METHOD FOR VALIDATING/INVALIDATING TARGET(S) AND PATHWAYS

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: METHOD FOR VALIDATING/INVALIDATING TARGET(S) AND PATHWAYS BACKGROUND OF THE INVENTION Field of the Invention This invention relates to a method for validating target genes, e. g. central nervous (CNS) genes which utilizes anti-sense oligonucleotides (oligos) of low adenosine (low A) or no adenosine (desA) content. This method aids in the screening of target genes and their functions by inhibiting the target gene's production of its product. This method is particularly suited for in vivo applications since many of a subject's functions are responsive to adenosine, and adenosine's effects may mask the specific target gene's detectable functions. Description of the Background The sequence of all human genes, approximately 100,000, is expected to become known within the next 1-3 years. This information will provide the opportunity to design new medicines for treatment of virtually all diseases which have in the past or presently afflict mankind. This massive accumulation of sequences, however, will not by itself be useful in the development of new medicines until the technology becomes available to discern the function of these genes, particularly with respect to the potential effects, i.e. therapeutic or toxicologic, of attenuating their function. For example, it is imperative that novel methods be designed for rapidly and accurately testing the function and, therefore, usefulness of newly discovered gene products as "targets" for drug discovery programs. In order to conserve drug discovery resources, such "targets" must be validated or invalidated as early as possible in the drug discovery process. Even so, the implementation of such methods will require the massive implementation of drug discovery programs to series of genes for which a sequence but not a function are known. One method of considerable potential to assess the value of newly discovered gene products as focal points for drug discovery programs is the use of anti-sense oligonucleotides to ablate the target gene function in vitro or in vivo. While this method has great theoretical importance, one problem is that anti-sense oligonucleotides have the potential to be degraded in vitro or in vivo, releasing their constitutive nucleotides. There is evidence that one of these products of oligonucleotide degradation, adenosine, is highly bioactive in certain tissues. Adenosine mediates pleiotropic effects including depression of neurotransmission, maintenance of thalamic spindle rhythms, sleep induction, antagonism of Dl and D2 dopamine receptors, anti-nociception, mediation of various effects of ethanol including motor incoordination, autonomic control of cardiac function, bronchoconstriction, negative chronotropy, inotropy and dromotropy, anti-β-adrenergic action, and renal sodium retention. Clearly, the liberation of even minute amounts of adenosine in certain tissues, e.g. CNS, the hyper-responsive asthmatic lung, heart, and kidney, among others, could locally activate adenosine receptors. This would make it impossible to interpret target validation data in a reliable, unambiguous fashion. Anti-sense oligonucleotides containing adenosine, thus, are not optimal to provide clear target validation data since their breakdown could cause pleiotropic adenosine-mediated effects. Basic neuroscience research during the past few years has established a relationship between excitatory amino acid (EAA) central nervous system (CNS) transmitters, such as glutamic and aspartic acid, and various pathological states, e.g. stroke and CNS trauma. For example, a major mechanism of neural tissue degeneration following cerebral ischaemia, stroke or trauma, seems to involve overactivity of the EAA system in the brain, i.e. excessive release of glutamate and aspartate. This process is called delayed excitatory toxicity, and certain neural cell populations are selectively sensitive to excitatory toxicity. Adenosine, among other activities, has been found to inhibit the release of EAA pre-synaptically and thus attenuate this excitatory toxicity, and its release would greatly mterfere with validation studies of this system. Because the effects of adenosine are generally mediated by extracellular receptors, the pharmacologically relevant pool of adenosine is, therefore, that which is outside the cell. Adenosine also has neuro-behavioral effects, and acts as a CNS depressant, i.e. inhibits neural activity It is also a natural anti-convulsant and sedative. New studies are revealing that, under normal circumstances adenosme promotes sleep and, therefore, may interfere with pathways involved in sleep related responses, such as sleep apnea Now mcreasmg evidence is confirming that adenosme is an important "fatigue factor" and may also mterfere with validation studies on the molecular underpinnings of the sleep cycle. For example m the bram, studies indicate that the key receptors reside on nerve cells m bram arousal networks. Some scientists believe that adenosme promotes sleepiness by targeting arousal networks m the bram such as the cholmergic system, such as the cholmergic basal forebrain and the mesopontine cholmergic nuclei which spur slumber. All these effects of adenosine mterfere with any validation study of targets m the systems and pathways where it acts. Accordmgly, there is a definite need for a rapid and efficient method to screen large numbers of genes and their expression products to determine their functions and, thus, their usefulness in the design of therapeutic agents for treating diseases and conditions associated with the target genes and/or their expressed products. Moreover, there is a need for a method which is suitable for testing individual gene functions while avoidmg triggering other gene functions which would obscure the interpretation of the results SUMMARY OF THE INVENTION The present invention relates to a method of vahdating\invalidating or 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. The method generally compπses obtaining oligonucleotides (oligos) consisting of up to about 15% adenosme (A), and which is anti-sense to a target selected from the group consistmg of target genes and their corresponding mRNAs, genomic and mRNA flanking regions selected from the group consisting of 3' and 5' mtron-exon borders and the juxta-section between coding and non-codmg regions, and all mRNA segments encodmg 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. Suitable applications for the present method are in the elucidation of genes or networks of genes which may be associated with diseases or conditions afflicting the lung, bram, heart, kidney, tumor, blood, immune system, skin, eye, nasal passages, scalp, testes, cervix, oral cavity, pharynx, esophagus, intestine (small and large), synovir tissues, muscle, ovaries, and ear canal, among others, and m general any cells that contain, or originate from, the target sites. The invention will now be descπbed in reference to specific drawings Other objects, advantages and features of the present invention will become apparent to those skilled m the art from the description BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 shows an experiment where saline (control), adenosme (o), and dAMP (P) were separately administered to rabbits. Saline had no effect, and both adenosine and dAMP affected a similar reduction compliance in a dose dependent manner. These results show that nucleosides such as dAMP either directly or following degradation and/or metabolism to adenosine, have the physiological effects of adenosine at adenosme receptors. Figure 2 accompanying this patent demonstrates that oligonucleotides (oligos) containing adenosme (A), but not those which do not have adenosme (desA) release bioactive adenosme. The released adenosme activates adenosine receptors and causes biological responses which may mterfere with signals to be observed in validation studies. Here, two 21-mer randomer phosphorothioate anti-sense oligonucleotides (oligos), one containing adenosme (triangles) and one desA oligo (circles), were administered to asthmatic rabbits The adenosme containing ohgonucleotide caused a significant loss of airway compliance, reflecting A receptor activity while the desA randomer ohgo did not Figures 3 and 4 demonstrate that anti-sense oligonucleotides may be utilized as effective agents m the validation of targets associated with pulmonary or airway diseases. Figure 3 illustrates the effects of oligonucleotides anti-sense to the adenosme A, receptor, and of mismatch control anti-sense oligonucleotides on the dynamic compliance of the bronchial airway m a rabbit model. Figure 3 illustrates the specificity of oligonucleotides anti-sense to the A, adenosme receptor as indicated by the A, and A 2 adenosme receptor number present in A, adenosine receptor anti-sense ohgonucleotide-treated airway tissue The invention will be better understood in reference to the following description of the preferred embodiments DESCRIPTION OF THE PREFERRED EMBODIMENTS This invention arose from a desire by the inventor to provide a novel technology for the rapid and efficient discovery of the function and, therefore, usefulness, of target genes and their products. The inventor surmised that he might successfully do this by applying his own pπor discovery that low adenosine anti-sense oligonucleotides (oligos) may be administered m vivo to subjects without eliciting undesirable side effects mediated by adenosme receptors The present method enables the assessment of the value of newly discovered genes and gene products as focal points for drug discovery programs with the aid of anti-sense oligonucleotides that ablate their function in vitro and/or m vivo While the utilization of anti-sense oligonucleotides to ablate gene expression has had theoretical importance m the past, the mventor's finding that anti-sense oligonucleotides are degraded in vitro and m vivo, and release their constitutive nucleotides and nucleosides has hampered their successful application, explaining their meffectiveness The present inventor has evidence that one of the oligonucleoside degradation products, adenosme monophosphate is, as adenosme itself, highly bioactive in vaπous tissues Adenosme itself is known to mediate pleiotropic effects mcludmg depression of neurotransmission, sleep induction, antagonism of Dl and D2 dopamine receptors, anti-nociception, mediation of vaπous effects of ethanol mcludmg motor mcoordmation, autonomic control of cardiac function, bronchoconstriction, negative chronotropy, inotrpy and dromotropy, anti-β-adrenergic action, and renal sodium retention, among others. Clearly, the liberation of even minute amounts of adenosme and as shown in the present examples adenosme nucleosides m certain tissues, e.g CNS, lung, heart, and kidney, among others, could activate adenosme receptors m the local environment This would make it impossible to interpret target validation data in a reliable, unambiguous fashion. Examples 30 and 31 and figures 1 and 2 accompanymg this patent illustrate the break down of adenosine-containing oligonucleotides to release bioactive adenosme nucleosides. The figures show that oligonucleotides (oligos) containing adenosme (A), but not those without adenosine (desA), release bioactive adenosme In the exemplary disclosure, two 21-mer randomer phosphorothioate anti-sense oligonucleotides (oligos), one containing adenosme (>) and one desA ohgo (o), were administered to asthmatic rabbits. The results showed that the adenosme containing ohgonucleotide caused a significant loss of airway compliance, reflecting adenosme receptor activity, while the desA randomer ohgo did not. See, Example 31 below In addition, an adenosme nucleoside (dAMP) was shown to have an effect similar to adenosme at an adenosme receptor. See, Example 30 below. The work descπbed here and results discussed m the examples accompanymg this patent clearly shows that target validation is successfully attained by applymg the method of the mvention to new targets as they are discovered and/or to known targets as they become associated with their functions. The experimental work further mdicates that the present method was found to be highly selective and effective at validating/invalidating targets when employing non-phosphodiester anti-sense oligonucleotides specifically targeted, by countering or reducing effects mediated by the target protems, and the like. That was shown for all the anti-sense oligos targetmg an adenosme A, receptor mRNA, the 1 anti-sense ohgo targeting an adenosme A, b receptor mRNA, the 2 anti-sense oligos targeting an A 3 receptor mRNA, and the 1 anti-sense ohgo targetmg a bradyki n receptor, were shown to counter effects mediated by the specific adenosme receptors elicited by exogenously administered adenosme. The method of this mvention, moreover, is specific in validating/invalidating the specifically selected target, and fails to inhibit other targets, as shown with the anti-sense oligos targeted to the adenosme A, and bradykmm genes and mRNAs. In addition, the results show that the method of the invention employing low or no adenosme containmg oligos results in extremely low or non-existent deleteπous side effects or toxicity This represents 100% success in providing a method that is highly effective and specific in the validation of targets, as shown here for the respiratory system. This mvention is broadly applicable in the same manner to all gene(s) and corresponding mRNAs encoding protems of the respiratory/pulmonary system, involved m or associated with airway diseases as well as targets of other systems associated with specific diseases or conditions which may be correlated with a specific function or end point, e.g. the CNS A companson was also made of the implementation of the method of the mvention with the aid of a phosphodiester ohgo, and a version of the same ohgonucleotide wherem the phosphodiester bonds are substituted with phosphorothioate bonds. The results of the application of the method of the mvention evidenced an unexpected supeπoπty for the substituted over the phosphodiester oligonucleotides. Anti-sense oligonucleotides with a high content of adenosine (33%) thus are not suitable to attain clear target validation data since their breakdown could cause pleiotropic adenosine-mediated effects. The low adenosme ohgomers utilized by the present method are clearly free of such side effects. This patent descπbes a method which may be utilized to perform unambiguous "target validation" that is enabling, for example, in the respiratory tract or pulmonary system, the CNS, and other organs or systems, where the liberation of adenosme may cause pleiotropic effects The method mvolves the use of low A or desA anti- sense oligos, i.e. oligos that have a low A content or lack adenosme altogether, and which may not, therefore, liberate amounts of significant bioactive adenosme upon degradation. The present technology is enabled for practice with virtually any gene, but has particular applicability for the following gene subsets: G-protem Coupled Receptors, Neurohormone Receptors, Neuropeptide Receptors, Neurotransmitter Receptors, G Protems, Calcium Channel Proteins, Sodium Channel Proteins, Potassium Channel Receptors, Chloπde Channel Receptors, i.e virtually any gene expressed in normal or diseased CNS, heart, lung, kidney, blood, immune system, and many more in addition to those listed below. Thus, the validation method descπbed in this patent may also be applied not only to genes and systems mentioned here, but to other genuses and subgenuses of genes and gene networks. Successful drug discovery programs depend on a fast, accurate assessment of the suitability of candidate gene products as drug design targets Traditionally, this process has consumed an mordinate amount of time, personnel and financial resources. This mvention provides a rapid, reliable method for target validation/invalidation m vanous biological systems that utilizes propπetary low or desAdenosine (desA) anti- sense oligonucleotides (collectively called here desA-ASONs). Using desA-ASONs, the present method may validate/invalidate potential gene targets with a level of speed and accuracy that has heretofore been impossible usmg traditional technologies. DesA-ASONs provide greater levels of accuracy and speed vs. classical methods. DesA-ASONS cannot break down and release bioactive adenosme which would obfuscate target validation by causing adenosine-induced side effects. Among the ASONS there is a subgroup mtended for respiratory admmistration herein refeπed to as RASONs. DesA-RASONs have been used by the inventor in practicing the method of this invention to validate adenosme A,, A 2 and A 3 targets. The present technology is also applicable to CNS targets involving biochemical, behavioral, physiological assessments following site specific functional gene ablation m any region of the bram with desA-BASONs, bram anti-sense oligonucleotides (administered in situ in the bram) which cannot break down to release adenosme, a major modulator of bram physiology. In addition to the exemplary validation of pulmonary targets, the present method is illustrated below for CNS associated targets, and generally compπses performing the following several steps for target validation using desA anti-sense oligonucleotides. The initial step requires the identification of a general system or area for the target validation, e.g. CNS, respiratory, renal, cardiac areas, etc. Then, the method turns to the aid of public hbraπes such as GenBank or other hbraπes in the public domain or pπvate hbraπes such as those propπetary to specific companies For example, a specific library such as one encompassmg all G-protem coupled receptors (GPCRs) found in public and pπvate data bases Currently, there are about 250 GPCRs in existence. The present target validation method, thus, is suitable for testing what physiological, biophysical, biological, behavioral, etc., events occur when the selected group of targets, e g. GPCRs, are individually attenuated usmg appropπate desA anti-sense oligos. For this, desA anti-sense oligos are designed as descπbed below, and synthesized for pre-selected target genes, e.g. the GPCRs refeπed to above. The desA anti-sense oligos are then tested, first m vitro, for example by using an appropπate cell line, primary cell culture, or other cell tissues. Such m vitro testing may be applied to determine which of several desA anti-sense oligos designed agamst a specific target are "most active" as anti-sense agents. That is, which one best down regulates or ablates gene expression This may be done by using a biophysical, biological, physiological, or other assay which may be coπelated with a specific activity of the cell. In other cases, knocking out the target gene in the in vitro system may itself provide useful target validation information. The most active desA anti-sense oligos are then selected and applied m vivo, for example, by direct instillation into any bram region for CNS studies, by admmistration mto the lung targets associated with the respiratory system, and the like This may be done by methods known m the art, such as via stereotactic implantation of cannulae for bra targets, via inhalation for respiratory targets, systemically for blood targets, by m situ admmistration for organs and other localized systems and for both parenchymal cells and vascular heart cells, and systemically via inhalation or via direct instillation for renal targets. Behavioral, biophysical, physiological, biochemical, immunological, and other tests may be applied and data obtamed on individual annuals by admmistration of the appropπate anti-sense oligonucleotides to ablate one or more target genes Behavioral functions or endpomts such as food mgestion, anxiety, libido, cognition, etc , or such physiological end points as temperature, electroencephalograms (ECG), electrocardiograms (EKG), glomerular filtration volume and content, ion retention, protem loss, etc., may be assessed by methods known in the art. These steps are illustrated for the CNS m the scheme shown below. Select appropπate bchm'iυrol as. i)'s fa perform on animals nitA demonstrated site-specific ablation of target gene Select TherapeMtle Areas of Interest - Ingctfivc behavior (noπnal insertion, raJcroElructural analysis of feeding biliaviors, clioicc tests, taste preferences) ANXIZTV/MOOn DISORDERS - Analgesia ( tall flick, Plantar test (Hargrβave'ε tesl) Sel ct Target Source - Locomotor behavior (photocell basod systems) SATIETY PARTNER CNS GENLJ- Sens/motor reflexes and gating processes DΛTΛ BASKS {prepυlse inhibition nnd hobituottoπ) PA IN rget Validation SOXIJΩI and reproductive behaviors 8SFGA * using EPI GENESIS CNS dr.tAdenosine Anti- ' Anxiety (Gcller-Seiftcr Conllicl Analysis; social COGNITION GENE DATA DΛSE sense Oligonuinteraction; light/dark exploration; elevated plus- cleotides ia the maze test; defensive burying) LIBIDO Learning and mαmorγ {radio! orm maze teβt; Mor¬ PUBLIC CNS GENE ris Walcr Mozc Task to test spatial momorγ and DATA BASE working memory) . Select Appropriate Physiological Assay SLEEP/ SLEEP DISORDERS Temperature rogulolbn (continuous temperature monitoring via deep core thermistor) TEMPERATURE REGULATION EpiGeπesis CNS Target Validation Cardiovascular Function ( via CordioMax2: car- dioc output; heart rate; meon arterial pressure; stroke volume; contractility [dPdD). Also, con¬ *7____ : Ti4 Srt*-ιpα.lβc fxtKtionαt ablation tinuous, noninvocivo blood pressure monitoring vie tail cuff. The present method relies on low A or desA-ASONs for application to respiratory (desA-RASONs), CNS (desA-BASONs), renal (desA-KASONs), cardiac (desA-CASONs), blood (desA-SASONs), immune system (desA-IASONs), malignant tissue (desA-MASONs), and other functions, without releasmg significant bioactive amounts of adenosme. In this fashion, the present method prevents the undesirable activation of adenosme receptors m the lung, CNS, kidney, heart, blood, immune system, malignant cell aggregates, etc. Results obtained with presently available methods are less interpretable because standard anti-sense constructs contam normal (high) levels of adenosine about 25%, and activate adenosme receptors. This effect blurs the results and confuses the interpretation of functionality. In the lung, for example, ohgo-released adenosme will cause changes m airway diameter (bronchoconstπction), inflammation, and secretion of surfactant, all effects that m the present case are unrelated to the validation of a different target. Such "side effects" make the data obtained with oligonucleotides or with πbozymes for that matter umnterpretable. Similarly when an A- containing anti-sense ohgonucleotide is used m the bram, its effects obscure a plurality of functions which may be used as end pomts to validate targets. For example, adenosme causes depression of neurotransmission, sleep induction, anti-nociception, mediates vanous effects of ethanol mcludmg lack of motor coordination, autonomic control of cardiac function, antagonism of dopamine Dl and D2 receptors, alterations m CNS blood flow, and a host of other effects The presence of significant amounts of adenosme clearly is contramdicated when anti-sense oligos are used in target validation studies m the hyper-responsive lung, CNS, and other systems which contain adenosme receptors or are otherwise responsive to adenosme. For these reasons, the present method provides a supeπor target validation tool. Particularly useful applications of the present method are to mvestigate therapeutic areas related to the respiratory tract, CNS, blood, malignant and uncontrollably growmg cells, and many other systems, which may be separately targeted, and where one or more functions associated with the targets are separately measured As the present method has been mvented at a smgular time m the history of biological sciences, it will permit a faster and more efficient utilization of the information obtained from the sequencmg of the human genome. Knowing the sequence of every gene m the human genome has been the Holy Grail of modern medicme. This accomplishment will enable the correlation of specific genes with their functions and thereafter the creation of entirely new types of medicines that strike closer to the heart of disease and lack the side effects of currently available drugs. The flood of sequences that is bemg banked currently provides important targets The present method provides an invaluable tool to identify genes that are important m body function and, thus, in disease, and to determine whether or not the inhibition of their function is therapeutically useful. The process of determining if inhibiting the function of a gene is therapeutic is called here "Target Validation". It is an important early step m the drug discovery process, and it helps determine whether or not cπtical resources are to be expended to develop new drugs for inhibiting the function of a target gene. In this context, it is just as important to validate a target as it is to invalidate it, by showmg that inhibiting its function is without functional (therapeutic) effect. An early invalidation of a target m the drug discovery process prevents the waste of resources in unfruitful drug discovery campaigns. This, in turn, will permit a more rapid and focused investigation of more productive targets. The present rapid and accurate target validation method may make the difference between success and failure m the therapeutic application of large volumes of information, e g. that was obtamed from the human genome project. The m vivo testing of the anti-sense oligos in the method of the mvention may be implemented, for example, m vitro and m animal models for important diseases, including models for respiratory diseases such as asthma, hormonal diseases, genetic diseases, obesity, and the like, including diseases of the CNS, renal, cardiac, blood diseases, etc. Assays known in the art may be utilized, such as whole body plethysmographic techniques in the conscious, unrestrained rodent, rabbit or pπmate, e.g. TruePπmateJ, or other species, applied to practice this invention The present method, thus, helps to determine 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 it The method itself generally comprises obtaining oligonucleotides (oligos) consisting of up to about 15% adenosme (A), 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' lntron-exon borders and the uxta-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 m vitro hybridization to the target mRNA, administering to a subject an amount of the selected ohgo effective for m 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 ohgo; wherein a change m 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 anti-sense oligos may be constructed by selecting fragments of a target having at least 4 contiguous nucleic acids selected from the group consisting of G and C and obtaining a first ohgonucleotide about 4, 6, 8, 10 to about 15, 25, 45, 60 nucleotides long which comprises the selected fragment and has a C and G content of about 0%, about 3%, about 5%, about 10%, about 12% up to about 15% Alternatively, the target fragments may be selected by their type and/or extent of activity, which may vary for specific purposes Any number of adenosines may be substituted, if present, from one to all, by a "universal" or alternative base such as heteroaromatic bases which bind to a thymidme base but have less than about 03 of the adenosine base agonist activity at the adenosine A,, A 2a , A 2b and A 3 receptors, and heteroaromatic bases which have no activity at the adenosine A 2a receptor The heteroaromatic bases may be pyπmidines and puπnes, which may be substituted, for example, by O, halo, NH 2 , SH, SO, S0 2 , S0 3 , COOH and branched and fused primary and secondary ammo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, which may be further substituted by O, halo, NH 2 , primary, secondary and tertiary amine, SH, SO, SO,, S0 3 , cycloalkyl, heterocycloalkyl and heteroaryl Other compounds and other substituents, however, are also suitable for use with the present method Typically, the pyπmidines and puπnes are substituted at positions 1, 2, 3, 4, 7 and 8, although other substitutions are also encompassed Examples of pyπmidines and purines are theophylhne, caffeme, dyphylhne, etophylhne, acephylhne piperazme, bamifylhne, enprofylhne and xanthine having the chemical formula O M I t I I i i wherein R' and R 2 are independently H, alkyl, alkenyl or alkynyl and R 3 is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O- cycloalkynyl, NH 2 -alkylamιno-ketoxyalkyloxy-aryl and mono and dialkylaminoalkyl-N-alkylamino-S0 2 aryl. Specific examples of universal or alternative bases are 3-mtropyrrole-2'-deoxynucleosιde, 5-nιtro-mdole, 2- deoxyπbosyl-(5-nιtromdole), 2-deoxyπbofuranosyl-(5-mtromdole), 2'-deoxymosιne, 2'-deoxynebulaπne, 6H, 8H-3,4-dιhydropyπmιdo [4,5-c] oxazme-7-one or 2-ammo-6-methoxyammopuπne, although others are also suitable. Most preferred are adenosme analogs which have no activity at adenosme receptors, i.e., which have neither agomst nor antagonist properties at any adenosme receptor. The method may also utilize, m another preferred embodiment, a methylated cytocme ( m C) substimted in or for at least one unmethylated C in a CpG dinucleotide if present m the ohgo(s), although many more or all may also be substituted. Other C-5 modifications at pyπmidmes are also useful, e.g , C-5 propyne, among others. For practicing this method, one or more or all linking residues of the anti-sense oligonucleotides are preferably substituted or modified with a residue selected from the group consisting of methylphosphonate, phosphotπester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamme, 2'- methylene(methymuno), (MMI), 2'-methoxymethyl (MOM), 2'-methoxyethyl (MOE), 2'-methyleneoxy (methylimino) (MOMA), 2'-methoxy methyl (MOM), 2'-0-methyl, phosphoramidate, and C-5 substitued (e. g C-5 propyne) residues and combinations thereof Anti-sense ohgos suitable for practicing this method are 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 long, although other lengths are also suitable. The method of the invention may also incorporate the use of an anti-sense ohgo that is linked to an agent that is internalized or up-taken by cells as well as cell targeting agents, such as transfemn, asialoglycoprotem and streptavidin, among others known m the art. In one embodiment the ohgo is linked to a vector, which may be prokaryotic or eukaryotic. Examples of vectors are known m the art and need not be descπbed further m this patent. The amount of anti-sense ohgo administered is generally one that is effective to reduce the production or availability, or to increase the degradation, of the mRNA, or to reduce the amount of the polypeptide present m situ For example, when the gene to be validated is associated with a respiratory function, the anti-sense ohgo may be administered directly to the lung (s). When the gene and the function are associated with another system, the nucleic acid is preferably administered m situ to the affected region, e. g. the bram, the heart, the kidney, the bladder, the gonads and the reproductive system in general, the respiratory and pulmonary systems, tumors m the case of cancer, the blood, the immune system, the lung, skin, eye, nasal passage, scalp, testes, cervix, oral cavity, larynx, esophagus, small and large intestine, synovial tissues, muscles, ovaπes, ear canal, and many more, such as any cells that oπgmate from a selected target site. In many cases, a disease or condition afflicts a certain system or area of a system, such as those descπbed above. For example, a respiratory ailment may be associated with an mcrease m bronchoconstπction, inflammation, IgE-mediated allergies, surfactant production, and other symptoms such as m asthma, allergic rhinitis, COPD, lung tumors, ARDS, etc. Where a disease or condition is associated with an lmmunological dysfunction, the target may be selected amongst lmmunoglobulins and antibody receptors, cytokmes and cytokine receptors, genes and other gene products, and corresponding mRNAs encodmg them and other associated functions, the genes and mRNA flanking regions and intron and exon borders, among others. When the disease or condition is associated with a malignancy or cancer, the target may be selected from cancer related gene products, genes and mRNAs encodmg them, genes and mRNAs associated with oncogenes, genomic and mRNA flanking regions and exon and intron borders, etc. The anti-sense ohgos for use with the present method may be produced by selection of a target from the group consisting of polypeptides associated with a dιsease(s) and/or condιtιon(s) afflicting lung airways, genes and RNAs encodmg them, the genomic and mRNA flanking regions and the gene(s) and mRNA(s) EPI-149 mtron and exon borders, then obtaining the sequence of a mRNA(s) selected from the group consisting of mRNAs corresponding to the target gene(s) and mRNAs encodmg the target polypeptιde(s), genomic and mRNA flankmg regions and the genes and mRNAs mtron and exon borders; selectmg at least one segment of the mRNA(s), synthesizmg one or more ohgo anti-sense to the selected mRNA segments), and substituting, if necessary, a universal or alternative base(s) for one or more A(s) to reduce the content of A present m the ohgo to up to about 10% of all nucleotides Specific examples of the encoded polypeptides associated with the pulmonary system are NficB Transcnption Factor, Interleukιn-8 Receptor (IL-8 R), Interleukin 5 Receptor (IL-5 R), Interleukin 4 Receptor (IL-4 R), Interleukin 3 Receptor (IL-3 R), Interleukin- lβ (IL-lβ), Interleukin lβ Receptor (IL- lβ R), Eotaxm, Tryptase, Major Basic Protein, β2-adrenergιc Receptor Kmase, Endothelm Receptor A, Endothelm Receptor B, Preproendothelin, Bradykinin B2 Receptor, IgE High Affinity Receptor, Interleukin 1 (IL-1), Interleukin 1 Receptor (IL-1 R), Interleukin 9 (IL-9), Interleukιn-9 Receptor (IL-9 R), Interleukin 11 (IL-11), Interleukin- 11 Receptor (IL- 11 R), Inducible Nitπc Oxide Synthase, Cyclooxygenase (COX), Intracellular Adhesion Molecule 1 (ICAM-1) Vascular Cellular Adhesion Molecule (VCAM), Rantes, Endothehal Leukocyte Adhesion Molecule (ELAM-1), Monocyte Activating Factor, Neutrophil Chemotactic Factor, Neutrophil Elastase, Defensm 1, 2 and 3, Muscaπnic Acetylcholine Receptors, Platelet Activating Factor, Tumor Necrosis Factor α, 5-lιρoxygenase, Phosphodiesterase IV, Substance P, Substance P Receptor, Histamine Receptor, Chymase, CCR-1 CC Chemokme Receptor, CCR-2 CC Chemokme Receptor, CCR-3 CC Chemokme Receptor, CCR-4 CC Chemokme Receptor, CCR-5 CC Chemokme Receptor, Prostanoid Receptors, GATA-3 Transcnption Factor, Neutrophil Adherence Receptor, MAP Kmase, Interleukιn-9 (IL-9), NFAT Transcnption Factors, STAT 4, MlP-lα, MCP-2, MCP-3, MCP-4, Cyclophillms, Phosphohpase A2, Basic Fibroblast Growth Factor, Metalloprotemase, CSBP/p38 MAP Kmase, Tryptose Receptor, PDG2, Interleukιn-3 (IL-3), Interleukin- lβ (IL-lβ), Cyclosponn A-Binding Protem, FK5-Bmdmg Protem, α4βl Selectin, Fibronectm, α4β7 Select , Mad CAM-1, LFA-1 (CD 11 a/CD 18), PECAM-1, LFA-1 Selectm, C3bι, PSGL-1, E-Selectin, P-Selectin, CD-34, L-Selectm, pi 50,95, Mac-1 (CD l ib/CD 18), Fucosyl transferase, VLA-4, CD-18/CDl la, CDl lb/CD18, ICAM2 and ICAM3, C5a, CCR3 (Eotaxm Receptor), CCR1, CCR2, CCR4, CCR5, LTB-4, AP-1 Transcnption Factor, Protem kmase C, Cystemyl Leukotπene Receptor, Tachychinnen Receptors (tach R), IκB Kmase 1 & 2, STAT 6, c-mas and NF-Interleukm-6 (NF-IL-6) Examples of polypeptides or genes associated with the CNS, ophthalmic, cardiovascular and cardiopulmonary systems are the family of G-protem coupled receptors (aproximately 250 known, and approximately 750-1,000 more postulated and yet to be sequenced), Neuropeptide genes, Neuropeptide receptor genes, Excitatory ammo acid receptor genes, Chloπde channel genes, Calcium channel genes, Puπnergic receptor genes, Adrenergic receptor genes, Serotonin receptor genes, Serotonm transporter genes, Excitatory ammo acid transporter genes, Potassium channel genes, Tyrosme kmases, Phosphorylases, Acetylcholine receptors, Cholecystokimn receptors, Nitric Oxide synthase, Dopamine receptors, Cholmergic receptors, Angiotensm, Angiotensm receptors, Ion Channels mcludmg Potassium Channels, Structural protems mcludmg those related to myelmation/demyelmation and axonic and dendritic structures, Neurotransmitter release mediators and structures, Opthalmic disorders, especially of the retma and asociated structures, Calcitonin and its receptors, Calcmeuπn and its receptors, CGRP and its receptors, Atnal natnuretic peptide and its receptors, Bram natπuretic peptide and its receptors, Bradykmin and its receptors, Baroreceptors, GABA/GABA receptors, Benzodiazepme receptors, Chohnesterases, Cannabinoid receptors, Calmoduhn and its receptors, Calcium Sodim exchange pump Carbonic anhydrase, Catecholamines and their receptors, Histamine receptors, Muscannic receptors, Opioid receptors, Chemokines, Cholme acetyltransferase, Cholecalciferol, Mediators of inflammation mcludmg cytokmes, mterleukms, interferons and their receptors, enzymes of the poxygenase pathway, proteases, DP(PGD2) receptors, Inositol phosphate associated enzymes, Endothelins and their receptors, Enkephalmase, Enkephalins, Benzodiazepine receptors, GABA transaminase, Galanin and its receptors, Gastnn releasmg factor, Growth factors, Growth factor inhibitors, Cyclms, Nucleoside kinases, Nucleotide kmases, Oncogenes' Receptors, 5-hydroxytryptamme (5HT) receptors, ADH, IGF, Insulin receptors, Lactamases, Kamate receptors, Kallikreins and their recepotrs, Leukotnene-associated enzymes, Lipocortms, L-NMMA and receptors, Melanocyte stimulating hormone, Steroid transporters and metabolism enzymes and synthases, NMDA and receptors, Morphine receptors, MPTP, Neurokimns and their receptors, Nicotmic receptors, Phosphohpases, Platelet activating factors, Signal transduction protems, PDGF, Dynorph s, Prostacyclms, Prolactm and its receptors, Prolactm release lnhibitmg factor, Prohormones, Prostanoids, Prostaglandms and their receptors, Thrombm, Prothrombm, Pteroylglutamic acid and its receptors, Lysergic acid receptors, Snake and scorpion venom recertors, Renm angiotensm system components, Reverse transcnptase, Second messengers and associated enzymes, Sodium channels, Somatostatin and its receptors, Somatotropin and its receptors, Substance P, Substance k, Synatpic transmitters, Tachikinins, Tetrodotoxm receptors, Thromboxanes and their receotrs, Thyroid hormones, Hormones, Thyrotropm and receptors, Protirelin, T4, T3 Topoisomerases, Tumor necrosis factors and their receptrors, TGFs and their receptors, Xanthine oxidase, Viral messenger RNAs, Bacterial mRNAs, Oxytocin and its receptors, Chlecystokinin and its receptors, Vasoactive intestinal peptide and its receptors, Monoamine oxidase, Tyrosme-kmase linked receptors, and many more Other specific target genes are, for example, G-protems and G-protem coupled receptors, calcium channel protems and associated protein receptors, sodium channel protems and associated protein receptors, potassium channel protems and associated protem receptors, and chloπde channel protems and associated protem receptors, neurotransmitters and neurotransmitter receptors, neurohormones and neurohormone receptors, neuropephdes and neuropeptide receptors, and many others mcludmg the ones listed throughout this patent Other target genes are, for example, G-protems and G-protem coupled receptor, calcium channel protems and associated protein receptors, sodium channel protems and associated protein receptors, potassium channel protems and associated protein receptors, and chlonde channel proteins and associated protem receptors, neurotransmitters and neurotransmitter receptors, neurohormones and neurohormone receptors, neuropeptides and neuropeptide receptors, and many others In the present method, the composition may be administered m vitro, orally, lntracavitanly, intranasally, intraanally, lntravaginally, intrauterally, lntracranially, pulmonanly, intrarenally, lntranodularly, lntraarticularly, lntraotically, mtralymphatically, transdermally, lntrabucally, intravenously, subcutaneously, intramuscularly, intratumorously, mtraglandularly, intraocularly, intracranial, mto an organ, lntravascularly, mtrathecally, by implantation, by inhalation, mtradermally, mtrapulmonaπly, mto the ear, onto the skin or scalp or cervix (e g topically), into the heart, by slow release, by sustained release and by a pump, and the like. Examples of target genes and mRNAs associated with different systems and diseases are genes and mRNAs encodmg polypeptides such as transcnption factors, stimulating and activating factors, cytokmes and their receptors, mterleukms, interleukin receptors, chemokines, chemokme receptors, endogenously produced specific and non-specific enzymes, immunoglobulins, antibody receptors, central nervous system (CNS) and peπpheral nervous and non-nervous system receptors, CNS and peπpheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensines, growth factors, vasoactive peptides, peptide receptors and binding protem and genes and mRNAs corresponding to oncogenes. The admmistration of the ohgo may be conducted with an oral formulation havmg a liquid earner such as solutions, suspensions, and oil-in-water and water-m-oil emulsions, and or may be administered as a powder, dragees, tablets, capsules, sprays, aerosols, solutions, suspensions and emulsions When administered as a topical formulation, the earner may be selected from creams, gels, ointments, sprays, aerosols, patches, solutions, suspensions and emulsions When the formulation is injectable, the earner may be selected from aqueous and alcoholic solutions and suspensions, oily solutions and suspensions and oil-in-water and water-in-oil emulsions, among others. When as a rectal formulation, it may be in the form of a suppository, when m the form of a transdermal formulation, the earner is selected from aqueous and alcoholic solutions, oily solutions and suspensions and oil-in-water and water-in-oil emulsions, although others are also suitable The transdermal formulation, may be an lontophoretic transdermal formulation, wherein the earner is selected aqueous and alcoholic solutions, oily solutions and suspensions and oil-in-water and water-m-oil emulsions, and the formulation may also contain a transdermal transport promoting agent, of which many are known in the art Also suitable for prolonged admmistration are implantable capsules or cartridges contammg the formulation. In this case, the earner may also be selected from aqueous and alcoholic solutions and suspensions, oily solutions and suspensions and oil- m-water and water-in-oil emulsions, be a hydrophobic earner, such as hpid vesicles or particles, e. g. hposomes made of N-(l-[ 2, 3-dιoleoxyloxι] propyl) -N,N,N- tnmethyl- ammonium methylsulfate. and/or other hpids, and microcrystals For pulmonary applications, the formulation is preferably a respirable or inhalable formulation, e. g in the form of an aerosol For prolonged exposure of a target area, the ohgo may be delivered through a localized implant, suppository, sublmgual formulation, and the like, all of which are known m the art A factor which proves this method supenor to other technology is the ability to obtam data of greater reliability and accuracy Anti-sense nbozyme technology is unstable in in vivo environments, and the presence of adenosme m nbozyme and other oligonucleotides prevents the attainment of reliable data, for instance m the hyperactive respiratory tract, and other systems having a substantial number of adenosme receptors No other method has proven, up to the present time, capable of unambiguously attenuating targets in A-contammg systems, e g the respiratory tract while providing reliable and accurate correlations. In addition, the present method may be utilized to the elucidation of gene networks, e.g. neuronal gene networks, a quantum leap above the identification of single genes m isolation of their broader context This may be done by selectmg more than one target linked in a metabolic pathway and testmg them separately and in conjunction with the others, that is by administration of one anti-sense ohgo at a time, then m twos, m threes, etc., and comparing the results to ascertain whether or not there is linkage, they work sequentially etc The present method permits the creation of a gene network data base suitable to supplement a more elaborate pharmoincentive discovery process to meet cntical medical needs m areas such as cognition, memory, pain, anxiety, behavioral disturbances, ingestive behavior, hunger and satiety, and neurological disease, among others relating to the CNS The present technology encompasses four basic areas functional genomics applied to discerning neuronal gene networks of relevance to new drug discovery, m situ hybπdization to understand the distribution of these networks within the bram, propnetary Site Specific Functional Gene Ablation (SSFGA) to determine the function of individual genes within network, Multifactoπal Behavioral Analysis for qualitative and quantitative analysis of the participation of genes and gene networks m vanous behaviors of medical relevance, and physiological, biophysical, biochemical, etc. analysis to discern the effects of genes and gene networks upon extrapyramidal systems such as the cardiovascular and pulmonary systems. The application of this method to the CNS may encompass areas such as pam, satiety, anxiety/mood disorders, libido, cognition/cognitive disorders, and sleep/sleep disorders, among many others.The ability to discern functional significance of identified novel genes and gene networks relies on an assessment of their spatial representation In situ hybridization, for example, may be is used to determine the precise three dimensional (3-D) location of selected genes and gene networks within the bram, and to enable the accurate targeting of gene ablation studies for assessmg their significance as candidates for drug discovery programs. Site-specific functional gene ablation (SSFGA) provides a means to selectively attenuate the expression of any target gene m any desired region of a system, e.g the bram. SSFGA for CNS target validation may be performed with the aid of the low A or desA anti-sense ohgonucleotide designed as descnbed below. Other approaches utilizing anti-sense oligonucleotides provide ambiguous data because the oligonucleotides used breakdown and release adenosme, one of the most bioactive autocoids, e g. m the CNS and m other target systems containing adenosme receptors The release of adenosme upon break-down of oligonucleotides either depresses or facilitates neurotransmission dependmg upon the system and the specific area, e.g. the bram region, where it is released, mduces sleep, affects nociception, alters thalamic spmdle rhythms, mitigates or potentiates mynad drug effects, affects autonomic control of cardiovascular function and respiration, inhibits Ca+ currents and presynaptic function of GABA, depresses both spontaneous and evoked neuronal firing, inhibits the release of neurotransmitters, decreases postsynapfac excitability, inhibits long-term potentiation postulated to be an underlymg event in learning and memory, and induces pleiotropic effects by causmg cephalic bronchodilation Clearly, the release of significant amounts of adenosme via break down of oligonucleotides in the brain, or elsewhere, of the experimental annual is contramdicated in target validation studies. Neurological and behavioral tests suitable for application as end pomts for CNS target validation are known m the art. For example, tests for memory, three dimensional or spatial ability, cognition, motor control, sensitivity and responsiveness to exogenous triggers, vision, eye coordination, skin sensory ability, gustation and olfactory recognition, and many more The testing of physiological parameters is also known m the art and may rely on the measurement of electncal conductivity such as EKGs and EEGs, or other bodily functions such as heart rate and rhythmicity, water voiding, sleep patterns, etc. In many cases, side effects, particularly cardiopulmonary, renal, and other side effects, constitute a major reason for disqualifying potential drug discovery CNS targets And vice versa, CNS side effects often disqualify otherwise suitable therapeutic cardiopulmonary, renal, and other agents It thus would be advantageous to obtain evidence of cardiopulmonary effects induced by attenuation of CNS targets as early as possible m the development of a drug. When such effects are discovered late in a development program they cause the cancellation of a program at a much later stage The present method utilizes state of the art analysis, significant surgical, electrophysiological, and other types of tests, to assess any detrimental effects, e g. cardiovascular effects of attenuating respiratory targets, and vice versa any detnmental side effects of attenuating respn atory targets on CNS or the cardiovascular system, among others For example, lung function studies performed m conscious, unrestrained annuals, and cardiac function studies may provide further msight on potential effects of attenuating candidate CNS targets Target validation may proceed via SSFGA as descnbed above usmg low A or desA anti-sense oligonucleotides targeting a variety of known receptors with suspected function m human pathology, e.g neuropeptide Y (NPY)/leptin receptors and ingestive behavior, as well as novel targets found from CNS gene hbranes. Animal models may be subjected to SSFGA targeting of a specific receptor and then assessed for changes m vanous behaviors, such as m ingestive behavior, analgesia, locomotor behavior, sensimotor reflexes, gating processes, sexual and reproductive behaviors, anxiety, learning and memory, among many others. This broad-based behavioral battery of tests provides a sensitive measure of the effect of attenuating specific CNS targets, and a cπtical knowledge in the initial stages of the drug development decision process. Combmed with a physiological/biophysical biochemical analysis, this method provides for a rapid, intensive determination of the potential of a candidate target as a worthwhile focus for further drug discovery efforts. The present method improves on prior methods for validation of gene and protem targets in that desadenosme anti-sense oligos targeted to genes associated with different systems are used to inhibit the expression of a gene product and to test the effects and symptomatology and changes these procedures. The present mvention is premised on the recent discovery by the inventor that, when oligonucleotides are metabolized m vivo to their mononucleohdes, bioactive adenosme metabolites are released. Adenosme (A)-contaιnιng oligonucleotides break down and release adenosme metabolites which, m turn, activate adenosme receptors which, for example, m the lungs cause bronchoconstnction, inflammation, and the like. The present technology relies on the design of anti-sense ohgos targeted to genes and mRNAs associated with systems involved m different functions, ailments, and pathology(ιes) The ohgos are modified to reduce their adenosme content to minimize the occurrence of undesirable side effects caused by its release upon breakdown. Domg so improves the statistical significance of the results observed, particularly where adenosme receptors may be mvolved in effects similar, or opposite to those bemg observed, or indirectly by producing changes m the experimental model's homeostasis of the system bemg observed. In this manner, the inventor targets a specific gene to design one or more anti-sense ohgonucleotide(s) (oligos) that selectively bmd(s) to the correspondmg mRNA and, if necessary, reduces their content of adenosme via substimtion with universal or alternative base or an adenosine analog mcapable of activating adenosine A, A 2a , A 2b or A 3 receptors. Based on his pπor expenence m the field, the inventor reasoned that in addition to "downregulating" or ablating specific genes, he could mcrease the accuracy of the results by either selecting segments of RNA that are devoid, or have a low content, of thymidme (T) or, alternatively, substitute one or more adenosme(s) present m the designed ohgonucleotide(s) with other nucleotide bases, so called universal or alternative bases, which bind to thymidme but lack the ability to activate adenosme receptors and otherwise exercise the effect of adenosine m the lungs, etc. Given that adenosine (A) is a nucleotide base complementary to thymidme (T), when a T appears in the RNA, the anti-sense ohgo will have an A at the same position. For consistency's sake, all RNAs and oligonucleotides are represented in this patent by a smgle strand m the 5' to 3' direction, when read from left to right, although their complementary sequence(s) is (are) also encompassed within the four corners of the invention. In addition, all nucleotide bases and ammo acids are represented utilizmg the recommendations of the IUPAC-IUB Biochemical Nomenclattire Commission, or by the known 3-letter code (for ammo acids). The method of the present invention may be used to validate or invalidate any number of target genes, from smgle genes associated with one function m a subject, to multiple single genes associated with a network or pathway within a system By validation/invalidation it is meant a demonstration through experimental evidence whether a specific gene is mvolved in any one of a number of biophysical, biochemical, physiological, behavioral, etc., functions. Even if a gene appears initially not to have an effect, it may be tested m conjunction with another gene target, as there may be a requirement for the simultaneous obliteration of their expression to observe an effect. The adenosme content of the anti-sense agent(s) of the mvention have a reduced A content to prevent its liberation upon m vivo degradation of the agent(s). For example, if the system is the pulmonary or respiratory system, a large number of genes is mvolved m different functions, mcludmg those listed in Table 1 below Table 1 : Pulmonary Disease or Condition Pulmonary and Inflammation Targets NficB Transcnption Factor Interleukιn-8 Receptor (IL-8 R) Interleukιn-5 Receptor (IL-5R) Interleukm-4 Receptor (IL-4R) Interleukιn-3 Receptor (IL-3R) Interleukin- 1 β (IL- 1 β) Interleukin- 1 β Receptor (IL- 1 βR) Eotaxm Tryptase Major Basic Protein β2-adrenergιc Receptor K ase Endothelm Receptor A Endothelm Receptor B Preproendothelm Bradykimn B2 Receptor (B2BR) IgE (High Affinity Receptor) Interleukin- 1 (IL-1) Interleukin 1 Receptor (IL-1 R) Interleukm-9 (IL-9) Interleukm-9 Receptor (IL-9 R) Interleukin- 11 (IL- 11) Interleukin- 11 Receptor (IL-11 R) Inducible Nitπc Oxide Synthase Cyclooxygenase (COX) Intracellular Adhesion Molecule 1 (ICAM-1) Vascular Cellular Adhesion Molec.Subst.P (NC AM) Rantes Endothehal Leukocyte Adhesion Molecule Endothelm ETA Receptor (ELAM-1) Cyclooxygenase-2 (COX-2) GM-CSF, Endothelm- 1 Monocyte Activating Factor Neutrophil Chemotactic Factor Neutrophil Elastase Defensm 1,2,3 Muscaπnic Acetylcholine Receptors Platelet Activating Factor Tumor Necrosis Factor α 5-hpoxygenase Phosphodiesterase IV Substance P Substance P Receptor Histamine Receptor Chymase CCR-1 CC Chemokme Receptor Interleukm-2 (IL-2) Interleukιn-4 (IL-4) Interleukin- 12 (IL-12) Interleukm-5 (IL-5) Interleukm-6 (IL-6) Interleukm-7 (IL-7) Interleukm-8 (IL-8) Interleukin- 12 Receptor (IL-12R) Interleukm-7 Receptor (IL-7R) Interleukin- 1 (IL-1) Interleukin- 14 Receptor (IL-14R) Interleukin- 14 CCR-2 CC Chemokme Receptor CCR-3 CC Chemokme Receptor CCR-4 CC Chemokme Receptor CCR-5 CC Chemokme Receptor Prostanoid Receptors GATA-3 Transcnption Factor Neutrophil Adherence Receptor MAP Kmase Interleukin- 15 (IL-15) Interleukin- 15 Receptor (IL-15R) Interleukin- 1 1 (IL-11) Interleukin- 11 Receptor (IL-11R) NFAT Transcnption Factors STAT 4 MlP-l MCP-2 MCP-3 MCP-4 Cyclophilhn (A, B, etc ) Phosphohpase A2 Basic Fibroblast Growth Factor Metalloprotemase CSBP/p38 MAP Kmase Tryptase Receptor PDG2 Interleukιn-3 (IL-3) Interleukm-lO (IL-lO) Cyclospoπn A - Binding Protem FK506-Bιndιng Protem oc4βl Selectm Fibronectm α4β7 Selectm cMad CAM-1 LFA-1 (CD 11 a/CD 18) PECAM-1 LFA-1 Selectm C3bι PSGL-1 E-Selectin P-Selectm CD-34 L-Selectm pi 50,95 Mac- 1 (CD l ib/CD 18) Fucosyl transferase VLA-4 STAT-1 STAT-2 CD-18/CDl la CD l ib/CD 18 ICAM2 and ICAM3 C5a CCR3 (Eotaxm Receptor) CCR1, CCR2, CCR4, CCR5 LTB-4 AP-1 Transcnption Factor Protem kmase C Cysteinyl Leukotπene Receptor Tachyk nen Receptors (tach R) IκB Kmase 1 & 2 Interleukm-2 Receptor (IL-2R) (e g , Substance P, NK-1 & NK-3 Receptors) STAT 6 c-mas NF-Interleukιn-6 (NF-IL-6) Interleukin- 10 Receptor (IL-1 OR) Interleukm-3 (IL-3) Interleukm-2 Receptor (IL-2R) Interleukm-13 (IL-13) Interleukin- 12 Receptor (IL-12R) Interleukιn-14 (IL-14) Interleukιn-6 Receptor (IL-6R) Interleukin- 16 (IL-16) Interleukm-13 Receptor (IL-13R) Medullasm Interleukin- 16 Receptor (IL-16R) Adenosme A, Receptor (A, R) Tryptase-I Adenosme A 2b Receptor (A 2b R) Adenosme A 3 Receptor (A 3 R) β Tryptase STAT-3 Adenosme A 2a Receptor (A 2a R) IgE Receptor β Subunit (IgE R β) Fc-epsilon receptor CD23 antigen IgE Receptor α Subunit (IgE R α) IgE Receptor Fc Epsilon Receptor (IgERFceR) Substance P Receptor Histidme decarboxylase Tryptase- 1 Prostaglandin D Synthase Eosmophil Canonic Protein Eosmophil Deπved Neurotoxm Eosmophil Peroxidase Endothehal Nitnc Oxide Synthase Endothehal Monocyte Activating Factor Neutrophil Oxidase Factor Cathepsin G Macrophage Inflammatory Protein -1- Interleukm-8 Receptor a Subunit (IL-8 Rα) Alpha/Rantes Receptor Endothelm Receptor ET-B These genes, and others, are involved in the normal functioning of respiration as well as m diseases associated with respiratory pathologies, including cystic fibrosis, asthma, pulmonary hypertension and vasoconstπction, chronic obstructive pulmonary disease (COPD), chronic bronchitis, respiratory distress syndrome (ARDS), allergic rhinitis, lung cancer and lung metastatic cancers and other airway diseases, mcludmg those with inflammatory response Anti-sense ohgos to the adenosine A,, A 2a , A 2b , and A 3 receptors, CCR3 (chemokme receptors), bradykmm 2B, CAM (vascular cell adhesion molecule), and eosmophil receptors, among others, have been shown to be effective in down-regulating the expression of their genes Some of these act to alleviate the symptoms or reduce respiratory ailments and or inflammation, for example, by "down regulation" of the adenosme A„ A 2a , A 2b , and or A 3 receptors and CCR3, bradykinin 2B, VCAM (vascular cell adhesion molecule) and eosmophil receptors These agents may be utilized by the present method alone or m conjunction with anti-sense ohgos targeted to other genes to validate pathway and or networks m which they are mvolved For better results, the ohgos are preferably admmistered directly mto the respiratory system, e g , by inhalation or other means, of the experimental animal, so that they may reach the lungs without widespread systemic dissemination This permits the use of low agent doses as compared with those admmistered systemically or by other generalized routes and, consequently, reduces the number and degree of undesirable side effects resultmg from the agent's widespread distnbution m the body The agent(s) of this invention has (have) been shown to reduce the amount of receptor protein expressed by the tissue These agents, thus, rather than merely interacting with their targets, e g a receptor, lower the number of target protems that other drugs may interact with In this manner, the present agent(s) afford(s) extremely high efficacy with low toxicity The receptors discussed above are mere examples of the high power of the present technology In fact, a large number of genes may be targeted in a similar manner by practicmg the present methods, to significantly down-regulate or obliterate protem expression and observe any changes wrought to one or more functions within a system, e g the respiratory, CNS, cardiovascular, renal and other systems By means of example, in the respiratory system, the functions tested may be ease of breathing, bronchoconstnction, inflammation, chronic bronchitis, surfactant production, and the like, and others associated with diseases and conditions such as chronic obstructive pulmonary disease (COPD), inhalation burns, Acute Respiratory Distress Syndrome (ARDS), cystic fibrosis, pulmonary fibrosis, radiation pulmonitis, tonsilitis, emphysema, dental pain, oral inflammation, joint pain, esophagitis, lung cancer and esophageal cancer, among others. These functions are of great interest because of their association with respiratory dysfunction, as is the case in asthma, allergies, allergic rhinitis, pulmonary bronchoconstriction and hypertension, chronic obstructive pulmonary disease (COPD), allergy, asthma, cystic fibrosis, Acute Respiratory Distress Syndrome (ARDS), cancer, which either directly or by metastasis afflict the lung, the present method may be applied to a list of potential target mRNAs, which includes the targets listed in Table 1 above, among others. In the CNS system, functions that may be selected are food ingestion/satiety, mood variation, anxiety, libido/sexual dysfunction, cognition, sexual function dysfunction, brain trauma, Alzheimer's mediators, aneurism, etc. The oligos of this invention may be obtained by first selecting fragments of a target nucleic acid having at least 4 contiguous nucleic acids selected from the group consisting of G and C and/or having a specific type and or extent of activity, and then obtaining a first ohgonucleotide 4 to 60 nucleotides long which comprises the selected fragment and has a thymidine (T) nucleic acid content of up to and including about 15%, preferably, about 12%, about 10%, about 7%, about 5%, about 3%, about 1%, and more preferably no thymidine. The latter step may be conducted by obtaining a second ohgonucleotide 4 to 60 nucleotides long comprising a sequence which is anti-sense to the selected fragment, the second ohgonucleotide having an adenosine base content of up to and including about 15%, preferably about 12%, about 10%), about 7%, about 5%, about 3%, about 1%, and more preferably no adenosine. When the selected fragment comprises at least one thymidine base, an adenosine base may be substituted in the corresponding anti-sense nucleotide fragment with a universal or alternative base selected from the group consisting of heteroaromatic bases which bind to a thymidine base but have less than about bout 10%, preferably less than about 1%, and more preferably less than about 0.3% of the adenosine base agonist activity at the adenosine A,, A 2a , A 2b and A 3 receptors, and heteroaromatic bases which have no activity at the adenosine A 2a receptor, when validating in the respiratory system. Other adenosine activities in other systems may be determined in other systems, as appropriate. The analogue heteroaromatic bases may be selected from all pyrimidines and purines, which may be substituted by O, halo, NH 2 , SH, SO, S0 2 , S0 3 , COOH and branched and fused primary and secondary amino, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, which may be further substituted by O, halo, NH 2 , primary, secondary and tertiary amine, SH, SO, S0 2 , S0 3 , cycloalkyl, heterocycloalkyl and heteroaryl. The pyrimidines and purines may be substituted at all positions as is known in the art, but preferred are those which are substituted at positions 1 , 2, 3, 4, 7 and/or 8. More preferred are pyrimidines and purines such as theophylline, caffeine, dyphylline, etophylline, acephylline piperazine, bamifylline, enprofylline and xantine having the chemical formula wherein R 1 and R 2 are independently H, alkyl, alkenyl or alkynyl and R 3 is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O- cycloalkynyl, NH 2 -alkylamιno-ketoxyalkyloxy-aryl, mono and dialkyl_minoalkyl-N-alkylamino-S0 2 aryl, among others. Similar modifications m the sugar are also embodiments of this mvention. Reduced adenosme content of the anti-sense ohgos correspondmg to the thymidines (T) present in the target RNA serves to prevent the breakdown of the ohgos mto products that free adenosme mto the system, e.g. the lung, bram, heart, kidney, etc., tissue environment and, thereby, to prevent any unwanted effects due to it By means of example, the NfKB transcnption factor may be selected as a target, and its mRNA or DNA searched for low thymidme (T) or desthyimdme (desT) fragments. Only desT segments of the mRNA or DNA are selected which, m turn, will produce desA anti-sense as their complementary strand When a number of RNA desT segments are found, the sequence of the anti-sense segments may be deduced. Typically, about 10 to 30 and even larger numbers of desA anti-sense sequences may be obtained. These anti- sense sequences may include some or all desA anti-sense ohgonucleotide sequences correspondmg to desT segments of the mRNA of the target, such as anyone of those shown m Table 1 above, in Table 2 below, and others associated with functions of the bram, cardiovascular and renal systems, and many others When this occurs, the anti-sense oligonucleotides found are said to be 100% A- free For each of the oπgmal desA anti- sense ohgonucleotide sequences correspondmg to the target gene, e g. the NFKB transcnption factor, typically about 10 to 30 sequences may be found within the target gene or RNA which have a low content of thymidme (RNA). In accordance with this mvention, the selected fragment sequences may also contam a small number of thymidine (RNA) nucleotides within the secondary or tertiary or quaternary sequences. In some cases, a large adenosme content may suffice to render the anti-sense ohgonucleotide less active or even inactive agamst the target In accordance with this invention, these so called "non-fiilly desA" sequences may preferably have a content of adenosine of less than about 15%, about 12%, about 10%, about 7%, about 5%, and about 2% adenosme. Most preferred is no adenosme content (0%). In some mstances, however, a higher content of adenosme is acceptable and the oligonucleotides still fail to show detrimental "adenosme activity" A particular important embodiment is that where the adenosme nucleotide is "fixed" or replaced by a "Universal or alternative" base that may base-pair with similar or equal affinity to two or more of the four nucleotide present m natural DNA A, G, C, and T A universal or alternative base is defined m this patent as any compound, more commonly an adenosme analogue, which has substantial capacity to hybndize to thymidme, reduced, or substantially lacks ability to bmd adenosme receptors or other molecules through which adenosme may exert an undesirable side effect m the experimental animal or m a cell system. Alternatively, adenosme analogs which completely fail to activate adenosme receptors, such as the adenosme A„ A^, A 2b and or A 3 receptors, most preferably A, receptors, may be used One example of a universal or alternative base is α-deoxyπbofuranosol-(5- mtromdole), and an artisan will know how to select others This "fixing" step generates further novel sequences, different from those anti-sense to the ones found m nature, that permits the anti-sense ohgonucleotide to bmd, preferably equally well, with the target RNA. Other examples of universal or alternative bases are 2-deoxynbosyl-(5-nιtromdole). Other examples of universal or alternative bases are 3- mtropyrrole-2'-deoxynucleosιde, 5-nιtro-ιndole, 2-deoxynbosyl-(5-mtromdole), 2-deoxynbofuranosyl-(5- nitroindole), 2'-deoxyιnosme, 2'-deoxynebulanne, 6H, 8H-3,4-dιhydropyπmιdo [4,5-c] oxazme-7-one and 2- ammo-6-methoxyammopunne In addition to the above, Universal or alternative bases which may be substituted for any other base although with somewhat reduced hybndization potential, mclude 3-nιtropyrrole 2'-deoxynucleosιde 2-deoxynbofuranosyl-(5-nιtroιndole), 2'-deoxymosιne and 2'-deoxynebulaπne (Glen Research, Sterling, VA) More specific mismatch repairs may be made usmg "P" nucleotide, 6H, 8H-3, 4-dihydropynmιdo[4,5-c] [1,2] oxazιn-7-one, which base pairs with either guanme (G) or adenme (A) and "K" nucleotide, 2-aπuno-6-methoxyammopunne, which base pairs with either cytidine (C) or thymidme (T), among others. Others which are known m the art or will become available are also suitable. See, for example, Loakes, D. and Brown, D. M., Nucl .Acids Res. 22:4039-4043 (1994); Ohtsuka, E. et al., J. Biol. Chem.260(5):2605-2608 (1985); Lm, P.K.T. and Brown, D. M., Nucleic Acids Res. 20(19):5149-5152 (1992; Nichols, R. et al, Nature 369(6480)- 492-493 (1994); Rahmon , M. S. and Humayun, N. Z., Mutation Research 377 (2): 263-8 (1997); Amosova, O., et al., Nucleic Acids Res. 25 (!0): 1930-1934 (1997); Loakes D. & Brown, D. M., Nucleic Acids Res. 22 (20): 4039-4043 (1994), the entire sections relating to universal or alternative bases and their preparation and use m nucleic acid binding bemg incorporated herein by reference. When non-fully desT sequences are found in the naturally occurring target, they typically are selected so that about 1 to 3 universal or alternative base substitutions will suffice to obtain a 100% "desA" anti-sense ohgonucleotide Thus, the present method provides either anti-sense oligonucleotides to different targets which are low in, or devoid of, A content, as well as anti-sense oligonucleotides where one or more adenosme nucleotides, e. g about 1 to 3, or more, may be "fixed" by replacement with a "Universal or alternative" base. Universal or alternative bases are known in the art and need not be listed herem. An artisan will know which bases may act as universal or alternative bases, and replace them for A As used herem, the term "validate" or "validating" a target within a certain system such as the respiratory, mflammatory, CNS, cardiopulmonary, renal immune, and other systems, refers to a process which starts by selecting a therapeutic area within the system, for example, m the CNS or the cardiovascular or cardiopulmonary systems, among others. Areas that may be selected for study within those systems are those of anxiety, mood disorders, satiety and regulation of appetite, pam, cognition, sleep mduction and disorders, regulation of temperature, and many others that are controlled by or regulated through the bram, and others exemplified in Table 2 below. The next step is to select a gene sequence data base correspondmg to the appropnate system, e.g. a CNS, lung, cardiac system, kidney/renal system, blood, immune system, pulmonary and respiratory system, sexual function/dysfunction, skm, eye, nasal passages, scalp, testes, cervix, oral cavity, pharynx, esophagus, small and large intestine, synvial tissues, ovanes, ear canal, and other systems. Target genes are selected amongst those known to be associated with the CNS, or with a certain area of the CNS, and those of unknown functions. Genes whose functions are known and might occur in the system may also be selected. Anti-sense oligos are then designed for these target genes or mRNA fragments as descπbed here Finally, ohgos may be selected for their ability to ablate or significantly reduce the expression of the target gene by m vitro hybndization to cell and tissue DNA and RNA. Ohgos that show high m vitro down regulation, ablation or expression inhibition are then applied m m vivo tests, preferably by site specific administration, e.g. to a region of the bram, heart, kidney, lung, etc., and pre-determined behavioral, biophysical, biochemical, cognitive, motor, sensory, physiological, and other functions, assessed to establish whether or not a correlation exists between the target and the function. In the respiratory system, for example, a correlation may be shown be decreasmg the likelihood that the subject admmistered such treatment will manifest symptoms of a respiratory or mflammatory lung disease or other lung conditions, such as a malignancy. As applied here, the term "down-regulate" refers to ducing a decrease in production, secretion or availability (and thus a decrease m concentration) of the targeted intracellular protem, which may include a complete ablation. Table 2: Examples of Diseases & Conditions Associated with Targets & Networks Dementia Stroke Anxiety Anhnociception Analgesia Cardiopulm. Funct. Behavioral Traumatic Organic Bram Autonomic Control Disorders Bram Injury Disease Degen. Encephalopathy Developmental Drug Sensitivity CNS Disord (Viral, etc.) Abnorm.&Deform. (Legal & Illegal) Vision Cognition Satiety Food Alcohol Sensitivity Heart Attacks Learning Depression Ingestion Bram Inflammation Anesthesia Hearing Olfaction Hypoxia Schizophrenia Bram Cancer Cranial Def. Memory Neuropathy Neurogemc Pam Dental Pam Headache Sensation Motor Coord. Mood disorders Mood Elevat. Bipolar Dis. Eating Dis. Cachexia AneunsmsCardiac & Vase. Congestive Cardiac & Vase. Cardiac & Vase. Exudation Heart Disease Pam Inflammation Stroke Angina Heart Failure Ischemia Plaque FormationRestenosis Viral Infec Arrythmias Vascular Permeab Arteπal Degener Libido Angiogen. & Inhib Structural & Biochem Defects Anger Transplant Reject The present mvention is concerned pnmaπly with target validation in vertebrates, and within this group, of mammals, mcludmg human and non-human simians, wild and domesticated animals, manne and land animals, household pets, and zoo animals, for example, felines, canmes, equmes, pachiderms, cetaceans, and still more preferably to human subjects. One particularly suitable application of this technology is for veterinary purposes, and includes all types of small and large animals m the care of a vetennanan, mcludmg wild animals, marme animals, household animals, zoo animals, and the like. Targeted genes and protems are preferably mammalian, and the sequences targeted are preferably of the same species as the subject bemg treated. Although in many instances, targets of a different species are also suitable, particularly those segments of the target RNA or gene that display greater than about 45% homology, preferably greater than about 85% homology, still more preferably greater than about 95% homology, with the recipient's sequence. A preferable group of agents is composed of desA anti-sense oligos. Another preferred group is composed of non-fully desA oligonucleotides, where one or more adenosme bases are replaced with universal or alternative bases. The terms "anti-sense" oligonucleotides generally refers to small, synthetic oligonucleotides, resemblmg single-stranded DNA, which m this patent are applied to the inhibition of gene expression by inhibition of a target messenger RNA (mRNA) See, Milhgan, J. F. et al., J. Med. Chem 36(14), 1923-1937 (1993), the relevant portion of which is hereby incorporated in its entirety by reference. The present method utilizes anti-sense agents to inhibit gene expression of target genes, mcludmg those listed in Table 1 above. This is generally attained by hybndization of the anti-sense oligonucleotides to codmg (sense) sequences of a targeted messenger RNA (mRNA), as is known in the art. The exogenously administered agents of the mvention decrease the levels of mRNA and protem encoded by the target gene and or cause changes m the growth characteπstics or shapes of the thus treated cells. See, Milhgan et al. (1993); Helene, C. and Toulme, J. Biochim. Biophys. Acta 1049, 99-125 (1990); Cohen, J. S. D., Ed., Ohgodeoxynucleotides as Anti-sense Inhibitors of Gene Expression; CRC Press: Boca Raton, FL (1987), the relevant portion of which is hereby incorporated m its entirety by reference. As used herem, "anti-sense ohgonucleotide" is generally a short sequence of synthetic nucleotide that (1) hybridizes to any segment of a mRNA encoding a targeted protein under appropriate hybridization conditions, and which (2) upon hybridization causes a decrease in gene expression of the targeted protein. The terms "desAdenosine" (desA) and "des-thymidine" (desT) refer to oligonucleotides substantially lacking either adenosine (desA) or thymidine (desT). In some instances, the des T sequences are namrally occurring, and in others they may result from substitution of an undesirable nucleotide (A) by another one lacking its undesirable activity. In the present context, the substimtion is generally accomplished by substitution of A with a "universal or alternative base", as is known in the art. The mRNA sequence of the targeted protein may be derived from the nucleotide sequence of the gene expressing the protein, whether for existing targets or those to be found in the future. Sequences for many target genes of different systems are presently known. See, GenBank data base, NIH, the entire sequences of which are incorporated here by reference. The sequences of those genes, whose sequences are not yet available, may be obtained by isolating the target segments applying technology known in the art. Once the sequence of the gene, its RNA and or the protein are known, anti-sense oligonucleotides are produced as described above and utilized to validate the target by in vivo administration and testing for a reduction of the production of the targeted protein in accordance with standard techniques, and of specific functions. In one aspect of this invention, the anti-sense ohgonucleotide has a sequence which specifically binds to a portion or segment of an mRNA molecule which encodes a protein associated with a disease or condition of a specific system, e.g. CNS, respiratory, pulmonary, motor, sensory, hormone regulatory, cardiac, renal, immune, blood, cancer genes, and the like. One effect of this binding is to reduce or even prevent the translation of the corresponding mRNA and, thereby, reduce the available amount of target protein in the subject's lung. In one preferred embodiment of this invention, one or more of the phosphodiester residues of the anti-sense ohgonucleotide are modified or substituted. Chemical analogs of oligonucleotides with modified or substituted phosphodiester residues, e.g., to the methylphosphonate, the phosphotriester, the phosphorothioate, the phosphorodithioate, or the phosphoramidate, which increase the in vivo stability of the ohgonucleotide are particularly prefened. The namrally occurring phosphodiester linkages of oligonucleotides are susceptible to some degree of degradation by cellular nucleases. Many of the residues proposed herein, on the contrary, are highly resistant to nuclease degradation. See Milligan et al., and Cohen, J. S. D., supra. In another preferred embodiment of the invention, the oligonucleotides may be protected from degradation by adding a "3'-end cap" by which nuclease-resistant linkages are substituted for phosphodiester linkages at the 3' end of the ohgonucleotide. See, Tidd, D. M. and Warenius, H.M., Be. J. Cancer 60: 343-350 (1989); Shaw, J.P. et al. Nucleic Acids Res. 19: 747-750 (1991), the relevant section of which are incorporated in their entireties herein by reference. Phosphoramidates, phosphorothioates, and methylphosphonate linkages all function adequately in this manner for the purposes of this invention. The more extensive the modification of the phosphodiester backbone the more stable the resulting agent, and in many instances the higher their RNA affinity and cellular permeation. See Milligan, et al, supra. Thus, the number of residues which may be modified or substituted will vary depending on the need, target, and route of administration, and may be from 1 to all the residues, to any number in between. Many different methods for replacing the entire phosphodiester backbone with novel linkages are known. See, Millikan et al, supra. Prefened analogue residues for the base, the internucleotide linkage, or the sugar include phosphorothioate, methylphosphonate, phosphotriester, thioformacetal, phosphorodithioate, phosphoramidate, formacetal boranophosphate, 3'- thioformacetal, 5'-thioether, carbonate, 5'-N-carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, 2'-0 methyl, sulfoxide, sulfide, hydroxylamine, methoxy methyl (MOM), methoxy ethyl (MOE), methylene(methylimino) (MMI), and methyleneoxy(methylimino) (MOMI) residues. Phosphorothioate and methylphosphonate-modified oligonucleotides are particularly prefened due to their availability through automated ohgonucleotide synthesis. See, Millikan et al, supra. Where appropπate, the agent of this mvention may be admmistered m the form of a pharmaceutically acceptable salt, or as a mixture of the anti-sense ohgonucleotide and a salt. In another embodiment of this mvention, a mixture of different anti-sense oligonucleotides or their pharmaceutically acceptable slats is admmistered. The agents of this mvention have the capacity to attenuate the expression of one target mRNA and or to enhance or attenuate the activity of one pathway. By means of example, the present method may be practiced by identifying all possible deoxyπbonucleotide segments which are low in thymidme (T) or deoxynucleotide segments low m adenosme (A) of about 7 or more mononucleotides, preferably up to about 60 mononucleotides, more preferably about 10 to about 36 mononucleotides, and still more preferably about 12 to about 21 mononucleotides, m a target mRNA or a gene, respectively. This may be attained by searchmg for mononucleotide segments within a target sequence which are low in, or lack thymidme (RNA), a nucleotide which is complementary to adenosme, or that are low m adenosine (gene), that are 7 or more nucleotides long. In most cases, this search typically results m about 10 to 30 such sequences, i.e. naturally lackmg or havmg less than about 40% adenosme, anti-sense oligonucleotides of varying lengths for a typical target mRNA of average length, I e , about 1800 nucleotides long. Those with high content of T or A, respectively, may be fixed by substitution of a universal or alternative base for one or more As The agent(s) of this mvention may be of any suitable length, mcludmg but not limited to, about 7 to about 60 nucleotides long, preferably about 12 to about 45, more preferably up to about 30 nucleotides long, and still more preferably up to about 21, although they may be of other lengths as well, dependmg on the particular target and the mode of delivery. The agent(s) of the mvention may be directed to any and all segments of a target RNA. One preferred group of agent(s) mcludes those directed to an mRNA region containing a junction between an mtron and an exon. Where the agent is directed to an mtron/exon junction, it may either entirely overlie the junction or it may be sufficiently close to the junction to inhibit the sphcing-out of the intervening exon during processmg of precursor mRNA to mature mRNA, e.g. with the 3' or 5' terminus of the anti-sense ohgonucleotide bemg positioned within about, for example, within about 2 to 10, preferably about 3 to 5, nucleotide of the intron exon junction. Also prefened are anti-sense oligonucleotides which overlap the initiation codon, and those near the 5' and 3' termini of the codmg region The anti-sense ohgo may have an adenosme content of about 0, about 3%, about 5%, about 7% to about 8%, about 10%, about 12%, about 15%, and any intermediate amounts and ranges of adenosine content. In the present method, one or more or all A may be substituted by a universal or alternative base such as heteroaromatic bases which bmd to thymidme but have less than about 0.5, about 0.3, about 0.1 of the agonist or antagonist activity of adenosme at the adenosme A„ A 2a , A 2b and A 3 receptors, and heteroaromatic bases which have no activity at the adenosme A 2a receptor. The alternative base may be 3-mtropyrrole-2'-deoxynucleosιde, 5-nιtro-mdole, 2- deoxynbosyl-(5-nιtromdole), 2-deoxynbofuranosyl-(5-nιtroιndole), 2'-deoxyιnosιne, 2'-deoxynebulanne, 6H, 8H-3,4-dιhydropyπmιdo [4,5-c] oxazme-7-one or 2-amιno-6-methoxyamιnopuπne, among others. The ohgo may have a methylated cytosine ( m C) substituted for one or more, or all unmethylated C in a CpG dinucleotide (s), if the latter is (are) present m the ohgo(s). Other C 5 modifications to pyrimidines are also embodunents of the current mvention, e.g. C 5 propyne, etc. One or more, or all nucleotide linking residues of the ohgos suitable for use with the present method may be methylphosphonate, phosphotπester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methyιmιno), (MMI), methoxymethyl (MOM), methoxyethyl (MOE), methyleneoxy (methylimino) (MOMA), methoxy methyl (MOM), 2'-0-methyl, phosphoramidate, and C-5 substituted (C-5 propyne) residues and combmations thereof The ohgo used m the present method may be linked to a vector, such as a prokaryotic or eukaryotic vector, many of which are known m the art The method of the mvention prescπbes the admmistration of an amount of anti-sense ohgo effective to reduce the production or availability, or to mcrease the degradation, of the mRNA, or to reduce the amount of the polypeptide present m the lungs The admmistration is preferably done m situ, e.g directly mto the respiratory system or nasal passage, e g. by inhalation or applied to the subject's lungs, for respiratory and pulmonary targets. The anti- sense ohgo may be admmistered directly mto the bram, heart, kidney, tumor, testes, eyes, ear passage, cervix, nasal passage, scalp, oral cavity, muscle, pharynx, esophagus, intestines, rectum, synovial tissue, ovaπes, and other localized tissues by injection, by stereotactic insertion, or in vitro, among other methods In addition, the ohgo may also be admmistered mto the blood when the target is part and parcel of the circulatory and immune systems. In the latter case, wherem the disease or condition is associated with an lmmunological dysfunction, the target may be lmmunoglobuhns, antibody receptors, cytokmes, cytokine receptors, gene(s) and the corresponding mRNA(s) encodmg them, the genes and mRNA flankmg regions and mtron and exon borders, among others. Wherem the disease or condition is associated with a malignancy or cancer, the target may be selected from growth regulation associated enzyme and other protems, lmmunoglobulins and antibody receptors, gene(s) and mRNA(s) encodmg them, genes and mRNAs associated with oncogenes, and genomic and mRNA flankmg regions and exon and mtron borders. Additionally, certain genes of normal cells that are involved m the cancer process, such as angiogenesis factors, adhesion molecules and protease enzymes involved in metastases and others are also part of the mvention The method may be practiced, for example, by administering the composition m vitro, orally, lntracavitanly, intranasally, intraanally, intravagmally, intrauterally, intracranially, pulmonaπly, mtrarenally, mtranodularly, lntraarhcularly, intraofacally, lntralymphatically, transdermally, lntrabucally, intravenously, subcutaneously, intramuscularly, lntratumorously, mtraglandularly, mtraocularly, intracranial, into an organ, intravascularly, mtrathecally, by implantation, by inhalation, intradermally, mtrapulmonaπly, mto the ear, mto the heart, by slow release, by sustained release and by a pump Other examples of targets are genes and mRNAs encodmg polypeptides selected from the group consisting of transcnption factors, stimulating and activating factors, cytokmes and their receptors, mterleukms, interleukin receptors, chemokines, chemokme receptors, endogenously produced specific and non-specific enzymes, lmmunoglobulins, antibody receptors, central nervous system (CNS) and peπpheral nervous and non-nervous system receptors, CNS and peπpheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensmes, growth factors, vasoactive pephdes, peptide receptors and bmdmg protem, and genes and mRNAs coπesponding to oncogenes, G-protem coupled receptors, etc The anti-sense ohgo(s) may be produced by selection of a target from polypeptides associated with diseases and conditions afflicting lung airways, such as difficult respiratory activity and malignancies, mcreased or decreased surfactant secretion, and many others, genes and RNAs encodmg them, the genomic and mRNA flankmg regions and the gene(s) and mRNA(s) mtron and exon borders; obtaining the sequence of a mRNA(s) selected from the group consisting of mRNAs conespondmg to the target gene(s) and mRNAs encodmg the target ρolypephde(s), genomic and mRNA flanking regions and the genes and mRNAs mtron and exon borders; selecting at least one segment of the mRNA(s); synthesizmg one or more ohgo anti-sense to the selected mRNA segments), and substituting, if necessary, an alternative base(s) capable of hybπdizing to thymidme (T) but havmg reduce or no agonist capacity at the adenosme receptors (no or reduced adenosme receptor activation) for one or more A(s) to reduce the content of A present m the ohgo to up to about 15% of all nucleotides. As already indicated, suitable targets are target protems, genes and mRNAs encoding polypeptides selected from transcnption factors, stimulating and activating factors, mterleukms, interleukin receptors, chemokines, chemokme receptors, endogenously produced specific and non-specific enzymes, lmmunoglobulins, antibody receptors, central nervous system (CNS) and peπpheral nervous and non-nervous system receptors, CNS and peπpheral nervous and non-nervous system peptide transmitters and their receptors, adhesion molecules, defensmes, growth factors, vasoactive peptides and their receptors, and bmdmg proteins, and target genes and mRNAs conespondmg to oncogenes, and their flankmg regions and mtron and exon borders The encoded polypeptides may be selected from NfKB Transcnption Factor, Interleukιn-8 Receptor (IL-8 R), Interleukin 5 Receptor (IL-5 R), Interleukin 4 Receptor (IL-4 R), Interleukin 3 Receptor (IL-3 R), Interleukin- lβ (IL-lβ), Interleukin lβ Receptor (IL- lβ R), Eotaxm, Tryptase, Major Basic Protem, β2-adrenergιc Receptor Kinase, Endothelm Receptor A, Endothelm Receptor B, Preproendothelin, Bradykmm B2 Receptor, IgE High Affinity Receptor, Interleukin 1 (IL-1), Interleukin 1 Receptor (IL-1 R), Interleukin 9 (IL-9), Interleukm-9 Receptor (IL-9 R), Interleukin 11 (IL-11), Interleukin- 11 Receptor (IL- 11 R), Inducible Nitnc Oxide Synthase, Cyclooxygenase (COX), Intracellular Adhesion Molecule 1 (ICAM-1) Vascular Cellular Adhesion Molecule (VCAM), Rantes, Endothehal Leukocyte Adhesion Molecule (ELAM-1), Monocyte Activating Factor, Neutrophil Chemotactic Factor, Neutrophil Elastase, Defensin 1, 2 and 3, Muscaπmc Acetylcholine Receptors, Platelet Activating Factor, Tumor Necrosis Factor α, 5-hpoxygenase, Phosphodiesterase IV, Substance P, Substance P Receptor, Histamine Receptor, Chymase, CCR-1 CC Chemokme Receptor, CCR-2 CC Chemokme Receptor, CCR-3 CC Chemokme Receptor, CCR-4 CC Chemokme Receptor, CCR-5 CC Chemokme Receptor, Prostanoid Receptors, GATA-3 Transcnption Factor, Neutrophil Adherence Receptor, MAP Kmase, Interleukm-9 (IL-9), NFAT Transcnption Factors, STAT 4, MlP-lα, MCP-2, MCP-3, MCP-4, Cyclophillms, Phosphohpase A2, Basic Fibroblast Growth Factor, Metalloproteinase, CSBP/p38 MAP Kmase, Tryptose Receptor, PDG2, Interleukιn-3 (IL-3), Interleukin- lβ (IL-lβ), Cyclosponn A-Binding Protein, FK5-Bιndmg Protem, α4βl Selectm, Fibronectin, α4β7 Selectm, Mad CAM-1, LFA-1 (CD 11 a/CD 18), PECAM-1, LFA-1 Selectm, C3bι, PSGL-1, E-Selectin, P-Selectm, CD- 34, L-Selectin, pl50,95, Mac-1 (CDl lb/CD18), Fucosyl transferase, VLA-4, CD-18/CDl la, CDl lb/CD18, ICAM2 and ICAM3, C5a, CCR3 (Eotaxm Receptor), CCR1, CCR2, CCR4, CCR5, LTB-4, AP-1 Transcnption Factor, Protem kmase C, Cystemyl Leukotnene Receptor, Tachychinnen Receptors (tach R), IKB Kmase 1 & 2, STAT 6, c-mas and NF-Interleukιn-6 (NF-IL-6), and their flankmg regions and mtron and exon borders However, this mvention is pnmaπly intended for application to newly discovered genes not yet available in public data bases It is for this group of genes that target validation is of most importance, for without it their role in disease remains unknown Table 3 below provides a short list of targets to which the agents of the invention are effectively apphed. These are by way of example only The method is applicable to any system in which target validation would be discussed by virtue of bioactive adenosme release during ohgonucleotie degradation This is of importance because adenosme is anti-inflammatory and its release would obscure cancer target validation results Table 3: Cancer Targets Transforming Therapy Oncogenes Targets ras Thymidylate Synthetase src Thyπudylate Synthetase myc Dihydrofolate Reductase bcl-2 Thymidme Kmase Deoxycytidine Kmase Ribonucleotide Reductase Angiogenesis factors Adhesion Molecules Oncogenes Folate Pathway Enzymes DNA repair genes (One Carbon Pool) Telomerase HMG CoA Reductase Farnesyl Transferase Glucose-6-Phosphate Transferase A group of prefened targets for the validation of cancer targets are genes associated with different types of cancers, or those generally known to be associated with malignancies, whether they are regulatory or involved m the production of RNA and/or proteins. Examples are transforming oncogenes, targets which are shown, among others, in Table 3 above. Other targets which present cancer target validation agents are directed to are various enzymes, pπmaπly, although not exclusively, thymidylate synthetase, dihydrofolate reductase, thymidine kmase, deoxycytidine kmase, nbonucleotide reductase, other gene products more abundantly manufactured in cancer cells than in normal cells, and the like. The present technology is particularly useful in the validation/invalidation of cancer target genes given that traditional cancer therapies are not effective in selectively killing cancer cells while preserving normal living cells from the devastating effects of treatments such as chemotherapy, radiotherapy, and the like. That is, present cancer treatments cannot be selectively targeted to malignant cells. Any target validated by the present method will provide the ability of selectively attenuating a desired gene product and attenuating or enhancing function, and its pathway. This approach provides a significant advantage over standard cancer treatments because it permits the selection of a system, and withm the system a pathway mcludmg multiple targets, e.g. primary, secondary and possibly tertiary targets, which may not be generally expressed simultaneously m normal cells and validate them separately and jointly Thus, the present method will provide targets for therapy. Once a target is validated by the present method, a selective agent acting on the target may be admmistered to a subject to cause a selective increase in toxicity within tumor cells that, for instance, express three targets while normal cells that may expresses only one or two of the targets will be significantly less affected or even spared. The present method administers agents which are preferably designed to be anti-sense to target genes and/or mRNAs related m ongm to the species to which it is to be admmistered. When for validating targets in humans, the agents are preferably designed to be anti-sense to a human gene or RNA. The agents of the invention encompass oligonucleotides which are anti-sense to naturally occurring DNA and or RNA sequences, fragments thereof of up to a length of one (1) base less than the targeted sequence, preferably at least about 7 nucleotides long, ohgos havmg only over about 0.1%, about 1%, about 4% up to about 5%, about 10%, about 15%, about 30%, or lackmg adenosme altogether, and ohgos in which one or more of the adenosme nucleotides have been replaced with so-called universal or alternative bases, which may pair up with thymidme nucleotides but fail to substantially tngger adenosme receptor activity. Examples of human sequences and fragments, which are not limiting, of anti-sense ohgonucleotide of the mvention are the following fragments as well as shorter segments of the fragments and of the full gene or mRNA codmg sequences, exons and mtron-exon junctions encompassmg preferably 7, 10, 15, 18 to 21, 24, 27, 30, n-1 nucleotides for each sequence, where n is the sequence's total number of nucleotides. These fragments may be selected from any portion of the longer ohgo, for example, from the middle, 5'- end, 3'- end or starting at any other site of the ongmal sequence. Of particular importance are fragments of low adenosine nucleotide content, that is, those fragments containing less than or about 30%, preferably less than or about 15%, more preferably less than or about 10%, and even more preferably less than or about 5%, and most preferably those devoid of adenosme nucleotide, either by choice or by replacement with a universal or alternative base m accordance with this invention The agent of the invention includes as a most prefened group sequences and their fragments where one or more adenosmes present m the sequence have been replaced by a universal or alternative base (B), as exemplified here. Similarly, also encompassed are all shorter fragments of the B- contammg fragments designed by substitution of B(s) for adenosme(s) (A(s)) contamed m the sequences, fragments thereof or segments thereof, as described above The present method may utilize the agents by themselves or in the form of pharmaceutical compositions compπsing an amount of the anti-sense ohgonucleotide as given above effective to reduce the expression of a target protem The anti-sense ohgo must first pass through a cell membrane to bmd specifically with mRNA encodmg the protein in the cell and prevent its translation Such compositions are provided m a suitable pharmaceutically acceptable earner, e.g. sterile pyrogen-free saline solution The agent of the mvention may be formulated with a hydrophobic earner capable of passing through a cell membrane, e.g. m a hposome, with the hposomes earned m a pharmaceutically acceptable aqueous carrier, optionally and alternatively with surfactant or lipid In addition, the oligonucleotides may be coupled to an agent which inactivates mRNA, such as a nbozyme to attain a more complete inhibition of translation The pharmaceutical formulation may also compnse chimenc molecules where the anti-sense ohgos are attached to molecules which are known to be internalized by cells These ohgonucleotide conjugates utilize cellular up-take pathways to mcrease the intracellular concentrations of the ohgonucleotide Examples of molecules used in this manner are macromolecules including transfernn, asialoglycoprotein (bound to oligonucleotides via polylysine) and streptavidin, among others known in the art The present method may also utilize anti-sense compounds m a pharmaceutical formulation, e g withm a hpid particle or vesicle, such as a hposome or microcrystal. The particles may be of any suitable structure, such as unilamellar or plunlamellar. The one prefened embodiment, the anti-sense ohgonucleotide is compnsed withm the hposome Positively charged hpids such as N-[l-(2, 3 -dioleoyloxi) propyl] -N, N, N-tπmethylammoniumethylsulfate, or "DOTAP," are particularly preferred for such particles and vesicles. However, others are also suitable and may m fact be more suitable. The preparation of such hpid particles is well known See, e.g, US Patent Nos. 4,880,635 to Janoff et al, 4,906,477 to Kurono et al, 4,911,928 to Wallach, 4,917,951 to Wallach, 4,920,016 to Allen et al, 4,921,757 to Wheatley et al, the relevant sections of all of which are herein incorporated m their entireties by reference. The method of the invention provides for the admmistration of the anti-sense oligos by any means, preferably those which afford the least transport, e.g. in situ m the bram, lungs, kidneys, heart, testes, etc. The admmistration of the agent(s) to the lungs may be done by any suitable means, but preferably through the respiratory system as a respirable formulation, more preferably in the form of an aerosol compπsmg respirable particles which, in rum, compnse the agent for respiration or inhalation by the subject. The respirable particles may be in gaseous, liquid or solid form, and they may, optionally, contain other therapeutic ingredients and formulation components. The particles of the present mvention are preferably particles of respirable size, preferably of a size sufficiently small to pass, upon inhalation, through the mouth and larynx and into the bronchi and alveoli of the lungs. In general, particles rangmg from about 0.5 to 10 microns m diameter are respirable. However, other sizes may also be suitable. Particles of non-respirable size, of considerably larger diameter, which are mcluded in the respirable formulation tend to deposit m the throat and may be swallowed. Accordmgly, it is desirable to minimize the quantity of non-respirable particles m the aerosol. For nasal admmistration, a particle size m the range of 10-500 :m is prefened to ensure their retention m the nasal cavity. Aerosols of liquid particles compnsmg the agent may be produced by any suitable means, such as with as insufflator or nebulizer. See, e.g, US Patent No. 4,501,729. Suitable propellants mclude solvents such as certain chlorofluorocarbon compounds, for example, dichlorodifluoromethane, tnchlorofluoromethane, dichlorotetrafluoroethane and or mixtures thereof. Other propellants are suitable and may be preferable when better suited for particular applications. The formulation may additionally compnse one or more co-solvents, for example, ethanol, surfactants, such as oleic acid or sorbitan tπoleate, antioxidants and suitable flavoring agents The anti-sense ohgos may be admmistered to the bram by stereotoxic procedures or by injection to target isolated areas of the CNS, all methods known in the art. Alternatively, the ohgos may be administered as a formulation that will cross the blood-bram barner, as is known m the art, e. g conjugates of streptavidm and a monoclonal antibody directed to the transfernn receptor may be employed as a umversal earner for the delivery of mono-biotinylated peptides, anti-sense oligos (3'-bιotinylation of phosphodiesters or other denvatives) and peptide-ohgos to the bram. See, for example, Levy, R M et al, J. Neuroviral 3 Suppl 574-75 (1997), Wu-Pong and Gewirtz, BioPharm, pp. 32-38 (Jan 1999); Boado, R. J, et al, J. Pharm. Sci 87 (11) 1308-1315 (1998) The administration to the heart, liver and kidneys as well as other organs, may be conducted by in situ admmistration techniques such as cathetenzation, injection, and regional diffusion, all of which are known in the art. See, for example, Lewis, K.J et al , J. Drug Target, 5(4): 291 (1998), Aynn, M.A. et al , Cathet Cardiovasc Diagn, 41(3). 232-240 (1997); Luft, F.C., J. Molec. Med. 76(2): 75 (1998) These administrations are typically conducted with liquid, solid or gaseous pharmaceutical compositions of the agent, that may be prepared by combining the anti-sense ohgo with a suitable vehicle or earner, such as stenle pyrogen-free water, hpid, and/or other known pharmaceutically or veteπnanly acceptable earner Other therapeutic compounds may be mcluded as well as other formulation ingredients as is known m the art Solid particulate compositions compnsmg dry particles of, e.g. the micronized agent of the mvention may be prepared by gπndmg the dry anti-sense compound with a mortar and pestle, and then passmg the thus ground, e g. micronized composition through a screen, e.g. 400 mesh screen, to break up or separate large agglomerates of particles. A solid particulate composition compnsmg the anti-sense compound may optionally also compnse a dispersant and other known agents, which serve to facilitate the formation of a mist or aerosol. A suitable dispersant is lactose, which may be blended with the anti-sense compound m any suitable ratio, about 1 : 1 w/w. Other ratios may be utilized as well, and other therapeutic and formulation agents may also be mcluded The relevant sections of the references cited m this patent are mtended for incorporation to this text by referene, particularly of those publications and patents which facilitate the enablement and wntten descπption of the vaπous aspects of the mvention. The dosage of the anti-sense compound admmistered generally vanes with the target, the function and its amplification, and disease being investigated, the condition of the subject, the particular formulation, the route and site of admmistration, the timing of admmistration, etc. In general, it is desirable to attain intracellular concentrations of the ohgonucleotide of from 0.05 to 50 :M, or more particularly 0.2 to 5 :M. However, a dose-response curve may suitably be determined to establish a proper dose to observe a clear response. The dosage utilized may be vaπed, e.g. from about 0.001, about 0.01, about 1 mg/kg to about 50, about 100, and about 150 mg/kg are typically employed. Higher and lower doses may also be admmistered as an artisan will see suitable for specific application. These amounts may be admmistered once or over a penod of time, e.g. every 24 hrs where needed, although other regimens are also suitable. The following examples are provided to illustrate the present mvention, and should not be construed as limiting thereon. In these examples, :M means micromolar, ml means milhhters, :m means micrometers, mm means millimeters, cm means centimeters, EC means degrees Celsius, :g means micrograms, mg means milligrams, g means grams, kg means kilograms, M means molar, and hrs. means hours. EXAMPLES Example 1: Design and Synthesis of Anti-sense Oligonucleotides The design of anti-sense oligonucleotides agamst target receptors may require the solution of the complex secondary strucmre of the target receptor mRNA. After generating this strucmre, anti-sense nucleotide are designed which target regions of mRNA which might be construed to confer functional activity or stability to the mRNA and which optimally may overlap the initiation codon. Other target sites are readily usable. As a demonstration of specificity of the anti-sense effect, other oligonucleotides not totally complementary to the target mRNA, but contammg identical nucleotide compositions on a w/w basis, are included as controls m anti-sense experiments For example, the mRNA secondary strucmre of the adenosme A, receptor was analyzed and used as descπbed above, to design a phosphorothioate anti-sense ohgonucleotide. The anti-sense ohgonucleotide which was synthesized was designated HAdA,AS and had the following sequence: 5' -GAT GGA GGG CGG CAT GGC GGG-3' (SEQ ID NO:l) As a control, a mismatched phosphorothioate anti-sense nucleotide designated HAdAlMMl was synthesized with the following sequence- 5' -GTA GCA GGC GGG GAT GGG GGC-3' (SEQ ID NO:2) Each ohgonucleotide had identical base content and general sequence structure. Homology searches in GENBANK (release 85.0) and EMBL (release 40.0) indicated that the anti-sense ohgonucleotide was specific for the human and rabbit adenosme A, receptor genes, and that the mismatched control was not a candidate for hybπdization with any known gene sequence. The secondary strucmre of the adenosme A 3 receptor mRNA was similarly analyzed and used as described above to design two phosphorothioate anti-sense oligonucleotides The first anti-sense ohgonucleotide (HAdA3ASl) synthesized had the following sequence- 5' -GTT GTT GGG CAT CTT Gees' (SEQ ID NO:3) As a control, a mismatched phosphorothioate anti-sense ohgonucleotide (HAdA3MMl) was synthesized, havmg the following sequence. 5' -GTA CTT GCG GAT CTA GGC-3' (SEQ ID NO:4). A second phosphorothioate anti-sense ohgonucleotide (HAdA3AS2) was also designed and synthesized, havmg the following sequence: 5' -GTG GGC CTA GCT CTC GCC-3' (SEQ ID NO:5) Its control ohgonucleotide (HAdA3MM2) had the sequence: 5' -GTC GGG GTA CCT GTC GGC-3' (SEQ ID NO:6). Phosphorothioate oligonucleotides were synthesized on an Applied Biosystems Model 396 Ohgonucleotide Synthesizer, and puπfied usmg NENSORB chromatography (DuPont, MD). Example 2: In Vivo Testing of Adenosine Aj Receptor Anti-sense Oligos The anti-sense ohgonucleotide agamst the human A, receptor (SEQ ID NO:l) descπbed above, was tested for efficacy m an in vitro model utilizmg lung adenocarcinoma cells HTB-54. HTB-54 lung adenocarcinoma cells were demonstrated to express the A, adenosme receptor usmg standard northern blotting procedures and receptor probes designed and synthesized m the laboratory HTB-54 human lung adenocarcinoma cells (106/100 mm tissue culture dish) were exposed to 5.0 :M HAdAlAS or HAdAlMMl for 24 hours, with a fresh change of media and oligonucleotides after 12 hours of incubation Following 24 hour exposure to the oligonucleotides, cells were harvested and their RNA extracted by standard procedures. A 21- mer probe conespondmg to the region of mRNA targeted by the anti-sense (and therefore havmg the same sequence as the anti-sense, but not phosphorothioated) was synthesized and used to probe northern blots of RNA prepared from HAdAlAS-treated, HAdAlMMl -treated and non-treated HTB-54 cells. These blots showed clearly that HAdAlAS but not HAdAlMMl effectively reduced human adenosme receptor mRNA by >50%. This result showed that HAdAlAS is a good candidate for an anti-asthma drug since it depletes intracellular mRNA for the adenosine A, receptor, which is mvolved m asthma. Example 3: In Vivo Efficacy of Adenosine Aj Receptor Anti-sense Oligos A fortuitous homology between the rabbit and human DNA sequences withm the adenosme A, gene overlappmg the initiation codon permitted the use of the phosphorothioate anti-sense oligonucleotides initially designed for use agamst the human adenosme A, receptor in a rabbit model. Neonatal New Zealand white Pasteurella-free rabbits were immunized mtrapentoneally within 24 hours of birth with 312 antigen units/ml house dustmite (D. faπnae) extract (Berkeley Biologicals, Berkeley, CA), mixed with 10% kaolm. Immunizations were repeated weekly for the first month and then biweekly for the next 2 months. At 3-4 months of age, eight sensitized rabbits were anesthetized and relaxed with a mixture of ketamine hydrochloπde (44 mg/kg) and acepromazine maleate (0.4 mg/kg) admmistered intramuscularly The rabbits were then laid supine m a comfortable position on a small molded, padded animal board and intubated with a 4 0-mm mtratracheal mbe (Mallmkrodt, Inc , Glens Falls, NY). A polyethylene catheter of external diameter 2.4 mm with an attached latex balloon was passed into the esophagus and mamtamed at the same distance (approximately 16 cm) from the mouth throughout the expeπments. The mtratracheal tube was attached to a heated Fleisch pneumotachograph (size 00; DOM Medical, Richmond, VA), and flow was measured usmg a Vahdyne differential pressure transducer (Model DP-45161927, Vahdyne Engmeeπng Corp, Northndge, CA) driven by a Gould earner amplifier (Model 11-4113; Gould Electronic, Cleveland, OH). The esophageal balloon was attached to one side of the differential pressure transducer, and the outflow of the mtratracheal tube was connected to the opposite side of the pressure transducer to allow recordmg of transpulmonary pressure. Flow was mtegrated to give a continuous tidal volume, and measurements of total lung resistance (RL) and dynamic compliance (Cdyn) were calculated at lsovolumetnc and flow zero points, respectively, usmg an automated respiratory analyzer (Model 6, Buxco, Sharon, CT) Animals were randomized and on Day 1 pretreatment values for PC50 were obtained for aerosolized adenosme Anti-sense (HAdAlAS) or mismatched control (HAdAlMM) oligonucleotides were dissolved in stenle physiological salme at a concentration of 5000 -g (5 mg) per 1 0 ml. Animals were subsequently admmistered the aerosolized anti- sense or mismatch ohgonucleotide via the mtratracheal mbe (approximately 5000 :g in a volume of 1.0 ml), twice daily for two days. Aerosols of either salme, adenosme, or anti-sense or mismatch oligonucleotides were generated by an ultrasonic nebulizer (DeVilbiss, Somerset, PA), producmg aerosol droplets 80% of which were smaller than 5 :m m diameter In the first arm of the experiment, four randomly selected allergic rabbits were admmistered anti-sense ohgonucleotide and four the mismatched control ohgonucleotide. On the morning of the third day, PC50 values (the concentration of aerosolized adenosme m mg/ml required to reduce the dynamic compliance of the bronchial airway 50% from the baselme value) were obtamed and compared to PC50 values obtained for these animals pnor to exposure to ohgonucleotide. Following a 1 week interval, animals were crossed over, with those previously admmistered mismatch control ohgonucleotide now admmistered anti-sense ohgonucleotide, and those previously treated with anti-sense ohgonucleotide now admmistered mismatch control ohgonucleotide. Treatment methods and measurements were identical to those employed in the first arm of the experiment It should be noted that in six of the eight animals treated with anti-sense ohgonucleotide, adenosme-mediated bronchoconstnction could not be obtamed up to the limit of solubility of adenosine, 20 mg/ml. For the purpose of calculation, PC50 values for these animals were set at 20 mg/ml. The values given therefore represent a minimum figure for anti-sense effectiveness. Actual effectiveness was higher. The results of this expenment are illustrated m Table 4 below. Table 4: Effect of Adenosine Ai Receptor Anti-sense Oligo upon PC50 Values in Asthmatic Rabbits The results are presented as the mean (n=8) ± SEM The significance was determined by repeated-measures analysis of variance (ANOVA), and Tukey's protected test **Sιgnιficantly different from all other groups, p<0 01 In both arms of the experiment, animals receiving the anti-sense ohgonucleotide showed an order of magnitude mcrease m the dose of aerosolized adenosme required to reduce dynamic compliance of the lung by 50%. No effect of the mismatched control ohgonucleotide upon PC50 values was observed. No toxicity was observed m any animal receiving either anti-sense or control inhaled ohgonucleotide These results show clearly that the lung has exceptional potential as a target for anti-sense ohgonucleotide-based therapeutic intervention in lung disease They further show, m a model system which closely resembles human asthma, that down regulation of the adenosine A, receptor largely eliminates adenosme-mediated bronchoconstnction in asthmatic airways Bronchial hypenesponsiveness m the allergic rabbit model of human asthma is an excellent endpomt for anti-sense intervention smce the tissues mvolved m this response he near to the pomt of contact with aerosolized oligonucleotides, and the model closely simulates an important human disease Example 4: Specificity of A] -adenosine Receptor Anti-sense Oligonucleotide At the conclusion of the cross-over expenment of Example 3 above, airway smooth muscle from all rabbits was quantitatively analyzed for adenosme A, receptor number As a control for the specificity of the anti-sense ohgonucleotide, adenosine A, receptors, which should not have been affected, were also quantified. Airway smooth muscle tissue was dissected from each rabbit and a membrane fraction prepared accordmg to the method of Klemstem et al. (Klemstein, J. and Glossmann, H, Naunyn-Schmiedeberg's Arch. Pharmacol 305: 191-200 (1978)), the relevant portion of which is hereby incorporated in its entirety by reference, with slight modifications. Crude plasma membrane preparations were stored at 70EC until the time of assay. Protem content was determined by the method of Bradford (M. Bradford, Anal. Biochem. 72, 240-254 (1976), the relevant portion of which is hereby incorporated m its entirety by reference). Frozen plasma membranes were thawed at room temperature and were incubated with 0.2 U/ml adenosme deammase for 30 mmutes at 37EC to remove endogenous adenosine The bmdmg of [ 3 H] DPCPX (A, receptor-specific) or [ 3 H] CGS- 21680 (A, receptor-specific) was measured as previously descnbed by Ah et al. (Ah, S et al, J. Pharmacol. Exp. Ther. 268, Am. J. Physiol 266, L271-277 (1994), the relevant portion of which is hereby incorporated m its entirety by reference) The animals treated with adenosme A, anti-sense ohgonucleotide in the cross-over expenment had a nearly 75% decrease m A, receptor number compared to controls, as assayed by specific bmdmg of the A, -specific antagonist DPCPX There was no change m adenosme A 2 receptor number, as assayed by specific bmdmg of the A, receptor-specific agomst 2- [p- (2-carboxyethyl)-ρhenethylamιno] -5' - (N-ethylcarboxamido) adenosine (CGS-21680) This is illustrated m Table 5 below The results below illustrate the effectiveness of anti-sense oligonucleotides m treating airway disease. Smce the anti-sense oligos descπbed above eliminate the receptor systems responsible for adenosme-mediated bronchoconstnction, it may be less imperative to elimmate adenosme from them However, it would be preferable to elimmate adenosme from even these oligonucleotides to reduce the dose needed to attain a similar effect Descnbed above are other anti-sense oligonucleotides targetmg mRNA of protems mvolved m inflammation Adenosme has been eliminated from their nucleotide content to prevent its liberation durmg degradation Table 5; Specificity of Action of Adenosine Aj Receptor Anti-sense Oligonucleotide Example 5: Anti-sense Oligos Directed to Other Target Nucleic Acids This work was conducted to demonstrate that the present mvention is broadly applicable to anti-sense oligonucleotides ("oligos") specific to nucleic acid targets broadly The following experimental studies were conducted to show that the method of the invention is broadly suitable for use with anti-sense ohgos designed as taught by this application and targeted to any and all adenosme receptor mRNAs For this purpose, vanous anti-sense ohgos were prepared to adenosme receptor mRNAs exemplified by the adenosme A,, A 2b and A 3 receptor mRNAs Anti-sense Ohgo I was disclosed above (SEQ ID NO: 1) Five additional anti-sense phosphorothioate ohgos were designed and synthesized as mdicated above 1- Ohgo II (SEQ ID NO: 7) also targeted to the adenosme A, receptor, but to a different region than Ohgo I 2- Ohgo V (SEQ ID NO: 10) targeted to the adenosme A 2b receptor 3- Ohgos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) targeted to different regions of the adenosme A 3 receptor 4- Ohgo I-PD (SEQ ID NO: ll)(a phosphodiester ohgo of the same sequence as Ohgo I) These anti-sense ohgos were designed for therapy on a selected species as descπbed above and are generally specific for that species, unless the segment of the target mRNA of other species happens to contam a similar sequences All anti-sense ohgos were prepared as descnbed below, and tested m vivo m a rabbit model for bronchoconstnction, inflammation and allergy, which have breathing difficulties and impeded lung airways, as is the case m ailments such as asthma, as descnbed in the above-identified application Example 6: Design & Sequences of Other Anti-sense Oligos Six ohgos and their effects in a rabbit model were studied and the results of these studies are reported and discussed below Five of these oligos were selected for this study to complement the data on Ohgo I (SEQ ID NO: 1) provided m Examples 1 to 4 above. This ohgo is anti-sense to one region of the adenosme A, receptor mRNA. The oligos tested are identified as anti-sense Oligos I (SEQ ID NO: 1) and II (SEQ ID NO: 7) targeted to a different region of the adenosme A, receptor mRNA, Ohgo V (SEQ ID NO: 8) targeted to the adenosme A 2b receptor mRNA, and anti-sense Ohgos III and IV (SEQ ID NOS: 9 and 10) targeted to two different regions of the adenosine A 3 receptor mRNA. The sixth ohgo (Ohgo I-PD) is a phosphodiester version of Ohgo I (SEQ ID NO: 1) The design and synthesis of these anti-sense ohgos was performed m accordance with Example 1 above (I) Anti-sense Oligo I The anti-sense ohgonucleotide I refeπed to m Examples 1 to 4 above is targeted to the human A, adenosme receptor mRNA (EPI 2010) Anti-sense ohgo I is 21 nucleotide long, overlaps the mitiation codon, and has the following sequence: 5'- GAT GGA GGG CGG CAT GGC GGG -3' (:SEQ ID NO: l). The ohgo I was previously shown to abrogate the adenosme-induced bronchoconstnction m allergic rabbits, and to reduce allergen-induced airway obstruction and bronchial hypenesponsiveness (BHR), as discussed above and shown by Nyce, J. W. & Metzger, W J , Nature, 385:721 (1977), the relevant portions of which reference are incorporated in their entireties herem by reference (II) Anti-sense Oligo II A phosphorothioate anti-sense ohgo (SEQ ID NO: 7) was designed m accordance with the mvention to target the rabbit adenosme A, receptor mRNA region +936 to +956 relative to the initiation codon (start site) The anti-sense ohgo II is 21 nucleotide long, and has the following sequence-5'-CTC GTC GCC GTC GCC GGC GGG-3' (SEQ ID NO: 7) (III) Anti-sense Oligo III A phosphorothioate anti-sense ohgo other than that provided m Example 1 above (SEQ ID NO: 8) was designed in accordance with the invention to target the anti-sense A 3 receptor mRNA region +3 to + 22 relative to the mitiation codon start site. The anti-sense ohgo III is 20 nucleotide long, and has the following sequence- 5'-GGG TGG TGC TAT TGT CGG GC-3' (SEQ ID NO: 8). (IV) Anti-sense Oligo IV Yet another phosphorothioate anti-sense ohgo (SEQ ID NO: 9) was designed m accordance with the mvention to target the adenosme A 3 receptor mRNA region + 386 to + 401 relative to the mitiation codon (start site). The anti-sense ohgo IV is 15 nucleotide long, and has the following sequence- 5'-GGC CCA GGG CCA GCC-3' (SEQ ID NO: 9). (V) Anti-sense Oligo V A phosphorothioate anti-sense ohgo (SEQ ID NO: 10) was designed m accordance with the invention to target the adenosme A 2b receptor mRNA region -21 to -1 relative to the mitiation codon (start site). The anti-sense ohgonucleotide V is 21 nucleotide long, and has the following sequence: 5'-GGC CGG GCC AGC CGG GCC CGG-3' (SEQ ID NO: 10). (VI) Aj Mismatch Oligos Two different mismatched oligonucleotides having the following sequences were used as controls for anti-sense ohgo I (SEQ ID NO: 1) descπbed in Example 5 above: A, MM2 5'-GTA GGT GGC GGG CAA GGC GGG-3' (SEQ ID NO: 12), and A, MM3 5'-GAT GGA GGC GGG CAT GGC GGG-3' (SEQ ID NO: 13). Anti-sense ohgo I and the two mismatch anti-sense ohgos had identical base content and general sequence structure. Homology searches m GENBANK (release 85.0) and EMBL (release 40.0) indicated that the anti-sense ohgo I was specific, not only for the human, but also for the rabbit, adenosme A, receptor genes, and that the mismatched controls were not candidates for hybridization with any known human or animal gene sequence (Vπ) Anti-sense Oligo Aχ-PD (Oligo VI) A phosphodiester anti-sense ohgo (Oligo VI; SEQ ID NO: 11) havmg the same nucleotide sequence as Ohgo I was designed as disclosed m the above-identified application. Anti-sense ohgo I-PD is 21 nucleotide long, overlaps the initiation codon, and has the following sequence- 5'- GAT GGA GGG CGG CAT GGC GGG -3' (SEQ ID NO: 11). (VIII) Controls Each rabbit was administered 5.0 ml aerosolized stenle salme following the same schedule as for the anti-sense oligos in (II), (III), and (IV) above The above are given as examples of G-protem coupled receptors. However, the method of reducmg adenosme content is generally applicable to any gene and any target validation system in which the release of bioactive adenosme could obscure experimental data for actualizing adenosine receptors Example 7: Synthesis of Anti-sense Oligos Phosphorothioate anti-sense ohgos havmg the sequences descnbed m (a) above, were synthesized on an Applied Biosystems Model 396 Ohgonucleotide Synthesizer, and puπfied using NENSORB chromatography (DuPont, DE) TETD (tetraethylthiuram disulfide) was used as the sulfuπzing agent during the synthesis. Anti-sense ohgonucleotide II (SEQ ID NO: 7), anti-sense ohgonucleotide III (SEQ ID NO: 8) and anti-sense ohgonucleotide IV (SEQ ID NO: 9) were each synthesized and punfied in this manner. Example 8: Preparation of Allergic Rabbits Neonatal New Zealand white Pasturella-free rabbits were immunized mtrapeπtoneally withm 24 hours of birth with 0 5 ml of 312 antigen units/ml house dust mite (D farinae) extract (Berkeley Biologicals, Berkeley, CA) mixed with 10% kaolin as previously descnbed (Metzger, W J , m Late Phase Allergic Reactions, Dorsch, W , Ed, CRC Handbook, pp. 347-362, CRC Press, Boca Raton (1990); Ah, S, Metzger, W. J. and Mustafa, S. J, Am J Resp Cπt. Care Med. 149- 908 (1994)), the relevant portions of which are incorporated in their entireties here by reference. Immunizations were repeated weekly for the first month and then biweekly until the age of 4 months. These rabbits preferentially produce allergen-specific IgE antibody, typically respond to aeroallergen challenge with both an early and late-phase asthmatic response, and show bronchial hyper responsiveness (BHR). Monthly mtrapentoneal admmistration of allergen (312 units dust mite allergen, as above) continues to stimulate and maintain allergen-specific IgE antibody and BHR. At 4 months of age, sensitized rabbits were prepared for aerosol admmistration as descπbed by Ah et al. (Ah, S, Metzger, W. J. and Mustafa, S J , Am. J. Resp. Cnt. Care Med. 149 (1994)), the relevant section bemg incorporated m its entirety here by reference. DOSE-RESPONSE STUDIES Example 9: Experimental Setup Aerosols of either adenosine (0-20 mg/ml), or anti-sense or one of two mismatch oligonucleotides (5 mg/ml) were separately prepared with an ultrasonic nebulizer (Model 646, DeVilbiss, Somerset, PA), which produced aerosol droplets, 80% of which were smaller than 5:m in diameter. Equal volumes of the aerosols were admmistered directly to the lungs via an mtratracheal mbe. The animals were randomized, and admmistered aerosolized adenosme. Day 1 pre-treatment values for sensitivity to adenosme were calculated as the dose of adenosme causmg a 50% loss of compliance (PC 50 Adenosme). The animals were then admmistered either the aerosolized anti-sense or one of the mismatch anti-sense ohgos via the mtratracheal mbe (5 mg/1.0 ml), for 2 mmutes, twice daily for 2 days (total dose, 20 mg). Post-treatment PC 50 values were recorded (post-treatment challenge) on the morning of the third day. The results of these studies are provided m Example 21 below. Example 10: Cross-over Experiments For some expenments utilizing anti-sense ohgo I (SEQ ID NO: 1) and a conespondmg mismatch control oligonucleotide A1MM2, following a 2 week interval, the animals were crossed over, with those previously admmistered the mismatch control A,MM2, now receivmg the anti-sense ohgo I, and those previously treated with the anti-sense ohgo I, now receivmg the mismatch control A,MM2 ohgo. The number of animals per group was as follows For mismatch A,MM2 (Control 1), n=7, smce one animal was lost m the second control arm of the expenment due to technical difficulties, for mismatch A,MM3 n=4 (Control 2) and for A,AS anti-sense ohgo I, n=8. The A,MM3 ohgo-treated animals were analyzed separately and were not part of the cross-over experiment The treatment methods and measurements employed following the crossover were identical to those employed in the first arm of the experiment In 6 of the 8 animals treated with the anti-sense ohgo I (SEQ ID NO: 1), no PC J0 value could be obtained for adenosine doses of up to 20 mg/ml, which is the limit of solubility of adenosme. Accordmgly, the PC 50 values for these animals were assumed to be 20 mg/ml for calculation purposes The values given, therefore, represent a minimum figure for the effectiveness of the anti-sense oligonucleotides of the mvention. Other groups of allergic rabbits (n=4 for each group) were admmistered 0.5 or 0.05 mg doses of the anti-sense ohgo I (SEQ ID NO: 1), or the A,MM2 ohgo m the manner and accordmg to the schedule descπbed above (the total doses being 2.0 or 0.2 mg). The results of these studies are provided in Example 22 below. Example 11: Anti-sense Oligo Formulation Each one of anti-sense ohgos were separately solubilized m an aqueous solution and admmistered as descπbed for anti-sense ohgo I (SEQ ID NO: 1) in (e) above, in four 5 mg ahquots (20 mg total dose) by means of a nebulizer via endorracheal mbe, as descnbed above. The results obtamed for anti-sense ohgo I and its mismatch controls confirmed that the mismatch controls are equivalent to salme, as descnbed m Example 19 below and m Table 1 of Nyce & Metzger, Nature 385, 721-725 (1997), the contents of which are mcorporated herem by reference. Because of this finding, salme was used as a control for pulmonary function studies employmg anti-sense oligos II, III and IV (SEQ ID NOS: 7, 8 and 9). Example 12: Specificity of Oligo I for Adenosine Aj Receptor (Receptor Binding Studies) Tissue from airway smooth muscle was dissected to primary, secondary and tertiary bronchi from rabbits which had been admmistered 20 mg ohgo I (SEQ ID NO: 1) m 4 divided doses over a penod of 48 hours as descnbed above. A membrane fraction was prepared accordmg to the method of Ah et al. (Ah, S, et al. Am. J. Resp. Cπt. Care Med 149: 908 (1994), the relevant section relating to the preparation of the membrane fraction is mcorporated in its entirety hereby by reference). The protem content was determined by the method of Bradford and plasma membranes were mcubated with 0.2 U/ml adenosine deammase for 30 mmutes at 37EC to remove endogenous adenosme. See, Bradford, M. M. Anal. Biochem. 72, 240-254 (1976), the relevant portion of which is hereby mcorporated m its entirety by reference. The bmdmg of [ 3 H]DPCPX, [ 3 H]NPC17731, or [ H]CGS-21680 was measured as descnbed by Jarvis et al. See, Jarvis, M.F, et al, Pharmacol. Exptl. Ther. 251, 888-893 (1989), the relevant portion of which is fully mcorporated herem by reference Similar amounts of an ohgo targeted to the bradykinin receptor 5'- GGTGATGTTGAGCATTTCGGC-3' (SEQ ID NO: 14) were admmistered to another group of annuals. The results of this study are shown m Table 6 and discussed m Example 20 below. Example 13: Pulmonary Function Measurements (Compliance CDY ant * Resistance) At 4 months of age, the immunized animals were anesthetized and relaxed with 1.5 ml of a mixture of ketamine HC1 (35 mg/kg) and acepromazme maleate (1.5 mg/kg) admmistered intramuscularly. After induction of anesthesia, allergic rabbits were comfortably positioned supme on a soft molded animal board. Salve was applied to the eyes to prevent drying, and they were closed The animals were then mtubated with a 4.0 mm intermediate high-low cuffed Murphy 1 endotracheal tube (Mallmckrodt, Glen Falls, NY), as previously descπbed by Zavala and Rhodes. See, Zavala and Rhodes, Proc Soc Exp Biol Med. 144: 509- 512 (1973), the relevant portion of which is mcorporated herem by reference in its entirety. A polyethylene catheter of OD 2 4 mm (Becton Dickmson, Clay Adams, Parsippany NJ) with an attached thin-walled latex balloon was passed mto the esophagus and mamtamed at the same distance (approximately 16 cm) from the mouth throughout the expenment. The endotracheal tube was attached to a heated Fleisch pneumotach (size 00; DEM Medical, Richmond, VA), and the flow (v) measured usmg a Vahdyne differential pressure transducer (Model DP-45-16-1927, Vahdyne Engmeenng, Northπdge, CA), dnven by a Gould earner amplifier (Model 11-4113, Gould Electronics, Cleveland, OH) An esophageal balloon was attached to one side of the Vahdyne differential pressure transducer, and the other side was attached to the outflow of the endotracheal mbe to obtam transpulmonary pressure (P,,,). The flow was mtegrated to yield a contmuous tidal volume, and the measurements of total lung resistance (R_) and dynamic compliance (C dyrι ) were made at isovolumetπc and zero flow points The flow, volume and pressure were recorded on an eight channel Gould 2000 W high-frequency recorder and C^.. was calculated usmg the total volume and the difference m P,,, at zero flow, and . R, was calculated as the ratio of Ptp and V at midtidal lung volumes. These calculations were made automatically with the Buxco automated pulmonary mechanics respiratory analyzer (Model 6, Buxco Electronics, Sharon, CT), as previously descπbed by Giles et al See, Giles et al. Arch. Int Pharmacodyn. Ther. 194 213-232 (1971), the relevant portion of which descnbmg these calculations is mcorporated m toto hereby by reference. The results obtamed upon admmistration of ohgo II on allergic rabbits are shown and discussed in Example 26 below Example 14: Measurement of Bronchial Hyperresponsiveness (BHR) Each allergic rabbit was admmistered histamine by aerosol to determine their baselme hypenesponsiveness. Aerosols of either salme or histamine were generated usmg a DeVilbiss nebulizer (DeVilbiss, Somerset, PA) for 30 seconds and then for 2 mmutes at each dose employed. The ultrasonic nebulizer produced aerosol droplets of which 80% were <5 micron m diameter. The histamine aerosol was admmistered m mcreasmg concentrations (0.156 to 80 mg/ml) and measurements of pulmonary function were made after each dose. The B4R was then determined by calculating the concentration of histamine (mg/ml) required to reduce the C^ 50% from baselme (PC 50 H . s u m J- Example 15: Cardiovascular Effect of Anti-sense Oligo I The measurement of cardiac output and other cardiovascular parameters usmg CardiomaxJ utilizes the principal of thermal dilution in which the change in temperature of the blood exiting the heart after a venous injection of a known volume of cool salme is monitored. A smgle rapid mjechon of cool salme was made mto the right atrium via cannulahon of the nght jugular vem, and the conespondmg changes m temperature of the mixed lnjectate and blood m the aortic arch were recorded via cannulahon of the carotid artery by a temperature-sensing mimprobe. Twelve hours after the allergic rabbits had been treated with aerosols of ohgo I (EPI 2010; SEQ ID NO: 1) as descπbed m (d) above, the animals were anesthetized with 0.3 ml/kg of 80% Ketamine and 20% Xylazme. This time point coincides with previous data showmg efficacy for SEQ ID NO: 1, as is clearly shown by Nyce & Metzger, (1997), supra, the pertinent disclosure bemg mcorporated in its entirety here by reference. A thermocouple was then inserted into the left carotid artery of each rabbit, and was then advanced 6.5 cm and secured with a silk ligature. The right jugular vem was then cannulated and a length of polyethylene tubing was inserted and secured. A thermodilution curve was then established on a CardiomaxJ II (Columbus Instruments, Ohio) by injecting steπle salme at 20EC to determine the coπectness of positioning of the thermocouple probe. After establishing the coπectness of the position of the thermocouple, the femoral artery and vem were isolated. The femoral vein was used as a portal for drug injections, and the femoral artery for blood pressure and heart rate measurements. Once constant baseline cardiovascular parameters were established, CardiomaxJ measurements of blood pressure, heart rate, cardiac output, total penpheral resistance, and cardiac contractility were made. Example 16: Duration of Action of Oligo I (SEQ ID NO: 1) Eight allergic rabbits received initially mcreasmg log doses of adenosine by means of a nebulizer via an lntra-tracheal mbe as descπbed in (f) above, beginning with 0.156 mg/ml until compliance was reduced by 50% (PCj o Adenosme ) t0 establish a baselme. Six of the rabbits then received four 5 mg aerosolized doses of (SEQ ID NO: 1) as descnbed above Two rabbits received equivalent amounts of salme vehicle as controls. Beginning 18 hours after the last treatment, the PC 50 Adαιos , nc values were tested again. After this pomt, the measurements were continued for all animals each day, for up to 10 days. The results of this study are discussed m Example 25 below Example 17: Reduction of Adenosine A2b Receptor Number by Anti-sense Oligo V Sprague Dawley rats were admmistered 2.0 mg respirable anti-sense ohgo V (SEQ ID NO: 10) three times over two days usmg an inhalation chamber as descnbed above Twelve hours after the last admmistration, lung parenchymal tissue was dissected and assayed for adenosme A 2b receptor bmdmg usmg [311]-NECA as descπbed by Nyce & Metzger (1997), supra. Controls were conducted by admmistration of equal volumes of salme. The results are significant at p<0.05 usmg Student's paired t test, and are discussed in Example 28 below. Example 18: Comparison of Oligo I & Corresponding Phosphodiester Oligo VI (SEQ ID NO: 11) Ohgo I (SEQ ID NO: 1) countered the effects of adenosine and eliminated sensitivity to it for adenosme amounts up to 20 mg adenosιne/5.0 ml (the limit of solubility of adenosme) Ohgo VI (SEQ ID NO: 11), the phosphodiester version of the oligonucleotide sequence, was completely ineffective when tested m the same manner. Both compounds have identical sequence, differmg only in the presence of phosphorothioate residues in Ohgo I (SEQ ID NO: 1), and were delivered as an aerosol as descnbed above and in Nyce & Metzger (1997), supra. Significantly different at p<0 001, Student's paired t test. The results are discussed m Example 29 below RESULTS OBTAINED FOR ANTI-SENSE OLIGO I - (SEQ ID NO: 1) Example 19: Results of Prior Work The nucleotide sequence and other data for anti-sense ohgo I (SEQ ID NO: 1), which is specific for the adenosme A, receptor, were provided above. The experimental data showing the effectiveness of ohgo I m down regulating the receptor number and activity were also provided above. Further information on the charactenstics and activities of anti-sense ohgo I is provided in Nyce, J. W. and Metzger, W J, Nature 385:721 (1997), the relevant parts of which relating to the following results are incorporated m their entireties herem by reference. The Nyce & Metzger (1997) publication provided data showing that the anti-sense ohgo I (SEQ ID NO: 1): (1) The anti-sense ohgo I reduces the number of adenosme A, receptors m the bronchial smooth muscle of allergic rabbits m a dose-dependent manner as may be seen m Table 6 below. (2) Anti-sense Ohgo I attenuates adenosme-induced bronchoconstnction and allergen- induced bronchoconstnction (3) The Ohgo I attenuates bronchial hypenesponsiveness as measured by PC 50 histamine, a standard measurement to assess bronchial hypenesponsiveness This result clearly demonstrates anti- inflammatory activity of the anti-sense ohgo I as is shown in Tables 4, 5 and 6 (4) As expected, because it was designed to target it, the anti-sense ohgo I is totally specific for the adenosine A, receptor, and has no effect at all at any dose on either the very closely related adenosme A, receptor or the related bradykmm B, receptor This is seen in Table 6 below. (5) In contradistinction to the above effects of the Ohgo I, the mismatch control molecules MM2 and MM3 (SEQ ID NO: 12 and SEQ ID NO: 13) which have identical base composition and molecular weight but differed from the anti-sense ohgo I (SEQ ID NO: 1) by 6 and 2 mismatches, respectively. These mismatches, which are the minimum possible while still retaining identical base composition, produced absolutely no effect upon any of the targeted receptors (A,, A 2 or B 2 ) These results, along with a complete lack of pnor art on the use of anti-sense oligonucleotides, such as ohgo I, targeted to the adenosme A, receptor, are unexpected results. The showmgs presented m this patent clearly enable and demonstrate the effectiveness, for its intended use, of the validation method employmg agents and targeted to genes or mRNA associated with a function or end pomt associated with pulmonary functions, such as airway blockage, bronchoconstnction, pulmonary inflammation and allergy(ιes), and the like. Example 20: Oligo I Significantly Reduces Response to Adenosine Challenge The receptor bmdmg experiment is descπbed m Example 12 above, and the results shown m Table 6 below which shows the bmdmg charactenstics of the adenosme A, -selective ligand [ 3 H]DPCPX and the bradykmm B 2 -selective ligand [ 3 H]NPC 17731 in membranes isolated from airway smooth muscle of A, adenosme receptor and B 2 bradykmm receptor anti-sense- and mismatch-treated allergic rabbits. Table 6: Binding Characteristics of Three Anti-Sense Oligos Treatment' A, receptor B 2 receptor " K3 " ~E_ " KΗ " ^ Adenosine A, Receptor 20 mg 0.36+0.02 nM 19± 1.52 fmoles* 0.39±0.031 nM 14.8±0.99fmoles 2 mg 0.38±0.030 nM 32±2.56 fmoles* 0.41±0.028 nM 15.5± 1.08 fmoles 0.2 mg 0.37±0.030 nM 49±3.43 fmoles 0.34±0.024 nM 15.0±1.06 fmoles A,MM1 (Control) 20 mg 0.34±0.027 nM 52.0±3.64 fmoles 0.35±0.024 nM 14.0± 1.0 fmoles 2 mg 0.37±0.033 nM 51.8±3.88 fmoles 0.38±0.028 nM 14.6±1.02 fmoles B 2 A (Bradykinin Receptor) 20 mg 0.36±0.028 nM 45.0±3.15 fmoles 0.38±0.027 nM 8.7±0.62 fmoles* 2 mg 0.39±0.035 nM 44.3±2.90 fmoles 0.34±0.024 nM 11.9±0.76 fmoles** 0.2 mg 0.40±0.028 nM 47.0±3.76 fmoles 0.35±0.028 nM 15.1 ±1.05 fmoles B j MM (Control) 20 mg 0.39±0.031 nM 42.0±2.94 fmoles 0.41±0.029 nM 14.0±0.98 fmoles 2 mg 0.41±0.035 nM 40.0±3.20 fmoles 0.37±0.030 nM 14.8±0.99 fmoles 0.2 mg 0.37±0.029 nM 43.0±3.14 fmoles 0.36±0.025 nM 15. l± 1.35 fmoles Salme Control 0.37±0.041 46.0±5.21 0.39±0.047 nM 14.2± 1.35 fmoles 1 Refers to total oligo administered in four equivalently divided doses over a 48 hour period. Treatments and analyses were performed as descπbed in methods Significance was determined by repeated-measures analysis of vaπance (ANOVA), and Tukey's protected t test, n = 4-6 for all groups. * Significantly different from mismatch control- and saiine-treated groups, p<0.001 ; "Significantly different from mismatch control- and saline-treated groups, p<0.05. Example 21: Dose-response Effect of Oligo I Anti-sense ohgo I (SEQ ID NO: 1) was found to reduce the effect of adenosine admmistration to the animal m a dose-dependent manner over the dose range tested as shown m Table 7 below Table 7: Dose-Response Effect to Anti-sense Oligo I Total Dose "^50 Adenosine (mg) (mg Adenosine) Anti-sense Oligo I 0.2 8.32"7.2 2.0 14.0"7.2 20 19.5"0.34 A,MM2 oligo (control) 0.2 2.51 ±0.46 2.0 3.13± 0.71 20 3.25± 0.34 The above results were studied with the Student's paired t test and found to be statistically different, p=0 05 The oligo I (SEQ ID NO: 1), an anti-adenosine A, receptor oligo, acts specifically on the adenosine A, receptor, but not on the adenosine A 2 receptors. These results stem from the treatment of rabbits with anti- sense oligo I (SEQ ID NO: 1) or mismatch control oligo (SEQ ID NO: 12; A,MM2) as described in Example 9 above and in Nyce & Metzger (1997), supra (four doses of 5 mg spaced 8 to 12 hours apart via nebulizer via endotracheal tube), bronchial smooth muscle tissue excised and the number of adenosine A, and adenosine A 2 receptors determined as reported in Nyce & Metzger (1 97), supra. Example 22: Specificity of Oligo I (SEQ ID NO: 1) for Target Gene Product Oligo I (SEQ ID NO: 1) is specific for the adenosine A, receptor whereas its mismatch controls had no activity. Figure 1 depicts the results obtained from the cross-over experiment described in Example 10 above and in Nyce & Metzger (1997), supra. The two mismatch controls (SEQ ID NO: 12 and SEQ ID NO: 13) evidenced no effect on the PC 50 Adni0S i„ e value. On the contrary, the administration of anti-sense oligo I (SEQ ID NO: 1) showed a seven-fold increase in the PC 50 Aden∞ i ne value. The results clearly indicate that the anti-sense oligo I (SEQ ID NO: 1) reduces the response (attenuates the sensitivity) to exogenously administered adenosine when compared with a saline control. The results provided in Table 6 above clearly establish that the effect of the anti-sense oligo I is dose dependent (see, column 3 of Table 6). The Oligo I was also shown to be totally specific for the adenosine A, receptor, (see, top 3 rows of Table 6), inducing no activity at either the closely related adenosine A 2 receptor or the bradykinin B 2 receptor (see, lines 8-10 of Table 6 above). In addition, the results shown in Table 6 establish that the anti-sense oligo I (SEQ ID NO: 1) decreases sensitivity to adenosine in a dose dependent manner, and that it does this in an anti-sense oligo- dependent manner since neither of two mismatch control oligonucleotides (A,MM2: SEQ ID NO: 12 and A,MM3: SEQ ID NO: 13) show any effect on PC 50 Adπι∞ine values or on attenuating the number of adenosine A, receptors. Example 23: Effect on Aeroallergen-induced Bronchoconstnction & Inflammation The Oligo I (SEQ ID NO: 1) was shown to significantly reduce the histamine- induced effect in the rabbit model when compared to the mismatch oligos. The effect of the anti-sense Oligo I (SEQ ID No:l) and the mismatch oligos (A,MM2, SEQ ID NO: 12 and A,MM3, SEQ ID NO: 12) on allergen-induced airway obstruction and bronchial hypenesponsiveness was assessed in allergic rabbits. The effect of the anti-sense oligo I (SEQ ID NO: 1) on allergen- induced airway obstruction was assessed. As calculated from the area under the plotted curve, the anti-sense oligo I significantly inhibited allergen-induced airway obstruction when compared with the mismatched control (55%, p<0.05; repeated measures ANOVA, and Tukey's t test). A complete lack of effect was induced by the mismatch oligo A,MM2 (Control) on allergen induced airway obstruction. The effect of the anti-sense oligo I (SEQ ID NO: 1) on allergen-induced BHR was determined as above. As calculated from the PC 50 H i_u m i ne value , the anti-sense oligo I (SEQ ID NO: 1) significantly inhibited allergen-induced BHR in allergic rabbits when compared to the mismatched control (61%, p<0.05; repeated measures ANOVA, Tukey's t test). A complete lack of effect of the A,MM mismatch control on allergen-induced BHR was observed. The results indicated that anti-sense oligo I (SEQ ID NO: 1) is effective to protect against aeroallergen-induced bronchoconstnction (house dust mite). In addition, the anti- sense oligo I (SEQ ID NO: 1) was also found to be a potent inhibitor of dust mite-induced bronchial hyper responsiveness, as shown by its effects upon histamine sensitivity which indicates anti- inflammatory activity for anti-sense oligo I (SEQ ID NO: 1). Example 24: Low A Content Anti-sense Oligo I is Free of Deleterious Side Effects The Ohgo I (SEQ ID NO: 1) was shown to be free of side effects that might be toxic to the recipient. No changes m arteπal blood pressure, cardiac output, stroke volume, heart rate, total penpheral resistance or heart contractility (dPdT) were observed following admmistration of 2.0 or 20 mg ohgo I (SEQ ID NO: 1). The addition, the results of the measurement of cardiac output (CO), stroke volume (SV), mean arteπal pressure (MAP), heart rate (HR), total peripheral resistance (TPR), and contractility (dPdT) with a CardiomaxJ apparatus (Columbus Instruments, Ohio) were assessed. These results evidenced that ohgo I (SEQ ID NO: 1) has no detnmental effect upon critical cardiovascular parameters. More particularly, this ohgo does not cause hypotension This finding is of particular importance because other phosphorothioate anti-sense oligonucleotides have been shown m the past to induce hypotension m some model systems. Furthermore, the adenosme A, receptor plays an important role in smoatnal conduction withm the heart. Attenuation of the adenosine A, receptor by anti-sense ohgo I (SEQ ID NO: 1) might be expected to result, therefore, in deletenous extrapulmonary activity m response to the down regulation of the receptor This is not the case. The anti-sense ohgo I (SEQ ID NO: 1) does not produce any deletenous lntrapulmonary effects and renders the admmistration of the low doses of the present anti-sense ohgo free of unexpected, undesirable side effects. This demonstrates that when ohgo I (SEQ ID NO: 1) is admmistered directly to the lung, it does not reach the heart in significant quantities to cause deletenous effects This is m contrast to traditional adenosme receptor antagonists like theophylline which do escape the lung and can cause deletenous, even life- threatening effects outside the lung Example 25: Long Lasting Effect of Oligo I The Ohgo I (SEQ ID NO: 1) evidenced a long lastmg effect as evidenced by the PC 50 and Resistance values obtained upon its admmistration pnor to adenosme challenge. The duration of the effect was measured for with respect to the PC 50 of adenosine anti-sense ohgo I when administered in four equal doses of 5 mg each by means of a nebulizer via an endotracheal mbe, as descnbed above The effect of the agent is significant over days 1 to 8 after administration When the effect of the anti-sense ohgo I (SEQ ID NO: 1) had disappeared, the animals were administered saline aerosols (controls), and the PC 50 Adenos , ne values for all animals were measured again Saline-treated animals showed base line PC 50 adenosine values (n=6) The duration of the effect (with respect to Resistance) was measured for six allergic rabbits which were admmistered 20 mg of anti-sense ohgo I (SEQ ID NO: 1) as described above, upon airway resistance measured as also described above The mean calculated duration of effect was 8 3 days for both PC 50 adenosme (p<0.05) and resistance (p<0 05) These results show that anti-sense ohgo I (SEQ ID NO: 1) has an extremely long duration of action, which is completely unexpected. Example 26: Adenosine-free Anti-sense Oligo II Better than Anti-sense Oligo I Anti-sense ohgo II, targeted to a different region of the adenosme A, receptor mRNA, was found to be highly active agamst the adenosine A, -mediated effects. The experiment measured the effect of the admmistration of anti-sense ohgo II (SEQ ID NO: 7) upon compliance and resistance values when 20 mg anti-sense ohgo II or salme (control) were administered to two groups of allergic rabbits as descnbed above. Compliance and resistance values were measured following an administration of adenosme or salme as descπbed above in Example 13 The effect of the anti-sense ohgo of the mvention was different from the control m a statistically significant manner, p<0 05 usmg paired t-test, compliance; ρ<0 01 for resistance. The results showed that anti-sense ohgo II (SEQ ID NO: 7), which targets the adenosme A, receptor and which contams no adenosme, effectively mamtams comphance and reduces resistance upon adenosme challenge In fact, the adenosine-free anti-sense Ohgo II is more potent than the low adenosine anti-sense Ohgo I (SEQ ID NO: 1) Because it contams no adenosme, it will not liberate adenosine during degradation, and hence it will not contnbute to activating adenosme receptors Example 27: Anti-sense Oligos III and IV Ohgos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) were shown to be m fact specifically targeted to the adenosme A 3 receptor by their effect on reducmg inflammation and the number of inflammatory cells present upon separate admmistration of 20 mg of the anti-sense ohgos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) to allergic rabbits as descπbed above The number of inflammatory cells was determined m their bronchial lavage fluid 3 hours later by counting at least 100 viable cells per lavage The effect of anti-sense ohgos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) upon granulocytes, and upon total cells m bronchial lavage were assessed following exposure to dust mite allergen The results showed that the anti-sense ohgo IV (SEQ ID NO: 9) and anti-sense o go III (SEQ ID NO: 8) are very potent anti-mflammatory agents m the asthmatic lung following exposure to dust mite allergen As is known in the art, granulocytes, especially eosinophils, are the primary inflammatory cells of asthma, and the administration of anti-sense ohgos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) reduced their numbers by 40% and 66%, respectively Furthermore, anti-sense ohgos IV (SEQ ID NO: 9) and III (SEQ ID NO: 8) also reduced the total number of cells m the bronchial lavage fluid by 40% and 80%, respectively This is also an important mdicator of anti- inflammatory activity by the present anti-adenosme A 3 agents of the mvention Inflammation is known to underlie bronchial hypenesponsiveness and allergen-mduced bronchoconstnction m asthma Both anti-sense oligonucleotides III (SEQ ID NO: 8) and IV (SEQ ID NO: 9), which are targeted to the adenosine A 3 receptor, are representative of an important new class of anti-mflammatory agents which may be designed to specifically target the lung receptors of each species Example 28: Anti-sense Oligo V The anti-sense ohgo V (SEQ ID NO: 10) , targeted to the adenosme A 2b adenosme receptor mRNA was shown to be highly effective at countering adenosme A 2b -medιated effects and at reducmg the number of adenosme A 2b receptors present to less than half Example 29: Unexpected Superiority of Substituted over Phosphodiester-residue Oligo I-DS (SEQ ID NO: 11) Ohgos I (SEQ ID NO: 1) and I-DS (SEQ ID NO: 11) were separately admmistered to allergic rabbits as descnbed above, and the rabbits were then challenged with adenosme The phosphodiester ohgo I- DS (SEQ ID NO: 11) was statistically significantly less effective m countering the effect of adenosme whereas ohgo I (SEQ ID NO: 1) showed high effectiveness, evidencmg a PC 50 Adenos , nc of 20 mg Example 30: Adenosine Containing Mononucleotides have Adenosine Receptor Activity This example demonstrates that in vivo break down products of anti-sense oligonucleotides such as πbonucleoside monophosphates, e g dAMP act at adenosine receptors When adenosine and adenosine monophosphate (dAMP) were separately admmistered to experimental animals at different doses of up to 10 mg/ml, both compounds have a similar effect in reducmg % comphance as shown m Figure 1 The effect m both cases increases with the dose whereas a saline control shows no effect. These results show that adenosine nucleosides as well as adenosine itself, interact with adenosine receptors. Example 31: Breakdown of Adenosine Containing Nucleic Acid Produce Adenosine Receptor Activity As a further test, randomer phosphorothioate anti-sense oligonucleosides were administered to rabbits to determine if they were degraded in vivo and released adenosine nucleosides capable of interacting with adenosine receptors. Asthmatic rabbits were separately administered saline (control), an adenosine containing randomer (?) and a desAdenosine randomer (C). The randomers used were a desAdenosine randomer consisting of random sequences of guanine, cytosine and thymidine and an adenosine containing randomer consisting of guanine, cytosine and adenosine. The results shown in Figure 2 clearly indicate that adenosine containing oligonucleotides release adenosine and/or adenosine nucleosides upon degradation, and that the adenosine compounds interact with adenosine receptors, while desAdenosine oligonucleotides do not. The release of adenosine nucleosides as degradation products of anti-sense oligonucleotides, thus, would confuse experimental results when assessing the effects of anti-sense knock out experiments for Target Validation. This experiment shows the necessity of using desAdenosine anti-sense (no A or low A) oligonucleotides in Target Validation studies. The foregoing examples are illustrative of the present invention, and are not to be construed as limiting thereof. The invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims: WHAT IS CLAIMED AS NOVEL AND UNOBVIOUS IN UNITED STATES LETTERS PATENT IS: 1. A method of determining the existence of a conelation between a function of a disease or condition and a gene or mRNA encodmg a target polypeptide suspected of bemg associated with a disease or condition, compnsmg obtammg oligonucleotides (oligos) consisting of up to about 15% adenosme (A), and which is anti- sense to a target selected from the group consisting of target genes and their conespondmg mRNAs, genomic and mRNA flankmg regions selected from the group consisting of 3' and 5' mtron-exon borders and the juxta- section between codmg and non-coding regions, and all mRNA segments encodmg polypeptides associated with a pre-selected disease or condition; selecting amongst the ohgos one that significantly inhibits or ablates expression of the polypeptide encoded by the mRNA upon in vitro hybndization to the target mRNA; administering to a subject an amount of the selected ohgo effective for in vivo hybndization to the target mRNA; and assessmg a subject's function that is associated with the disease or condition before and after admmistration of the ohgo; wherem a change m the function's value greater than about 70% indicates a positive conelation, between about 40 and about 70% a possible conelation, and below about 30% a lack of conelation. 2. The method of claim 1, wherem the anti-sense oligos are constructed by selecting target fragments havmg at least 4 contiguous nucleic acids selected from the group consisting of G and C and obtammg a first ohgonucleotide 4 to 60 nucleotides long which compnses the selected fragment and has a C and G content of up to about 15%, and/or having a desirable type and/or extent of activity. 3 The method of claim 1, further compnsmg, when the anti-sense fragment compnses at least one A, substimtmg at least one A with an alternative base (B) selected from the group consisting of heteroaromatic bases which bind to thymidme (T) but have less than about 0.3 of A's adenosme A,, A 2a , A 2b and A 3 receptor agonist or antagonist activity 4 The method of claim 3, where the heteroaromatic bases are selected from the group consisting of pyπmidines and punnes, which may be substituted by O, halo, NH 2 , SH, SO, S0 2 , S0 3 , COOH and branched and fused primary and secondary ammo, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, which may be further substituted by O, halo, NH 2 , primary, secondary and tertiary amine, SH, SO, S0 2 , S0 3 , cycloalkyl, heterocycloalkyl and heteroaryl. 5 The method of claim 4, wherem the pyrimidines and punnes are substituted at positions 1 , 2, 3, 4, 7 and 8. 6 The method of claim 4, wherem the pyπmidines and puπnes are selected from the group consisting of theophylhne, caffeine, dyphylline, etophylline, acephylline piperazme, bamifylhne, enprofylline and xanthme having the chemical formula wherein R 1 and R 2 are independently H, alkyl, alkenyl or alkynyl and R 3 is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O- cycloalkynyl, NH 2 -alkylamino-ketoxyalkyloxy-aryl and mono and dialkylaminoalkyl-N-alkylamino-S0 2 aryl. 7. The method of claim 1, wherein the anti-sense oligo has an adenosine content of about 0 to about 12%. 8. The method of claim 7, wherein the oligo consists of up to about 5% A. 9. The method of claim 8, wherein the oligo is A-free. 10. The method of claim 1, wherein one A is substituted by an alternative base selected from the group consisting of heteroaromatic bases which bind to a thymidine base but have antagonist or agonist activity of less than about 0.5 of the adenosine base agonist or antagonist activity at the adenosine A,, A 2a , A 2b and A 3 receptors. 11. The method of claim 10, wherein all As are substituted by an alternative base selected from the group consisting of heteroaromatic bases which bind to a thymidine base but have activity less than about 0.3 of the adenosine base agonist or antagonist activity at the adenosine A,, A 2a A 2b and A 3 receptors. 12. The method of claim 6, wherein the universal base is selected from the group consisting of 3-nitropyrrole-2'-deoxynucleoside, 5-nitro-indole, 2-deoxyribosyl-(5-nitroindole), 2-deoxyribofuranosyl-(5- nitroindole), 2'-deoxyinosine, 2'-deoxynebularine, 6H, 8H-3,4-dihydropyrimido [4,5-c] oxazine-7-one or 2- amino-6-methoxyaminopurine. 13. The method of claim 1, where a methylated cytosine ( m C) is substituted instead of C in at least one CpG dinucleotide if present in the oligo(s). 14. The method of claim 1, wherein at least one nucleotide residue of the anti-sense oligonucleotide(s) is a residue selected from the group consisting of methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methyimino), (MMI), methoxymethyl (MOM), methoxyethyl (MOE), methyleneoxy (methylimino) (MOMA), methoxy methyl (MOM), 2'-0-methyl, phosphoramidate, and C-5 substituted residues, and combinations thereof. 15. The agent of claim 14, wherein all nucleotide linking residues are substituted. 16. The method of claim 1, wherein the anti-sense oligo comprises about 7 to 60 mononucleotides. 17. The method of claim 1, wherein the anti-sense oligo is linked to an agent selected from the group consisting of cell internalized or up-taken agent(s) and cell targeting agents. 18. The method of claim 17, wherein the cell internalized or up taken agent is selected from the group consisting of transfenin, asialoglycoprotein and streptavidin. 19. The method of claim 18, wherein the nucleic acid is linked to a vector. 20. The method of claim 19, wherein the vector comprises a prokaryotic or eukaryotic vector. 21. The method of claim 1, wherein the anti-sense oligo is administered to the lung, brain, heart, kidney, tumor, blood, skin, eye, scalp, nose panages, testes, cervix, oral cavity, pharynx, esophagus, small or large intestine, synovial tissue, muscle tissue, ovaries, ear canal or in vitro. 22 The method of claim 1, wherem the disease or condition is a disease or condition afflicting the lung, bram, heart, kidney, tumor, blood, immune system, skm, eye, scalp, nose panages, testes, cervix, oral cavity, pharynx, esophagus, small or large intestine, synovial tissue, muscle tissue, ovanes, and ear canal 23 The method of claim 22, wherem the disease or condition is a disease or condition afflicting the lung 24 The method of claim 22, wherem the disease or condition is associated with bronchoconstnction, lung inflammation and/or allergy(ιes) 25 The method of claim 22, wherem the disease or condition is a disease or condition afflicting the bram, or associated with bram activity 26 The method of claim 22, wherem the disease or condition is associated with immune dysfunction 27 The method of claim 26, wherem the target is selected from the group consisting of lmmunoglobulins, antibody receptors, cytokmes, cytokme receptors, gene(s) and the conespondmg mRNA(s) encodmg them, the genes and mRNA flankmg regions and mtron and exon borders 28 The method of claim 22, wherein the disease or condition is a disease or condition afflicting the cardiovascular system 29 The method of claim 22, wherem the disease or condition is a disease or condition associated with the gastrointestinal system 30 The method of claim 22, wherem the disease or condition is associated with a malignancy or cancer. 31 The method of claim 30, wherem the target is selected from the group consisting of lmmunoglobulins and antibody receptors, gene(s) and mRNA(s) encodmg them, genes and mRNAs associated with oncogenes, and genomic and mRNA flankmg regions and exon and mtron borders 32 The method of claim 1, wherem the composition is administered in vitro, orally, lntracavitaπly, intranasally, intraanally, intravaginally, intrauterally, lntrachranially, pulmonaπly, mtrarenally, intranodularly, lntraarticularly, mtraotically, intralymphatically, transdermally, lntrabucally, intravenously, subcutaneously, intramuscularly, intratumorously, lntraglandularly, intraocularly, lntracranial, into an organ, lntravascularly, intrathecally, by implantation, by inhalation, lnrradermally, mtrapulmonanly, mto the ear, mto the heart, by slow release, by sustained release and by a pump 33 The method of claim 1, wherem the target is selected from the group consisting of genes and mRNAs encodmg polypeptides selected from the group consisting of transcnption factors, stimulatmg and activating factors, cytokmes and their receptors, mterleukms, interleukin receptors, chemokmes, chemokme receptors, endogenously produced specific and non-specific enzymes, lmmunoglobulins, antibody receptors, central nervous system (CNS) and penpheral nervous and non-nervous system receptors, CNS and penpheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensmes, growth factors, vasoactive peptides, peptide receptors and bmdmg protem, and genes and mRNAs conespondmg to oncogenes 34 The method of claim 1 , wherem the anti-sense ohgo(s) are produced by selectmg a target from the group consisting of polypeptides associated with a dιsease(s) and/or condιtion(s) afflicting lung airways, genes and RNAs encodmg them, the genomic and mRNA flankmg regions and the gene(s) and mRNA(s) mtron and exon borders, obtammg the sequence of a mRNA(s) selected from the group consistmg of mRNAs conespondmg to the target gene(s) and mRNAs encoding the target polypeptιde(s), genomic and mRNA flankmg regions and the genes and mRNAs mtron and exon borders, selecting at least one segment of the mRNA(s); synthesizing one or more ohgo anti-sense to the selected mRNA segments); and substituting, if necessary, an alternative base(s) for one or more A(s) to reduce the content of A present m the ohgo to up to about 15% of all nucleotides. 35. The method of claim 1, wherem the target gene is selected from the group consisting of target genes and mRNAs encodmg polypeptides selected from the group consisting of transcnption factors, stimulating and activating factors, mterleukms, interleukin receptors, chemokmes, chemokme receptors, endogenously produced specific and non-specific enzymes, lmmunoglobulins, antibody receptors, central nervous system (CNS) and peripheral nervous and non-nervous system receptors, CNS and penpheral nervous and non-nervous system peptide transmitters and their receptors, adhesion molecules, defensmes, growth factors, vasoactive peptides and their receptors, and bmdmg protems, and target genes and mRNAs conespondmg to oncogenes, and their flankmg regions and mtron and exon borders. 36. The method of claim 35, wherem the encoded polypeptides are selected from the group consisting of Nfi B Transcnption Factor, Interleukιn-8 Receptor (IL-8 R), Interleukin 5 Receptor (IL-5 R), Interleukin 4 Receptor (IL-4 R), Interleukin 3 Receptor (IL-3 R), Interleukin- lβ (IL-lβ), Interleukin lβ Receptor (IL- lβ R), Eotaxm, Tryptase, Major Basic Protem, β2-adrenergιc Receptor Kmase, Endothelm Receptor A, Endothelm Receptor B, Preproendothelin, Bradykmm B2 Receptor, IgE High Affinity Receptor, Interleukin 1 (IL-1), Interleukin 1 Receptor (IL-1 R), Interleukin 9 (IL-9), Interleukm-9 Receptor (IL-9 R), Interleukin 11 (IL-11), Interleukin- 11 Receptor (IL-11 R), Inducible Nitnc Oxide Synthase, Cyclooxygenase (COX), Intracellular Adhesion Molecule 1 (ICAM-1) Vascular Cellular Adhesion Molecule (VCAM), Rantes, Endothehal Leukocyte Adhesion Molecule (ELAM-1), Monocyte Activating Factor, Neutrophil Chemotactic Factor, Neutrophil Elastase, Defensin 1, 2 and 3, Muscarmic Acetylcholme Receptors, Platelet Activating Factor, Tumor Necrosis Factor a, 5-hpoxygenase, Phosphodiesterase IV, Substance P, Substance P Receptor, Histamme Receptor, Chymase, CCR-1 CC Chemokme Receptor, CCR-2 CC Chemokme Receptor, CCR-3 CC Chemokme Receptor, CCR-4 CC Chemokme Receptor, CCR-5 CC Chemokme Receptor, Prostanoid Receptors, GATA-3 Transcnption Factor, Neutrophil Adherence Receptor, MAP Kmase, Interleukm-9 (IL-9), NFAT Transcnption Factors, STAT 4, MlP-lα, MCP-2, MCP-3, MCP-4, Cyclophillms, Phosphohpase A2, Basic Fibroblast Growth Factor, Metalloproteinase, CSBP/p38 MAP Kmase, Tryptose Receptor, PDG2, Interleukιn-3 (IL-3), Interleukm-lβ (IL-lβ), Cyclosponn A-Binding Protem, FK5-Bιndιng Protem, α4βl Selectm, Fibronectm, α4β7 Selectm, Mad CAM-1, LFA-1 (CD 11 a/CD 18), PECAM-1, LFA-1 Selectm, C3bι, PSGL-1, E-Selectm, P-Selectin, CD-34, L-Selectm, pl50,95, Mac-1 (CDl lb/CD18), Fucosyl transferase, VLA-4, CD-18/CDl la, CDl lb/CD18, ICAM2 and ICAM3, C5a, CCR3 (Eotaxm Receptor), CCR1, CCR2, CCR4, CCR5, LTB-4, AP-1 Transcnption Factor, Protem kmase C, Cystemyl Leukotnene Receptor, Tachychinnen Receptors (tach R), IκB Kmase 1 & 2, STAT 6, c-mas and NF-Interleukm-6 (NF-IL-6), and their flankmg regions and mtron and exon borders. 37. The method of claim 1, wherem the target gene encodes a G-protem or a G-protem coupled receptor. 38. The method of claim 1, wherem the target gene encodes a calcium channel protem or receptor, a sodium channel protein or receptor, a potassium channel protem or receptor, or a chlonde channel protem or receptor. 39. The method of claim 1, wherein the target gene encodes a neurotransmitter receptor or a neurohormone receptor. 40. The method of claim 1, wherein the target gene encodes a neuropeptide or neuropeptide receptor. 41. The method of claim 1, further comprising repeating all steps with the separate administration of further oligo(s) which are anti-sense to further targets whose functions are suspected of being associated with the first target; repeating the administration and assessment steps with the joint administration oligos targeted to the first and further targets; and comparing the results obtained with those obtained separately for each target; wherein when the combined oligo effects are about 20% or more greater than that with each oligo, it may be said there is a positive association between the first and further oligo(s), when the results are within about 20% of that of one oligo it may be said that there is no association, and when the results are less than about 20% lower than with an individual oligo it may be said that there is a negative association between them.

Inventors: NYCE JONATHAN W, NYCE, JONATHAN, W.
Applicants: EPIGENESIS PHARMACEUTICALS INC, NYCE JONATHAN W, EPIGENESIS PHARMACEUTICALS, INC., NYCE, JONATHAN, W.
Application year: 2000
Main classification: A61K31
IPC codes: A61K38, C12N15, C12Q1