) (for example, P material, NK-1 ﹠ amp; The NK-3 acceptor) STAT6 c-masNF-interleukin-6, (NF-IL-6) interleukin-10 acceptor, (IL-10R) interleukin-3, (IL-3) Interleukin 2 Receptor, (IL-2R) interleukin-13, (IL-13) IL-12 acceptor, (IL-12R) IL-14, (IL-14) interleukin-6 acceptor, (IL-6R) IL-16, (IL-16) interleukin-13 acceptor, (IL-13R) Medullasin IL-16 acceptor, (IL-16R) adenosine A1Receptor (A 1R) trypsinlike enzyme-I adenosine A 2bReceptor (A 2bR) adenosine A 3Receptor (A 3R) β trypsinlike enzyme STAT-3 adenosine A 2aReceptor (A 2aR) the IgE acceptor Fc of the Fc-epsilon receptor CD23 of IgE receptor β subunit (IgE R β) antigen I gE receptor alpha subunit (IgE R α) acceptor (IgERFc ε R) the Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2 acceptor histidine decarboxylase trypsinlike enzyme-1 Prostaglandin D synthase eosinocyte cationic protein eosinocyte neurotoxin eosinocyte peroxidase endothelial nitric oxide synthase endothelial mononuclear cell activating factor neutrophil oxidizing ferment factor cathepsin G macrophage inflammatory protein of the deriving-α of 1-interleukin-8 receptor alpha subunit (IL-8R α)/Rantes acceptor endothelin-receptor ET-B
The normal function of these genes and other gene and breathing and relevant with breathing pathological disease, comprise cystic fibrosis, asthma, pulmonary hypertension and vasoconstriction, chronic obstructive pulmonary disease (COPD), chronic bronchitis, respiratory distress syndrome (ARDS), allergic rhinitis, pulmonary carcinoma and metastatic lung cancer and other airway disorders, comprise the disease that those have inflammatory reaction.Confirmed adenosine A 1, A 2a, A 2bAnd A 3The antisense oligonucleotide of receptor, CCR3 (chemokine receptors), Kallidin I 2B, CAM (vascular cell adhesion molecule) and eosinocyte receptor etc. can be regulated these expression of gene effectively downwards.Wherein some can relief of symptoms or are alleviated and breathe slight illness and/or inflammation, for example, and by " regulating downwards " adenosine A 1, A 2a, A 2bAnd/or A 3Receptor and CCR3, Kallidin I 2B, CAM (vascular cell adhesion molecule) and eosinocyte receptor.These materials can use separately in the method for the invention, or are used in combination with the antisense oligonucleotide of targeting in other gene, and are effective to confirm approach and/or the network relevant with them.In order to obtain better result, preferably with oligonucleotide by sucking or other method is applied directly to the respiratory system of laboratory animal, thereby can make oligonucleotide reach pulmonary and can be in whole body wide dispersion.Be administered systemically or compare by other whole body administration, do like this and can use lower dosage, therefore can reduce because the quantity and the degree of the material caused adverse side effect of extensive distribution in vivo.Confirmed that material of the present invention can reduce the amount of the receptor protein of tissue expression.Therefore, these materials not only can with their target spot acceptor interaction for example, and can reduce the quantity that other medicines also can target protein interactional with it.By this mode, material of the present invention provides high effectiveness and toxicity is very low.
Receptor discussed above only is some strong examples of technical capability of the present invention.In fact, can by method of the present invention in an identical manner targeting in a large amount of genes, to regulate downwards or to eliminate protein expression significantly and to observe any change of one or more functions in for example breathing of system, CNS, cardiovascular, kidney and other system.For example, in respiratory system, the function of test can be the production of alleviating of breathing, bronchoconstriction, inflammation, chronic bronchitis, surfactant etc., and other aspect relevant with disease and disease, for example chronic obstructive pulmonary disease (COPD), suck burn, adult respiratory distress syndrome (ARDS), cystic fibrosis, pulmonary fibrosis, radiation pneumonia, tonsillitis, emphysema, have a toothache, oral inflammation, arthralgia, esophagitis, pulmonary carcinoma and the esophageal carcinoma etc.These functions are very valuable, because they are relevant with respiratory dysfunction, for example at asthma, allergy, allergic rhinitis, the contraction of pulmonary branches trachea and pulmonary hypertension, chronic obstructive pulmonary disease (COPD), allergy, asthma, cystic fibrosis, adult respiratory distress syndrome (ARDS), directly or infect by transfer under the situation of cancer of lung, method of the present invention can be used for a series of potential said target mrnas, is included in target spot listed in the table 1.In the CNS system, the function that can select is food intake/satiety, emotion changes, anxiety, libido/sexual dysfunction, cognition, sexual function/dysfunction, brain trauma, alzheimer's medium (Alzheimer ' s mediator), aneurysm etc.
Antisense oligonucleotide of the present invention can make by the following method: select earlier to contain at least 4 continuous nucleic acid that are selected from G and C and/or have the fragment of the target nucleic acid of specific active type and/or intensity, obtain then the long content that contains selected fragment and thymidine (T) nucleic acid of 4 to 60 nucleotide mostly be most (containing) about 15%, preferred about 12%, about 10%, about 7%, about 5%, about 3%, about 1%, more preferably do not contain first oligonucleotide of thymidine.Back one step can be undertaken by obtaining long second oligonucleotide that contains the sequence of selected fragment antisense of 4 to 60 nucleotide, the adenosine base contents of second oligonucleotide mostly be most (containing) about 15%, preferred about 12%, about 10%, about 7%, about 5%, about 3%, about 1%, more preferably do not contain adenosine.When selected fragment contains at least one thymidine base, can be with general or alternate base replacement adenosine base in corresponding antisense oligonucleotide fragment, when when respiratory system is confirmed, described general or alternate base is selected from and can combines with the thymidine base but in adenosine A 1, A 2a, A 2bAnd A 3Receptor have less than about 10%, preferably less than about 1%, as to be more preferably less than about 0.3% adenosine base agonist activity heteroaromatic base or in adenosine A 2aReceptor does not have active heteroaromatic base.As needs, can measure adenosine activity in other systems in other systems.
Similarly the heteroaromatic base can be selected from all pyrimidines and purine compound, and it can be by O, halogen, NH 2, SH, SO, SO 2, SO 3, COOH and side chain and condensed primary and secondary amino, alkyl, alkenyl, alkynyl group, cycloalkyl, Heterocyclylalkyl, aryl, heteroaryl, alkoxyl, alkenyloxy, acyl group, ring acyl group, aryl-acyl, chain oxy-acetylene, cycloalkyloxy, aroyl, arylthio, aryl-sulfonyl oxygen, halogenated cycloalkyl, alkyl-cycloalkyl, alkenyl cycloalkyl, alkynyl group cycloalkyl, halogenated aryl, alkylaryl, alkenyl aryl, alkynyl group aryl, aryl alkyl, aromatic yl alkenyl, aryl alkynyl chain, cycloalkyl aryl replace, it again can be by O, halogen, NH 2, primary, the second month in a season and tertiary amine, SH, SO, SO 2, SO 3, cycloalkyl, Heterocyclylalkyl and heteroaryl replace.Pyrimidine and purine can be substituted in all positions known in the art, but preferably are substituted at 1,2,3,4,7 and/or 8.Preferred pyrimidine and purine are theophylline, caffeine, diprophylline, etofylline, acefylline piperazine, bamifylline, enprofylline and the xanthine with following chemical structural formula
Figure A0080675900351
R wherein 1And R 2Be H, alkyl, alkenyl or alkynyl group independently of one another, R 3Be H, aryl, bicyclic alkyl, bicycloenyl, two cycloalkynyl radicals, cycloalkyl, cycloalkenyl group, cycloalkynyl radical, O-cycloalkyl, O-cycloalkenyl group, O-cycloalkynyl radical, NH 2-alkyl amino-ketone oxygen base (ketoxy) alkoxyl-aryl, list and dialkyl aminoalkyl-N-alkyl amino-SO 2Aryl etc.Similar modification in sugar also is embodiment of the present invention.Corresponding to the adenosine content in the antisense oligonucleotide of existing thymidine (T) among the target RNA reduce can prevent oligonucleotide be degraded into can to system for example lung, brain, heart, kidney etc., tissue around it discharge the product of adenosine, thereby prevention is because its caused any undesired effect.
For example, can select Nf κ B transcription factor, seek its mRNA or the low thymidine (T) among the DNA or take off thymidine (taking off T (desT)) fragment as target spot.That only selects mRNA or DNA takes off the T part, takes off the complementary strand of A antisense as them by its generation then.When having found that a large amount of RNA take off the T part, can infer the sequence of antisense part.Usually, can obtain about 10 to 30 even the more substantial A of taking off antisense sequences.These antisense sequences can comprise corresponding to the mRNA of target spot take off T part partly or entirely take off the A Antisensedigonucleotsequence sequence, for example at shown in last table 1 and the following table 2 those, and other those relevant with the function of brain, cardiovascular and kidney system.When this situation occurred, the antisense oligonucleotide of being found was called as 100% and does not contain A.For each original A Antisensedigonucleotsequence sequence that takes off, can in target gene or RNA, find about 10 to 30 sequences usually with low thymidine (RNA) content corresponding to target gene, for example NF κ B transcription factor.According to the present invention, selected fragment sequence can also contain a spot of thymidine (RNA) nucleotide in second, third or the 4th sequence.In some cases, high adenosine content may be enough to make antisense oligonucleotide to reduce even do not have an activity target spot is active.According to the present invention, these so-called " not exclusively taking off A " sequence preferences contain and are lower than about 15%, about 12%, about 10%, about 7%, about 5% and the adenosine content of about 2% adenosine.First-selection does not contain adenosine (0%).But in some cases, higher adenosine content is an acceptable and oligonucleotide still can not show deleterious " adenosine activity ".The embodiment of particular importance is that adenosine nucleoside acid is " fixed " or quilt can be replaced with two or more " the general or alternate " bases of carrying out base pairing in the four kinds of nucleotide (A, G, C and T) that exist in similar or identical affinity and the n DNA.
In the present invention, general or alternate base be meant can be with thymidine hybridization but with adenosine receptor or adenosine by its in laboratory animal or cell system, produce the bonded ability of molecule of adverse side effect more weak or basically can not bonded with it any chemical compound, neplanocin more commonly.Perhaps, can use and to activate for example adenosine A of adenosine receptor fully 1, A 2a, A 2bAnd/or A 3Receptor, first-selected A 1The neplanocin of receptor.An example general or alternative base is α-desoxyribofuranose base-(a 5-nitroindoline), the technical staff can know how to select other, should " fixing " step produce another kind of new sequence, be different from sequence with naturally occurring sequence antisense, this makes antisense oligonucleotide to combine with target RNA, and preferred combination gets good equally.Another example general or alternative base is 2-deoxyribosyl base-(a 5-nitroindoline).Another example general or alternative base be 3-nitro-pyrrole-2 '-deoxynucleoside, 5-nitro-indole, 2-deoxyribosyl base-(5-nitroindoline), 2-desoxyribofuranose base-(5-nitroindoline), 2 '-deoxyinosine, 2 '-deoxidation nebularine, 6H, 8H-3, the 4-dihydro-pyrimidin also [4,5-c] oxazine-7-ketone and 2-amino-6-methoxyl group amidopurin.In addition, can replace though the ability of its hybridization decreases the general or alternative base of any other base comprise 3-nitro-pyrrole 2 '-deoxynucleoside 2-desoxyribofuranose base-(5-nitroindoline), 2 '-deoxyinosine and 2 '-deoxidation nebularine (Glen Research, Sterling, VA).More specific mispairing reparation can be used " P " nucleotide, 6H, 8H-3, the 4-dihydro-pyrimidin also [4,5-c] oxazine-7-ketone (its base and guanine (G) or adenine (A) pairing) and " K " nucleotide, 2-amino-6-methoxyl group amidopurin (its base and cytosine (C) or thymidine (T) match).Also can use other material that maybe can obtain known in the art.Referring to, for example, Loakes, D. and Brown, D.M., Nucl.Acids Res.22:4039-4043 (1994); Ohtsuka, E. etc., J.Biol.Chem.260 (5): 2605-2608 (1985); Lin, P.K.T. and Brown, D.M., NucleicAcids Res.20 (19): 5149-5152 (1992; Nichols, R. etc., Nature 369 (6480): 492-493 (1994); Rahmon, M.S. and Humayun, N.Z., Mutation Research377 (2): 263-8 (1997); Amosova, O. etc., Nucleic Acids Res.25 (10): 1930-1934 (1997); Loakes D.﹠amp; Brown, D.M., Nucleic Acids Res.22 (20): 4039-4043 (1994), its part about general or alternate base and preparation and the purposes in the nucleic acid combination is incorporated herein by reference.
When in natural target spot, having found not exclusively to take off the T sequence, usually it is selected so that about 1 to 3 general or alternate base is replaced the antisense oligonucleotide that will be enough to obtain 100% " taking off A ".Therefore, it is low or do not contain the antisense oligonucleotide of A to the invention provides A content at different target spots, and one or more adenosine nucleoside acid are wherein arranged, and for example about 1 to 3 or more, by the antisense oligonucleotide of " having fixed " with the base substitution institute of " general or alternative ".General or alternate base is known in the art, need not to list at this.Those skilled in the art can know which base can be used as general or alternate base and replaces A with it.
Used term confirms that target " effectively " or engineering noise are meant following process in the literary composition in for example breathing of certain system, inflammation, CNS, cardiopulmonary, kidney immunity and other system: earlier in system treatment field of selection in CNS or cardiovascular or the cardiorespiratory system for example.The field that can select to be used to study in these systems is anxiety, dysthymic disorder, satiety and appetite regulation, pain, cognition, sleep derivation and obstacle, thermoregulation etc. by brain control or the field regulated, and following table 2 exemplified those.
Step subsequently is the geneseq database of selecting corresponding to suitable system, for example CNS, lung, cardiac system, kidney system, blood, immune system, lung and respiratory system, sexual function/malfunction, skin, eye, nasal passage, scalp, testis, cervix uteri, oral cavity, pharynx, esophagus, small intestinal and large intestine, synovial tissue, ovary, auditory meatus and other system.Target gene is selected from known those relevant with the specific region of CNS or CNS, and those of Unknown Function.Also can selection function the known and gene that may in this system, occur.Then according to these target genes of design described herein or the segmental antisense oligonucleotide of mRNA.At last, by with cell and tissue DNA and the external hybridization of RNA, according to the ability of eliminating or obviously reduce expression of target gene oligonucleotide is selected.Then the external oligonucleotide that demonstrates high downward adjusting, elimination or expression inhibiting effect is used for the body build-in test, preferred sites is applied to for example zones such as brain, heart, kidney, lung specifically, and predict its behavioristics, biophysics, biochemistry, cognition, motion, sensation, physiology and other function, whether these are assessed is relevant in order to determine to exist between target spot and function.For example, in respiratory system, may show dependency, the probability that the individuality that carries out this treatment will show the symptom of respiratory tract or inflammatory lung disease or other lung disorder such as malignant tumor reduces.Term used herein " regulate downwards " is meant that the proteic production of the intramolecularly that causes targeting, secretion or availability reduce the reduction of concentration (thereby cause), comprise fully and eliminating.
Table 2: with target spot and network diseases associated and examples of disorders
The medicaments insensitive sexual abnormality of the anti-nociception analgesia of dull-witted apoplexy anxiety cardio-pulmonary function Autonomous Control behavior disorder traumatic brain injury organ pipe disease of brain degenerative brain diseases CNS disease progression and vision (viral etc.) distortion (legal with illegal)
The heart attack of cognition food satiety alcohol sensitivity
The study depression encephalitis of ingesting
Anesthesia audition olfactory sensation hypoxia schizophrenia
Brain cancer cranium defective memory neuropathy neuropathic pain
Toothache headache sensorimotor coordination
Dysthymic disorder's emotion is inspired the bipolar disorder eating disorder
Cachexia aneurysm heart and congestion of blood vessel heart and blood vessel heart and blood vessel ooze out heart disease pain
Inflammation apoplexy angina pectoris heart failure
The ischemia platelet forms the restenosis viral infection
The angry transplant rejection of arrhythmia vascular permeability arteriasis libido angiogenesis and inhibition structure and biochemistry defective
The present invention is chiefly directed to and confirms in the mammiferous vertebrates that target is effective, the animal of this group comprises the animal in animal, house pet and the zoo of people and inhuman troglodyte, wild and domestic animal, ocean and land, for example, felid, dog, horse, pachyderm, whale, but more preferably human.A kind of suitable especially application of this technology is the purpose that is used for the veterinary, comprises all types of small-sized and larger animal that the veterinary pays close attention to, and comprises the animal in wild animal, marine animal, domestic animal, zoo etc.Target gene and albumen preferred mammal, target sequence preferably belongs to same species with the individuality of being treated.Although in many cases, the target of different plant species also suits, particularly the sequence of those and receptor show greater than about 45% homology, be preferably greater than about 85% homology, more preferably greater than the target RNA of about 95% homology or the part of gene.One group of preferred material comprises and takes off the A antisense oligonucleotide.Another is preferably organized and comprises wherein having one or more adenosine bases not exclusively to be taken off the A oligonucleotide by what general or alternate base had replaced.
Term " antisense " oligonucleotide typically refers to little, synthetic oligonucleotide, is similar to single stranded DNA, and it is used in this patent by suppressing the expression that target messenger RNA (mRNA) comes suppressor gene.Referring to Milligan, J.F. etc., J.Med.Chem.36 (14), 1923-1937 (1993), its relevant part is incorporated herein by reference in full.Method of the present invention uses the antisense material to suppress target gene expression, comprises listed those in the table 1.This material obtains by coding (justice is arranged) sequence hybridization with antisense oligonucleotide and messenger RNA known in the art (mRNA) usually.Exogenously use material of the present invention and can reduce by coded mRNA and the proteic level of target gene and/or cause the change of handled cell growth characteristics or shape.Referring to, Milligan etc. (1993); Helene, C. and Toulme, J.Biochim.Biophys.Acta 1049,99-125 (1990); Cohen, J.S.D. compiles, and the oligonucleotide Deoxydization nucleotide is as the antisense inhibitor of gene expression; CRC Press:Boca Raton, FL (1987), its relevant part is incorporated herein by reference in full.Used " antisense oligonucleotide " the short sequence of synthetic nucleotide normally in the literary composition, this sequence (1) can be under suitable hybridization conditions and the arbitrary portion hybridization of the mRNA of coding target protein, and (2) can cause the reduction of target protein gene expression after hybridization.Term " takes off adenosine ", and (taking off A) and " taking off thymidine " (taking off T) is meant the oligonucleotide that does not contain adenosine (taking off A) or thymidine (taking off T) substantially.In some cases, it is naturally occurring taking off the T sequence, and under the other situation, they can be by replacing undesired nucleotide (A) to obtain with another kind of its undesired active nucleotide that lacks.Herein, this is usually by replacing A to finish with " general or alternate base " known in the art.
The mRNA sequence of target protein can obtain from the nucleotide sequence of the gene of expressing protein, no matter is the target spot that existed or may be at the target spot of finding in the future.The sequence of the target gene of many different systems is known.Referring to the GenBank data base, NIH, its all sequence all is incorporated herein by reference.For those sequences still sequence of unknown gene, can be by partly obtaining from target spot with technical point known in the art.In case it is known that gene order, its RNA and/or albumen become, just can as described above produce antisense oligonucleotide and be used for by using in the body and detecting the minimizing of target protein production and the decline of specific function confirms that target is effective according to routine techniques.In one aspect of the invention, antisense oligonucleotide have can with coding and the particular system for example part or the bonded sequence of fragments specific of the mRNA molecule of the diseases of CNS, breathing, lung, motion, sensation, hormonal regulation, heart, kidney, immunity, blood, oncogene etc. or disease proteins associated.One of this bonded effect is to reduce even prevent the translation of corresponding mRNA, reduces the content of target protein in individual lung thus.
In an embodiment preferred of the present invention, one or more di-phosphate ester residues of antisense oligonucleotide are modifications or replace.Especially preferably can increase the chemical analog of the oligonucleotide that has the di-phosphate ester residue of modifying or replacing of oligonucleotide body internal stability, for example be modified into methyl phosphonate, phosphotriester, thiophosphate, phosphorodithioate or phosphoramidate.The natural phosphodiester key of oligonucleotide is caused to a certain degree degraded by the nuclease of cell easily.In contrast, many residues that this paper proposed are height endurabilities for the degraded of nuclease.Referring to Milligan etc., and Cohen, J.S.D., the same.In another embodiment preferred of the present invention, can prevent the degraded of oligonucleotide by adding " 3 '-terminal medicated cap ", wherein, with the phosphodiester bond of the key replacement oligonucleotide 3 ' end of anti-nuclease.Referring to, Tidd, D.M. and Warenius, H.M., Be.J.Cancer 60:343-350 (1989); Shaw, J.P. etc., Nucleic Acids Res.19:747-750 (1991), its relevant part is incorporated herein by reference in full.Phosphoramidate, thiophosphate and methylphosphonic acid ester bond all can similarly be realized purpose of the present invention in this mode.Modify manyly more to phosphodiester backbone, formed material is stable more, and their RNA affinity and cell permeability are high more in many cases.Referring to Milligan, etc., the same.Therefore, the quantity of the residue that can be modified or replace will be as required, target spot, route of administration and change, and can be to any amount between all residues from 1.Known have many different new keys of usefulness to replace the method for whole phosphodiester backbone.Referring to Millikan etc., the same.Base, the preferred residue analog of key between nucleotide or sugar comprises thiophosphate, methyl phosphonate, phosphotriester, thioformacetal, phosphorodithioate, sulfamate, formacetal, the bora phosphate ester, 3 '-thioformacetal, 5 '-thioether, carbonic ester, 5 '-the N-carbamate, sulfuric ester, sulphonic acid ester, sulfonamide, sulfone, sulfite, the 2-O-methyl, sulfoxide, sulfide, hydroxylamine, methoxy (MOM), methoxy ethyl (MOE), methylene (methyl-imino) (MMI) and (MOMI) residue of methylene oxygen base (methyl-imino).Preferred especially thiophosphate and methyl phosphonate are because they can obtain by automatic oligonucleotide is synthetic.Referring to, Millikan etc., the same.If suitably, can be with material of the present invention with the form of officinal salt or with the form administration of the mixture of antisense oligonucleotide and salt.In another embodiment of the invention, the mixture of different antisense oligonucleotides or its officinal salt is carried out administration.Material of the present invention can reduce a kind of expression and/or enhancing of said target mrna or weaken a kind of activity of approach.For example, method of the present invention be used in said target mrna or the gene identify all possible about 7 or more a plurality of mononucleotide, preferred about 60 mononucleotides at most, more preferably from about 10 to about 36 mononucleotides, deoxyribonucleotide part or the low deoxynucleoside acid moieties of adenosine (A) content that still more preferably from about 12 thymidines to about 21 mononucleotides (T) content is low.This can mononucleotide part (RNA) or the low mononucleotide part (gene) of adenosine content low by long thymidine (with the complementary nucleotide of the adenosine) content of 7 of searchings or more mononucleotides in target sequence or the shortage thymidine finish.Under most of situation, this searching usually produces about 10 to 30 such sequences, and promptly natural shortage or adenosine content are lower than about 40% sequence, has average length, the i.e. antisense oligonucleotide of the different length of the long exemplary target mRNA of about 1800 nucleotide.Sequence with high T or A content can be by fixing with one of general or alternate base replacement or single A.
Material of the present invention can have any suitable length, include but are not limited to about 7 long to about 60 nucleotide, preferred about 12 to about 45, more preferably about 30 nucleotide are long at most, still more preferably at most about 21, but they also can be other length, and this depends on the concrete target spot and the mode of conveying.Material of the present invention can be at any and all target RNA.One group of preferred material comprises at the mRNA zone of containing contact between intron and exon.When this material during at intron/exon contact, it can be stacked on the contact fully, perhaps can enough in the process that precursor mRNA is processed into ripe mRNA, be sheared with contact near the exon that inserts with inhibition, for example, make 3 of antisense oligonucleotide ' or 5 ' terminal between about 2 to 10, preferred about 3 to 5 nucleotide of for example intron/exon contact.Start codon and those have also preferably been covered near 5 of coding regions ' and 3 ' terminal antisense oligonucleotide.Antisense oligonucleotide can have about 0, about 3%, about 5%, about 7% to about 8%, about 10%, about adenosine content of 12%, about 15%, and the quantity of any intermediary adenosine content and scope.In the method for the invention, can there be one or more or whole A to be replaced, for example can combines but in adenosine A with thymidine by general or alternate base 1, A 2a, A 2bAnd A 3Receptor has less than the heteroaromatic base of about 0.5, about 0.3%, about 0.1% adenosine agonists or antagonist activities or in adenosine A 2aReceptor does not have active heteroaromatic base.Alternate base can be 3-nitro-pyrrole-2 '-deoxynucleoside, 5-nitro-indole, 2-deoxyribosyl base-(5-nitroindoline), 2-desoxyribofuranose base-(5-nitroindoline), 2 '-deoxyinosine, 2 '-deoxidation nebularine, 6H, 8H-3, the 4-dihydro-pyrimidin also [4,5-c] oxazine-7-ketone or 2-amino-6-methoxyl group amidopurin etc.Oligonucleotide can with methylated cytosine ( mC) come one or more or whole unmethylated C in the replaced C pG dinucleotide, if the latter exists in oligonucleotide.The present invention can also adopt other C to pyrimidine 5Modify, for example C 5Propine etc.One or more or the whole oligonucleotide connection residues that are applicable to the inventive method can be methyl phosphonates, phosphotriester, thiophosphate, phosphorodithioate, the bora phosphate ester, formacetal, thioformacetal, thioether, carbonic ester, carbamate, sulfuric ester, sulphonic acid ester, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene (methyl-imino), (MMI), methoxy (MOM), methoxy ethyl (MOE), methylene oxygen base (methyl-imino) (MOMA), methoxy (MOM), 2 '-the O-methyl, (C-5 propine) residue and combination thereof that phosphoramidate and C-5 replace.Being used for oligonucleotide of the present invention can link to each other with carrier, the carrier of protokaryon or eucaryon for example, and many examples of carrier are known in the art.Method of the present invention has been specified to use and a certain amount ofly can effectively have been reduced production or the availability of mRNA or increase its degraded or reduce the antisense oligonucleotide of content of peptides in the lung.Administration is preferably carried out in position, for example, for breathing and the lung target spot, can directly administration in respiratory system or nasal passage by sucking or be applied to individual lung.Antisense oligonucleotide can insert or directly organizes administration to brain, heart, kidney, tumor, testis, eye, auditory meatus, cervix uteri, nasal passage, scalp, oral cavity, muscle, pharynx, esophagus, intestinal, rectum, synovial tissue, ovary and other are localized external by injection, three-dimensional location.In addition, when target spot is a blood circulation and an immune part, also can be with oligonucleotide administration in blood.Under latter event, when disease or disease were relevant with immunodeficiency disease, target spot can be immunoglobulin, antibody receptor, cytokine, cytokine receptor, encode their gene and corresponding mRNA, gene and mRNA flanking region and exon and intron border etc.When disease or disease and malignant tumor or related to cancer, target spot can be selected from the enzyme of growth regulating dependency and other albumen, immunoglobulin and antibody receptor, their gene and the mRNA that encode, gene and mRNA and genome and mRNA flanking region and exon and the intron border relevant with oncogene.In addition, some Normocellular gene relevant with the cancer process for example angiogenesis factor, adhesion molecule and with cancerometastasis proteins associated enzyme etc. also be a part of the present invention.For example, this method can be by using composition outward, oral, intracavity is used, intranasal administration, anal is used, intravaginal is used, intrauterine is used, intracranial is used, pulmonary administration, use in the kidney, (intranodularly) uses in the joint, intraarticular is used, intraotically uses, use in the lymph, applied dermally, use in the cheek, intravenous is used, subcutaneous administration, intramuscular is used, use in the tumor, use in the gland, ophthalmic is used, intracranial is used, use in the organ, use in the blood vessel, use in the sheath, by implanting, suction is used, intradermal is used, use in the lung, in ear is used, use in the heart, pass through slow release, continue to discharge or use and finish by pump.Other example of target spot is the gene and the mRNA of coded polypeptide, and described peptide is selected from transcription factor, stimulate and activation factor, cytokine and receptor thereof, interleukin, interleukin-2-receptor, chemotactic factor, chemokine receptors, specificity and non-specific enzyme that endogenous produces, immunoglobulin, antibody receptor, central nervous system (CNS) and peripheral nervous and non-nervous system receptor, CNS and peripheral nervous and non-nervous system peptide mediator, adhesion molecule, sozin, somatomedin, vasoactive peptide, peptide receptor and conjugated protein, gene and mRNA corresponding to oncogene, g protein coupled receptor etc.Antisense oligonucleotide can be produced by the following method: from the disease that influences lung airway and disease for example relevant peptide such as the secretion increase of respiratory activity difficulty and malignant tumor, surfactant or minimizing, encode their gene and RNA, genome and mRNA flanking region and gene and mRNA exon and intron border selection target spot; Obtain the mRNA sequence, described mRNA is selected from mRNA, genome and mRNA flanking region and gene and mRNA exon and the intron border corresponding to the mRNA of target gene and coding target polypeptide; Select the fragment of at least a mRNA; Synthetic one or more oligonucleotide to the fragment antisense of selected mRNA; And, as needs, with a kind of can be with thymidine (T) hybridization but in the agonist ability of adenosine receptor lower or do not have the alternative base of agonist ability (do not have or the adenosine receptor that reduces activates) to replace one or more A so that in the oligonucleotide content of A be reduced to maximum about 15% of whole nucleotide.As already pointed out, suitable target spot is a target protein, (described peptide is selected from transcription factor for the gene of coded polypeptide and mRNA, stimulate and activation factor, interleukin, interleukin-2-receptor, chemotactic factor, chemokine receptors, specificity and non-specific enzyme that endogenous produces, immunoglobulin, antibody receptor, central nervous system (CNS) and peripheral nervous and non-nervous system receptor, CNS and peripheral nervous and non-nervous system peptide mediator and receptor thereof, adhesion molecule, sozin, somatomedin, vasoactive peptide and receptor thereof and conjugated protein), target gene and mRNA corresponding to oncogene, and flanking region and exon and intron border.Encoded polypeptides can be selected from Nf κ B transcription factor, interleukin-8 receptor (IL-8R), interleukin-15 receptor (IL-5R), interleukin 4 receptors (IL-4R), interleukin-13 receptor (IL-3R), il-1 β (IL-1 β), interleukin-11 beta receptor (IL-1 β R), the eotaxin, trypsinlike enzyme, main basic protein, the beta-2-adrenoreceptor kinases, endothelin-receptor A, endothelin-receptor B, the anterior endothelium angiogenic peptide that contracts is former, bradykinin b 2 receptor, the IgE high-affinity receptor, interleukin-11 (IL-1), interleukin 1 receptor (IL-1R), interleukin 9 (IL-9), interleukin-9 receptor (IL-9R), interleukin 11 (IL-11), interleukin-11 receptor (IL-11R), the inducible nitric oxide synthase, cyclo-oxygenase (COX), adhesion molecule 1 (ICAM-1) vascular cell adhesion molecule (VCAM) in the cell, Rantes, endothelial leucocyte adhesion molecule (ELAM-1), the monocyte activation factor, chemotactic factor for neutrophil, the neutrophil elastoser, sozin 1,2 and 3, the muscarine acetylcholinergic receptor, platelet activating factor, tumor necrosis factor, the 5-lipoxygenase, phosphodiesterase IN, the P material, P material receptor, histamine receptor, chymase, the CCR-1 CC-chemokine receptor, the CCR-2 CC-chemokine receptor, the CCR-3 CC-chemokine receptor, the CCR-4 CC-chemokine receptor, the CCR-5 CC-chemokine receptor, the prostaglandin receptoroid, the GATA-3 transcription factor, the neutrophil adhesion receptor, map kinase, interleukin-9 (IL-9), the NFAT transcription factor, STAT 4, MIP-1 α, MCP-2, MCP-3, MCP-4, cyclophilin, phospholipase A2, basic fibroblast growth factor, metalloproteases, the CSBP/p38 map kinase, Trypsin
Figure A0080675900451
Receptor, PDG2, interleukin-3 (IL-3), il-1 β (IL-1 β), cyclosporin A-conjugated protein, FK5-is conjugated protein, α 4 β 1 select albumen, fibronectin, α 4 β 7 select albumen, Mad CAM-1, LFA-1 (CD11a/CD18), PECAM-1, LFA-1 selects albumen, C3bi, PSGL-1, E-selects albumen, P-selects albumen, CD-34, L-selects albumen, p150,95, Mac-1 (CD11b/CD18), fucosyltransferase, VLA-4, CD-18/CD11a, CD11b/CD18, ICAM2 and ICAM3, C5a, CCR3 (eotaxin's receptor), CCR1, CCR2, CCR4, CCR5, LTB-4, the AP-1 transcription factor, Protein kinase C, cysteinyl leukotriene receptor, Tachychinnen receptor (tach R), I kappa b kinase 1﹠amp; 2, STAT 6, c-mas and NF-interleukin-6 (NF-IL-6), its flanking region and exon and intron border.But the present invention is used for the newfound gene that can't obtain at public database.Because target is confirmed to be very important for this group gene, otherwise their effects in disease will remain the unknown.
Following table 3 has been listed the target spot that material of the present invention can effectively be used simply.This only is an illustrative.This method can be used for wherein target and confirms to depend on any system that the release of bioactive adenosine in the oligonucleotide degradation process is discussed.This is very important, because adenosine is an antiinflammatory, its release will make the affirmation result of cancer target spot become to be difficult to be explained.
Table 3: cancer target spot
Transform the cancer gene therapy target spot
The ras thymidylate synthetase
The src thymidylate synthetase
The myc dihydrofolate reductase
The bcl-2 thymidine kinase
Deoxycytidine kinase
Ribonucleotide reductase
The angiogenesis factor adhesion molecule
The enzyme of oncogene folic acid approach
DNA-repair gene (a carbon storehouse)
Telomerase
HMG CoA reductase
Farnesyl transferase
G-6-P transesterify enzyme
One group is used to preferably to confirm that the effective target spot of cancer target spot is the gene with dissimilar related to cancer, or those it has been generally acknowledged that the target spot relevant with malignant tumor, and no matter they are to regulate or participate in RNA and/or proteic production.Its example is to transform oncogene, promptly goes up the target spot shown in the table 3 etc.Cancer target of the present invention confirm material institute at other target spot be various enzymes, mainly be (but being not limited only to) thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, ribonucleotide reductase, in cancer cell content than other gene outcome of in normal cell, enriching etc.Technology of the present invention be specially adapted to confirm the cancer target gene effectively/invalid because traditional cancer therapy can not be effectively kill cancer cell and preventing simultaneously optionally because the caused destructive influence of treatment of chemotherapy, radiotherapy etc. to normal living cells.That is to say, existing cancer therapy optionally targeting in malignant cell.Any target spot of confirming with method of the present invention can both optionally weaken required gene outcome and reduction or enhancement function and approach thereof.This method obviously is better than conventional cancer therapy, because it allows to select a system, and in system, select a kind of approach that comprises a plurality of target spots (for example first, second and the 3rd possible target spot, these target spots generally can not expressed simultaneously) in normal cell and respectively and confirm jointly with them.Therefore, the method for the present invention target spot that will be provided for treating.In case confirm that by method of the present invention target spot is effective, just can use the optionally material that acts on this target spot to individuality optionally increases to cause the toxicity in the tumor cell of for example expressing three target spots, will be obviously less even not influenced and only express the influence that the normal cell of one or two target spot is subjected to.The material that method of the present invention is used is preferably designed for the target gene relevant with the species for the treatment of administration and/or the material of mRNA antisense.When being used for when the mankind confirm that target spot is effective, it is antisense that described material is preferably designed for human gene or RNA.Material of the present invention comprise to n DNA and/or RNA sequence or its length mostly be most than target sequence lack a base, preferably at least about the oligonucleotide of the long fragment antisense of 7 nucleotide, contain only about 0.1%, about 1%, about 4% to about 5%, about 10%, about 15%, about 30% or do not contain the oligonucleotide of adenosine, and one or more adenosine nucleoside acid by can with the thymidine oligonucleotide ligand to but can not activate the oligonucleotide that the general or alternate base of the active what is called of adenosine receptor has replaced substantially.The human sequence of antisense oligonucleotide of the present invention and segmental example are following fragment and the complete genome of described fragment and coded sequence, exon and intron-exon contact or the shorter part of mRNA, preferably in each sequence, contain 7,10,15,18 to 21,24,27,30, a n-1 nucleotide, wherein n is the nucleotide sum of sequence.These fragments can be selected from any part of long oligonucleotide, for example middle part, 5 '-terminal, 3 '-end, or from any other site of original series.Particularly importantly hang down the fragment of adenosine nucleoside acid content, be those contain be lower than or about 30%, preferably be lower than or about 15%, more preferably less than or about 10%, still more preferably less than or about 5%, the first-selected fragment that does not contain adenosine nucleoside acid, can be by selecting or replacing with general or alternate base according to the present invention.Material first-selection of the present invention comprises sequence and the fragment thereof that wherein has one or more adenosines that are present in the sequence to be replaced by general or alternate base (B) that this paper exemplified.Equally, the present invention also comprises design all shorter fragments that contain B fragment, its fragment or part with adenosine (A) contained in the B replacement sequence, referring to above description.
Method of the present invention can separately be used these materials or its form with the pharmaceutical composition that contains the above-described antisense oligonucleotide that can effectively reduce the amount that target protein expresses is used.Antisense oligonucleotide must could combine and stop its translation with proteic mRNA in the Codocyte by cell membrane earlier specifically.Said composition is present in suitable pharmaceutically suitable carrier, for example aseptic pyrogen-free saline solution.Material of the present invention can for example be prepared in liposome with preparing by the hydrophobic carrier of cell membrane, and described liposome is in the pharmaceutically acceptable aqueous carrier, and optionally contains surfactant or lipid.In addition, also can be with oligonucleotide and for example ribozyme coupling of material that can deactivation mRNA, to suppress translation more completely.Pharmaceutical preparation can also contain chimeric molecule, and wherein, antisense oligonucleotide can be connected by the molecule of cell internalizing with known.These oligonucleotide conjugatess utilize the cellular uptake approach to increase the IC of oligonucleotide.The example of the molecule of using in this mode is a macromole, comprises transferrins, asialoglycoprotein (being connected by many lysins with oligonucleotide) and streptavidin and other macromole known in the art.Method of the present invention is also used the antisense compounds in pharmaceutical preparation, for example in lipid granule or capsule, and for example liposome or crystallite.Granule can be any suitable structure, for example monolayer or multiwalled structure.In a kind of embodiment preferred, antisense oligonucleotide is included in the liposome.For these granules and capsule, preferred especially positively charged lipid, for example N-[1-(2,3-two oily acyloxy) propyl group]-N, N, N-trimethyl ammonium Methylsulfate, or be called " DOTAP ".But other lipid also is fit to, and in fact may be more suitable.The preparation of described lipid granule is known.Referring to, for example, United States Patent (USP) 4,880,635 (authorizing Janoff etc.), 4,906,477 (authorizing Kurono etc.), 4,911,928 (authorizing Wallach), 4,917,951 (authorizing Wallach), 4,920,016 (authorizing Allen etc.), 4,921,757 (authorizing Wheatley etc.), its relevant part is incorporated herein by reference in full.Method of the present invention can be used antisense oligonucleotide by any method, preferably can make to carry minimum method, for example in original position administrations such as brain, lung, kidney, heart, testis.
Can finish by any suitable method to the lung application of substances, but preferably pass through the respiratory system administration with the form of inhalable formulations, more preferably to contain the form administration that can suck particulate aerosol, the described granule that sucks contains the material that is useful on by patient's suction.Can suck granule can be gas, liquid or solid form, and they can optionally contain other therapeutic component and preparation composition.Granule of the present invention preferably can suck the granule of size, thus preferred enough little can be when suction by oral cavity and larynx and enter into the bronchus and the alveolar of pulmonary.Usually, the granule in about 0.5 to 10 micrometer range can suck.But other size also suits.Being included in the bigger granule that can not suck size of diameter in the inhalable formulations might be deposited on venturi and may be swallowed.Therefore, preferably reduce to minimum with sucking particulate amount in the aerosol.For nose administration, the granule in the preferred 10-500 mu m range can rest on nasal cavity to guarantee them.The aerosol that contains the liquid particles of described material can produce by any suitable method, for example uses insufflator or nebulizer.Referring to, for example U.S. Patent number 4,501, and 729.Suitable propellant comprises solvent, for example some cfc chemical compound, for example dichlorodifluoromethane, Arcton 11, dichlorotetra-fluoroethane and/or their mixture.Other propellant also is fit to, and when it was more suitable for concrete application, they may be preferred.Preparation can also contain for example ethanol of one or more cosolvents; Surfactant is oleic acid or sorbitan trioleate for example; Antioxidant and suitable correctives.Antisense oligonucleotide can be injected to the brain administration by stereotactic method or by the target area of isolation to CNS, and all these methods all are known in the art.Perhaps, can be with oligonucleotide with the dosage form administration that can pass through blood brain barrier known in the art, for example, can be with the conjugates of the monoclonal antibody of streptavidin and anti-TfR as universal support from peptide-oligonucleotide to brain that carry single biotinylated peptide, antisense oligonucleotide (3 of di-phosphate ester '-biotinylation or other derivant) and.Referring to, for example, Levy, R.M. etc., J.Neuroviral 3Suppl:574-75 (1997); Wu-Pong and Gewirtz, BioPharm, pp.32-38 (Jan.1999); Boado, R.J., etc., J.Pharm.Sci.87 (11): 1308-1315 (1998).Can be undertaken by the original position medicine-feeding technology to heart, liver and kidney and other organ pipe administration, for example conduit insertion, injection and local diffusion, all these all are known in the art.Referring to, for example, Lewis, K.J. etc., J.Drug Target, 5 (4): 291 (1998); Ayrin, M.A. etc., Cathet.Cardiovasc.Diagn, 41 (3): 232-240 (1997); Luft, F.C., J.Molec.Med.76 (2): 75 (1998).These administrations are carried out with liquid, solid or the gaseous medication compositions of described material usually, and these pharmaceutical compositions can prepare by antisense oligonucleotide and suitable solvent or carrier such as aseptic pyrogen-free water, lipid and/or other pharmacy or veterinary's acceptable carrier are mixed.Can also contain other treatment chemical compound and other preparation composition known in the art.The solid particle composition that contains the dried particles of micronized material of the present invention can prepare by the following method: exsiccant antisense compounds is ground the micronized compositions that will grind then (for example 400 mesh sieves) bulk with fragmentation or separating particles of sieving with mortar and pestle.The solid particle composition that contains antisense compounds can also optionally contain dispersant and other known substances, and its effect is to help to form mist or aerosol.Suitable dispersant is a lactose, it can with antisense compounds with any suitable mixed, be about 1: 1w/w.Also other ratio can be used, and other therapeutic agent and preparation composition can be contained.The relevant portion of the list of references of quoting in this patent all is incorporated herein by reference, and particularly those help to realize publication and patent with written description each side of the present invention.
The dosage of antisense compounds is usually with target, function and amplification thereof, the disease of being studied, individual condition, concrete preparation, approach and position, the administration time etc. of administration.Usually, preferably obtain 0.05 to 50 μ M or the more preferably oligonucleotide IC of 0.2 to 5 μ M.But, preferably measure dose-response curve so that determine appropriate dosage and observe clear and definite reaction.Used dosage can change, and for example, adopts about 0.001, about 0.01, about 1mg/kg to about 50, about 100 and the dosage of about 150mg/kg usually.It will be understood by those skilled in the art that for special applications, also can adopt higher and lower dosage.These dosage can be administered once or (for example per 24 hours) administration in a period of time as required, but also can adopt other dosage regimen.With following embodiment the present invention is described, should regard these embodiment as limitation of the invention.In these embodiments, μ M represents the micromole, and mL represents milliliter, and μ m represents micron, and mm represents millimeter, and cm represents centimetre, ℃ expression degree centigrade, and μ g represents microgram, and mg represents milligram, and g represents gram, and kg represents kilogram, and M represents mole, and hrs represents hour.EXAMPLE Example 1: the design of antisense oligonucleotide and synthetic
The secondary structure that may need to understand the complexity of target receptor mrna at the design of the antisense oligonucleotide of target receptor.After obtaining this structure, the antisense nucleotide in design targeting mRNA zone, described zone can be considered to give mRNA functional activity or stability, and preferably can be overlapping with start codon.Other target sites are wieldy.As the specific excess syndrome of antisense effect, other are not exclusively complementary with said target mrna but contain the oligonucleotide that identical nucleotide is formed on the w/w basis, are included in contrast during antisense tests.For example, analyze adenosine A 1Receptor mRNA secondary structure, and as the above-mentioned design D2EHDTPA antisense oligonucleotide of using it for.Synthetic antisense oligonucleotide is named as HAdA 1AS has following sequence: 5 '-GAT GGA GGG CGG CAT GGC GGG-3 ' (SEQ ID NO:1).In contrast, the D2EHDTPA antisense nucleotide of synthetic mispairing, it is named as HAdA1MM1, has following sequence: 5 '-GTA GCA GGC GGG GAT GGG GGC-3 ' (SEQ IDNO:2).Each oligonucleotide has identical base contents and general sequential structure.Carry out the homology search in GENBANK (discharging 85.0) and EMBL (discharging 40.0), the result shows that the antisense oligonucleotide specificity is at the adenosine A of people and rabbit 1Acceptor gene, and the contrast of mispairing is not the material standed for of the hybridization carried out with any known sequence.Adenosine A 3The secondary structure of receptor mrna is analyzed similarly, and as above-mentionedly is used to design the phosphordithiic acid antisense oligonucleotide.Synthetic first antisense oligonucleotide (HAdA3AS1) has following sequence: 5 '-GTT GTT GGG CAT CTT GCC-3 ' (SEQ ID NO:3).In contrast, the D2EHDTPA antisense oligonucleotide (HAdA3MM1) of a synthetic mispairing has following sequence: 5 '-GTA CTT GCG GAT CTA GGC-3 ' (SEQ ID NO:4).Also designed and synthesized second D2EHDTPA antisense oligonucleotide (HAdA3AS2), it has following sequence: 5 '-GTGGGC CTA GCT CTC GCC-3 ' (SEQ ID NO:5).Its control oligonucleotide (HAdA3MM2) has sequence: 5 '-GTC GGG GTA CCT GTC GGC-3 ' (SEQ IDNO:6).Phosphorothioate oligonucleotide is synthetic with Applied Biosystems 396 type oligonucleotide synthesizers, and (DuPont MD) carries out purification to utilize the NENSORB chromatography.Embodiment 2: adenosine A 1Measure in the body of receptor antisense oligonucleotide
Utilize lung adenocarcinoma cell HTB-54, measure above-mentioned at people A 1The effect of the antisense oligonucleotide of receptor (SEQ ID NO:1) in external model.Utilize standard rna trace method and, confirm HTB-54 lung adenocarcinoma cell expression A at laboratory design and synthetic acceptor probe 1Adenosine receptor.HTB-54 human lung adenocarcinoma cell (tissue culture's ware of 106/100mm) is exposed to 5.0 μ M HAdA1AS or HAdA1MM1 24 hours, behind incubation, changed fresh culture medium and oligonucleotide in 12 hours.Be exposed to oligonucleotide after 24 hours, collecting cell extracts its RNA with standard method.Synthetic corresponding to (and therefore having the sequence identical with described antisense sequences by antisense sequences, but be not D2EHDTPAization) the poly-probe of A 21-in the mRNA zone of targeting, and using it for the RNA trace of detecting RNA, described RNA obtains from the HTB-54 cell preparation through HAdA1AS processing, HAdA1MM1 processing and non-processing.These traces clearly illustrate, HAdA1AS rather than HAdA1MM1 reduce effectively＞and people's adenosine receptor mRNA of 50%.This result shows that HAdA1AS is a good material standed for of antasthmatic, because its scarcity is at the adenosine A that participates in asthma 1MRNA in the cell of receptor.Embodiment 3: adenosine A 1Effect in the body of receptor antisense oligonucleotide
With the eclipsed adenosine A of start codon 1In the gene, rabbit and human DNA sequence's accidental homology can use the D2EHDTPA antisense oligonucleotide, and described D2EHDTPA antisense oligonucleotide is to be designed to antagonism people adenosine A in rabbit model at first 1Receptor.(CA), the newborn New Zealand white rabbit to no pasteurella in postnatal 24 hours is carried out the intraperitoneal immunity for BerkeleyBiologicals, Berkeley with the dermatophagoides pteronyssinus that is mixed with 10% kaolinic 312 antigen units/ml (D.farinae) extract.In first month, repeat immunity weekly once, at ensuing 2 months, immunity every other week was once then.With the mixture of ketamine hydrochlorate (44mg/kg) and acetylpromazine maleate (0.4mg/kg), through the intramuscular administration, the rabbit anesthesia that makes 8 3-4 months big sensitization is with lax.Then, rabbit faced up with a comfortable posture to be placed on the little molding filling type animal plate, and (Glens Falls NY) inserts for Mallinkrodt, Inc. the endotracheal tube of 4.0-mm.The one outer polyethylene catheter through 2.4mm that is connected with latex balloon is inserted in the esophagus, and in whole experiment, keeps identical distance (approximately 16cm) with mouth.Fleisch pneumotachograph (size 00 with endotracheal tube and unlatching; DOM Medical, Richmond VA) links to each other, and utilizes by Gould carrier amplifier (11-4113 type; Gould Electronic, Cleveland, OH) Validyne differential pressure pickup (the DP-45161927 type of Qu Donging; Validyne Engineering Corp., Northridge CA) measures throughput.Oesophageal bulb is linked to each other with a side of differential pressure pickup, and the flow export of endotracheal tube is connected to the opposite side of pressure transducer, to allow the pressure reduction between record lung and the lung inner chamber.The synthetic gas stream amount provides a successive timing fluctuation amount, and utilizes Self-breathing analyser (6 types; Buxco, Sharon CT) is waiting appearance and air-flow to calculate the measured value of total pulmonary resistance (RL) and dynamic retractility rate (Cdyn) zero point respectively.Select animal at random, obtain at the 1st day PC before the processing of aerosol thinner adenosine 50Value.Contrast (HAdA1MM) oligonucleotide of antisense (HAdA1AS) or mispairing is dissolved in the physiological saline solution, and concentration is 5000 μ g (5mg)/1.0ml.With after endotracheal tube gives the antisense or the mispairing oligonucleotide (about 5000 μ g, volume is 1.0ml) of aerosol thinner, every day twice, totally two days to animal.(PA) aerosol of generation saline, adenosine or antisense or mispairing oligonucleotide produces aerosol droplets for DeVilbiss, Somerset, and wherein 80% diameter is less than 5 μ m by ultrasound atomizer.In first experiment, give antisense oligonucleotide to 4 allergy rabbits of selecting at random, to other 4 control oligonucleotide that give mispairing.In the 3rd day the morning, obtain PC 50Value (the dynamic retractility rate of bronchial catheter is reduced by 50% necessary aerosol thinner adenosine concentration from baseline value, mg/ml), and with the PC of these animals before being exposed to oligonucleotide 50Value is compared.After 1 week of interval, to animal exchange administration, give antisense oligonucleotide now to those animals that before given the mispairing control oligonucleotide, give the mispairing control oligonucleotide now to those animals that before given antisense oligonucleotide.Processing method and measurement are with employed identical in first experiment.It should be noted that under the solubility limit 20mg/ml that reaches adenosine at the most, in 8 animals of handling, 6 bronchoconstrictions that can not produce by the adenosine mediation are arranged with antisense oligonucleotide.For computation purpose, the PC of these animals 50Value is set to 20mg/ml.Therefore, these values that provide have been represented the minima of antisense effect.Actual effect is higher.This result of experiment is listed in the table below in 4.
Table 4: adenosine A 1The receptor antisense oligonucleotide is to the PC in the asthma rabbit 50The influence of value
Mispairing contrast A 1The receptor antisense oligonucleotide
Give before the oligonucleotide Give after the oligonucleotide Give before the oligonucleotide Give after the oligonucleotide
3.56+1.02 5.16+1.03 ???2.36+0.68 ??＞19.5+0.34 **
The result is expressed as meansigma methods (n=8) ± SEM.
By the replicate analysis (ANOVA) and the TukeyShi protection experiment of deviation, determine significance.
*Significant difference is arranged, p＜0.01 with other all groups.
In two batches of experiments, the animal performance of the accepting antisense oligonucleotide lung dynamic retractility rate of sening as an envoy to reduces by 50% required aerosol thinner adenosine dosage and increases greatly.The control oligonucleotide of not observing mispairing is to PC 50The influence of value.Accepting antisense or sucking in the animal of control oligonucleotide, do not observe toxicity.These results clearly illustrate that in pneumonopathy, lung has the unusual potential as the target of antisense oligonucleotide base therapeutic intervention.They also show, with people's asthma very similarly in the model system, downward modulation adenosine A 1Receptor is eliminated the bronchoconstriction that is mediated by adenosine in the asthma air flue greatly.In the allergy rabbit model of people's asthma, the extraordinary reaction of bronchus is that fabulous antisense is intervened the end of the final point, this be because participate in the tissue of this reaction be positioned at aerosol thinner oligonucleotide contact point near, and this model critically imitates an important human diseases.Embodiment 4:A 1The specificity of-adenosine receptor antisense oligonucleotide
When the exchange test of the foregoing description 3 finishes, the adenosine A of the airway smooth muscle of all rabbits of quantitative analysis 1Acceptor number.As the specific contrast of antisense oligonucleotide, this should affected adenosine A 2Receptor also is quantized.From each rabbit, downcut asm tissue, method (Kleinstein according to Kleinstein etc., J. and Glossmann, H., Naunyn-Schmiedeberg ' s Arch.Pharmacol.305:191-200 (1978)) and in addition minority is revised and is prepared the film fraction, and the relevant portion of the document is incorporated herein for referencial use thus in full.Rough plasma membrane goods are kept at 70 ℃ until mensuration.Method (240-254 (1976), the relevant portion of the document are incorporated herein for referencial use thus in full for M.Bradford, Anal.Biochem.72) with Bradford is determined protein content.Refrigerated plasma membrane at room temperature thaws, and with the 0.2U/ml ADA Adenosine deaminase 37 ℃ of incubations 30 minutes, to remove the endogenous adenosine.[ 3H] DPCPX (A 1Receptor-specific) or [ 3H] CGS-21680 (A 1Receptor-specific) combination, utilize method (Ali, the S. etc. of Ali etc. as mentioned above, J.Pharmacol.Exp.Ther.268, Am.J.Physiol266, L271-277 (1994) measures, the relevant portion of the document is incorporated herein for referencial use thus in full).Compared with the control, in exchange test, use adenosine A 1The animal that antisense oligonucleotide is handled is at A 1Reduce on the acceptor number about 75%, as utilizing A 1The specificity of-specific antagonists DPCPX is in conjunction with measured.Adenosine A 2Acceptor number does not change, as utilizing A 2Receptor-specific agonist 2-[is to (2-carboxyethyl)-phenethyl amino]-5 '-specificity of (N-ethyl carboxamido) adenosine (CGS-21680) is in conjunction with measured.This results are shown in the following table 5.The effect of following presentation of results antisense strategy airway disorders.Because above-mentioned antisense oligonucleotide is eliminated the receptor system that the bronchoconstriction by the adenosine mediation is worked, therefore may be very unnecessary from wherein eliminating adenosine.But, preferably in these oligonucleotide, eliminate adenosine, obtain the required dosage of similar effect to reduce.Other antisense oligonucleotides of the proteic mRNA of targeting participation inflammation as mentioned above.From its contained nucleotide, eliminate adenosine, in degradation process, discharge to prevent it.
Table 5: adenosine A 1The specificity of receptor effect of antisense
Mispairing contrast A 1Antisense
The oligonucleotide oligonucleotide
??A 1The combination of-specificity ????1105+48 ** ????293+18
??A 2The combination of-specificity ????302+22 ????442+171
The result is expressed as meansigma methods (n=8) ± SEM.
By the replicate analysis (ANOVA) and the TukeyShi protection experiment of deviation, determine significance.
*With the mispairing contrast significant difference, p＜0.01 are arranged.
Embodiment 5: the antisense oligonucleotide that points to other target nucleic acids
Carrying out this work is in order to prove that the present invention is widely used in the extensive special antisense oligonucleotide (" oligonucleotide ") of nucleic acid target.Carry out following experimentation and be in order to show that method of the present invention is widely used in using together with designed antisense oligonucleotide as described in the present application, and any adenosine receptor mRNA of targeting.For this purpose, at adenosine receptor mRNA such as adenosine A 1, A 2bAnd A 3Receptor mrna has prepared multiple antisense oligonucleotide.Antisense oligonucleotide I (SEQ ID NO:1) as mentioned above.Other 5 kinds of antisense phosphorothioate oligonucleotide such as above-mentioned designing and synthesizing.
1-oligonucleotide II (SEQ ID NO:7) is the targeting adenosine A also 1Receptor, but with the different zone of oligonucleotide I targeting.
2-oligonucleotide V (SEQ ID NO:10) targeting adenosine A 2bReceptor.
3-oligonucleotide III (SEQ ID NO:8) and IV (SEQ ID NO:9) targeting adenosine A 3The zones of different of receptor.
4-oligonucleotide I-PD (SEQ ID NO:11) (phosphodiester oligonucleotide) with sequence identical with oligonucleotide I.
These antisense oligonucleotides are designed to treat selected species, and generally special to these species, unless the sections of the said target mrna of other species contains similar sequence by chance.All antisense oligonucleotides of preparation as described below, and in rabbit model, measure it in the body to bronchoconstriction, inflammation and allergic effect, it causes dyspnea and lung airway to be obstructed, as cases such as asthma, described in above-mentioned application.Embodiment 6: the design of other antisense oligonucleotides and sequence
6 kinds of oligonucleotide have been studied and in the effect of rabbit model, the following report of the result of these researchs and discussion.Select 5 kinds in these oligonucleotide to be used for this research, so that the data of the oligonucleotide I (SEQ ID NO:1) that provides in the foregoing description 1 to 4 to be provided.This oligonucleotide is to adenosine A 1A zone of receptor mrna is an antisense.The oligonucleotide of being surveyed is named as the targeting adenosine A 1Antisense oligonucleotide I of receptor mrna zones of different (SEQ ID NO:1) and II (SEQ ID NO:7), the targeting adenosine A 2bOligonucleotide V of receptor mRNA (SEQ IDNO:8) and targeting adenosine A 3The antisense oligonucleotide III of two zoness of different of receptor mrna and IV (SEQ ID NO:9 and 10).The 6th kind of oligonucleotide (oligonucleotide I-PD) is the di-phosphate ester form of oligonucleotide I (SEQ ID NO:1).According to the foregoing description 1, carry out the design of these antisense oligonucleotides and synthetic.
(I) antisense oligonucleotide I
The foregoing description 1 to 4 described antisense oligonucleotide I targeting people A 1Adenosine receptor mRNA (EPI 2010).Long 21 nucleotide of antisense oligonucleotide I, overlapping with start codon, and have following sequence: 5 '-GAT GGA GGG CGG CAT GGC GGG-3 ' (: SEQID NO:1).Oligonucleotide I had before demonstrated and had suppressed the bronchoconstriction that brought out by adenosine in the allergy rabbit, and reduced airway obstruction and the extraordinary reaction of being brought out by allergen of bronchus (BHR), as above-mentioned and by Nyce, and J.W.﹠amp; Metzger, W.J., Nature, 385:721 (1977) is described, and the relevant portion of the document is incorporated herein for referencial use in full.
(II) antisense oligonucleotide II
Design monothio phosphoric acid antisense oligonucleotide (SEQ ID NO:7), its targeting rabbit adenosine A according to the present invention 1The receptor mrna district :+936 to+956, with respect to start codon (starting point).Long 21 nucleotide of antisense oligonucleotide II, and have following sequence: 5 '-CTC GTC GCCGTC GCC GGC GGG-3 ' (SEQ ID NO:7).
(III) antisense oligonucleotide III
Design a D2EHDTPA antisense oligonucleotide that (the SEQ ID NO:8) that provided in the foregoing description 1 be provided, its targeting antisense A according to the present invention 3The receptor mrna district :+3 to+22, with respect to the start codon starting point.Long 20 nucleotide of antisense oligonucleotide III, and have following sequence: 5 '-GGG TGG TGC TAT TGT CGG GC-3 ' (SEQ ID NO:8).
(IV) antisense oligonucleotide IV
Design another D2EHDTPA antisense oligonucleotide (SEQ ID NO:9), its targeting adenosine A according to the present invention 3The receptor mrna district :+386 to+401, with respect to start codon (starting point).Long 15 nucleotide of antisense oligonucleotide IV, and have following sequence: 5 '-GGC CCA GGGCCA GCC-3 ' (SEQ ID NO:9).
(V) antisense oligonucleotide V
Design monothio phosphoric acid antisense oligonucleotide (SEQ ID NO:10), its targeting adenosine A according to the present invention 2bThe receptor mrna district :-21 to-1, with respect to start codon (starting point).Long 21 nucleotide of antisense oligonucleotide V, and have following sequence: 5 '-GGC CGG GCCAGC CGG GCC CGG-3 ' (SEQ ID NO:10).
(VI) A 1The mispairing oligonucleotide
The two kinds of contrast with different mispairing oligonucleotide of following sequence as top embodiment 5 described antisense oligonucleotide I (SEQ ID NO:1): A 1MM2 5 '-GTA GGT GGCGGG CAA GGC GGG-3 ' (SEQ ID NO:12) and A 1MM3 5 '-GAT GGA GGCGGG CAT GGC GGG-3 ' (SEQ ID NO:13).Antisense oligonucleotide I has identical base composition and general sequential structure with two kinds of mispairing antisense oligonucleotides.Carry out the homology search in GENBANK (discharging 85.0) and EMBL (discharging 40.0), the result shows that antisense oligonucleotide I is not only to people's adenosine A 1Acceptor gene is special, and to the rabbit adenosine A 1Acceptor gene is also special, and the contrast of mispairing is not the material standed for that is used for any known mankind or animal gene sequence hybridization.
(VII) antisense oligonucleotide A 1-PD (oligonucleotide VI)
Disclosed as above-mentioned application, design and oligonucleotide I have di-phosphate ester antisense oligonucleotide (the oligonucleotide VI of identical sequence; SEQ ID NO:11).Long 21 nucleotide of antisense oligonucleotide I-PD, overlapping with start codon, and have following sequence: 5 '-GAT GGAGGG CGG CAT GGC GGG-3 ' (SEQ ID NO:11).
(VIII) contrast
According to give (II), (III) and (IV) in the identical scheme of antisense oligonucleotide, give 5.0mL aerosol thinner Sterile Saline to each rabbit.More than be that example as g protein coupled receptor provides.But the method that reduces adenosine content is applicable to that generally any gene and any target confirm system, and wherein the release of the biological activity adenosine experimental data that can be used in the real embodiment adenosine receptor becomes unclear.Embodiment 7: antisense oligonucleotide synthetic
Utilize Applied Biosystems 396 type oligonucleotide synthesizers, synthetic D2EHDTPA antisense oligonucleotide with the sequence described in top (a), and (DuPont DE) carries out purification to utilize the NENSORB chromatography.In building-up process, TETD (tetraethylthiuram disulfide) is used as vulcanizing agent.Use this method, synthetic respectively and purification of antisense oligonucleotides II (SEQ IDNO:7), antisense oligonucleotide III (SEQ ID NO:8) and antisense oligonucleotide IV (SEQ IDNO:9).Embodiment 8: preparation allergy rabbit
(Metzger, W.J., in Late Phase Allergic Reactions, Dorsch, W., Ed., CRC Handbook, pp.347-362, CRC Press, BocaRaton (1990) as previously mentioned; Ali, S., Metzger, W.J. and Mustafa, S.J., Am.J.Resp.Crit.Care Med.149:908 (1994)), in birth 24 hours, with dermatophagoides pteronyssinus (D.farinae) extract (BerkeleyBiologicals, the Berkeley that are mixed with 10% kaolinic 0.5mL, 312 antigen units/ml, CA) the newborn New Zealand white rabbit of no Pasteurella is carried out the intraperitoneal immunity, the relevant portion of described document is incorporated herein for referencial use thus in full.In first month, repeat immunity weekly once, immunity every other week is once big until 4 months then.These rabbits real estate of selecting the superior is given birth to the allergen specific IgE antibody, and it generally reacts former generation to the source of the gas paraphilia replys, antagonism asthma reaction in early stage and late period, and demonstrate the extraordinary reaction of bronchus (BHR).Every menstruation intraperitoneal gives allergen, and (312 unit dirt demodicid mite allergens, as mentioned above), this continues to stimulate and keep allergen specific IgE antibody and BHR.As (Ali, S., Metzger, W.J. and Mustafa, S.J., Am.J.Resp.Crit.Care Med.149 (1994)) as described in Ali etc., prepare 4 months big sensitization rabbits and be used for aerosol drug delivery, the relevant portion of described document is incorporated herein for referencial use in full.Dose-response research
Embodiment 9: experimental establishment
(Somerset PA), prepares the aerosol of one of adenosine (0-20mg/ml) or antisense or two kinds of mispairing oligonucleotide (5mg/ml) separately for 646 types, DeVilbiss, and 80% diameter is less than 5 μ m in the aerosol droplets of generation with ultrasound atomizer.Directly give the aerosol of equivalent to lung through endotracheal tube.Select animal at random, and give aerosol thinner adenosine.Preceding 1 day value to the sensitivity of adenosine of computing is as causing that expansion and contraction reduces by 50% adenosine dosage (PC 50Adenosine).Then, give one of the antisense of aerosol thinner or mispairing antisense oligonucleotide (5mg/1.0ml) 2 minutes to animal through endotracheal tube, every day twice, totally 2 days (accumulated dose, 20mg).At the 3rd day morning, PC after the recording processing 50Value (handling the back attacks).The result of these researchs is provided among the embodiment 21 below.
Embodiment 10: exchange test
For some experiments that utilize antisense oligonucleotide I (SEQ ID NO:1) and corresponding mispairing control oligonucleotide A1MM2, after 2 weeks of interval, animal exchange administration gives mispairing contrast A to before 1The animal of MM2 gives antisense oligonucleotide I now, gives mispairing contrast A now to those animals that before given antisense oligonucleotide I 1The MM2 oligonucleotide.Every group number of animals is as follows.For mispairing A 1MM2 (contrast 1), n=7 is because lose 1 animal because of technological difficulties in the 2nd batch of control experiment; For mispairing A 1MM3, n=4 (contrast 2); For A 1AS antisense oligonucleotide I, n=8.Separate analysis A 1The acid-treated animal of MM3 oligonucleoside, not a part of in return testing.The measurement method that use method of handling and exchange back with test at first in those of application identical.To reaching under the adenosine solubility limit 20mg/ml, in 8 animals of handling, there are 6 can not obtain PC at adenosine dosage with antisense oligonucleotide I (SEQ ID NO:1) 50Value.Therefore, for computation purpose, with the PC of these animals 50Value is assumed to be 20mg/ml.Therefore, the value that provides has been represented the minima of antisense oligonucleotide effect of the present invention.According to such scheme, give 0.5 or the antisense oligonucleotide I (SEQ ID NO:1) or the A of 0.05mg dosage to the allergy rabbit (every group of n=4) of other groups in this way 1MM2 oligonucleotide (accumulated dose be 2.0 or 0.2mg).The result of these researchs is provided among the embodiment 22 below.Embodiment 11: the preparation of antisense oligonucleotide
Each antisense oligonucleotide is dissolved in the aqueous solution respectively, and as in the above in (e) at the description of antisense oligonucleotide I (SEQ ID NO:1), utilize nebulizer through the endotracheal tube administration, totally 4 equal portions, every part of 5mg (accumulated dose 20mg), as mentioned above.Result at antisense oligonucleotide I and its mispairing contrast confirms that the mispairing contrast is equal to saline, as the following examples 19 and Nyce; Metzger, Nature 385, and shown in the table 1 of 721-725 (1997), the content of described document is incorporated herein for referencial use.Based on this discovery, saline is used to use in contrast the pulmonary function research of antisense oligonucleotide II, III and IV (SEQ ID NO:7,8 and 9).Embodiment 12: oligonucleotide I is to adenosine A 1The specificity of receptor (receptors bind research)
As mentioned above, from the rabbit that in 48 hours, separately gives 20mg oligonucleotide I (SEQ IDNO:1) for 4 times, scale off tissue, until one-level, secondary and tertiary bronchus from airway smooth muscle.According to the method for Ali etc. (Ali, S., etc., Am.J.Resp.Crit.Care Med.149:908 (1994), relevant film fraction preparation chapters and sections be incorporated herein for referencial use in full with it) preparation film fraction.Utilize the method for Bradford to determine protein content, plasma membrane and 0.2U/ml ADA Adenosine deaminase are 37 ℃ of incubations 30 minutes, to remove the endogenous adenosine.Referring to Bradford, M.M.Anal.Biochem.72,240-254 (1976), its relevant portion are incorporated herein for referencial use thus in full.As described in Jarvis etc., measure [ 3H] DPCPX, [ 3H] NPC17731 or [ 3H] combination of CGS-21680.Referring to Jarvis, M.F. etc., Pharmacol.Exptl.Ther.251,888-893 (1989), its relevant portion is incorporated herein for referencial use in full.With the oligonucleotide 5 of the targeting bradykinin receptor of analog quantity '-GGTGATGTTGAGCATTTCGGC-3 ' (SEQ ID NO:14) gives another treated animal.This research the results are shown in table 6, and discuss among the embodiment below 20.Embodiment 13: pulmonary function is measured (expansion and contraction c DYNAnd resistance)
Give the 1.5mL mixture of ketamine hydrochlorate (35mg/kg) and acetylpromazine maleate (1.5mg/kg) through intramuscular, make the rabbit anesthesias of big immunity in 4 months and lax.After bringing out anesthesia, the allergy rabbit is placed on the flexible, molded animal plate with the comfortable appearance of facing upward.Ointment is applied to eye to prevent drying, they are closed up.Then, as above Zavala and Rhodes are described, and (Mallinckrodt, Glen Falls NY) insert animal with Murphy 1 endotracheal tube of medium-low big envelope of 4.0mm.Referring to Zavala and Rhodes, Proc.Soc.Exp.Biol.Med.144:509-512 (1973), its relevant portion is incorporated herein for referencial use in full.The polyethylene catheter (BectonDickinson, Clay Adams, Parsippany NJ) that is connected with the OD 2.4mm of heavy wall latex balloon is inserted in the esophagus, and in whole experiment, keeps identical distance (approximately 16cm) with mouth.Fleisch pneumotachograph (size 00 with endotracheal tube and startup; DEM Medical, Richmond, VA) link to each other, utilization is by Gould carrier amplifier (11-4113 type, Gould Electronics, Cleveland, OH) Validyne differential pressure pickup (the DP-45-16-1927 type of Qu Donging, ValidyneEngineering, Northridge CA) measures throughput (v).Oesophageal bulb is connected to a side of differential pressure pickup, and the flow export of endotracheal tube is connected to opposite side, to obtain the pressure reduction (P between lung and the lung inner chamber Tp).The synthetic gas stream amount obtains successive tidal volume, and is waiting appearance and zero air-flow point measurement total pulmonary resistance (R t) and dynamic retractility rate (C Dyn).With 8 passage Gould2000W high frequency recording instrument record throughput, volume and pressure, utilize the P of cumulative volume and 1 air-flow point TpDifference is calculated C Dyn, the ratio of the lung volume V in Ptp and fluctuation mid-term be can be regarded as R tAs above Giles etc. is described, and (Sharon CT) carries out these calculating automatically for 6 types, BuxcoElectronics with the automatic pulmonary of Buxco mechanics breath analyzer.Referring to Giles etc., Arch.Int.Pharmacodyn.Ther.194:213-232 (1971), its relevant portion of describing these calculating is incorporated herein for referencial use in full.In rabbit gives the result embodiment 26 below of oligonucleotide II, provide and discuss.Embodiment 14: measure the extraordinary reaction of bronchus (BHR)
Utilize aerosol to give histamine, to determine the baseline of its extraordinary reaction to each allergy rabbit.(PA) aerosol of generation saline or histamine is 30 seconds, keeps under each using dosage 2 minutes then for DeVilbiss, Somerset to utilize the DeVilbiss nebulizer.Ultrasound atomizer produces aerosol droplets, wherein 80% diameter＜5 micron.Give histamine aerosol (0.156 to 80mg/ml) with the concentration that increases progressively, giving to measure pulmonary function behind each dosage.Then, by calculating with C DynReduce by 50% required histamine concentration (mg/ml) (PC from baseline 50 histamine), determine BHR.Embodiment 15: the cardiovascular effect of antisense oligonucleotide I
Utilize heat dilution principle, use CardiomaxJ to measure heart output and other cardio-vascular parameters, wherein the variations in temperature that flows out the blood of heart behind the cool brine of intravenous injection known volume is monitored.Through right jugular vein intubate, in right atrium, utilize temperature sensitive micro detector relevant temperature of blended injection and blood in carotid artery intubate record aortic arch to change fast injection of cool brine.(EPI 2010 using oligonucleotide I as mentioned above; SEQ ID NO:1) aerosol was handled the allergy rabbit after 12 hours, used 0.3ml/kg 80% ketamine and 20% xylazine with Animal Anesthesia.This time point and the previous data consistent that shows SEQ ID NO:1 effect are as Nyce ﹠amp; It is such that Metzger (1997, the same) clearly illustrates, relevant open source literature is incorporated herein for referencial use in full.Then, a thermocouple is inserted in the left neck artery of each rabbit, is increased to 6.5cm then and also fixes with the silk binder.Then, carry out right jugular vein intubate, with one section polyethylene catheter insertion and fixing.Then, by injecting Sterile Salines at 20 ℃, (Columbus Instruments Ohio) determines hot dilution curve, to determine the correctness of thermocouple probe position with CardiomaxJ II.After the correctness of determining the thermocouple probe position, isolate femoral artery and vein.With the inlet of femoral vein, femoral artery is used for the measurement of blood pressure and heart rate as the medicine injection.In case set up constant baseline cardio-vascular parameters, the CardiomaxJ that just carries out blood pressure, heart rate, heart output, total peripheral resistance and cardiac contractility measures.Embodiment 16: the persistency of oligonucleotide I (SEQ ID NO:1) effect
Described in top (f), menophania endotracheal tube utilizes nebulizer to increase progressively the adenosine of batch dosage to 8 allergy rabbits, begins to reduce by 50% (PC until expansion and contraction with 0.156mg/ml 50 adenosines), thereby set up baseline.Then, give the oligonucleotide I (SEQ ID NO:1) of the aerosol thinner of 4 5mg dosage as mentioned above to 6 rabbits wherein.The saline excipient that gives equivalent to 2 rabbits in contrast.In the end preceding 18 hours of processing, measure PC once more 50 adenosinesValue.After this point, continuously all animals are measured every day, to nearly 10 days.Discuss among the result of this research embodiment 25 below.Embodiment 17: with antisense oligonucleotide V reduction adenosine A 2bAcceptor number
Utilize above-mentioned suction chamber, in 2 days, give the antisense oligonucleotide V (SEQ ID NO:10) 3 times that 2.0mg can suck to Sprague Dawley rat.After the administration in the end 12 hours, downcut lung parenchyma tissue, and as Nyce ﹠amp; Metzger (1997, the same) described utilizations [311]-NECA measures adenosine A 2bThe combination of receptor.By giving the saline of equivalent, contrast.Utilize the paired t of Si Shi check, the result time has significance in p＜0.05, discusses among this result embodiment 28 below.Embodiment 18: relatively oligonucleotide I and phosphodiester oligonucleotide VI (SEQ IDNO:11) accordingly
Oligonucleotide I (SEQ ID NO:1) is opposite with the effect of adenosine, and it eliminates the sensitivity to adenosine, and described adenosine is to reaching 20mg adenosine/5.0ml (adenosine solubility limit).When measuring in the same way, oligonucleotide VI (SEQ ID NO:11) is that the di-phosphate ester form of described oligonucleotide sequence is fully invalid.Two kinds of chemical compounds have identical sequence, and only for there to be thiophosphate residue in oligonucleotide I (SEQ ID NO:1), they are as top and Nyce for difference; Metzger (1997, the same) is described to be transmitted as aerosol.In the paired t of Si Shi check, o'clock has significant difference in p＜0.001.Discuss among the result embodiment 29 below.Acetonideexample 19 at antisense oligonucleotide I-(SEQ ID NO:1) acquisition: the result who formerly works
Provide specificity at adenosine A in the above 1The nucleotide sequence of the antisense oligonucleotide I of receptor (SEQ IDNO:1) and other data.The experimental data that shows the effect of oligonucleotide I in downward modulation acceptor number order and activity also is provided in the above.At Nyce, J.W. and Metzger, W.J. provides characteristic and active other information of relevant antisense oligonucleotide I among the Nature 385:721 (1997), and the relevant following result's of described document part is incorporated herein for referencial use in full.Nyce ﹠amp; The data show antisense oligonucleotide I (SEQID NO:1) that Metzger (1997) publication provides:
(1) antisense oligonucleotide I reduces the A in the bronchus adenosine smooth muscle of allergy rabbit in dosage dependence mode 1Acceptor number can see table 6.
(2) antisense oligonucleotide I weakens bronchoconstriction that is brought out by adenosine and the bronchoconstriction that is brought out by allergen.
(3) oligonucleotide I weakens the extraordinary reaction of bronchus, as uses PC 50Histamine is measured, and it is for estimating the canonical measure method of the extraordinary reaction of bronchus.This result clearly proves the anti-inflammatory activity of antisense oligonucleotide I, as shown in table 4,5 and 6.
(4) as expected, because it is designed to the targeting target, so antisense oligonucleotide I is to adenosine A 1Receptor is fully special, and under any dosage the adenosine A to being closely related 2Receptor or relevant Kallidin I B 2Receptor is without any effect.See table 6.
(5) different with the above-mentioned effect of oligonucleotide I, mispairing contrast molecule MM2 and MM3 (SEQ ID NO:12 and SEQ ID NO:13) have identical base composition and molecular weight but are different from antisense oligonucleotide I (SEQ ID NO:1) because of 6 and 2 mispairing respectively.These mispairing have minimum probability under the situation that still keeps identical base composition, they to any by the receptor (A of targeting 1, A 2Or B 2) absolute not influence.
There is not relevant antisense oligonucleotide fully, for example the targeting adenosine A 1Under the prior art situation that the oligonucleotide I of receptor uses, these results are unexpected results.Disclosed result clearly gives and proves the effect of effective ways on desired use of drug application and target gene or mRNA in this patent, and described gene or mRNA are associated with relevant with lung functions such as airway obstruction, bronchoconstriction, pneumonia and allergy an etc. function or end of the final point.Embodiment 20: oligonucleotide I significantly reduces the reaction to adenosine
Described the receptors bind experiment among the superincumbent embodiment 12, the results are shown in the following table 6, this table has shown from through A 1Adenosine receptor and B 2Adenosine A in the airway smooth muscle of bradykinin receptor antisense and mispairing processing allergy rabbit in the isolating film 1Selective ligands [ 3H] DPCPX and Kallidin I B 2Selective ligands [ 3H] binding characteristic of NPC 17731.
Table 6: the binding characteristic of three kinds of antisense oligonucleotides is handled 1A 1Receptor B 2Receptor
Kd B MaxKd B MaxAdenosine A 1Receptor 20mg 0.36 ± 0.029nM 19 ± 1.52fmoles *0.39 ± 0.031nM 14.8 ± 0.99fmoles2mg 0.38 ± 0.030nM 32 ± 2.56fmoles *0.41 ± 0.028nM 15.5 ± 1.08fmoles0.2mg 0.37 ± 0.030nM 49 ± 3.43fmoles 0.34 ± 0.024nM 15.0 ± 1.06fmolesA 1MM1 (contrast) 20mg 0.34 ± 0.027nM 52.0 ± 3.64fmoles 0.35 ± 0.024nM 14.0 ± 1.0fmoles2mg 0.37 ± 0.033nM 51.8 ± 3.88fmoles 0.38 ± 0.028nM 14.6 ± 1.02fmolesB 2A (bradykinin receptor) 20mg 0.36 ± 0.028nM 45.0 ± 3.15fmoles 0.38 ± 0.027nM 8.7 ± 0.62fmoles *2mg 0.39 ± 0.035nM 44.3 ± 2.90fmoles 0.34 ± 0.024nM 11.9 ± 0.76fmoles *0.2mg 0.40 ± 0.028nM, 47.0 ± 3.76fmoles, 0.35 ± 0.028nM, 15.1 ± 1.05fmolesB 2MM (contrast) 20mg 0.39 ± 0.031nM 42.0 ± 2.94fmoles 0.41 ± 0.029nM 14.0 ± 0.98fmoles2mg 0.41 ± 0.035nM 40.0 ± 3.20fmoles 0.37 ± 0.030nM 14.8 ± 0.99fmoles0.2mg 0.37 ± 0.029nM 43.0 ± 3.14fmoles 0.36 ± 0.025nM 15.1 ± 1.35fmoles salt solution 0.37 ± 0.041 46.0 ± 5.21 0.39 ± 0.047nM 14.2 ± 1.35fmoles contrast
1Be meant and in 48 hours, divide the total oligonucleotide that gives for 4 times.Described in method, handle and analyze.Significance is determined in repeated measure analysis (ANOVA) and TukeyShi protection t check that utilization is carried out deviation.For all groups, n=4-6.
*Contrast with mispairing-significant difference is arranged, p＜0.001 with the saline treatment group;
*Contrast with mispairing-significant difference is arranged, p＜0.05 with the saline treatment group.Embodiment 21: the effect of oligonucleotide I dose-response
As shown in following table 7, in the dosage range of being surveyed, antisense oligonucleotide I (SEQ IDNO:1) is found the effect that reduces the adenosine that gives animal in the dose dependent mode.
Table 7: at the dose-response effect of antisense oligonucleotide I
Accumulated dose PC 50 adenosines
(mg) (mg adenosine)
Antisense oligonucleotide I
0.2????????????????????????8.32″7.2
2.0????????????????????????14.0″7.2
20?????????????????????????19.5″0.34
A 1MM2 oligonucleotide (contrast)
0.2????????????????????????2.51±0.46
2.0????????????????????????3.13±0.71
20?????????????????????????3.25±0.34
The above results is studied with the paired t of Si Shi check, and is found and has significant difference, p=0.05
Oligonucleotide I (SEQ ID NO:1) is the antagonism adenosine A 1A kind of oligonucleotide of receptor, it specifically with adenosine A 1Receptor acting, but not with adenosine A 2Receptor acting.As top embodiment 9 and Nyce; Metzger (1997, the same) described (4 dosage, each 5mg, 8-12 hour at interval, through endotracheal tube through the nebulizer administration), with antisense oligonucleotide I (SEQID NO:1) or mispairing control oligonucleotide (SEQ ID NO:12; A 1MM2) handle rabbit, downcut the bronchial smooth muscle tissue also as Nyce ﹠amp; The described definite adenosine A of Metzger (1997, the same) 1And adenosine A 2The number of receptor, and then obtain above result.Embodiment 22: oligonucleotide I (SEQ ID NO:1) is to the specificity of target gene product
Oligonucleotide I (SEQ ID NO:1) is to adenosine A 1Receptor is a specificity, and its mispairing contrast does not then have activity.Fig. 1 shows the result who obtains from exchange test, described exchange test is set forth in top embodiment 10 and Nyce ﹠amp; Among the Metzger (1997, the same).Two mispairing contrasts (SEQ ID NO:12 and SEQ ID NO:13) are to PC 50 adenosinesValue is influence not.On the contrary, give antisense oligonucleotide I (SEQ ID NO:1) and then demonstrate PC 50 adenosinesValue increases by 7 times.The result illustrates that clearly when comparing with saline control, antisense oligonucleotide I (SEQ ID NO:1) reduces the reaction (weakening sensitivity) to the adenosine of external source administration.Result shown in the last table 6 illustrates that clearly the effect of antisense oligonucleotide I is dose dependent (referring to the 3rd row of table 6).Oligonucleotide I also demonstrates adenosine A 1Receptor special fully (referring to preceding 3 row of table 6), not adenosine A to being closely related 2Receptor or Kallidin I B 2Any activity of receptor (it is capable that ginseng sees the above table 6 8-10).In addition, the presentation of results shown in the table 6, antisense oligonucleotide I (SEQ ID NO:1) reduces sensitivity to adenosine in the dose dependent mode, and it brings into play this effect in antisense oligonucleotide dependency mode, because two mispairing control oligonucleotide (A 1MM2:SEQID NO:12 and A 1MM3:SEQ ID NO:13) none demonstrates PC in 50 adenosinesValue has any influence or reduces adenosine A 1Acceptor number.Embodiment 23: to the influence by source of the gas paraphilia former bronchoconstriction that brings out of reaction and inflammation
In rabbit model, when comparing with the mispairing oligonucleotide, oligonucleotide I (SEQ IDNO:1) demonstrates remarkable reduction by the inductive effect of histamine.In the allergy rabbit, estimate antisense oligonucleotide I (SEQ ID No:1) and mispairing oligonucleotide (A 1MM2, SEQ ID NO:12 and A 1MM3, SEQ ID NO:12) to the airway obstruction that brings out by allergen and the effect of the extraordinary reaction of bronchus.Estimated antisense oligonucleotide I (SEQ ID NO:1) effect to the airway obstruction that brings out by allergen.As what calculate from the curve zone down of marking and drawing, when the contrast with mispairing compares, antisense oligonucleotide I significantly the airway obstruction that brings out by allergen of inhibition (55%, p＜0.05; Repeated measure ANOVA and TukeyShi t check).Mispairing oligonucleotide A 1MM2 (contrast) is invalid fully to the airway obstruction that is brought out by allergen.As above determined antisense oligonucleotide I (SEQ ID NO:1) effect to the BHR that brings out by allergen.As from PC 50 histamineValue calculates, when the contrast with mispairing compares, the BHR that antisense oligonucleotide I (SEQ ID NO:1) significantly suppresses to be brought out by allergen in the allergy rabbit (61%, p＜0.05; Repeated measure ANOVA, TukeyShi t check).Observe A 1MM mispairing contrast is invalid fully to the BHR that is brought out by allergen.The result shows that antisense oligonucleotide I (SEQ ID NO:1) is for preventing that by the source of the gas paraphilia former bronchoconstriction that brings out of reaction (dermatophagoides pteronyssinus) be effective.In addition, also find the potent inhibitor of the extraordinary reaction of bronchus that antisense oligonucleotide I (SEQ ID NO:1) is brought out by the dirt demodicid mite, to shown in the influence of histamine sensitivity, described histamine sensitivity shows the anti-inflammatory activity of antisense oligonucleotide I (SEQ ID NO:1) as it.The result shows that antisense oligonucleotide I (SEQ ID NO 1) is for preventing that by the source of the gas paraphilia former bronchoconstriction that brings out of reaction (dermatophagoides pteronyssinus) be effective.In addition, find that also antisense oligonucleotide I (SEQ ID NO 1) is the potent inhibitor of the extraordinary reaction of bronchus brought out by the dirt demodicid mite, to shown in the influence of histamine sensitivity, described histamine sensitivity shows the anti-inflammatory activity of antisense oligonucleotide I (SEQID NO:1) as it.Embodiment 24: the antisense oligonucleotide I of low A content is with no harmful side-effects
Oligonucleotide I (SEQ ID NO:1) demonstrates cannot be to the virose side effect of receptor.Give 2.0 or 20mg oligonucleotide I (SEQ ID NO:1) after, do not observe arteriotony, heart output, stroke volume, heart rate, total peripheral resistance or cardiac contractility (dPdT) and change.In addition, to (Columbus Instruments, the heart of Ohio) measuring output (CO), stroke volume (SV), mean arterial pressure (MAP), heart rate (HR), total peripheral resistance (TPR) and contractility (dPdT) result estimate with the CardiomaxJ instrument.These results prove that oligonucleotide I (SEQ ID NO:1) does not have injurious effects to the cardio-vascular parameters of key.More specifically, this oligonucleotide can not cause hypotension.This finds particular importance, brings out hypotension because other D2EHDTPA antisense oligonucleotides demonstrate in some model systems in the past always.In addition, adenosine A 1Play an important role in the antrum conduction of receptor in heart.Therefore, antisense oligonucleotide I (SEQ ID NO:1) reduces adenosine A 1Receptor, expection may cause the outer activity of deleterious lung occurring with the downward modulation of receptor.This is really not so.Antisense oligonucleotide I (SEQ IDNO:1) does not produce effect in any deleterious lung, and it makes the low dosage administration of antisense oligonucleotide of the present invention not produce side effect beyong contemplation, undesirable.This proof, as oligonucleotide I (SEQID NO:1) when directly being administered into lung, it can not arrive heart with the amount that effectively causes illeffects.This is opposite with traditional adenosine receptor antagonists such as theophylline, and traditional adenosine receptor antagonists leaves lung and can cause deleterious or even life-threatening effect at lung.Embodiment 25: the dauer effect of oligonucleotide I
Oligonucleotide I (SEQ ID NO:1) has proved a kind of dauer effect, as the PC that gives to obtain under the situation of oligonucleotide I before adenosine is attacked 50Proved with Resistance Value.When as mentioned above with 4 inferior dosage, each 5mg when endotracheal tube utilizes the nebulizer administration, at the PC of adenosine anti-sense oligonucleotide I 50, the persistency of measurement effect.After administration 1-8 days, this material effect was remarkable.When the effect of antisense oligonucleotide I (SEQ ID NO:1) disappears, give saline aerosol (contrast) to animal, measure the PC of all animals once more 50 adenosinesValue.Animal through saline treatment demonstrates baseline PC 50 adenosinesValue (n=6).Measure and to have given the persistency (at resistance) that acts in 6 allergy rabbits of 20mg antisense oligonucleotide I (SEQ ID NO:1) as mentioned above, also as mentioned above surveyingpin to the effect persistency of airway resistance.At PC 50The effect persistency intermediate value that adenosine (p＜0.05) and resistance (p＜0.05) calculate is 8.3 days.These results show that antisense oligonucleotide I (SEQ ID NO:1) has extremely long effect persistency, and this does not expect fully.Embodiment 26: no adenosine anti-sense oligonucleotide II is better than antisense oligonucleotide I
Antisense oligonucleotide II targeting adenosine A 1The zones of different of receptor mrna finds that it has activity highly and resists by adenosine A 1The effect of mediation.Experiment measuring when giving two groups of allergy rabbits with 20mg antisense oligonucleotide II or saline (contrast) as mentioned above, antisense oligonucleotide II (SEQ ID NO:7) administration is to the influence of expansion and contraction and Resistance Value.Described in top embodiment 13, give after adenosine or the saline, measure expansion and contraction and Resistance Value.The work of antisense oligonucleotide of the present invention is distinguished mutually in order to significant mode of statistics and contrast, for expansion and contraction, and p＜0.05 when utilizing paired t-to check; For resistance, p＜0.01.The result shows, the targeting adenosine A 1Receptor and do not contain the antisense oligonucleotide II (SEQ ID NO:7) of adenosine is kept expansion and contraction effectively and is reduced resistance under the situation that adenosine is attacked.In fact, the antisense oligonucleotide II of no adenosine is stronger than the antisense oligonucleotide I (SEQ ID NO:1) of low adenosine.Because it does not contain adenosine, so it does not discharge adenosine in degradation process, so it does not promote the activation to adenosine receptor.Embodiment 27: antisense oligonucleotide III and IV
In fact, giving under the situation of 20mg antisense oligonucleotide III (SEQ ID NO:8) and IV (SEQ ID NO:9) to the allergy rabbit respectively as mentioned above, oligonucleotide III (SEQ IDNO:8) and IV (SEQ ID NO:9) demonstrate targeting adenosine A specifically by its effect to the inflammatory cell number of minimizing inflammation and existence 3Receptor.After 3 hours, in its bronchial perfusate, determine the inflammatory cell number, each at least 100 living cells of lavation by counting.After being exposed to dirt demodicid mite allergen, estimate antisense oligonucleotide III (SEQ ID NO:8) and IV (SEQID NO:9) influence to the granulocyte in the bronchial lavage and total cell number.The result shows, for the asthma pulmonary that is exposed to behind the dirt demodicid mite allergen, antisense oligonucleotide IV (SEQ IDNO:9) and antisense oligonucleotide III (SEQ ID NO:8) are very strong antiinflammatories.As known in the field, granulocyte, particularly eosinocyte are the elementary inflammatory cells of asthma, and, give antisense oligonucleotide III (SEQ ID NO:8) and IV (SEQ ID NO:9), make its decreased number 40% and 66% respectively.In addition, antisense oligonucleotide IV (SEQ ID NO:9) and III (SEQ ID NO:8) also make the total cellular score in the bronchial perfusate reduce 40% and 80% respectively.This also is the anti-adenosine A of the present invention 3The important indication of material anti-inflammatory activity.Known inflammation is the reason of the bronchoconstriction that brought out by allergen in extraordinary reaction of bronchus and the asthma.The targeting adenosine A 3Antisense oligonucleotide III of receptor (SEQ ID NO:8) and IV (SEQ ID NO:9) all represent the important new anti-inflammatory agent of a class, and they can be designed to pulmonary's receptor of the various species of targeting specifically.Embodiment 28: antisense oligonucleotide V
The targeting adenosine A 2bThe antisense oligonucleotide V of adenosine receptor mRNA (SEQ ID NO:10) demonstrates for antagonism by adenosine A 2bThe effect of mediation and for the adenosine A that will exist 2bAcceptor number reduces to that to be less than 50% be highly effective.Embodiment 29: the unexpected superiority of the oligonucleotide I-DS that the di-phosphate ester residue is replaced (SEQ ID NO:11)
Give the allergy rabbit with oligonucleotide I (SEQ ID NO:1) and I-DS (SEQ ID NO:11) as mentioned above respectively, attack rabbit with adenosine then.At the antagonism adenosine on, the effectiveness of phosphodiester oligonucleotide I-DS (SEQ ID NO:11) is significantly relatively poor statistically, and oligonucleotide I (SEQ ID NO:1) demonstrates higher effective, and proof PC 50 adenosinesBe 20mg.Embodiment 30: the adenosine that contains mononucleotide has the adenosine receptor activity
This embodiment proves that the vivo degradation product of antisense oligonucleotide is ribonucleotide list phosphoric acid for example, and for example dAMP acts on the adenosine receptor.So that when nearly the various dose of 10mg/ml gives laboratory animal respectively, two chemical compounds have similar effect aspect the % expansion and contraction reducing, as shown in Figure 1 when adenosine and adenosine monophosphate (dAMP).Effect under two kinds of situations is all strengthened with the increase of dosage, and saline control is then without any effect.These results show that adenosine nucleoside and adenosine self and adenosine receptor interact.Embodiment 31: the decomposition that contains adenosine nucleic acid produces the adenosine receptor activity
As another test, give rabbit with the D2EHDTPA antisense oligonucleotide nucleoside of selecting at random, with determine they whether degradation in vivo and discharge can with the interactional adenosine nucleoside of adenosine receptor.Give saline (contrast) respectively, contain the thing at random (κ) of adenosine and take off the thing at random (C) of adenosine to the asthma rabbit.Employed thing at random is one not contain the thing at random of adenosine, and its random sequence by guanosine, cytidine and thymidine is formed and a thing at random that contains adenosine, and it is made up of guanosine, cytidine and adenosine.Result shown in Fig. 2 clearly illustrates that, the oligonucleotide that contains adenosine discharges adenosine and/or adenosine nucleoside when degraded, adenosine chemical compound and adenosine receptor interaction, and the oligonucleotide that does not contain adenosine is not then.Therefore, when estimating the do time spent of antisense knock-out experiment for the target affirmation, adenosine nucleoside discharges as the catabolite of antisense oligonucleotide, will obscure experimental result.This experiment shows, uses the necessity of antisense (no A or the low A) oligonucleotide that does not contain adenosine in target is confirmed to study.
Above embodiment is in order to demonstrate the invention, rather than as its restriction.The present invention is limited by claims.

Claims (41)

1. whether have the method for dependency between the gene of the target polypeptide that the function that is determined at disease or disease and coding are relevant with disease or disease by inference or the mRNA, this method comprises:
Obtain and contain maximum about 15% adenosines (A) and the oligonucleotide of target spot antisense, described target spot is selected from target gene and corresponding mRNA thereof, be selected from 3 ' and 5 ' intron-exon border and coding and noncoding region between the genome and the mRNA flanking region of part arranged side by side, and all mRNA fragments of the coding polypeptide relevant with previously selected disease or disease;
From described oligonucleotide, select and a kind ofly when the hybridization of external and said target mrna, obviously suppress or eliminate the oligonucleotide of expressing by the mRNA encoded polypeptide;
To individuality use can be effectively in vivo with the selected oligonucleotide of the amount of said target mrna hybridization; With
Be evaluated at and use oligonucleotide front and back and described disease or the relevant individual function of disease; Wherein, the change of function value is greater than about 70% expression positive correlation, and it is relevant to express possibility between about 40 to about 70%, and it is uncorrelated to be lower than about 30% expression.
2. the process of claim 1 wherein that described antisense oligonucleotide makes up by the following method:
Selection contain at least 4 successive G of being selected from and C nucleic acid the target fragment and obtain the long content that contains selected fragment and C and G of 4 to 60 nucleotide and be about 15% first oligonucleotide at most, and/or
Selection has the target fragment of ideal activity type and/or degree.
3. the method for claim 1 also comprises, when described antisense fragment contains at least one A, at least one A is combined with thymidine (T) but has A adenosine A less than about 0.3 with being selected from 1, A 2a, A 2bAnd A 3The alternative base (B) of the heteroaromatic base of receptor stimulating agent or antagonist activities is replaced.
4. the method for claim 3, wherein said heteroaromatic base is selected from pyrimidine and purine compound, and it can be by O, halogen, NH 2, SH, SO, SO 2, SO 3COOH and side chain and condensed primary and secondary amino; alkyl; alkenyl; alkynyl group; cycloalkyl; Heterocyclylalkyl; aryl; heteroaryl; alkoxyl; alkenyloxy; acyl group; the ring acyl group; aryl-acyl; chain oxy-acetylene; cycloalkyloxy; aroyl; arylthio; aryl-sulfonyl oxygen; halogenated cycloalkyl; alkyl-cycloalkyl; the alkenyl cycloalkyl; the alkynyl group cycloalkyl; halogenated aryl; alkylaryl; the alkenyl aryl; the alkynyl group aryl; aryl alkyl; aromatic yl alkenyl; aryl alkynyl chain; cycloalkyl aryl replaces, and the above group again can be by O; halogen; NH 2, primary, the second month in a season and tertiary amine, SH, SO, SO 2, SO 3, cycloalkyl, Heterocyclylalkyl and heteroaryl replace.
5. the method for claim 4, wherein said pyrimidine and purine compound are substituted at 1,2,3,4,7 and 8.
6. the method for claim 4, wherein said pyrimidine and purine compound are selected from theophylline, caffeine, diprophylline, etofylline, acefylline piperazine, bamifylline, enprofylline and have the xanthine of following chemical structural formula
R wherein 1And R 2Be H, alkyl, alkenyl or alkynyl group independently of one another, R 3Be H, aryl, bicyclic alkyl, bicycloenyl, two cycloalkynyl radicals, cycloalkyl, cycloalkenyl group, cycloalkynyl radical, O-cycloalkyl, O-cycloalkenyl group, O-cycloalkynyl radical, NH 2-alkyl amino-ketone oxygen base alkoxyl-aryl and single and dialkyl aminoalkyl-N-alkyl amino-SO 2Aryl.
7. the process of claim 1 wherein that the adenosine content of described antisense oligonucleotide is about 0 to about 12%.
8. the method for claim 7, wherein said oligonucleotide is made up of the A of as many as about 5%.
9. the method for claim 8, wherein said oligonucleotide does not contain A.
10. the process of claim 1 wherein that an A is selected from and combined but adenosine A with thymidine 1, A 2a, A 2bAnd A 3Receptor has less than the alternative base of the heteroaromatic base of about 0.5 adenosine agonists or antagonist activities to be replaced.
11. the method for claim 10, wherein all A all are selected to be combined with thymidine but to adenosine A 1, A 2a, A 2bAnd A 3Receptor has less than the alternative base of the heteroaromatic base of about 0.3 adenosine agonists or antagonist activities to be replaced.
12. the method for claim 6, wherein said universal base be selected from 3-nitro-pyrrole-2 '-deoxynucleoside, 5-nitro-indole, 2-deoxyribosyl base-(5-nitroindoline), 2-desoxyribofuranose base-(5-nitroindoline), 2 '-deoxyinosine, 2 '-deoxidation nebularine, 6H, 8H-3, the 4-dihydro-pyrimidin also [4,5-c] oxazine-7-ketone or 2-amino-6-methoxyl group amidopurin.
13. the process of claim 1 wherein, if in described oligonucleotide, there is at least one CpG dinucleotide, with methylated cytosine ( mC) replace C at least one CpG dinucleotide.
14. the method for claim 1, wherein, at least one nucleotide residue of described antisense oligonucleotide is selected from methyl phosphonate, phosphotriester, thiophosphate, phosphorodithioate, the bora phosphate ester, formacetal, thioformacetal, thioether, carbonic ester, carbamate, sulfuric ester, sulphonic acid ester, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene (methyl-imino), (MMI), methoxy (MOM), methoxy ethyl (MOE), methylene oxygen base (methyl-imino) (MOMA), methoxy (MOM), 2 '-the O-methyl, residue and combination thereof that phosphoramidate and C-5 replace.
15. the material of claim 14, wherein all nucleotide connects residue and has all replaced.
16. the process of claim 1 wherein that described antisense oligonucleotide contains 7 to 60 mononucleotides of having an appointment.
17. the process of claim 1 wherein that described antisense oligonucleotide is connected with the material that is selected from by the material of cell internalizing or picked-up and cell targeted material.
18. the method for claim 17, wherein said material by cell internalizing or picked-up is selected from transferrins, asialoglycoprotein and streptavidin.
19. the method for claim 18, wherein said nucleic acid is connected with carrier.
20. the method for claim 19, wherein said carrier comprise prokaryote or eukaryote carrier.
21. the process of claim 1 wherein described antisense oligonucleotide to lung, brain, heart, kidney, tumor, blood, skin, eye, scalp, nasal passage, testis, cervix uteri, oral cavity, pharynx, esophagus, small intestinal or large intestine, synovial tissue, muscular tissue, ovary, auditory meatus administration or treated in vitro.
22. the process of claim 1 wherein that described disease or disease are disease or the diseases that influences lung, brain, heart, kidney, tumor, blood, immune system, skin, eye, scalp, nasal passage, testis, cervix uteri, oral cavity, pharynx, esophagus, small intestinal or large intestine, synovial tissue, muscular tissue, ovary and auditory meatus.
23. the method for claim 22, wherein said disease or disease are disease or the diseases that influences lung.
24. the method for claim 22, wherein said disease or disease are relevant with bronchoconstriction, pneumonia and/or allergy.
25. the method for claim 22, wherein said disease or disease be influence brain or with cerebration diseases associated or disease.
26. the method for claim 22, wherein said disease or disease are relevant with immunodeficiency disease.
27. the method for claim 26, wherein said target spot are selected from immunoglobulin, antibody receptor, cytokine, cytokine receptor, encode their gene and corresponding mRNA, gene and mRNA flanking region and exon and intron border.
28. the method for claim 22, wherein said disease or disease are disease or the diseases that influences cardiovascular system.
29. the method for claim 22, wherein said disease or disease are and gastrointestinal system diseases associated or disease.
30. the method for claim 22, wherein said disease or disease and malignant tumor or related to cancer.
31. the method for claim 30, wherein said target spot be selected from immunoglobulin and antibody receptor, their gene and the mRNA that encode, gene and mRNA and genome and mRNA flanking region and exon and the intron border relevant with oncogene.
32. the process of claim 1 wherein described composition is used outward, oral, intracavity is used, intranasal administration, anal is used, intravaginal is used, intrauterine is used, intracranial is used, pulmonary administration, use in the kidney, use in the joint, intraarticular is used, intraotically uses, use in the lymph, applied dermally, use in the cheek, intravenous is used, subcutaneous administration, intramuscular is used, use in the tumor, use in the gland, ophthalmic is used, intracranial is used, be administered in the organ, use in the blood vessel, use in the sheath, use by implantation, use by suction, intradermal is used, use in the lung, be administered in ear, be administered in the heart, pass through slow release, continue to discharge or use by pump.
33. the method for claim 1, wherein said target is selected from the gene and the mRNA of coded polypeptide, and described polypeptide is selected from transcription factor, stimulate and activation factor, cytokine and receptor thereof, interleukin, interleukin-2-receptor, chemotactic factor, chemokine receptors, specificity and non-specific enzyme that endogenous produces, immunoglobulin, antibody receptor, central nervous system (CNS) and peripheral nervous and non-nervous system receptor, CNS and peripheral nervous and non-nervous system peptide mediator, adhesion molecule, sozin, somatomedin, vasoactive peptide, peptide receptor and conjugated protein, gene and mRNA corresponding to oncogene.
34. the process of claim 1 wherein that described antisense oligonucleotide can produce by the following method:
From the polypeptide relevant with disease with the disease that influences lung airway, encode they gene and RNA, genome and mRNA flanking region and gene and mRNA exon and intron border select target;
Obtain the mRNA sequence, described mRNA is selected from mRNA, genome and mRNA flanking region and gene and mRNA exon and the intron border corresponding to the mRNA of target gene and coding target polypeptide;
Select at least one fragment of described mRNA;
Synthetic one or more oligonucleotide to selected mRNA fragment antisense; And,
As needs, with a kind of alternative base replace one or more A so that in the oligonucleotide content of A be reduced to and account for maximum about 15% of complete nucleotide.
35. the process of claim 1 wherein that described target gene is selected from: coding is selected from transcription factor, stimulate and activation factor, interleukin, interleukin-2-receptor, chemotactic factor, chemokine receptors, specificity and non-specific enzyme that endogenous produces, immunoglobulin, antibody receptor, central nervous system (CNS) and peripheral nervous and non-nervous system receptor, CNS and peripheral nervous and non-nervous system peptide mediator and receptor thereof, adhesion molecule, sozin, somatomedin, the target gene of vasoactive peptide and receptor thereof and protein-bonded polypeptide and mRNA, target gene and mRNA corresponding to oncogene, its flanking region and exon and intron border.
36. the method for claim 35, the wherein said polypeptide that is encoded are selected from Nf κ B transcription factor, interleukin-8 receptor (IL-8R), interleukin-15 receptor (IL-5R), interleukin 4 receptors (IL-4R), interleukin-13 receptor (IL-3R), il-1 β (IL-1 β), interleukin-11 beta receptor (IL-1 β R), the eotaxin, trypsinlike enzyme, main basic protein, the beta-2-adrenoreceptor kinases, endothelin-receptor A, endothelin-receptor B, the anterior endothelium angiogenic peptide that contracts is former, bradykinin b 2 receptor, the IgE high-affinity receptor, interleukin-11 (IL-1), interleukin 1 receptor (IL-1R), interleukin 9 (IL-9), interleukin-9 receptor (IL-9R), interleukin 11 (IL-11), interleukin-11 receptor (IL-11R), the inducible nitric oxide synthase, cyclo-oxygenase (COX), adhesion molecule 1 (ICAM-1) vascular cell adhesion molecule (VCAM) in the cell, Rantes, endothelial leucocyte adhesion molecule (ELAM-1), the monocyte activation factor, chemotactic factor for neutrophil, the neutrophil elastoser, sozin 1,2 and 3, the muscarine acetylcholinergic receptor, platelet activating factor, tumor necrosis factor, the 5-lipoxygenase, phosphodiesterase IN, the P material, P material receptor, histamine receptor, chymase, the CCR-1 CC-chemokine receptor, the CCR-2 CC-chemokine receptor, the CCR-3 CC-chemokine receptor, the CCR-4 CC-chemokine receptor, the CCR-5 CC-chemokine receptor, the prostaglandin receptoroid, the GATA-3 transcription factor, the neutrophil adhesion receptor, map kinase, interleukin-9 (IL-9), the NFAT transcription factor, STAT 4, MIP-1 α, MCP-2, MCP-3, MCP-4, cyclophilin, phospholipase A2, basic fibroblast growth factor, metalloproteases, the CSBP/p38 map kinase, Trypsin
Figure A0080675900081
Receptor, PDG2, interleukin-3 (IL-3), il-1 β (IL-1 β), cyclosporin A-conjugated protein, FK5-is conjugated protein, α 4 β 1 select albumen, fibronectin, α 4 β 7 select albumen, Mad CAM-1, LFA-1 (CDlla/CDl8), PECAM-1, LFA-1 selects albumen, C3bi, PSGL-1, E-selects albumen, P-selects albumen, CD-34, L-selects albumen, p150,95, Mac-1 (CDllb/CDl8), fucosyltransferase, VLA-4, CD-18/CDlla, CDllb/CD18, ICAM2 and ICAM3, C5a, CCR3 (eotaxin's receptor), CCR1, CCR2, CCR4, CCR5, LTB-4, the AP-1 transcription factor, Protein kinase C, cysteinyl leukotriene receptor, Tachychinnen receptor (tach R), I kappa b kinase 1﹠amp; 2, STAT 6, c-mas and NF-interleukin-6 (NF-IL-6) and flanking region thereof and exon and intron border.
37. the process of claim 1 wherein described target gene coding G albumen or g protein coupled receptor.
38. the process of claim 1 wherein described target gene coding calciphorin or receptor, sodium channel protein or receptor, potassium channel protein or receptor or chloride channel albumen or receptor.
39. the process of claim 1 wherein described target gene coding neurotransmitter receptor or neuro hormone receptor.
40. the process of claim 1 wherein described target gene coding neuropeptide or neuropeptide receptor.
41. the method for claim 1 also comprises by giving that additionally other oligonucleotide of other the target spot antisense relevant with described first target spot of its function is by inference repeated all steps; By giving targeting comes repeat administration and assessment in the oligonucleotide of first target spot and other target spot step jointly; To compare for the result that each target spot obtains respectively; Wherein, when the oligonucleotide effect that merges than the effect of each oligonucleotide by force about 20% or when higher, can think and between first oligonucleotide and other oligonucleotide, have positive correlation, when the result single oligonucleotide effect about 20% within the time, can think uncorrelated, when the result is low more about 20% the time than the effect of single oligonucleotide, can think to have negative correlation between them.
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CN102169121A (en) * 2010-02-25 2011-08-31 北京诺赛基因组研究中心有限公司 New application of human kinase SBK1 (SH3-binding domain kinase 1)
CN102169121B (en) * 2010-02-25 2013-12-04 北京诺赛基因组研究中心有限公司 New application of human kinase SBK1 (SH3-binding domain kinase 1)
CN114480406A (en) * 2021-09-16 2022-05-13 广东翠点生物科技有限公司 IL-1 signal path response element and application thereof
CN114480406B (en) * 2021-09-16 2024-01-30 广东翠点生物科技有限公司 IL-1 signal path response element and application thereof

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