KR20010030622A - Multiple Target Hybridizing Nucleic Acids, Their Preparation, Compositions, Formulation, Kits and Applications - Google Patents

Abstract

Antisense oligonucleotides bound to two or more RNA targets are disclosed.

Description

Multiple Target Hybridizing Nucleic Acids, Their Preparation, Compositions, Formulation, Kits and Applications

Respiratory illnesses associated with various ailments and pains are very common in the general population, especially in certain minority groups such as African Americans. In some cases, such disease involves inflammation that worsens the condition of the lungs. For example, asthma is one of the most common low-cost diseases in industrial countries. Asthma accounts for 1% of all medical costs in the United States. Significant increases in asthma and deaths have been recorded over the past decade, and asthma is likely to become a serious occupational lung disease in the next decade. The increase in asthma deaths in industrial countries is due to high confidence in beta agonists, the cure for the disease, but the underlying cause of asthma is little known.

Antisense oligonucleotides have been of great theoretical interest as useful pathological agents of human disease. However, practical application as a practical model of human disease was rather difficult. One difficulty in the effective application of them has been to find suitable routes of administration for transport to the site of action. Many in vivo experiments have been conducted in which antisense oligonucleotides are directly administered to specific regions of the brain. However, these applications have been inevitably limited in clinical usefulness by their invasive nature.

As a substantial inherent problem in targeting disease-involved tissues, systemic administration of antisense oligonucleotides has also shown significant problems. In contrast, the lung is an excellent target for the direct administration of antisense oligonucleotides and has a non-invasive and tissue-specific route. The transport of antisense agents into the lung has not been studied much.

Adenosine in the lung is an important mediator in a variety of diseases including bronchial asthma. Asthma patients had a significant brochoconstriction response to aerosolized adenosine, and studies with nonacute patients suggested a possible role for adenosine. The dust mite allergic rabbit model, an asthma rabbit animal model for the study of human asthma, showed a significant bronchial stenosis response to similar aerosolized adenosine, whereas non-asthma There was no reaction in rabbits. A more recent study with this animal model suggested that adenosine-induced bronchial stenosis and asthmatic bronchial hyperresponse primarily mediate through stimulation of adenosine receptors. Adenosine also appears to exhibit adverse effects, including death, when first administered therapeutically to other diseases and pain in patients with undiagnosed overreactive airways.

Although generally limited, small amounts of drugs have been used to treat respiratory diseases and pain. Diophylline, an important drug in the treatment of asthma, is known as an antagonist of adenosine receptors that has been recorded to eliminate adenosine-mediated bronchial stenosis in asthmatic rabbits. An optional adenosine A1 receptor antagonist, 8-cyclopentyl-1.3-dipropylzantine (DPCPX), can inhibit adenosine-mediated bronchial contraction and bronchial hyperresponsiveness in allergic rabbits. Nevertheless, the therapeutic and prophylactic application of currently available adenosine receptor A ₁ -specific antagonists is limited because of the toxicity of antagonists. For example, diophylline is widely used for the treatment of asthma, but it is disproportionate to a narrow therapeutic range and shows considerable toxicity. Although many studies have progressed over the last decade, certain adenosine receptor specific antagonists are not clinically available due to the general toxicity of these agents.

Antisense oligonucleotides are of considerable theoretical interest for use as pathological agents in human diseases. However, due to the large amount of administration, very few practical and effective applications of these agents are found in practical models of human disease. Another important concern in the pathological application of the things molecule is the route of administration. Many applications in vivo only involve the direct administration of antisense oligonucleotides to defined portions of the brain. However, such applications are penetrating in nature, limiting their clinical utility.

Systemic administration of antisense oligonucleotides as a pathological agent has been an important problem because of the difficulty in targeting the tissues containing the disease. That is, in order to obtain a therapeutic amount by intravenous injection or mouth, there is a difficulty in sufficiently diluting antisense oligonucleotides in the circulatory system. In the case of oral administration, bioavailability is less than 5%.

Many people, including the inventors, have used antisense oligonucleotides in the clinic, and we have already successfully treated various diseases and pains by directly administering these agents to the lungs. In many cases, others have a lot of difficulty in positioning DNA molecules as desired targets. Therefore, the medication route is very important for treating general illness and pain, as well as the location of the disease and pain.

Therefore, there is a need for an effective method for implementing antisense therapies, and more particularly, for the treatment of diseases and pains in complex conditions to be highly effective. As the latter example, complex pathways and many endogenous substances, alone or in combination with each other, cause pathological pain in patients.

The present invention relates to multiple target antisense oligonucleotides (MTA oligos) with little or no adenosine content. The agent is effective for the prevention and treatment of diseases and pains associated with inflammation, including pulmonary diseases, impaired airway, and secondary diseases that injure the lungs of patients. The agent is a specific gene, genomic flaking regions, initiation codons, intron-exon borders, and the like, or multiple media that act on certain proteins, particularly various related pathways. It targets the coding and non-coding regions of RNA that encode proteins associated with the disease. Examples include allergies, asthma, respiratory distress, pain, cystic fibrosis and cancers such as colon cancer, leukemias, and the like. The agent can be easily administered prophylactically and therapeutically in combination with other therapies, or can be used in place of therapies with significant and negative side effects.

[Summary of invention]

FIELD OF THE INVENTION The present invention relates to antisenseTM oligonucleotides or multiple target antisense oligos (MAT oligos) that form hydrides with two or more mRNAs. For example, target genes, gene contiguous regions, initiation codons, intron-axon boarders, and the like, and the coding region and 5 'cap or terminus of the mRNA, such as a 3' terminus such as a poly-A fraction. Such as antisense oligos corresponding to RNAs of the entire sequence, including RNAs, and RNAs encoding proteins associated with one or more diseases or disorders or combinations thereof. For example, mRNA may be transcription factors, stimuli and activators, interleukins, interleukin receptors, chemokines, chemokine receptors, intrinsically produced specific and non-specific enzymes, immunoglobulins, Antibody receptors, immunoglobulins, antibody receptors, central and peripheral and non-neural receptors, central and peripheral and non-neutral peptide transporters, adsorption molecules, defensins, growth factors, blood vessels Peptide (s), such as functional peptides and receptors, and binding proteins, may be encoded, or may encode mRNA corresponding to an oncogene. Agents of the invention contain up to about 15% adenosine (A) and in many cases contain no adenosine at all. In addition, the agents of the present invention may be presented in the form of a composition, and may be present in the form of a kit provided with capsules and cartridges, various formulations, transporters and instructions for use. The antisense-oligos of the present invention may have a reduced adenosine content by substituting with adenosine-like bond substituents as a general base. The kit may also have components for other therapeutic agents and compositions. In addition, the agent may be provided in the form of a host cell operably linked to or transfected with the vector.

The agents, compositions and formulations of the present invention may be applied to the treatment of diseases and disorders associated with the presence of mRNA corresponding to at least one of the target genes, gene contiguous regions or proteins, the production of at least one target mRNA and An amount of MTA oligo of the present invention is administered to a patient that is effective to reduce utility or increase degradation. In a preferred embodiment, the agent may be administered in an effective amount to reduce the productivity and availability of at least two target mRNAs or to increase their degradation. Typical diseases and disorders of the present invention relate to respiratory disorders and inflammation, including lung diseases, diseases and pain that have a negative effect on the lungs of a patient. Examples of diseases and conditions that can be treated prophylactically and therapeutically with the agents of the invention include pulmonary vasoconstriction, inflammation, allergic rhinitis, acute asthma, allergy, asthma, respiratory disorders, dyspnea syndrome, pain, cystic For example, colon cancer, breast cancer, as well as all types of cancer that have metastasized or are metastasizing to the lung, including fibrosis, pulmonary hypertensin, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), leukemia, lymphoma, breast cancer and prostate cancer Carcinomas such as lung cancer, pancreatic cancer, subcutaneous cancer, kidney cancer, melanoma, liver metastasis, and the like. The agent (s) may also be administered before or during other treatments, including radiotherapy, chemotherapy, antibody therapy, prototherapy, cancer and other surgical therapies. In addition, the present agents, which can be effectively administered for the prevention and treatment, are also suitable for other treatments that have parallel or other treatments, treat the disease on its own without other treatments, or have significant and negative effects.

Compositions of the present invention include those injected into the mouth, intranasal, intranasal, anal, vaginal, transdermal, intradermal, buccal, hematopoietic, subcutaneous, intramuscular and intratumoral, generally intraarterial, vascular, inhaled By intra-cranial, capillary, intratracheal, for example, liver and other organs, including through the sideways, and through implantation, intraocular administration of other animals, including vertebrates such as humans and mammals. Can be. The therapy of the present invention may be prophylactic and therapeutic. In a preferred embodiment, the agent of the invention can be administered directly to the patient's respiratory tract so that the agent reaches the lung directly in an effective amount to reduce or inhibit the effect in the lung of the targeted disease and disorder.

Agents of the invention may include genes, contiguous regions, indron-axon borders, etc., or RNA encoding the entire sequence of RNA, including non-coding RNAs, and proteins known to be associated with one or more diseases and disorders. Selecting two or more targets such as; Obtaining RNA selected from the group consisting of RNAs corresponding to genes and genomic contiguous regions and RNA encoding a target protein; Selecting a fraction of at least about 60%, preferably 80%, more preferably 90% and even nearly 100% homologous first RNA to the second RNA; And from synthesizing one or more antisense oligonucleotides for one or more RNA segments. Target RNAs include precursors, spliced mRNA and fractions superimposed side by side with other RNA molecules, coding- and noncoding-sequences containing the 5'- and 3'-ends and the coding region. In a preferred form of the invention, two or more targets may be located in the same molecule. For example, for mRNA encoding a protein with multiple subunits, MTA oligos can be found in the RNA fraction encoding other protein subunits.

In addition, MTA oligos are provided that can be produced according to the methods of the present invention.

Detailed description of the invention

The present invention enhances the discovery that the inventors themselves or others have previously discovered, ie previously antisense oligonucleotides can be used therapeutically in treating a disease or condition with multiple related pathways. Is derived from the inventor's hope. Through the present invention, the present inventors can enhance the inventor's previous success in mitigating or enhancing the effects of one specific pathway by designing antisense oligonucleotides to targets with specific diseases and associated diseases. Inferred. Accordingly, the inventors have begun trying new and unclear strategies that are directly related to multiple targets. In the meantime, the inventors have overcome a number of disorders, particularly targeting, as well as overcoming the extensive search and selection necessary to locate genomic DNA, RNAs, or proteins, desired sequences associated with a particular disease. The inventors then demonstrated the invention by applying it to several specific diseases or disorders, provided various and preferred embodiments, and specifically constructed multiple target antisense oligonucleotide (MTO oligo) sequences.

The multiple target antisense (MTA) oligonucleotide FP tide of the present invention has the ability to alleviate the expression of one or more target mRNAs, or to enhance or alleviate the activity of one or more pathways. Through the methods of the embodiments, the method of the present invention comprises about 7, about 10, about 12, about 15, about 18, about 21 to about 28, about 30, about 35, about 40, about 45, about All potential desadenosine (desA) antisense sequences for 50, about 60, or more mononucleotides could be identified and identified for the first time. This was obtained by searching for fractions of 7 or more nucleotides in target sequences with little or no thymidine, a nucleotide complementary to adenosine. This typical search resulted in 10-30 desT fractions (ie, those naturally lacking thymidine) or low T contents (eg, up to about 15% T). From this, anti-sense oligonucleotides of various lengths can be designed for typical target mRNAs of average length, such as, for example, about 1800 nucleotides in length. Thereafter, a sense sequence that is strictly complementary to each of the desA antisense sequences obtained for a particular target can be inferred. Thus, the deduced sense sequence can be used to find the sequence of the desired secondary target. In addition, one or more sequence databases, such as GENBANK, may find another alternative secondary target. Thus, targeting can be performed in several ways, one of which is to select specific targets related to one or more diseases. In addition, the primary target may be selected initially, and an antisense oligonucleotide, preferably desA oligonucleotide, may be found, and then a second, tertiary or more target may be found. In a typical search, a list of preferred secondary targets or a list of databases, examples of homologous secondary targets of interest are identified. In other words, the techniques of the present invention allow for finding examples with natural homology between the primary, secondary and higher sequences. The findings can be used to treat certain diseases or conditions associated with the target macromolecules from which the MTAs are obtained.

The technique of the present invention is used in the fabrication of antisense oligos targeting mRNAs associated with diseases involving lung airway lesions, and for the intended purpose, is caused by the destruction and release of adenosine while maintaining activity and effectiveness. It is also used to modify the oligos to reduce the occurrence of undesired side effects. In this way, we design one or more antisense oligonucleotide (s) capable of selecting a particular gene and selectively binding to the corresponding mRNA and, if necessary, a general base or adenosine A ₁ , A _2b , The content of adenosine is reduced by replacing it with an adenosine analog that prevents A ₃ receptors from being activated. Based on prior experience in this field, the inventors have found that, in addition to certain genes that are “downregulated,” one or more of thymidine (T) is absent, low in content, or present in the designed oligonucleotide (s). RNA adenosine (s) is substituted with other nucleotide bases called so-called common bases that bind to thymidine but lack the ability to activate adenosine receptors or otherwise shrink the adenosine effect in the lung It has been found that selecting fractions can increase the effect of the administered agent (s). Considering that adenosine (A) is a nucleotide base complementary to thymidine, when T appears in the RNA, the antisense oligo will have A at the same position. Although complementary sequences are included within the four corners of the present invention, consistently, all RNA and oligonucleotides herein are represented by a strand of strands in the 5 'to 3' direction when read from left to right. Moreover, all nucleotide bases and amino acids are shown according to the regulations of the IUPAC-IUB biochemical Nomenclature Comminssion, or using known three letter codes (for amino acids).

The methods of the present invention can be used to treat diseases associated with reduced airway function in a patient whatever the cause. The adenosine content of the antisense agent of the invention reduced the content of A to prevent release of the agent in vivo. Examples of airway diseases that can be treated with the method of the present invention include cystic sexual asthma, asthma, full monolithic hypertensin, vasoconstriction, COPD, long-term frontalism, respiratory stress syndrome, lung cancer, lung metastatic cancer, and inflammatory response It includes other airway diseases, including.

Among other things, antisense oligos against adenosine A ₁ , A _2a , A _2b , A ₃ receptors, CCR3 (chemokine receptors), bradykinin 2B, CAM (blood cell adsorption molecule), eosinophil receptors are responsible for the expression of their genes. Effective for down-regulation Some of these may relieve symptoms or respiratory disease and / or inflammation, such as adenosine A ₁ , A _2a , A _2b , and / or A ₃ receptors and CRR3, bradykinin 2B, VCAM (vascular cell adsorption molecules). ), And "downregulation" of eosinophil receptors. Such agents are preferably administered directly into the respiratory system using, for example, inhalers or other means so that the lungs can be reached without extensive system metastasis. This makes it possible to administer substantially small amounts of the agents of the invention, as compared to administration via the prior art or other common routes, and consequently, undesirable effects caused by the wide distribution of agents in the body. It helps to reduce side effects. Agents of the invention have been known to reduce the amount of receptor protein expressed in tissues. Thus, for example, agents of the invention that interact only with targets, such as receptors, can reduce the number of target proteins that other drugs interact with. In this way the agent (s) of the present invention provide extremely high efficiency with low toxicity.

The adenosine receptors reviewed above are well-behaved examples of our technique. Indeed, a large number of genes have been targeted in a manner similar to the present invention to reduce or downregulate expression of proteins. By way of an embodiment, if the target disease or condition is respiratory disorder or respiratory depression, bronchial stenosis, chronic bronchitis, pulmonary bronchial stenosis and / or hypotension, chronic obstructive pulmonary disease (CODP), allergy, asthma, cystic fibrosis, respiratory distress syndrome In the case of cancer, which is directly or metastasized and has a negative effect on the lungs, the methods of the present invention may be applied to a list of possible target mRNAs, in particular the targets listed in Table 1 below.

Table 1: Lung Disease or Pain (Asthma / Inflammation) Targets

NfκB Transcription Factor Interleukin-5 Receptor (IL-5R) Interleukin-3 Receptor (IL-3R) Interleukin-1β Receptor (IL-1βR) Tryptaseβ ₂ -Adrenergic Receptor Kinase Endoserine Receptor B Bradykinin bradykinin) B ₂ receptor (B2B) interleukin-1 (IL-1) interleukin-9 (IL-9) interleukin-11 (IL-11) inducible nitric oxide synthase intracellular adsorption molecule 1 (ICAM-1; intracellular Adhesion Molecule 1) Rantes cyclooxygenase-2 (COX-2) monocyte Acting factor neutrophil elastase muscaric acetylcholine receptor tumor necrosis factor α (Tumor necrosis factor) phosphodiesterase IV Substance P Receptor chymase Interleukin-2 (IL-2) Interleukin-12 (IL-12) Interleukin-6 (IL-6) Interleukin-8 (IL-8) Interleukin-7 Receptor (IL-7R Interleukin-14 receptor (IL-14R) CCR-2 CC chemokine receptor CCR-4 CC chemokine receptor Interleukin-8 Receptor (IL-8R) Interleukin-4 Receptor (IL-4R) Interleukin-1β Receptor (IL-1βR) Eotaxin Major Basic Receptor Endotherial Receptor A Free PreproendothelinIgE (high affinity receptor) interleukin-1 receptor (IL-1R) interleukin-9 receptor (IL-9R) interleukin-11 receptor (IL-11R) cyclooxygenase (COX; Cyclooxygenase) Vascular celluar adhersion molecule (VCAM) Endothelial Leukocyte Adhesion Molecule (ELAM-1) GM-CSF, Endothelin-1 neutrophil chemitactic factor defensin 1, 2, 3 Platelet Activating factor 5-lipoxygenase substance P histamine receptor CCR-1 CC chemokine receptor interleukin-4 (IL-4) interleukin-5 (IL-5) interleukin-7 (IL-7 Interleukin-12 Receptor (IL-12) Interleukin-1 (IL-1) Interleukin-14 (IL-14) CCR-3 CC Chemokine Acceptance CCR-5 CC chemokine receptor

Table 1: Lung Disease or Pain (Asthma / Inflammation) Targets (Continued)

Prostanoid Receptor Neutrophil Adsorption Receptor Interleukin-15 (IL-15) Interleukin-11 (IL-11) NFAT Transcription Factor MIP-1α MCP-3 Cyclophilin (A, B, etc.) Basic Fibroblast Growth Factor CSBP / p38 MAP Kinase PDG2 Interleukin-10 (IL-10) FK506-binding protein fibronectin cMad CAM-1PECAM-1C3 biE-selectin CD-34p150,95 fucosyl transferase CD-18 / CD11aICAM2 and ICAM3CCR3 (eotaxin receptor) LTB- 4 protein kinase C tachykinninen receptor (tach R) interleukin-2 receptor (IL-2R) STAT 6NF-interleukin-6 (NF-IL-6) interleukin-3 (IL-3) interleukin-13 (IL Interleukin-14 (IL-14) Interleukin-16 (IL-16) Medullasin GATA-3 Transcription Factor MAP Kinase Interleukin-15 Receptor (IL-15R) Interleukin-11 Receptor (IL-11R) STAT 4MCP-2MCP-4 Phospholipase A2 Metalloproteinase Trypsin Receptor Interleukin-3 (IL-3) Sporin A-binding protein α4β1 selectin α4β7 selectin LFA-1 (CD11a / CD18) LFA-1 selectin PSGL-1P-selectin L-selectin Mac-1 (CD11b / CD18) VLA-4CD11b / CD18C5aCCR1, CCR2, CCR4, CCR5AP-1 Transcription Factor Cysteinyl Lucotriene Receptor IκB Kinases 1 and 2 (eg, Substance P, NK-1 & NK-3 Receptors) c-mas Interleukin-10 Receptor (IL-10R) Interleukin-2 Receptor (IL-2R) Interleukin-12 Receptor (IL-12R) Interleukin-6 Receptor (IL-6R) Interleukin-13 Receptor (IL-13R) Interleukin-16 Receptor (IL-16R)

Table 1: Lung Disease or Pain (Asthma / Inflammation) Targets (Continued)

Adenosine A ₁ receptor (A1R) Adenosine A _2b receptor (A _2b R) β tryptase adenosine A _2a receptor (A _2a R) Fc-epsilon receptor CD23 antigenIgE receptor Fc epsilon receptor (IgERFcξR) histidine decarboxylaseprostaglandin D Synthase Eosinophil Induction Neurotoxin Endothelial Nitric Oxide Synthase Neutrophil Oxidase Factor Macrophage Inflammatory Protein-1-alpha / Rantetsu Receptor Endoserine Receptor ET-B Tryptase-I adenosine A ₃ receptor (A ₃ R) IgE receptor β subunit (IgE Rβ) IgE receptor α subunit (IgE Rα) substance P receptor tryptase-1 eosinophil cationic protein eosinophil peroxidase endothelial Monocyte activator cathepsin G interleukin-8 receptor α subunit (IL-8 R α) substance Pendoserine ETA receptor

Selecting fragments of the target nucleic acid having at least four consecutive nucleic acids selected from the group consisting of G and C and from 4 to 60 nucleotides in length including the selected fragment and having a G and C nucleic acid content of up to about 15% From the step of obtaining the primary nucleotides, oligos of the invention can be obtained. The next step is to obtain secondary oligonucleotides having 4 to 60 nucleotides in length comprising sequences that are antisense for the selected fragment. The secondary oligonucleotide has an adenosine content of up to 15%. In addition, this method binds adenosine bases in the corresponding nucleotides of the antisense fragment to thymidine bases, but has antagonist activity when the selected fragment comprises at least one thymidine base, and has adenosine A ₁ , A _2b , A ₃ Substitution with a general base selected from the group consisting of heteroaromatic bases having an adenosine base agonist activity of about 0.3 at the receptors, and heteroaromatic bases having no agonist activity or agonist activity at the adenosine A _2a receptor It includes. Heteroaromatic base analogs include O, allo, NH ₂ , SH, SO, SO ₂ , SO ₃ , COOH and branched and fused primary and secondary amino, alkyl, alkenyl, alkynyl, cycloaryl, heterocycloalkyl, Aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsuloxyl, akynylallyl, arylacyl, arylalkenyl, arylaki And may be substituted with aryl, allylcycloacyl, and further substituted with O, halo, NH ₂ , primary, secondary, tertiary amine, SH, SO, SO ₂ , SO ₃ , cycloalkyl, heterocycloalkyl, and heteroaryl. Can be selected from both pyrimidines and purines. Pyrimidines and purines may be substituted at all positions known in the art, but are preferably substituted at positions 1,2,3,4,7, and / or 8. Furthermore, pyrimidines and purines, such as thiophylline, caffeine, diphylline, etophylline, acepiline piperazine, bamiphylline, enpropyline and xanthine, having the formula (1) are preferred.

Wherein R ₁ , R ₂ are independently H, alkyl, alkenyl or alkynyl, and R ₃ is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloakenyl, cyclo Alkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloacynyl, NH ₂ -alkylamino-ketoxyalkyloxy-aryl, mono and dialkylaminoacyl-N-acylamino-SO ₂ aryl to be.

In the absence of fractions with the desired T content, or when the desired fraction contains the T content, the inventors have reduced the adenosine content of the antisense oligod corresponding to the thymidines (T) in the target RNA, A less than about 15% or completely removed A from the nucleotide sequence, and in this way, their degradation products can freely move adenosine around the lung tissue, making the patient's disease worse or reducing the useful effects of the administered agent. It was not allowed.

By the method of the example, the NfκB transcription factor can be selected as the primary target and the destimidine (desT) fractions can be found. When numerous desTs have been found, antisense fractions can be inferred and perhaps about 20 or more desA antisense sequences can be obtained. Such antisense sequences represent, if possible, all desA antisense sequences found in the mRNA of the primary target and are homologous in a preferred list of secondary targets such as those shown in Table 1 or Table 2 above or in sequence data bases such as GENBAND. It can be used to start searching for sexual sequences. For each of about 20 native desA antisense sequences found for the NFκ transcription factor, typically about 10 to 30 homologous sequences can be found among other elements of the group shown in Table 1 above (secondary). , Tertiary, such a target). In some instances, search may be performed to find not only the secondary target (homologous between the sequence from the primary target and other target mRNAs) but also the tertiary target (eg, sequences from three different target mRNAs and 1 Homogeneity between primary targets) and homologous to primary targets. However, the latter case is extremely rare. If this occurs, it can be said that the antisense oligos found are 100% homologous. However, the sequences found contain several nucleotides that are not homologous at all in the secondary, tertiary, or quaternary sequences. In many cases, this mismatch can cause the antisense oligonucleotides to be sufficiently less active or not active at all to the target. In some cases, the presence of one nonhomologous nucleotide is sufficient to reduce the activity of the antisense oligonucleotide. When so-called "homologous" sequences are mismatched, up to 40%, preferably up to about 30%, more preferably up to 20%, more preferably up to 10% mismatched sequences can be tolerated. In some cases, higher% discrepancies are tolerated,

Non-homologous nucleotides are "common" that can pair bases with similar or identical affinity to two or more of the four bases (A, G, T, and C) present in natural DNA. Oligos are still active because they can be "fixed" or replaced with bases. The “fixing step” additionally creates a new sequence. The sequence differs from that found in nature and allows the antisense oligonucleotides to bind, preferably equally well, primary target, secondary target, tertiary target, and the like.

When the NfκB transcription factor is selected as the target, its mRNA or DNA is searched for low thymidine (T) or destimidine (desT) fragments. Only the desT fraction of the mRNA or DNA to produce desA antisense in turn as the complementary strand is selected. When numerous RNA desT fractions are found, the sequence of the antisense fraction is inferred. Typically, about 10-30 and even more desA antisense sequences can be obtained. Such antisense sequences may include some or all desA antisense oligonucleotide sequences corresponding to the desT fraction of the target mRNA, as shown in Table 1 or Table 2 below. When this occurs, it can be said that the antisense oligonucleotides found are 100% A free. For each of the original desAs, an antisense oligonucleotide sequence of about 10 to 30 sequences corresponding to a target gene, eg, an NFκB transcription factor, can be found in the target gene or RNA with a small amount of thymidine (RNA). In accordance with the present invention, selected fragment sequences may also comprise a small number of thymidine (RNA) in the secondary, tertiary or quaternary sequence. In some cases, high adenosine content may cause the antisense oligonucleotide FP nucleotides to be less activated or even inactivated at the target. According to the invention, these so-called 'overall desA' sequences preferably have an adenosine content of less than 15%, more preferably less than 10%, more preferably less than 5%, even 2 It is preferable to have smaller than%. In some cases higher adenosine content is tolerated, oligonucleotides are still active, in particular where adenosine nucleotides are similar or identical to two or more of the four nucleotides (A, G, T, and C) present in nature It can be repaired or substituted with 'normal' bases that can be base paired with afiniy. General bases have the ability to hybridize with thymidine, and are preferably defined in this patent as any compound, more generally adenosine analog, that reduces or lacks substantially the ability to bind adenosine receptors. In addition, adenosine analogs can be used that prevent the activation of adenosine receptors such as adenosine A ₁ , A _2b , and / or A ₃ receptors, most preferably A ₁ receptors. One example of a common base is α-diosi ribofuranosol- (5-nitroindole), and experts know how to choose other examples. This calibration step, which allows the antisense oligonucleotides to bind the target RNA, preferably identically, results in a newer sequence. An example of a common base is 2-dioxyribosyl- (5-nitroindole). Other examples of common bases include 3-nitropyrrole-2'-dioxynucleoside, 5-nitro-indole, 2-dioxyribosyl- (5-nitroindole), 2-dioxyribofuranolsil- (5-nitroindole ), 2'-deoxyinosine, 2'-deoxynebulin, 6H, 8H-3,4-dihydropyrimido [4,5c] oxakin-7-one and 2-amino-6-mesoxy Aminopurine. In addition to the above, common bases having reduced hybridization capacity but which can be substituted with another base are 3-nitropyrrole 2'dioxynucleoside, 2-dioxyribofuranosyl- (5-nitroindole), 2'deoxyinosine and 2'deoxynebulaline and the like (Glen Research, Sterling, VA). More specifically, the mismatch repair is 6H, 8H-3,4-dihydropyrimido [4,5c] [1,2, which is a base pair "P" nucleoside with guanine (G) or adenine (A) Axakin-7-one, and 2-amino-mesoxyaminopurine, which is a "K" nucleoside base paired with cystidine or thymidine. Things are also suitable. See, eg, Loakers, D. and Brown, DM, Nucl. Acids Res. 22: 4039-4043 (1994); Ohtsuka, E. et al., J. Biol. Chem. 260 (5): 2605-2608 (1985); Lin, PKT and Brown, DM, Nucleic Acids Res. 20 (19): 5149-5152 (1992); Nichols, R. et al., Nature 369 (6480): 492-493 (1994); Rahman, MS and Humayun, NZ, Mutation Research 377 (2): 263-268 (1997); Amosova, O., et al., Nucleic Acids Res. 25 (10): 1930-1934 (1997); Loakes D. & Brown, DM, Nucleic Acids Res. 22 (20): 4039-4043 (1994), the entire field of general bases and their preparation and their use in nucleic acid binding is shown in this reference.

When fully desT sequences are not naturally found at the target, they are typically selected so that a general base substitution of about 1 to 3 results in 100% "desA" antisense oligonucleotides. Thus, the method provides antisense oligonucleotides and other antisense nucleotides for one or more nucleotides, such as about 1 to 3, or more, to other targets with little or no A content, and have been calibrated by being replaced with "common" bases. General bases are known in the art and need not be listed here. The skilled person will know which base can be used as a general base and a substituent of A.

This approach in the production of antisense oligonucleotides has been shown to include several inflammatory diseases including cystic fibrosis, chronic obstructive pulmonary disease, chronic bronchitis, and bronchial hippertensin and breast cancers that have metastasized to the lungs, colon cancer, respiratory distress syndrome, prostate cancer, It can also be used for a variety of other diseases and pains, including cancers including cancers of pancreatic cancer, kidney cancer, lymphoma, melanoma, hepatocellular carcinoma and the like.

As used herein, the term "treatment" or "treating" of asthma or other respiratory and inflammatory diseases or diseases relates to a therapy in which patients receiving such therapy are less likely to exhibit respiratory or inflammatory lung disease or other lung pain. The term “down regulate” relates to inducing a decrease (ie, a decrease in concentration) in the production, secretion, or availability of a targeted intracellular protein.

The present invention is primarily directed to vertebrates, mammals, including human and non-human apes in their class, wild and livestock animals, aquatic and terrestrial animals, pet dogs, and zoo animals, including, for example, cats, dogs, horses, horns, and whales. With interest, most preferably human patients. One particularly suitable application of the present technology relates to veterinary medicine and includes all kinds of small and large animals such as wild animals, aquatic animals, livestock animals, zoo animals, and the like. The targeted genes and proteins are preferably of mammals, and the targeted sequences are of the same kind as those of the patient to be treated. Although there are many examples, most preferably at least 95% homogeneity of other species of target RNAs or genes exhibits at least 45% homology with the sequence of the agent, more preferably at least 85% homology. It is also suitable. A preferred class of agents is desA. Another preferred class also includes oligonucleotides that are not completely desA and one or more adenosine bases are substituted with regular bases.

In general, the term “antisense” relates to small, synthetic oligonucleotides and is applied in this patent to inhibit gene expression by inhibition of target messenger RNA (mRNA) (see Milligan, JF et al., J. Med. Chem. 36 (14), 1923-1937 (1993), see relevant portion thereof). Adenosine A ₁ , A _2a , A _2d , or A ₃ receptor, CCR3 (compound receptor 320, also known as eotaxin receptor), VCAM (vascular cell adsorption molecule), unophil receptor, bridykinin 2B receptor, and the above This agent inhibits gene expression of target genes, such as many others listed in Table 1. As is known in the art, this is generally obtained by hybridization with the coding (sense) sequence of the antisense oligonucleotide and the targeted messenger RNA (mRNA). Administration of the agent of the invention extracellularly reduces the level of mRNA and protein encoded by the target gene and / or causes changes in the growth properties or shape of the administered cells (see Milligan et al. ( 1993); helene, C. and Toulme, J. Biochim. Biophys.Acta 1049, 99-125 (1990); Cohen, JSD, Ed., Oligodeoxynucleotide as anti-sense Inhibition of Gene Expression; CRC Press: Boca RAton, FL (1987), see the relevant part of this). As used herein, an "antisense oligonucleotide" generally refers to (1) forming a hybrid with a fraction of an mRNA encoding a protein of interest in an appropriate hybridization environment, and (2) the amount of gene expression of a protein targeted by hybrid formation. Short sequence of synthetic nucleotides nucleotides.

The terms "des-adenosine" (desA) and "des-thymidine" (desT) refer to oligonucleotides that are substantially free of adenosine (desA) or thymidine (desT). In some cases, the desT sequence is formed naturally and in other cases is made by substituting an unwanted nucleotide (A) for something that is not active. As is known in the art, the substitutions herein are generally made by substituting A with a general base.

The mRNA of the target protein can be derived from the nucleotide sequence of the gene that expresses the protein. By way of example, the sequences of adenosine A ₁ receptors of human genes and the sequences of adenosine A ₃ receptors of mice and humans are known (see US Pat. No. 5,320,962; Zhou, F., et al., Proc. Nat'l). Acad. Sci. (USA) 89: 7432 (1992); Jacobson, MA, et al., UK Pat. Appl. No. 9304582.1). The sequence of the adenosine A _2b receptor gene is known (see, for example, Salvatore, CA, Luneau, CJ, Johnson, RG and Jacobson, M., Genomics (1995), relevant portions thereof). The sequences of many exemplary target genes are also known (see GenBank, NIH). Sequences of genes for which such sequences are not yet available can be obtained by separating the unfolded fractions by applying techniques known in the art. Once the sequence of a gene, its RNA, and / or protein is known, antisense oligonucleotides can be made according to the present invention above and can reduce expression of the target protein according to standard techniques.

In one aspect of the invention, the antisense oligonucleotides comprise a sequence that specifically binds to a portion or fraction of an mRNA molecule encoding a protein associated with respiratory failure, pulmonary inflammatory response, airway obstruction, bronchitis, and such diseases or pains. Have The effect of this binding can reduce or even prevent the transfer of the corresponding mRNA and reduce the available amount of target protein in the lungs of the patient.

In a preferred embodiment of the invention, the phosphodiester residues of the antisense oligonucleotides are modified or substituted. Oligonucleotides of modified or substituted phosphodiesters as in the example of methylphosphonates, phosphoresters, phosphorothioates, phosphorodithioates, or phosphoramidates which increase the stability of oligonucleotides in vivo Analogue chemicals of are particularly preferred. The phosphodiester linkage of naturally occurring oligonucleotides is susceptible to degradation by three nucleases in the cell. In contrast, many of the many residues proposed here have high resistance to nuclease degradation. (See Milligan et al., And Cohen, J. S. D., supra). In another preferred embodiment of the invention, the degradation is prevented by adding a "3'-terminal cap" to the oligonucleotide to replace the phosphodiester linkage at the 3 'end of the oligonucleotide with a nuclease-resistant linkage. (See Tidd, DM and Warenius, HM, Be. J. Cancer 60: 343-350 (1989); Shaw, JP et al., Nucleic Acids Res. 19: 747-750 (1991), here. See the relevant section). Phosphoramidate, phosphorothioate, and medylophosphonate linkages can all fully function in this manner for the purposes of the present invention. Expanding and modifying the phosphodiester backbone results in more stable agents formed and, in many instances, more affinity and cell permeability of RNA (see Milligan, et al., Supra). Thus, the number of residues that may be modified may vary, depending on the needs, targets, and route of administration, and may be any number from 1 to all residues, or in between. Many other methods are known for converting a complete phosphodiester framework into a new linkage (see Millikan et al., Supra). Airbone analog residues are phosphorothioate, methylphosphonate, phosphorus ester, thioformacetal, phosphorodithioate, phosphoramidate, formacetal, boranophosphate, 3'-thioformacetal, 5'- Thioether, carbonate, 5'-N-carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, 2'-0 methyl, sulfoxide, sulfide, hydroxyamide ??, methylene It is preferred to include mino) (MMI), and methyleneneo (methylimino) (MOMI) residues. Although oligonucleotides are synthesized automatically, phosphorothioate and methylphosphonate-modified oligonucleotides are particularly preferred for their availability (see Milikan et al., Supra). Suitably, the agents of the present invention may be administered in the form of a pharmaceutically acceptable salt and a mixture of antisense oligonucleotides and salts thereof.

Agents of the invention have the ability to diminish the expression of one target mRNA and / or have the ability to induce or diminish the activity of one pathway. By way of example, more preferably, the fraction of deoxynucleotides low in thymidine (T) or adenine (A) as the target mRNA or gene is at least about 7 mononucleotides, preferably up to about 60 mononucleotides Can be used in the method by identifying all possible deoxyribonucleotides that are mononucleotides from about 10 to 36, most preferably from about 12 to about 21 mononucleotides. This can be accomplished by searching for mononucleotides in the target sequence with low or no thymidine (RNA), nucleotides complementary to adenosine, or nucleotide lengths of 7 or more with low adenosine (genes). In most cases, this search results in a sequence of about 10 to 30 sequences, typically of less than about 40% naturally or lacking, and of various lengths of antisense oligonucleotides of typical target mRNAs of about 1800 nucleotides in length. appear. Those with a high content of T or A were each corrected by substituting one or more A's for a common base.

The agent of the present invention may also be of different lengths, depending on the particular target and transport method, but the agent of the present invention is not limited but may be from about 7 to about 60 nucleotides in length, preferably from about 12 to about 45 in length, more preferably Preferably up to about 30, most preferably up to about 21 in length. Agents of the invention can refer to any and all target RNA fractions. One preferred class of agents includes those that are mRNA regions comprising pointing junctions between introns and axons. When an agent points to an intron / axon gap, it is fully placed or close enough to its junction, eg, about 2 to 10, preferably 3 to 5, in the 3 'or 5' end of the antisense oligonucleotide Located in the nucleotide nucleotide of the axon junction, it inhibits splicing away of the axon that is interposed during the process of the precursor mRNA to the mature mRNA. Also preferred are antisense oligonucleotides comprising near the 5 'and 3' ends of the initiation codon and coding region.

Thus, this multi-target antisense (MTA) oligonucleotide approach applies to a variety of diseases and pains, including other inflammatory reactions such as cystic fibrosis, chronic obstructive pulmonary disease, organ bronchitis and the like. Other specific diseases and pains to which this technique is effectively applied include lung hypertensin and cancer.

Table 2 below shows a number of targets to which multi-target antisense (MTA) oligonucleotides can be effectively applied. Others can also be targeted.

Table 2: Cancer Targets

Transforming Oncogene Therapeutic target rassrcmycbcl-2 Thymidylate synthetase thymidylate synthetase dihydrofolate reductase thymidine kinase dioxycytidine kinase ribonucleotide reductase

A class of targets for cancer treatment are genes related to other forms of cancer, including those known to be associated with malligence, generally including the production of regulators or RNA and / or proteins. Examples include, but are not limited to, transgenic oncogenes such as ras, src, myc, and bcl-2. While other targets are not absolute, other targets indicate that cancer's chemotherapeutic agents preferentially vary, such as dimidylate synthetase, dihydrofolate reductase, thymidine kinase, dioxystidine kinase, ribonucleotide reductase. These can point to enzymes. While therapies such as chemotherapy and radiation therapy prevent the destruction of normal living cells, conventional cancer therapies face an unsolved problem of selectively killing cancer cells, so the present technology is very important. The technology has the ability to simultaneously reduce or induce multiple paths. This approach has significant benefits because it allows a mixed selection of multiple pathways, including primary, secondary and possibly tertiary targets, which are not simultaneously expressed in normal cells. Thus, the agents of the present invention, for example, can be administered to a patient to increase toxicity only in tumor cells that have expressed all three types of targets, and in normal cells, only one or two targets are expressed and are significantly less affected or Even most survive.

A class comprising a target gene, a contiguous region, a starting predominant point, an intron-axon border, and RNA complementary thereto, or an mNRA coding region, an uncoding RNA fraction, a 5'-end and a 3'-end, eg Complete sequences of precursor RNAs, including poly-A fractions and oligos that target a juxta-section between coding and non-coding regions, and proteins known to be associated with one or more diseases or disorders or mixtures thereof Agents comprising antisense oligonucleotides for two or more mRNAs selected from the coding RNAs are provided herein.

The agent administered according to the invention is preferably designed with a target gene and / or antisense associated with the species of origin to be administered. When treated in humans, the agent is preferably designed with antisense of a human gene or RNA. Agents of the invention are oligonucleotides that are naturally occurring DNA and / or RNA sequences, of which one (1) base is less than the targeted sequence, preferably fragments having a length of 7 nucleotides, about 0.02%, Preferably up to about 0.01%, more preferably up to about 1%, more preferably up to about 4%, having adenosine nucleotides up to about 30%, more preferably up to 15%, more preferably up to 10% And most preferably oligos having 5% adenosine nucleotides or lacking adenosine, and oligos in which one or more adenosine nucleotides are converted into so-called general bases to form base pairs with thymidine but tentatively induce adenosine receptor activity. . Examples of human sequences and fragments for the antisense oligonucleotides of the present invention include, but are not limited to, the entire genes preferably comprising 7, 10, 15, 18 to 21, 24, 27, 30, n-1 or A fragment of a sequence, an axon, and a fragment of the intron-axon junction and a shorter fragment that encodes the sequence, where n is the total number of nucleotides of the sequence. Middle, 5'-terminus, 3'-terminus, or the beginning of any other portion of the original sequence. It is important that the fragments have a low adenosine nucleotide content, and these fragments are less than 30%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, most preferably Can be made free of adenosine content, either selectively or by conversion to a common base in accordance with the present invention. Agents of the invention include the most preferred sequence classes and fragments replaced by a general base (B), as in the example where there is one or more adenosine in the sequence. Similarly, it includes fragments designed by substituting B for adenosine and also includes all shorter fragments of shorter B-containing fragments included in such sequences, fragments or fractions above. It is shown in. Some examples of antisense oligonucleotide sequence fragments target the initiation codon of each gene, and in some cases adenosine is substituted with a generic base adenosine analogue, denoted as "B," to bind the ability to bind adenosine A1 and / or A3 receptors. Scarcely. Many of the examples below show one such sequence and many fragments comprising an initiation codon, wherein the number n of nucleotides is about 7, about 10, about 12, about 15, about 18, about 21 and up to about 28, about 35, about 40, about 50, about 60 is preferred.

In one embodiment, at least one of the mRNAs targeted by the MTA oligos of the invention is a transcription factor, a facilitating and activating factor, an intracellular and extracellular receptor and a general peptide transmitter, interleukin, interleukin receptor, chemokine, chemokine receptor, endocrine specific and Nonspecific enzymes, immunoglobulins, antibody receptors, central nervous system (CNS) and peripheral and non-nerve receptors, CNS and peripheral and non-nerve peptide transmitters, adsorption molecules, defensins, growth factors, vasoactive peptides and receptors, and binding Proteins, such proteins; Or mRNA corresponds to an oncogene and other genes associated with various diseases and disorders. Examples of target proteins include, in particular, eotaxin, major base protein, preproendodylin, eusinofil cationic protein, P-selectin, STAT 4, MIP-1α, MCP-2, MCP-3, STAT 6, c- mas, NF-IL-6, cyclophylline, PDG2, cyclosporin A-binding protein, FK5-binding protein, fibronectin, LFA-1 (CD11a / CD18), PECAM-1, C3bi, PSGL-1, CD-34 , Southernton P, p150, 95, Mac-1 (CD11b / 18), VLA-4, CD-18 / CD11a, CD11b / CD18, C5a, CCR1, CCR2, CCR4, CCR5, and LTB-4. However, others may also be suitable examples.

In another embodiment, at least one of the mRNAs targeted by the MTA oligo is in particular sympathetic stimulating receptors, parasympathetic receptors, GABA receptors, adenosine receptors, bradykinin receptors, insulin receptors, glucagon receptors, prostaglandin receptors, thyroid receptors, androgens Intracellular and extracellular receptors and peptide delivery, such as receptors, anabolic receptors, estrogen receptors, progesterone receptors, receptors associated with coagulation cascades, adenohypophysiological receptors, adenohypophysiological peptide transmitters, and histamine receptors (HisR) Code the substance. But there may be others as well. Coded sympathetic and parasympathetic receptors include acetylcholinesterase receptors (AcChaseR), acetylcholine receptors (AChR), atropine receptors, muscarinic receptors, epinephrine receptors (EpiR), dopamine receptors (DOPAR), and nordose Epinephrine receptor (NEpiR) and the like. Examples of more encoding receptors are adenosine A ₁ receptor, adenosine A _2b receptor, adenosine A ₃ receptor, endoderin receptor A, endoderin receptor B, IgE high affinity receptor, muscaric acetylcholine receptor, substance P receptor, histamine receptor , CCR-1 CC chemokine receptor, CCR-3 CC chemokine receptor (Eotaxin receptor), interleukin-1β receptor (IL-1βR), interleukin-1 receptor (IL-1R), interleukin-1β receptor (IL -1βR), interleukin-3 receptor (IL-3R), CCR-4 CC chemokine receptor, cysteininyl leukotriene receptor, prosteinoid receptor, GATA-3 transcription factor receptor, interleukin-1 receptor (IL-1R ), Interleukin-4 receptor (IL-4R), interleukin-5 receptor (IL-5R), interleukin-8 receptor (IL-8R), interleukin-9 receptor (IL-9R), interleukin-11 receptor (IL-11R) ), Bradykinin B2 receptor, sympathetic stimulating receptor, parasympathetic stimulating receptor, GABA receptor, adenosine Solution, Bradykey receptor, insulin receptor, glucagon receptor, prostaglandin receptor, thyroid receptor, androgen receptor, anabolic receptor, estrogen receptor, progesterone receptor, receptor associated with coagulation cascade, adenohypophysiological receptor, adenohypophobic Ear peptide transmitters, and histamine receptors (HisR). There may also be others not listed here.

Enzymes encoded for the development of the MTA oligo in the present invention are synthetase, kinase, oxidase, phosphatase, reductase, polysaccharide, triglyceride, and protein hydrolase, esterase, erra Staases, and polysaccharides, triglycerides, lipids, protein syntheses, and the like. Examples of target enzymes include tryptase, induced nitric oxide synthase, cyclooxygenase (Cox), map kinase, eusinphil peroxidase, β2-adrenergic receptor kinase, leukotrien c-4 Synthase, 5-lipooxyase, phosphodiesterase IV, metalloproteinase, tryptase, CSBP / p35 map kinase, neutrophyll elastase, phospholipase A2, cyclooxynase 2 (Cox -2), fucosyl transferases, chimases, frontein kaanases C, thymitylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, and Ribonuclease reductase. However, enzymes involved in illness or pain are suitable as targets of the present invention.

Suitable encoding factors for the present application include, in particular, the NfκB transcription factor, granulosite macrophage colony stimulating factor (CM-CSF), AP-1 transcription factor, GATA-3 transcription factor, monosite activator, Neutrofil Commotitatic factor, granulosite / macrophage colony-forming-factor (G-CSF), NFAT transcription factor, platelet activating factor, tumor necrosis factor α (TNFα), and basic pipeloblast growth factor (BFGF) There is this. Although not mentioned in detail, there may be other factors in the present invention. Suitable adsorption molecules used in the present invention include intracellular adsorption molecules 1 (ICAM-1), 2 (ICAM-2), and 3 (ICAM-3), vascular cell adsorption molecules (VCAM), endothetical leucosite adsorption molecules- 1 (ELAM-1), Neutrophil Adsorption Receptor, and CAM-1, and the like. Other known and unknown perceptions (at this point) may also be targeted herein. Interleukin-1 (IL-1), Interleukin-1β (IL-1β), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Interleukin-5 among cytokines, lymphokines and chemokines (IL-5), interleukin-8 (IL-8), interleukin-8 (IL-8), interleukin-11 (IL-11), CCR-5 CC chemokines, and Rantes are preferred. . However, other diseases known to be included for the specific disease or disorder to be treated or for generic activity such as inflammation can also be targeted. Examples of defensins for use of the invention include defensin 1, defensins 2, and defensins 3, α4β1 selectin, α4β7 selectin, LFA-selectin, E-selectin, P-selectin, L-selectin as the selectin. Examples of oncozin, but not all, are ras, src, myc, and bcl-2r kdlT. However, others are also suitable for use in the present invention.

Agents to be administered according to the invention are preferably designed with antisense to target genes and / or mRNA related to the same species as the species to be administered. When treated in humans, the agent is preferably designed with antisense to human genes or RNA. Agents of the invention are oligonucleotides that are naturally occurring DNA and / or RNA sequences, of which one (1) base is less than the targeted sequence, preferably fragments having a length of 7 nucleotides, about 0.02%, Preferably up to about 0.01%, more preferably up to about 1%, more preferably up to about 4%, having adenosine nucleotides up to about 30%, more preferably up to 15%, more preferably up to 10% And most preferably oligos having 5% adenosine nucleotides or lacking adenosine, and oligos in which one or more adenosine nucleotides are converted into so-called general bases to form base pairs with thymidine but tentatively induce adenosine receptor activity. . Examples of human sequences and fragments for the antisense oligonucleotides of the present invention include, but are not limited to, the entire genes preferably comprising 7, 10, 15, 18 to 21, 24, 27, 30, n-1 or Fragments of sequences, axons, and indron-axon junctions encoding shorter sequences and shorter fragments, where n is the total number of nucleotides in the sequence. From the part, it is selected from the middle, 5'-end, 3'-end, or the beginning of any other part of the original sequence. It is important that the fragments have a low adenosine nucleotide content, and these fragments are less than 30%, more preferably less than 15%, more preferably less than 10%, more preferably less than 5%, most preferably Can be made free of adenosine content, either selectively or by conversion to a common base in accordance with the present invention. Agents of the invention include the most preferred sequence classes and fragments replaced by a general base (B), as in the example where there is one or more adenosine in the sequence. Similarly, it includes fragments designed by substituting B for adenosine and also includes all shorter fragments of the shorter B-containing fragments included in such sequences, fragments or fractions above. It is shown below.

Some examples of antisense oligonucleotide sequence fragments target the initiation codons of each gene, and in some cases adenosine is replaced with a common base adenosine analog, denoted as "B," to bind the ability to bind adenosine A1 and / or A3 receptors. Scarcely. Many of the examples below show one such sequence and many fragments comprising an initiation codon, wherein the number n of nucleotides is about 7, about 10, about 12, about 15, about 18, about 21 and up to about 28, about 35, about 40, about 50, about 60 is preferred.

Human Receptor-Related Antisense Polynucleotides

Human Enzyme-Related Antisense Polynucleotides

Human Factor Related Antisense Oligonucleotides

Human Adenosine A ₁ Receptor Antisense Oligonucleotide Fragments

Human Adenosine A2a Receptor Antisense Oligonucleotide Fragments

Human Adenosine A2b Receptor Antisense Oligonucleotide Fragments

Human Adenosine A3 Receptor Antisense Oligonucleotide Fragments

Human IgE Receptor β Antisense Oligonucleotide Fragments

Human Fc-ζ Receptor CD23 Antigen (IgE Receptor)

Antisense Oligonucleotide Fragments

Human IgE Receptor α Antisense Oligonucleotide Fragments

Human IgE Receptor (Fc Epsolon R) Antisense Oligonucleotide Fragments

Human histidine decarboxylase antisense oligonucleotide fragments

Human Beta Tryptase Antisense Oligonucleotide Fragments

Human Tryptase-I Antisense Oligonucleotide Fragments

Human Prostagladin-D Synthase Antisense Oligonucleotide Fragments

Human Cyclooxygenase-2 Antisense Oligonucleotide Fragments

Human eosinophil cationic protein antisense oligonucleotide fragments

Human eosinophil-induced neurourotoxin antisense oligonucleotide fragments

Human eosinophil peroxidase antisense oligonucleotide fragments

Human Intracellular Adenosine Molecule-1 (ICAM-1)

Antisense Oligonucleotide Fragments

Human Vascular Cell Adenosine Molecule-1 (VCAM-1) Antisense Oligonucleotide Fragments

Human endothelial leukocyte adsorption molecule

(ELAM-1) Antisense Oligonucleotide Fragments

Human P Selectin Fragments

Human endothelial monocyte activator antisense oligonucleotide fragments

Human IL3 ^* Antisense Oligonucleotide Fragments

Human IL3 Receptor Antisense Oligonucleotide Fragments

Human IL-4 Receptor Antisense Oligonucleotide Fragments

Human IL-4 Receptor Antisense Oligonucleotide Fragments

Human IL5 ^* Receptor Antisense Oligonucleotide Fragments

Human IL-5 Receptor Antisense Oligonucleotide Fragments

Human IL-6 Receptor Fragments

Human IL-6 Antisense Oligonucleotide Fragments

Human monocyte-induced neutrophil chemotactic factor antisense oligonucleotide fragments

Human neutrophil elastase (medulacin) factor antisense oligonucleotide fragments

Human neutrophil oxidase factor antisense oligonucleotide fragments

Human Kadeepsin G Antisense Oligonucleotide Fragments

Human Defensin 1 Antisense Oligonucleotide Fragments

Human Defensin 3 Antisense Oligonucleotide Fragments

Human Macrophage Inflammatory Protein-1 Alpha / Rantenz Receptor Factor Antisense Oligonucleotide Fragments

Lantents antisense oligonucleotide fragments

Human muscaric acetylcholine receptor HM1 ^* antisense oligonucleotide fragments

Human muscaric acetylcholine receptor HM3 ^* antisense oligonucleotide fragments

Human Fibronectin ^* Antisense Oligonucleotide Fragments

Human Interleukin-8 ^* Antisense Oligonucleotide Fragments

Human IL-8 Receptor Alpha Antisense Oligonucleotide Fragments

Human GM-CSF Antisense Oligonucleotide Fragments

Human Tumor Necrosis Factor α Antisense Oligonucleotide Fragments

Human leukotriene C4 synthase Antisense Oligonucleotide Fragments

Human Endoserine-1 Antisense Oligonucleotide Fragments

Endoserine Receptor ET-B Antisense Oligonucleotide Fragments

Endoserine ETA Receptor Antisense Oligonucleotide Fragments

Substance P Antisense Oligonucleotide Antisense Oligonucleotide Fragments

Substance P Receptor Antisense Oligonucleotide Fragments

Cimaze antisense oligonucleotide antisense oligonucleotide fragments

Endothelial Nitric Oxide Synthase Antisense Oligonucleotide Fragments

Inducible Nitric Oxide Synthase Antisense Oligonucleotide Fragments

NF-kB antisense oligonucleotide fragments

Human major basic protein antisense oligonucleotide fragments

Human eosinophil major basic protein fragments

BP-1 Antisense Oligonucleotide Fragments

C / EBPβ Antisense Oligonucleotide Fragments

Here, B is an adenosine or adenosine changed and a general base, and it is more preferable that it is an adenosine A _2a receptor agonist, an adenosine A _2b antagonist, an adenosine A ₃ receptor antagonist, or an adenosine A ₁ receptor antagonist.

Bradkinin Receptor Antisense Oligonucleotide Fragments

In a preferred embodiment, the linkage between neighboring mononucleotides f nucleotides is a phosphodiester linkage. In another preferred embodiment, at least one mononucleotide phosphodiester moiety of the antisense oligonucleotide (s) is methylphosphonate, phosphoroester, phosphorothioate, phosphorodithioate, boranophosphate, form Acetal, thioformacetal, diether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, 2'-0-methyl, methylene Imino), methyleneoxy (methylimido), phosphoramidate residues, and combinations thereof. MTA oligos having one or more phosphodiester moieties substituted with one or more other moieties are those whose residues are more resistant to hydrolysis than the phosphodiester moieties and thus last longer. In some cases, about 10%, about 30%, about 50%, about 75%, and even all phosphodiester residues (100%) may be completely substituted. Typically, multiple target antisense oligonucleotides (MTA oligos) of the invention comprise about 7 mononucleotides, in some cases up to 60 and more mononucleotides, preferably about 10 to about 36, more preferably about 12 to about 21 mononucleotides. However, depending on the length of the target polymer, other lengths may also be suitable. Examples of MTA oligos of the invention are shown in Table 3 below and comprise 96 sequences. (SEQ ID NOS: 2317 to 2411).

TABLE 3A MTA Oligos, Targeted Locations and Targets

TABLE 3b MTA Oligos, Targeted Locations and Targets

TABLE 3c MTA Oligos, Targeted Locations and Targets

TABLE 3d MTA Oligos, Targeted Locations and Targets

MTA Oligo Table 3 is suitable for use with two or more targets listed in Table 4 below.

Table 4: Targets of the MTA Oligos in Table 3

compound Target EPI 2010 Adenosine A ₁ Receptor EPI 2045 Adenosine A ₃ Receptor EPI 2873, EPI 2193 NFκB EPI 1873 Interleukin-1 EPI 1857 Interleukin-5 EPI 2945 Interleukin-4 EPI 2977 Interleukin-8 EPI 2031 5-lipoxygenase EPI 1898 Rukotriene C-4 Synthase EPI 1856 Eotaxin EPI 1131 ICAM EPI 1085 VCAM EPI 2085 TNFα EPI 1908 PAF EPI 1925 IL-4 receptor EPI 2643 β ₂ adrenergic receptor kinase EPI 2934 Tryptase EPI 2033 Main basic protein EPI 2795 Eosinophil peroxidase NfκB: nuclear factor κB (ICAB) ICAM: intracellular adhesion molecule VCAM: vascular cell adhesion molecule TNF: tumor necrosis factor PAF: platelet activator (platelet) activating factor)

In most preferred embodiments used in the lung, the invention of this MTA oligo is one or more of the overall base sequences consistent with the invention and antisense in the natural desthymidine sequence in the desadenosine oligonucleotide. It includes by substitution. Methods of substitution of nucleotide sequences, oligonucleotide synthesis of specific nucleotide sequences, and which of the bases to borrow are known from art and are known in artisan and need no longer be mentioned here. A more detailed example of an agent of the invention and an MTA oligo are linked to a molecule that is received by an agent or an actively living cell. In this way the absorption of the inventive agent is enhanced as is known in art. Examples of agents or molecules suitable for the use of MTA oligos in this invention are transferrin, asialoglycoprotein, and steptavidin. Others are of course appropriate.

The agents herein are of antisense nucleotide formulations, including those that have sufficient effect of inhibiting the expression of target mRNAs, and specifically bind to target mRNAs in cells or through cell membranes to inhibit the transfer of mRNA. Passing through is another aspect of this invention. Such configurations are provided by suitable pharmaceutical carriers, such as sterile pyrogen-free saline solutions. Agents of the invention can be synthesized with hydrophobic carriers that can cross cell membranes, such as liposomes, which can be pharmaceutically transported by carriers. The oligonucleotides may be disrupted with mRNA, such as inactivated ribozymes. Such oligonucleotides may be operated by the need for a role that inhibits the action of specific receptors, enzymes and / or proteins, and / or elements. Pharmaceutical synthesis also includes chemical molecules including antisense oligonucleotides attached to molecules known to be accepted by the cell. Such oligonucleotide complexes utilize cell receptor mechanisms to increase the intracellular concentration of oligonucleotides. Examples of molecules used in such methods include macromolecules, including transferrin, asialoglycoprotein and streptavidin. Antisense compounds belong to pharmaceutical compounds that contain vesicles of lipids such as liposomes or microcrystals. These particles are suitable for structures such as uni-lamellar and plural lamellae. In one preferred embodiment, antisense oligonucleotides are included in the vesicles. Positively polarized lipids such as, for example, N- [1- (2,3-dioleoyloxi) propyl] -N, N, N-trimethylammoniumethylsulfate or "DOTAP" are particularly desirable for such particles or vesicles. Others are also suitable. Preparations for these lipid particles are well known. (See, US Patent Nos. 4880635 to Janoff et al., 4906477 to Kurono et al., 4911928 to Wallach, 4917951 to Wallach, 4920016 to Allen et al., 4921757 to Wheatley et al. All of them are incorporated here by reference.)

The combination of this invention can be adjusted by any method of delivering the agent to the lungs. Current agents can be adjusted to the patient's lungs by any other suitable method, but are preferably through the respiratory tissue as a form suitable for breathing, and more preferably in the form of an aerosol that breathes less particles or comprises an inhalant. Inhalants are in the form of gases, liquids or solids and they contain additives or preparations that are beneficial to health as needed. The particles of the invention are small enough to be sized enough to pass through the breath or pass through the mouth and larynx, ascites, the alveoli of the lungs. Alternative diameters for respiration of the particles are on the order of 0.5 to 10 microns. But other sizes are possible. Particles of a relatively large diameter contained in the respirator that cannot be absorbed by the respirator accumulate in the neck and are swallowed. Therefore, it is desirable to minimize the amount of particles of a size that cannot be absorbed into the lungs. Particle sizes on the order of 10-500 micrometers are preferred for the induction of particles in the nose.

Sterile pyrogen-deficient water or other known suitable pharmaceutical carriers, such as, for example, aerosols with antisense oligos combined with suitable carriers, for the production as a respiratory agent. There is. Other pharmaceutical compounds as well as other ingredients known in the art are included. Components of solid particulates comprising respirable dried particles, such as miniaturized agents in the invention, are prepared by pulverizing dried antisense compounds with a mortar and pestle, followed by a synthesis such as 400mesh screen for crushing large chunks of particles. Pass the membrane. The composition of the solid particles comprising the antisense composite may also include other agents and dispersants known to play a role in inducing the formation of aerosols or mists. A suitable dispersant is lactose, which is mixed with the antisense compound in an appropriate proportion of about 1: 1 by mass. Other ratios are also used and include other pharmaceutical compounds and particle formers. Aerosols of liquid particles containing agents may be produced by methods such as insufflators or nebulizers. (See US Patent No. 4501729.) Sprays may also be used to commercialize a solution that converts the suspension of an aqueous solution or agent into a mist of aerosols, or alternatively to accelerate air or oxygen compressed gas, for example by narrow venturi tubes or ultrasonic stirrers. It is possible. Suitable agents for the use of sprays (insufflator or nebulizers), suitable synthesis comprising this invention, are preferably made into body components and isotonic solutions by adding salts and the like, with a water-soluble alcohol solution or a liquid carrier of typical water in less than 0.01% by mass. An amount of about 40% is preferred. Other carriers are also possible. Other additional additives include, for example, methylhydroxybenzoate, antioxydants, flavoring agents, volatile oils, preservatives, surfactants and buffers, other And preservatives when not sterilized.

The pharmaceutical compositions presented herein comprise the nucleoacid artisense oligonucleotides shown above and one or more surfactants. Suitable surfactants or surfactants that enhance the uptake of antisense oligonucleotides in the present invention are the surfactant proteins A, B, C, DE and di-saturated phosphatidylcholine and di palmitoyl force. Patidylcholine (dipalmitoylphophatidylcholine), phosphatidylcholine, phosphatidylglycerol, phosphatidyllinositol, phosphatidylethanolamine, phosphatidylethanolamine, lysophosphatidylcholydylcholine Palmitoyl-lysophosphatidylcholine, phosphatidylserine, phosphatidic acid, ubiquinones, dehydroepiandrosterone, dehydroepiandrosterone (dolichols), sulfatidic acid, glycerol-3-phosphate, dihydroxyacetone phosphate xyacetone phosphate, glycerol, glycero-3-phosphocholine, dihydroxyacetone, palmitate, cytidine diphosphide (CPD) (cytidine diphosphate diacylglycerol), CDP choline (choline), choline (choline), choline phosphate (choline phosphate); It also contains nonionic blocks of complexes of omega-3-fatty acids, polyenic acid, lecithin, palmitic acid, ethylene or propylene oxides. Natural artificial lamellar body, polypropylene, monomeric and polymeric polyoxyethylene, dextran or alkanoyl side, which are the natural carriers that carry the surfactant elements together. chains), surfactants synthesized with Brij 35, Triton X-100 ALEC, Exosurf, Survan and Atovaquone, and others. These surfactants can be used as a combination of one or several surfactant elements, the 3 'or 5' end and the antisense oligonucleotide. The constructs of this invention can be used by any other method of delivering antisense nucleotides and nucleolytic complexes to the lungs. Antisense compounds were distributed in the patient's lungs by any suitable method, such as by inhalation in the aerosol form of an inhalant containing antisense. These inhalants include liquids, solids, and certain types of ingredients that differ from other preparations or diagnostics. Examples of the use of other agents include acetaminophen, anireldine, aspirin, aspirin, buprenorphine, butabital, butorpphanol, choline salicylate , Codeine dezocine, declofenac, diflunisal, dihydrocodeine, elcatoninin, ectolac, etodolac, phenopropene (fenoprofen), hydrocodone, hydromorphone, ibuprofen, ketoprofen, ketoprofen, ketorolac, levophanol, magnesium salicylate salicylate, meclofenamate, mefenami acid, meperidine, meperidine, methadone, metotrimeprazine, morphine, nalbuphine, Naproxen, opium, oxycodone, oxymolphone (o Listed above are xymorphone pentazocine, phenobarbital, propoxyphene, salsalate, sodium salicylate, tramadol and nacotic analgesics To the old ones. Mosby's Physician's GenRx antidepressants also include alprazolam, bromagepam, buspirone, chlordiazepoxide, chlormezanone, and clorazepate. Diazepam halazepam, hydroxyzine, ketaszolam, lorazzepam, meprobamate, oxazepam and prezepam It is also useful to include other things. Antidepressants are related to mental depression, such as chlordiazepoxide, amitriptyline, loxapine maprotiline and perphenazine. Non-rheumatic aspirin, choline salicylate, diclofenac, ketoprofen, diflunisal, etodolac, phenoprene (penoprofen), floctafenine (floctafenine), flurbiporfen, ibuprofen, indomethacin, magnesium salicylate, meclofenamate, Mefenamic acid, nabumetone, naproxen, oxaprozin, phenylbutazone, phenylbutazone, piroxicam, salsalate, bovine Sodium salicylate, sulindac, tenoxicam, tiaprofenic acid, tolmetin, diclofenac, flurbiprofen, Indomethacin, ketorolac, rimexolone (usually used after surgery) Anti-inflammatory agents, such as, beclomethaxone, budesonide, dexamethasone, dexamethasone, flunisolide, triamcinolone for the application of non-infectious noses. Soporifics (sleep promoters), alprazolam, bromage palm, bromazepam, diazepam, diphenhydramine, doxylaamine, estazolam, flu Flurazepam, halazepam, ketazolam, logazepam, nitrazepam, prazepam, quazepam, temazepam It can be used to treat insomnia, such as triazolam, zolpidem and sopiclone. Sedatives such as diphenhydramine, hydroxyzine, metotrimeprazine, promethazine, propofol, melatonin, trimerprazine Include. Stabilizers and agents include amitriptyline HCl; Chlordiazepoxide, amobarbital; Secobarbital, aprobarbital, butabarbital, ethchiorvynol, glutethimide, L-tryptophan. Mephobabital, metohexital Na, midazolam HCl, oxazepam, pentobarbital Na, phenobarbital, secobarbital Na, It is used to treat epilepsy or tremors in other environments, such as thiiamylal Na. Enadoline HCl, cytoprotective agents, and menopausal symptoms such as ergotamine, belladonna alkaloid and phenobarbital, used to treat hair trauma, clonidine ( clonidine, synthesized estrogen, and medroxyprogesterone, estradiol, estradiol cypinate, estradiol valerate, estrogens, synthesized It is used to treat menopausal motor neuron symptoms such as estrogens, estifeid estrone, estropipate, and ethinyl estradiol. Examples of therapeutic agents for dysmenorrhea (PMS) include progesterone, progestin, gonadotrophic hormones, oral contraceptives, danazol, luprolide acetate, and vitamin B6. There is. Examples of agents used in psychotherapy include tricyclic antidepressants, amitriptyline HCl (elavil), amitriptyline HCl, perphenazine, and doxepin. Stabilizers, anti-depressants, and anti-anxiety agents include diazipam (valium), lorajipam (ativan), alprazipam (xanax), SSRI's (selective serotonin uptake inhibitors), fluoxetine HCl (prozac), and cetalin There are zoloft, paraxetine HCl (paxil), fluvoxamine malate (luvox), venlafaxine HCl (effexor), serotonin, serotonin fenfluramine, and other overall pharmaceuticals (OTCs).

The composition of this invention can be administered to include particles of a size that can be absorbed into the respirator. It is small enough to be inhaled through the eyes, mouth, and larynx and to pass through the alveoli of the bronchus and lungs, usually about 0.5 to 10 microns. The amount of inhalable particles contained in aerosols will accumulate on the neck and be swallowed, so the amount should be minimized. For the care of the nose the particle size is 10-500 micrometers is preferred for maintenance of the nasal cavity. Aerosols or mists of solid particles containing the inventive agent can be made into aerosols and mists into solid particles by any other mechanism. Aerosols and fog generators are suitable for the formation of solid particle medicaments. These machines produce respirable particles as described above and also include pre-determining the determination of drug quantities in the appropriate amounts for animals or humans. An exemplary aerosol former type of solid particles is an insufflator. It is contained in the form of finely ground powder by the blower through the nasal breathing and is formed to be suitable for operation. In this nebulizer, a dose of agent powder is placed in the capsule to effectively carry out the formulations herein. These are usually made of gelatin or plastic and can be cut or opened so that the powder is delivered by absorption of air through an inhaler operated by usage. The powder applied to the nebulizer consists of a pure agent or a mixture of agents, for example, lactose and appropriately diluted powder, and optional surfactants or other agents. Such agents will comprise from 0.01 to 100 mass ratio. The formation of an exemplary aerosol of the second type includes those quantified by inhaled particles. Quantified inhalants include solutions or suspensions of liquefied particles and activated urea maintained at a constant pressure by an aerosol spray. The use of such a machine is used for the delivery of quantitative volumes on the order of approximately 10 to 150 microliters as well as other suitable volumes for the production of purified spray particles containing the active ingredients. Suitable propellants are, for example, chlorofluorocarbon composites such as dichlorodifluoromethane, trichloroprololmethane, dichlorotetraproloedane mixtures. One or more additional mixtures are, for example, ethanol, surfactants, oleic acid or sorbitan trioleate, antioxidants and suitable spices. Aerosols, either solid or liquid, are produced by the aerosol generator at a rate of approximately 10-150 liters per minute, most preferably about 60 liters per minute. Aerosols containing significant amounts of drugs can be made faster.

As already pointed out, agents of the invention include agents of the invention as well as vehicles. The carrier should preferably be biologically acceptable and suitable for other transport routes depending on the intent of the pharmaceutically or efficient gas, liquid, solid or mixture. Its composition may include other diseases or environments, spices, antioxidants, colorants, fillers, volatile oils, buffers, diffusion agents, surfactants, RNA inactivators, propellants, preservatives or other known therapeutic mixtures. Housed in the field. One agent example of an mRNA inactivator is an enzyme such as ribozyme. The mixing usually includes antisense oligonucleotides of about 1-40 mass ratios, more preferably 5-20 mass ratios, preferably in a 0.01-99.99 mass ratio. However, other ingredients and amounts of other agents should be appropriate for the limitations of this invention. The agents of the invention also supply composites that are operated in a variety of different ways or calculated by various transport methods. Synthetic Absorbed into the skin, as well as other suitable skin, mouth, nose, vagina, anus, eyes, ears, other body blood, consistent supply, intracranial, intracellular, intravascular, inhalation, intraalveolar, intratracheal , By implantation, creams, gels, and the like, as well known in the art, with a combination of carriers and carriers with careful consideration. In one particular form, the agent is suspended or dissolved in solution. Other carriers include hydrophobic carriers such as lipid particles and vesicles such as liposomes or microcrystals. The preparation of all such written ingredients or compounds is as known in the art and need not be mentioned further. In a particularly preferred form of vehicle formation, the vehicle comprises a liposome comprising an antisense oligonucleotide. Lipid carriers are suitably used for the delivery of DNA to N- (1- [2,3-dioleolsiloxy propyl) -N, N, N-thyrimethyl-ammonium methylcellulose and target cells in the art. Includes other known things that may be. In this embodiment, the form includes a form suitable for respiration, such as an aerosol. The agents, and forms of the present invention, are made in the form of bulks and units, and in the form of implants, solutions or suspensions, capsules or cartridges, for ease of eating as is known in the art.

The cart is also provided including a transport device and a separate container, agent, composition or form of the present invention, and optional other agents, and kit inclusion instructions. In one preferred embodiment, the vehicle comprises a nebulizer for transporting one or multiple metered form doses. One metered amount of nebulizer is supplied as a disposable kit pre-mounted aseptically with sufficient agent in the present application. The nebulizer is supplied in the form of a blower and an easy to eat capsule or cartridge. In another embodiment, the transportation device includes a pressurized inhaler, and the agent is in the form of a turbid solution or solution. Kits may contain agents, antioxidants, flavored and colored agents, filters, volatile oils, buffers, dispersants, surfactants, cells incorporating agents, RNA inactivators, ante in a container and from a class comprising other therapeutic compounds. And may optionally include an agent selected from the class consisting of oxidants, flavors, propellants, and health agents that can be added in other forms as appropriate. When a solvent or inclusion of an agent is added, an organic solvent and an aquous solvent mixed with an organic solvent and one or more cosolvents may be used as known in the art.

Agents of the present invention may be provided in combination with a vector for transport purposes or to produce a large number of complexes of the agent. As is known in the art, an agent can be effectively linked to a vector. Agents can also be introduced into immersion cells for the purpose of increasing and storing MTA oligos.

Agents of the invention are themselves or of at least one target gene, of gene flanking regions, initiation codons, intron-axon borders, and the like, or of non-coding RNA fractions, and of coding and non-coding regions. Forms or various methods for the treatment of diseases or pain related to complete proRNAs comprising 3'-terminus such as 5'-terminus such as juxta-section and poly-A fraction, and protein encoding regions Can be utilized by administering a significant amount of antisense oligonucleotide to a patient affected by a disease or pain, thereby reducing the productivity and availability of at least one patient target mRNA and increasing fragmentation. Typically, the agent is administered effectively to reduce the productivity and availability of at least two target mRNAs, or to increase degradation. Alternatively, the agent may be administered directly to the patient's VP, and aerosols that are easy to breathe are preferred. Experts know how to measure the dose of agonist according to the weight of patients treated according to the contents of this patent, and the effective amount such that the concentration of the multiple target antisense oligonucleotides in the cell is from about 0.05 to about 10 μM. It is preferred to administer the agent, and more preferably, the concentration of the multiple target antisense oligonucleotides in the cell is up to about 5 μΜ.

The therapeutic industry according to the patent is suitable for the treatment of numerous diseases and pains, which application is limited only to the availability of the target molecule and its sequence. One form in which the present technology can be particularly well applied is lung disease and pain. For this type of application, at least one target mRNA encodes proteins such as adenosine A ₁ receptor, adenosine A _2b receptor, adenosine A ₃ receptor, and bradykinin B2 receptor. However, it can also be applied to other targets. In certain applications, the disease or pain is associated with obstruction of the patient's airways, more particularly with asthma and the like. While many others have been mentioned above, one of the preferred target proteins includes NfκB transcription factors. In another preferred application, the disease and pain are related to the inflammatory response. In this form of application, the at least one target mRNA preferably encodes a protein selected from the class comprising interleukins, chemokines, Rantes and their receptors. Another application is the treatment of a disease or pain associated with allergies. In this application, the mRNA preferably encodes a target selected from the class comprising the antibody and the antibody receptor. In the application of the present technology to diseases or pain related to malligency or cancer, it is preferred that the mRNA encodes a target selected from the classes comprising oncozin, cotton weak globulin, and antibody receptors.

Depending on the target organ or tissue, the agents of the present invention may be buccal to the skin or systemic route, more specifically into the mouth, into the cavity, into the anus, into the nose, into the vagina, through the skin, bucal Intramuscularly, intravascularly, subcutaneously, intramuscularly, intratumorally, glandically, by implantation, into the skin, and by one of many methods in the example of many different routes of administration.

In addition, the form can be implanted, slow released, released through the skin, sustained release, and covered with one or more polymers to prevent the agent from destroying before reaching the selected target. Patients that can be treated with the agent are humans and other common animals, in particular vertebrates, among them stray animals, more particularly humans, and large and small, wild and livestock, aquatic and farm animals, preferably humans And livestock and farm animals. In one aspect of the invention, the mRNA of at least one target and the patient's are of the same species, preferably they are human. However, in practice, the mismatched nucleotides have been replaced, so mismatched species can also be used.

The multiple target antisense oligonucleotides of the invention can be administered in a wide range. A dose of about 0.0005 to about 150 mg / kg body weight per dose is preferred, and the agent may be administered by syringe treatment in seven doses per day, and continuous administration may lead to the level of specific molecules. An amount of about 75 mg / kg body weight is preferred, and more preferably about 1 to 50 mg / kg body weight. The method is administered in a prophylactic or therapeutic way.

Agents of the invention may be selected by selecting two or more targets selected from the classes consisting of genes, gene blanking regions, mRNAs and proteins known to be associated with at least one disease or pain; Oligos targeting genes to gene flanking regions, initiation codons, intron-axon borders and the like, or to juxta-sections between non-coding RNA fractions, poly-A fractions and coding and non-coding regions By obtaining an RNA selected from the class constituting the RNA corresponding to the complete sequence of the RNA comprising the 5'-end and 3'-end, and an RNA fraction encoding the target protein; By selecting a primary RNA fraction having at least about 60% homology to the fraction of the secondary RNA; And one or more antisense oligonucleotides for one or more RNA segments. In a preferred embodiment, the method comprises replacing at least one with a common base and, in some cases, replacing all of the non-similar nucleotides of the antisense oligonucleotide, in another embodiment, the method is directed to an antisense oligonucleotide. Replacing the cytosine of at least one CpG with methylated stocin. This technique includes methylation as known in the art, but will not be mentioned here.

The specific length of the MTA is determined by the length of the target and its fraction comprising some thymidine, but the antisense oligonucleotides are preferably at least 7 nucleotides in length, and at most about 60 nucleotides in length. Specific backbone chemistry can be selected by an expert based on the teachings herein and considerable knowledge of the present technology. One factor that interferes with the choice of nucleotide crosslinking moiety is the degree of nuclease resistance required and the other factor is the specificity of one or another mode of administration. Another factor is the need for a location that minimizes or completely eliminates side effects that may occur after treatment with an agent.

The following examples illustrate the invention but are not limited thereto. In this embodiment, μM is micromolar, ml is milliliters, μm is micrometers, mm is millimeters, cm is centimeters, ° C is degrees Celsius, μg is micrograms, mg is in milligrams, g is in grams, kg is in kilograms , m is mole, and h is time.

Example 1 Design and Synthesis of Antisense Oligonucleotides

Designing the endense oligonucleotides for A ₁ and A ₃ adenosine receptors requires the interpretation of complex secondary structures of the target A ₁ receptor mRNA and target A ₃ receptor mRNA. After forming this structure, it can be interpreted as giving functional activity or stability to mRNA, and antisense oligonucleotides are designed for target regions of mRNA that can overlap with the start codon optimally. Other targeting sites can be readily used. As shown for the specificity of the antisense effect, another nucleotide that is not complementary to the target mRNA but includes the same nucleotide composition on a w / w basis is included as a control in the antisense experiment.

The mRNA secondary structure of the adenosine A ₁ receptor was analyzed and used as shown above to design phosphorothioate antisense oligonucleotides. The synthesized antisense oligonucleotide named HAdA ₁ AS and has the sequence 5'-CAT GGA GGG CGG CAT GGC GGG-3 '(SEQ ID NO: 1).

As a control, the mismatched phosphorothioate antisense nucleotide is named HAdA ₁ MM1 and has the sequence 5'-GTA GCA GGC GGG GAT GGG GGC-3 '(SEQ ID NO: 2).

Each oligonucleotide has the same base content and general sequence structure. Searching for similarities in GENBANK (Release 85.0) and EMBL (Release 40.0) revealed that antisense oligonucleotides are specific for adenosine A ₁ receptors in humans and rabbits, and mismatched controls are substances for hybridization with sequences of any known gene. Indicated that this could not be. The secondary structure of adenosine A ₃ receptor mRNA can be similarly analyzed and used, as in designing the two phosphothioate antisense oligonucleotides. The first antisense oligonucleotide (HAdA ₃ AS1) synthesized has the sequence 5'-GTT GTT GGG CAT GGG CAT CTT GCC-3 '(SEQ ID NO: 3).

As a control, mismatched phosphorothioate antisense oligonucleotides (HAdA ₃ MM1) are synthesized and have the sequence 5'-GTA CTT GCG GAT CTA GGC-3 '(SEQ ID NO: 4).

In addition, a second phosphorothioate antisense oligonucleotide (HAdA ₃ AS2) is designed and synthesized and has the sequence 5'-GTG GGC CTA GCT CTC GCC-3 '(SEQ ID NO: 5).

Its control oligonucleotide (HAdA ₃ MM2) has the sequence 5 'GTC GGG GTA CCT GTC GGC-3' (SEQ ID NO: 6).

Phosphothioate oligonucleotides were synthesized on an Applied Biosystem Model 396 Olgonucleotide Sunthesizer and purified using NENSORB chromatography (Dupont, MD).

Example 2: Test of Adenosine A ₁ Receptor Antisense Oligo In Vivo

Antisense oligonucleotides (SEQ ID NO: 1) for the human A ₁ receptor described above were tested for efficacy in an in vitro model using lung adeno cell HTB-54. It has been demonstrated that HTB-54 lung adenoma cells express A ₁ adenosine receptors using receptor probes designed and synthesized in normal Northern blotting experiments and laboratories. HTB-54 human lung adenoma cells (106/100 mm tissue culture dishes) were incubated with 5.0 μM HAdA ₁ AS or HAdA ₁ MM1 for 24 hours and fresh media and oligonucleotides were changed after 12 hours. After incubation with oligonucleotides for 24 hours, the cells were harvested and RNA extracted through normal laboratory procedures. 21-mer probes corresponding to mRNA regions targeted by antisense (ie, sequences like antisense but not phosphorothioate) were synthesized and treated with HAdA ₁ AS-treated, GAdA ₁ MM1-treated and untreated HTB- RNA prepared from 54 cells was used as a Northern blot probe. These blots clearly showed that HAdA ₁ AS effectively reduced the amount of human adenosine receptor mRNA by more than about 50%, but not HAdA ₁ MM1. These results indicate that HAdA ₁ AS depletes the intracellular mRNA of adenosine A ₁ receptors associated with asthma, making it a useful material for anti-asthma medicine.

Example 3 In Vivo Efficacy of Adenosine A1 Receptor Antisense Oligo

Overlapping with the initiation codon, the phosphorous thioate antisense oligonucleotides, designed for the first time for human adenosine A ₁ receptors, can be used in rabbit models due to the accidental similarities between rabbit and human DNA sequences in the adenosine A ₁ gene. . 312 antigen unit / ml House dust mite (D.farinae) extract (Berkeley Biologicals, Berkeley, Calif.) Mixed with about 10% kaolin is immunized into the peritoneum within 24 hours of age. The first month is immunized once a week and the next two months once every two weeks. At the age of 3-4 months, rabbits immunized eight times are anesthetized and relaxed by intramuscularly administering a mixture of ketamine hydrochloride (44 mg / kg) and acepromacin merate (0.4 mg / kg). It was placed in a comfortable position in a small, fluffy molded animal box (Mallinkrodt, Inc., Glen Falls, NY) and incubated in a 4.0 mm respiratory tract. A polyethylene catheter with an outer diameter of 2.4 mm with a latex balun is placed into the esophagus and maintained at the same distance (approximately 16 cm) at the entrance for this experiment. The catheter is attached to a heated Pish Pneumochograph (Size 00; DOM Medical, Richmond, VA), and the flow is volley derived from the Goul Carrier Empire (Model 11-4113; Gould Electronic, cleveland, OH). Measurement was performed using a Dean partial pressure inductor (model DP-45161927; Validyne Engineering Corp., Northridge, Calif.). The longitudinal flask of the esophagus is attached to one side of the partial pressure inducer, and the flow of the esophagus tube is connected to the opposite side of the partial pressure inducer to record the pressure of the bronchus. The flow continues to constitute a periodic volume, and the measurements of total lung resistance (RL) and dynamic compliance (C _dyn ) are the same using an autorespiratory analyzer (Model 6; Buxco, Shron, CT), respectively. Calculations were made at isovolumetric and flow zero points. Animals were randomly selected and pretreatment values of PC ₅₀ were obtained for aerosolized adenosine on the first day. Antisense (HAdA1AS) or mismatched control (HAdA1MM) oligonucleotides were dissolved in sterile physiological saline at a concentration of 5000 μg (5 mg) per 1.0 ml. The animals were administered twice daily with aerosolized antisense or mismatched oligonucleotides (5000 μg in a volume of about 1 ml) through the endotracheal tube twice daily. Aerosols of saline, adenosine, or antisense or mismatched oligonucleotides are droplets produced by ultrasonic nebulizers (DeVilbiss, So-merset, PA) and 80% of the droplets formed are smaller than 5 μm. As the first arm of the experiment, four allergic rabbits were randomly selected to receive antisense oligonucleotides and four controls were given mismatched oligonucleotides. On the third morning, a PC ₅₀ value (from baseline value, mg / ml concentration of aerosol adenosine required to reduce dynamic compliance at 50% bronchial airways) was obtained and obtained in animals prior to exposure to oligonucleotides. Compared to the value of PC ₅₀ . After a one week period, animals first cross each other with rabbits treated with antisense oligonucleotides and then administered with mismatched control oligonucleotides. Treatment methods and measurements are the same as those used in the first method of this experiment. In 6 of 8 animals treated with antisense oligonucleotides, it should be noted that adenosine-mediated bronchial stenosis cannot be obtained with a solubility of adenosine, a limit of 20 mg / ml. For calculation purposes, the value of PC ₅₀ for these animals is fixed at 20 ml / mg. Therefore, the stated values represent the minimum for the effectiveness of antisense. The actual effective value is higher. The results of this experiment are shown in Table 3 below.

Table 3: Effect of Adenosine Receptor A ₁ Antisense Oligo on PC ₅₀ Values in Asthmatic Rabbits

Discrepancy control A1 receptor antisense oligo Before oligonucleotide treatment After oligonucleotide treatment Before oligonucleotide treatment After oligonucleotide treatment 3.56 ± 1.02 5.16 ± 1.03 2.36 ± 0.68 〉 19.5 ± 0.34 ** The results are expressed as mean (m = 8) ± SEM. Significance was calculated by repaeted-measures analysis of variance (ANOVA) and Tukey's protected test. ** All other Significantly different from the group, p <0.01

In the two experimental methods, animals treated with antisense oligonucleotides showed that the doses of aerosolized adenosine required to reduce lung dynamic compliance by up to 50% were increased in order of magnitude. It was observed that the mismatched control oligonucleotides had no effect on the value of PC ₅₀ . Inhaled antisense or control oligonucleotides showed no toxicity in any animal.

These results indicate that lungs are a potential target for antisense oligonucleotide-based therapeutics in lung diseases. Furthermore, they appear that in a model system very similar to human asthma, downregulation of adenosine A ₁ receptors can eliminate adenosine-mediated bronchial stenosis, mainly in the asthma airway. Because this hypersensitivity is close to the point of contact of the tissues involved with the aerosolized oligonucleotides and the model is stimulated similarly to critical human disease, bronchial hypersensitivity in allergic rabbit models of human asthma is very good for antisense intervention. The result is.

Example 4: Specificity of A ₁ -Adenosine Receptor Antisense Oligonucleotide

As a conclusion of the crossover experiment of Example 3, the number of adenosine A ₁ receptors in airway smooth muscle from all rabbits was quantitatively analyzed. In addition, as a control for the specificity of antisense oligonucleotides, unaffected adenosine A ₂ receptor was quantified. The airway smooth muscle tissue obtained from each rabbit was dissected, and the cell membrane fraction was analyzed by Kleinstein et al. (Kleinstein, J. and Glossmann, H., Naunyn-Schmiedeberg's Arch.Pharmacol. 305: 191-200 (1978)). , Relevant parts of which are incorporated herein by reference in their entirety. The prepared natural cell membrane was stored at 70 ° C. until inspection. Protein content was determined by Bradford's method (M. Bradford, Anal. Biochem. 72, 240-254 (1976), the relevant parts of which are incorporated herein by reference in their entirety). The frozen cell membrane is dissolved at room temperature, 0.2U / ml adenosine deaminase is added, and incubated at 37 ° C. for 30 minutes to remove intrinsic adenosine. Binding of [ ³ H] DPCPX (A1 receptor-specific) or [ ³ H] CGS-21680 (A1 receptor-specific) is described in Ali et. ac. Method (Ali, S. et al., J. Pharmacol. Exp. Ther. 268, Am J. Physiol 266, L271-277 (1994), all of which are hereby incorporated by reference in their entirety.) Measured by. In the analysis of the specific binding of A1-specific antagonist DPCPX in the literature, animals treated with adenosine A1 antisense oligonucleotides in crossover experiments reduced the number of A1 receptors by 75% compared to the control group. . Analysis of specific reactive linkage of A ₂ receptor-specific antagonist 2- [p- (2-carboxyethyl) -phenethylamino] -5 '-(N-ethylcarboxamido) adenosine (CGS-21680) There was no change in the number of adenosine A ₂ receptors. This is shown in Table 4 below.

Table 4: Action Specificity of Adenosine A ₁ Recipient Antisense Oligonucleotides

Mismatched Control Oligonucleotides A ₁ antisense oligonucleotide A ₁ -specific combination 1105 ± 48 ** 293 ± 18 A ₂ -specific combination 302 ± 22 442 ± 171 The results are mean (m = 8) ± SEM. Significance was calculated by ANOVA (repaeted-measures analysis of variance) and Tukey's protected test (**), meaning significant differences from discordant controls. p <0.01

The results show the effectiveness of antisense oligonucleotides in treating airway disease. Since the antisense oligos described above eliminate the receptor system that causes adenosine-mediated bronchial stenosis, it is not necessary to remove adenosine from the receptor system. However, it is desirable to remove adenosine from these oligonucleotides to reduce the dosage required to achieve a similar effect. Other antisense oligonucleotides that target mRNA of proteins involved in inflammation are described above. To prevent the release of adenosine during the degradation, adenosine was removed from the nucleotides.

Example 5 Antisense Oligonucleotides Against Other Target Nucleic Acids

This work was performed to show that the present invention can be widely applied to antisense oligonucleotides (“oligodes”) that are broadly specific for nucleic acid targets. As the following experimental studies were carried out, the present invention showed that the use of antisense oligos designed as presented by the present invention and oligos targeting any and all adenosine receptor mRNAs could be widely applied. For this purpose, various antisense oligos have been prepared for the illustrated adenosine receptor mRNAs of adenosine A ₁ , A _2b , A ₃ receptor mRNAs. Antisense Oligo I was disclosed above (SEQ. ID NO: 1). Five antisense phosphorothioate oligos added were designed and synthesized as set forth above.

1-oligo II (SEQ. ID NO: 7), which also targets adenosine A1 receptor but differs from oligo I

2-oligo V (SEQ.ID NO: 10) Targeting Adenosine A _2b Receptor

3- oligo III (SEQ. ID NO: 8) and IV (SEQ. ID NO: 9) targeting other sites of the adenosine A ₃ receptor

4- oligo I-PD (SEQ. ID NO: 1681) (phosphodiester oligos of the same sequence as oligo I)

Such antisense oligos are designed for treatment in a selected species, as described above, and are generally specific to that species unless the fragments of the target mRNA of another species contain similar sequences. All antisense oligos were prepared as follows, and as described herein above in vivo with antisense oligos in a rabbit model for bronchial stenosis, inflammation, and allergies with respiratory disorders and respiratory failure as in the case of diseases such as asthma I did my test.

Example 6: Design and Sequence of Other Antisense Oligos

Six oligos and their action were studied in a rabbit model and the results of this study were recorded and reviewed below. Five of these oligos were selected for the study to supplement the data for oligo I (SEQ ID NO: 1) mentioned in Examples 1-4 above. The oligos tested were antisense oligos I (SEQ ID NO: 1) and II (SEQ ID NO: 7), targeting oligo V (SEQ ID NO: 8) targeting other regions of adenosine A1 receptor mRNA, and oligo V (SEQ ID NO: 8) targeting adenosine A _2b receptor mRNA. ), Oligos III and IV (SEQ ID NOs: 9 and 10), which target the other two regions of adenosine A3 receptor mRNA. The sixth oligo (oligo I-PD) is a phosphodiesterated version of oligo I (SEQ ID NO: 1). Preparation and synthesis of these antisense oligos was performed according to Example 1.

(I) antisense oligo I

The antisense oligonucleotide I mentioned in Examples 1-4 is targeted to human A1 adenosine receptor mRNA (EPI 2010). Antisense Oligo I is 21 nucleotides, overlaps the initiation codon and has the sequence 5'-GAT GCA GGG CGG CAT GGC GGG-3 '(: SEQ. ID No 1).

As previously reviewed, see Nyce, J.W. & Metzger, WJ, Nature, 385: 721 (1977) (the relevant part of which is incorporated herein by reference in its entirety). Oligo I is adenosine-induced bronchus of an allergic rabbit as described above. It has been shown to eliminate stenosis and to reduce allergen-induced airway disorders and bronchial hyperresponsiveness (BHR).

(II) antisense oligo II

Phosphorothioate antisense oligos (SEQ. ID NO: 7) were designed according to the invention to target rabbit adenosine A1 receptor mRNA at +936 to +956 relative to the start codon (start point). Antisense Oligo II is 21 nucleotides and has the sequence 5'-CTC GTC GCC GTC GCC GGC GGG-3 '(SEQ. ID NO: 7).

(III) antisense oligo III

Phosphorothioate antisense oligos (SEQ. ID NO: 8) different from those set forth in Example 1 above are used in the present invention to target mRNA regions at the antisense A ₃ receptors +3 to +22 relative to the initiation codon (starting point). Designed according to Antisense Oligo III is 20 nucleotides and has the sequence 5'-GGG TGG TGC TAT TGT CGG GC-3 '(SEQ. ID NO: 8).

(IV) antisense oligo IV

Another phosphorothioate antisense oligo (SEQ. ID NO: 9) was designed in accordance with the present invention to target adenosine A3 receptor mRNA at +386 to +401 relative to the start codon (start point). Antisense Oligo IV is 15 nucleotides and has the sequence 5'-GGC CCA GGG CCA GCC-3 '(SEQ. ID NO: 9).

(V) antisense oligo V

Phosphorothioate antisense oligos (SEQ. ID NO: 10) were designed according to the present invention to target adenosine A _2b receptor mRNA at -21--1 relative to the start codon (starting point). Antisense oligo V is 21 nucleotides and has the sequence 5'-GGC CGG GCC AGC CGG GCC CGG-3 '(SEQ. ID NO: 10).

(VI) raise A1 mismatch

Two different mismatched oligonucleotides with the following sequence were used as controls for the antisense oligo I (SEQ. ID NO: 1) described in Example 5 above:

A ₁ MM2 5′-GTA GGT GGC GGG CAA GGC GGG-3 ′ (SEQ. ID NO: 1682);

A ₁ MM3 5'-GAT GGA GGC GGG CAT GGC GGG-3 '(SEQ.ID NO: 1683)

Antisense oligo I and two mismatched antisense oligos had the same base content and general sequence structure. Similarity searches in GENBANK (Release 85.0) and EMBL (Release 40.0) show that antisense oligo I is specific for adenosine A ₁ receptor genes in humans and rabbits, and mismatched controls do not hybridize with known human or animal gene sequences. Showed that.

(VII) antisense oligo A1-PD (oligo VI)

Phosphodiester antisense oligos (Oligo VI; SEQ. ID NO: 1681) having the same nucleotide sequence as oligo I were designed as disclosed herein above. Antisense oligo I-PD is 21 nucleotides, overlaps the initiation codon, and has the sequence 5'-GAT GGA GGG CGG CAT GGC GGG-3 '(SEQ. ID NO: 1681).

(VIII) control

5.0 ml aerosolized sterile saline 5.0 ml was administered to each rabbit according to the same method as the antisense oligos in (II), (III) and (IV) above.

Example 7: Synthesis of Antisense Oligo

Phosphorothioate antisense oligos having the sequence described in (a) above were synthesized in an Applied Biosystem Model 396 Oligonucleotide Synthesizer and purified using NENSORB chromatography (DuPont, DE). TETD (tetraethylthiuram disulfide) was used during the synthesis as a vulcanizing agent. Antisense oligonucleotide II (SEQ. ID NO: 7), antisense oligonucleotide III (SEQ. ID NO: 8), and antisense oligonucleotide IV (SEQ. ID NO: 9) are each synthesized and purified in the same manner as described above. It was.

Example 8: Preparation of Allergic Rats

Within 24 hours of the birth of a newly born New Zealand White Pasteurella-overlapping rabbit, 10% kaolin (Metzger, WJ, in Late Phase Allergic Reactions, Dorsch, W., Ed., CRC Handbook, pp.347-362, 312 antigen units / ml House dust mite (D. farinae) extract (Berkeley) mixed with CRC Press, Boca Raton (1990); relevant parts of which are incorporated herein by reference in their entirety. Biologicals, Berkeley, CA) intraperitoneally immunized with 0.5 ml (Metzger, WJ, in Late Phase Allergic Reactions, Sorsch, W., Ed., CRC Handbook, pp. 347-362, CRC Press, Boca RAton (1990); Ali, S., Metzger, WJ and Mustafa, SJ, Am. J. Resp. Crit.Care Med. 149: 908 (1994), the relevant parts of which are incorporated herein by reference in their entirety. Immunization was repeated once a week for the first month and once every two weeks until the next four months. Preferably such rabbits produce allergen-specific IgE antibodies, typically respond to allergen exposure in both early and late asthma reactions, and bronchial hypersensitivity (BHR) was seen. Allergens (such as 312 unit dust mite allergens) may be administered monthly to the peritoneum to continuously stimulate and maintain allergen-specific IgE antibodies and BHR. As described by Alo et al., Aerosols are administered at the age of four months to produce immunized rabbits. (Ali., S., Metzger W. J. and Mustafa, S. J., Am. J. Resp. Crit. Care Med. 149 (1994), the relevant parts of which are incorporated herein by reference in their entirety).

Dose-response study

Example 9: Experimental Apparatus

Aerosols for adenosine (0-20 mg / ml) or one of the antisense oligonucleotides or two mismatched oligonucleotides (5 mg / ml) are prepared separately using an ultrasonic nebulizer (Model 646, DeVilbiss, Somerset, PA) The ultrasonic nebulizer produced aerosol droplets, of which 80% was smaller than 5 μm. An equal volume of aerosol was administered directly to the lungs through the internal respiratory tract. Animals were randomly selected and administered aerosolized adenosine. On the first day, the pretreatment value in response to adenosine is calculated as the dose of adenosine causing a 50% loss of compliance (PC ₅₀ adenosine). The animals were then administered to the animals, either aerosolized or mismatched antisense oligos, for 2 minutes twice a day for two days through a tube in the trachea (5 mg / ml) (total dose, 20 mg). Post-treatment PC ₅₀ values were recorded on the third morning (post-treatment dose). The results of this study are mentioned in Example 21 below.

Example 10 Crossover Experiment

In some experiments using antisense oligo I (SEQ ID NO: 1) and the corresponding mismatched control oligonucleotide A ₁ MM2, animals were crossed after 2 weeks. That is, those previously administered with the mismatched control A ₁ MM2 receive antisense oligo I and those previously treated with the antisense oligo I receive the mismatched control A ₁ MM2 oligo. The number of animals in each class is as follows. Since one animal died in the second arm of the experiment due to technical difficulties, the discrepancy A ₁ MM2 (control 1) was n = 7 and for n discrepancy A ₁ MM3 n = 4 (control 2) and A ₁ AS antisense oligo I was n = 8. A ₁ MM3 oligo-treated animals were analyzed separately and the crossover experiments were not. The treatments and measurements used after crossover were the same as those used in the first branch of the experiment.

In 6 of 8 animals treated with antisense oligo I (SEQ. ID NO: 1), no PC ₅₀ value was obtained at adenosine doses up to 20 mg / ml, the limit of solubility of adenosine. Therefore, the value of PC ₅₀ for these animals was assumed to be 20 mg / ml for calculation purposes. Therefore, a given value represents the minimum value for the effectiveness of the antisense oligonucleotides of the present invention. According to the method described above, 0.5 or 0.005 mg (2.0 or 0.2 mg total) of antisense oligo I (SEQ ID NO: 1) or A ₁ MM2 oligo were added to other groups of allergic rabbits (n = 4 per group). Administered. The results of this study are mentioned in Example 22 below.

Example 11: Prescription of Antisense Oligo

Each antisense oligo is isolated and dissolved in an aqueous solution, respectively, and four 5 mg portions using a nebulizer through a tube in the trachea, as described for antisense oligo I (SEQ. ID NO: 1) in (e) above. (Total amount of 20 mg). As shown in Example 19 and Table 1 below, the results for Antisense Oligo I and its Mismatch Control 3 confirmed that the mismatch control was identical to saline. Because of this finding, saline was used as a control in lung function studies using antisense oligos II, III, IV (SEQ. ID NO: 7, 8, 9).

Example 12 Specificity of Oligo I to Adenosine A1 Receptor (Receptor Binding Study)

As described above, airway smooth muscle tissue from rabbits that had been administered 20 mg oligo I (SEQ ID NO: 1) at four divided doses over 48 hours was dissected into primary, secondary and tertiary bronchus. Ali et al. (Ali., S., et al., Am. J. Resp. Crit. Care Med. 149: 908 (1994), sections relating to the swelling of cell membrane parts are hereby incorporated by reference in their entirety. The cell membrane part was prepared according to the method of. Protein content was measured using the Bradford method, the cell membrane was incubated for 30 minutes at 37 ℃ with adenosine deaminase of 0.2U / ml, to remove the inherent adenosine. Bradford, M.M. Anal. Biochem. 72, 240-254 (1976). Binding of [3H] DPCPX, [3H] NPC17731, or [3H] CGS-21680 was measured as described by Jarvis et al. (Jarvis et al. See, Jarvis, MF, et al, Pharmaco. Exptl. Ther. 251, 888-893 (1989), the relevant parts of which are incorporated herein by reference in their entirety.) Results of this study Are shown in Table 8 and Example 20 below.

Example 13: Pulmonary Function Measurement (Compliance C _DYN and Resistance)

At 4 months of age, the immunized animals were anesthetized and relaxed by intramuscular injection of 1.5 ml of a mixture of ketamine HCl (35 mg / kg) and acepromaline malate (1.5 mg / kg). After induction of anesthesia, the allergic rabbit was laid comfortably in a softly molded animal box. Ointment was applied to eyes to prevent drying, and eyes closed. It was then intubated with a 4.0 mm medium high-cuffed Murphy 1 endotracheal tube, as described by Zavala and Rhodes. (Mallinckrodt, Glen Fall, NY) (See, Zavala and Rhodes, Proc.) Exp. Biol. Med. 144: 509-512 (1973), the relevant part of which is incorporated herein by reference in its entirety.) OD 2.4 mm polyethylene catheter with thin-walled latex balun (Becton Dickinson) Clay Adams, Parsippany, NJ) were passed through the esophagus and maintained at the same distance from the mouth (about 16 cm) during the experiment. The tube in the trachea is attached to a heated fresh pneumotach (size 00; DEM Medical, Richmond, VA) and a Gould carrier amplifier (Model 11-4113, Gould Electronics, Cleveland, OH) The flow (v) was measured using a Vallyne partial pressure induction machine (Model DP-45-16-1927, Validyne Engineering, Northridge, Calif.). An esophageal balloon was connected to one side of the ballidine partial pressure inducer and the other side to the outlet of the endotracheal tube to obtain the lung pressure (Ptp). Flow was continuously integrated to produce periodic volumes, and total lung resistance (Rt) and dynamic compliance (C _dyn ) were measured at the same volume and flow point. Flow, volume, and pressure were recorded on 8 channel Gould 2000W high frequency recorders, and C _{dyn was} calculated using Ptp difference in total volume and zero flow, and Ptp and V in the lung volume of Ptp and intermediate periods. Rt was calculated as the ratio. As previously described by Giles et al., The above calculations were made automatically using a Buksco automated pulmonary mechanical breathing analyzer (Model 6, Buxco Electronics, Shron, CT; automated pulmonary mechinics respiratory analyzer). al., Arch. Int. Pharmacodyn. Ther. 194: 213-232 (1971), all of which are hereby incorporated by reference in their entirety.) The results obtained by administering oligo II to allergic rabbits are shown below. It is shown in Example 26 and is under consideration.

Example 14 Measurement of Bronchial Hypersensitivity (BHR)

Baseline hypersensitivity was measured by aerosolized histamine in each allergic rabbit. An aerosol of saline or histamine was made. When each dose was applied, histamine aerosol was made using a Devilbiss nebulizer (devilbiss, Somerset, PA) for 30 seconds and then 2 minutes. Ultrasonic nebulizers produce aerosol fines, 80% of which are smaller than 5 microns in diameter. Histamine aerosol was administered at increasing concentrations (from 0.156 to 80 mg / ml) and pulmonary function was measured after each administration. Then, B4R was determined by calculating the concentration of histamine (mg / ml) (PC ₅₀ histamine) required to reduce C _dyn 50% (PC _{30 Histamine} ) from the baseline.

Example 15 Cardiovascular Effect of Antisense Oligo I

The measurement of cardiac ejection and other cardiovascular parameters using Cardiomax ^™ incorporates a dilution principle of skin fever that monitors changes in blood temperature exiting the heart after vascular infusion of a known volume of cold saline. Cold saline was rapidly introduced into the right atrium through the right jugular vein, and the corresponding change in the temperature of the mixed input and blood of the aortic arch was recorded by a temperature-sensitive small probe through the carotid artery. Allergic rabbits were treated with oligo I (EPI 2010; SEQ. ID NO: 1) of aerosol as mentioned in (d) above, and after 12 hours, animals were 0.3 mg of 80% ketamine and 20% xyraine Anesthetized at / kg. As clearly shown in Nyce & Metzger (1997), spura (the appropriate disclosure of which is incorporated herein by reference in its entirety), this time point is shown in SEQ. Consistent with previous data indicating efficacy of ID NO: 1. Then, a thermocouple was inserted into the left carotid artery into each rat, advanced 6.5 cm, and tightly tightened with silk suture. The right jugular vein was plumbed, tipped into polyethylene tube length and tightened.

Then, sterile saline was added at 20 ° C. to obtain a thermal dilution curve in Cardiomax ^™ II (Columbus Instrument, Ohio) to determine the exact position of the thermocouple probe. After confirming the exact location of the thermocouple, the femoral artery and vein were isolated. The femoral vein was used as a portal for drug administration and the femoral artery was used for blood pressure and pulse measurements. Once constant baseline cardiovascular parameters were established, blood pressure, pulse rate, cardiac output, total peripheral resistance and cardiac stretch were measured with Cardiomax ^™ .

Example 16: Activity Period of Oligo I (SEQ. ID NO: 1)

As mentioned in (f) above, using the nebulizer through the endotracheal tube, the initial increase starts at 0.156 mg / ml until the compliance is reduced to 50% (PC _{50 adenosine} ) to find the reference point. Log doses were administered to eight allergic rabbits. Then, as described above, six of the rabbits were administered four times a 5 mg aerosolized amount of (SEQ. ID NO: 1). Two rabbits received the same amount of saline solution as a control. 18 hours after the last dose, the value of PC _{50 adenosine} was tested again. After this point, measurements were continued for all animals daily up to 10 days. The results of this study were reviewed in Example 25 below.

Example 17 Reduction of Adenosine A _2b Receptor Number by Antisense Oligo V

Three days every two days using the adsorption chamber, 2.0 mg of respirable antisense oligo V (SEQ ID NO: 10) was administered to Sprague Dawley rats. 12 hours after the last dose, lung glandular tissue was dissected and analyzed for adenosine A _2b receptor binding using [311] -NECA as described in Nyce & Metzger (1997). The control group received the same amount of saline. The results using Student's paired t test had a significance of p <0.05 and were reviewed in Example 28 below.

Example 18 Comparison of Oligo I with Corresponding Phosphodiester Oligo VI (SEQ. ID NO: 1681)

Oligo I (SEQ. ID NO: 1) reduced the effects of adenosine and eliminated the stimulus sensitivity to adenosine yall up to 20 mg adenosine / 5.0 ml (limit of adenosine solubility). Oligo VI (SEQ. ID NO: 1681), the sequence of oligonucleotides of the phosphodiester type, was completely ineffective when tested in the same manner. All of these compounds differ only in the presence or absence of phosphorothioate residues in oligo I (SEQ. ID NO: 1) and have the same sequence and were aerosolized and sprayed as described above and in Nyce & Metzger (1997). . The paired t test of the students differed significantly at p <0.001 and the results were reviewed in Example 29 below.

Results obtained for antisense oligo I (SEQ. ID NO: 1)

Example 19: Result of Predecessor

The nucleotide sequence and other data of antisense oligo I (SEQ. ID NO: 1) specific for adenosine A ₁ receptor have been provided above. In addition, experimental data showing the effectiveness of oligo I in downregulation of receptor number and activity were also provided above. More detailed information on the properties and activities of antisense oligo I is provided in Nyce, JW and Metzger, WJ, Nature 385: 721 (1997). (On the other hand, the parts related to the following results are incorporated in their entirety by reference herein.) Meanwhile, the publication of Nyce & Metzger (1997) is a data set that shows the following characteristics of antisense oligo (SEQ. ID NO: 1): Provided:

(1) As shown in Table 5 below, antisense oligo I reduces the number of adenosine A1 receptors in bronchial smooth muscle of allergic rabbits depending on the dose.

(2) Antisense Oligo I alleviates adenosine-induced bronchial stenosis and allergen-induced bronchial stenosis.

(3) Oligo I alleviates bronchial hyperresponsiveness, as measured by standard measurement PC ₅₀ histamine for assessing bronchial hyperresponsiveness. As shown in Table 5, this result shows the anti-inflammatory activity of antisense oligo I.

(4) As anticipated, since antisense oligo I was designed to target the adenosine A ₁ receptor, antisense oligo I is specific for the adenosine A ₁ receptor as a whole and is very closely linked to the adenosine A ₂ receptor at any dosage. Or has no effect on the associated bradykinin B ₂ receptor. This is shown in Table 5.

(5) In contrast to the above effects of oligo I, the mismatched control molecules MM2 and MM3 (SEQ. ID NO: 1682 and SEQ. ID NO: 1683) have the same base composition as antisense oligo I (SEQ. ID NO: 1) and It has a molecular weight, but differs from the antisense oligo I due to a mismatch of about 6 and 2, respectively. The minimal inconsistency that could continue to maintain the same base composition still remaining showed no effect at all on the targeted receptor (A1, A2, or B2).

Since there is no prior art for the use of antisense oligonucleotides such as oligo I, which target adenosine A ₁ receptors, these results are unexpected results. The results shown in this patent may intentionally show the effectiveness of the claimed agents and methods for treating diseases or conditions associated with pulmonary airways, such as bronchial stenosis, inflammation, allergies, and the like.

Example 20 Oligo I Effectively Reduces Response to Adenosine Attacks.

Receptor binding experiments are described in Example 12 above and the results shown in Table 5 below show adenosine in cell membranes isolated from airway smooth muscle of antisense- and mismatched-treated allergic rabbits of A ₁ adenosine receptor and B ₂ bradykinin receptor. A ₁ -selective ligand [ ³ H] DPCPX and bradykinin B ₂ -selective ligand [3H] NPC 17731 shows the binding properties.

Table 5: Binding Properties of Three Antisense Oligos

process A1 receptor B ₂ receptor Kd B _max Kd B _max Adenosine A ₁ A receptor 20 mg2 mg0.2 mgA1MM 120 mg2 mgB ₂ A (bradykinin receptor) 20 mg2 mg0.2 mgB ₂ MM (control) 20 mg2 mg0.2 mg saline 0.36 ± 0.029 nM0.38 ± 0.030 nM0.37 ± 0.030 nM (control) 0.34 ± 0.027 nM0.37 ± 0.033 nM0.36 ± 0.028 nM0.39 ± 0.035 nM0.40 ± 0.028 nM0.39 ± 0.031 nM0.41 ± 0.035 nM0.37 ± 0.029 nM0.37 ± 0.041 19 ± 1.52 fmoles * 32 ± 2.56 fmoles * 49 ± 3.43 fmoles52.0 ± 3.64 fmoles51.8 ± 3.88 fmoles45.0 ± 3.15 fmoles44.3 ± 2.90 fmoles47.0 ± 3.76 fmoles42.0 ± 2.94 fmoles40.0 ± 3.20 fmoles43. 0 ± 3.14 fmoles46.0 ± 5.21 0.39 ± 0.031 nM0.41 ± 0.028 nM0.34 ± 0.024 nM0.35 ± 0.024 nM0.38 ± 0.028 nM0.38 ± 0.027 nM0.34 ± 0.024 nM0.35 ± 0.028 nM0.41 ± 0.029 nM0.37 ± 0.030 nM0. 36 ± 0.025 nM 0.39 ± 0.047 nM 14.8 ± 0.99 fmoles15.5 ± 1.08 fmoles15.0 ± 1.06 fmoles14.0 ± 1.0 fmoles14.6 ± 1.02 fmoles8.7 ± 0.62 fmoles * 11.9 ± 0.76fmoles ** 15.1 ± 1.05 fmoles14.0 ± 0.98 fmoles14.8 ± 0.99 fmoles15 .1 ± 1.35 fmoles ¹ Total oligos administered in equal amounts over four times over 48 hours. Treatment and analysis were performed as mentioned in the method. Significance was calculated through repeated-measures analysis of variance (ANOVA) and Tukey's reported t test. N in all groups was significantly different from 4-6 * mismatched control- and saline-treated groups, reliability p <0.001; ** significant difference from mismatched control root- and saline-treated groups, reliability, p <0.05

Example 21 Action of Dose-Response of Oligo I

Antisense Oligo I (SEQ ID NO: 1) has been found to reduce the effects of adenosine administration in animals in a dose dependent manner over the range of doses tested as shown in Table 6 below.

Table 6: Dose-response effects of antisense oligo I

Total dose (mg) PC ₅₀ Adenosine (mg Adenosine) Antisense Oligo I 0.2 2.0 20A ₁ MM2 Oligo (Control) 0.2 2.0 20 8.32 ± 7.214.0 ± 7.219.5 ± 0.342.51 ± 0.463.13 ± 0.713.25 ± 0.34 The results were studied by the student's paired t test and the statistical difference was found to be p = 0.05.

The anti-adenosine A1 receptor oligo, oligo I (SEQ ID NO: 1), specifically acts on the A ₁ receptor but does not act on the adenosine A ₂ receptor. Antisense Oligo I (SEQ ID NO: 1), as described in Example 9 above and in Nyce & Metzger (1997), supra (administering a dose of 5 mg four times at 8-12 hour intervals via a nebulizer or endotracheal tube) ) Or number of adenosine A ₁ and adenosine A ₂ receptors determined as recorded in treatment with mismatched control oligo (SEQ ID NO: 1682; A ₁ MM2), incised bronchial smooth muscle tissue, and Nyce & Metzger (1997) The above result is obtained.

Example 22 Specificity of Oligo I (SEQ ID NO: 1) for Target Gene Production]

Oligo I (SEQ ID NO: 1) is specific for adenosine A ₁ receptor but the mismatched control has no activity. FIG. 1 shows the results obtained from the crossover experiments described in Example 10 and Nyce & Metzger (1997), supra. Administration of two mismatched controls (SEQ ID NO: 1682 and SEQ ID NO: 1683) had no effect on PC ₅₀ adenosine values. Conversely, administration of antisense oligo I (SEQ ID NO: 1) resulted in a 7-fold increase in PC ₅₀ adenosine values. These results indicate that antisense oligo I (SEQ ID NO: 1) was administered extracellularly compared to the saline control group. It is clearly shown to reduce the response to the adenosine (relieving sensitivity) The results provided in Table 2 above confirm that the effect of antisense oligo I is dose dependent (see, Table 1). In addition, oligo I is particularly specific for the adenosine A ₁ receptor (cf. line 3 in the table above), and thus exhibits no activity at the very closely related A ₂ receptor or the bradykinin B ₂ receptor (see the table above). Lines 8-10 of 2. Further, the results shown in Table 2 show that antisense oligo I (SEQ ID NO: 1) is determined in a dose dependent manner and in an antisense oligo-dependent manner. . Reduce didanosine a sensitive two mismatch control oligonucleotide _{(A 1 MM2; SEQ ID NO} : 1682 , and A ₁ MM3 SEQ ID NO: 1683) all of which no effect at all on the number of the PC ₅₀ adenosine value or the adenosine A ₁ receptor Because it does not appear.

Example 23: Effect on allogene-induced bronchial stenosis and inflammation

Oligo I (SEQ ID NO: 1) has been shown to significantly reduce histamine-induced action in rabbit models when compared to mismatched oligos. Allergic effects of antisense oligo I (SEQ ID NO: 1) and mismatch oligos (A ₁ MM2, SEQ ID NO: 1682 and A ₁ MM3 SEQ ID NO: 1682) on allergen-induced airway obstruction and bronchial hypersensitivity Evaluation was made in sexual rabbits. The effect of antisense oligo I (SEQ ID NO: 1) on allergen-induced airway obstruction was evaluated.

As calculated from the plotted curve area, antisense oligo I significantly inhibited allergen-induced airway obstruction compared to the inconsistent control (55%, P <0.05; repeated ANOVA, and Turkey). t test) Mismatched oligo A ₁ MM2 (control) had no effect on the effect on allergen-induced airway obstruction. The effect of antisense oligo I (SEQ ID NO: 1) on allergen-induced BHR was determined as above. As calculated from the PC ₅₀ histamine value, antisense oligo I (SEQ ID NO: 1) significantly inhibited allergen-induced BHR compared to the inconsistent control (61%, p <0.05; repeated measures). ANOVA, t test in Turkey) A ₁ MM mismatched control was observed to have no effect on allergen-induced BHR.

These results suggest that antisense oligo I (SEQ ID NO: 1) is effective in protecting against allergen-induced bronchial stenosis (house dust). In addition, as shown by the effect on histamine sensitivity, which exhibits anti-inflammatory activity against antisense oligo I (SEQ ID NO: 1), antisense oligo I (SEQ ID NO: 1) is responsible for the induction of dust-induced bronchial hypersensitivity. It has been found to be a possible inhibitor.

Example 24 Antisense Oligo I Has No Toxic Side Effects

Oligo I (SEQ ID NO: 1) has been shown to be free of side effects that may be toxic to the agent. When 2.0 or 20 mg of Oligo I (SEQ ID NO: 1) was administered, there was no change in arterial blood pressure, cardiac output, heart rate, pulse rate, total terminal resistance or cardiac contractility (dPdT). In addition, cardiac output (CO), pulse rate (SV), mean arterial blood pressure (MAP), pulse rate (HR), total terminal resistance (TRP), and contractility (dPdT) were measured using a Cardiomax ^TM instrument (Columbus Instrument, Ohio). The measurement results were evaluated.

These results demonstrate that oligo I (SEQ ID NO: 1) does not have any detrimental effect on important cardiovascular parameters. More specifically, this oligo does not cause hypotension. This result is particularly important because other phosphorothioate antisense oligonucleotides have been shown to induce hypotension in some model systems in the past. Moreover, adenosine A ₁ receptors play an important role in isotonic conduction in the heart. It can be expected to deplete adenosine A ₁ receptors by antisense oligo I (SEQ ID NO: 1), resulting in detrimental extrapulmonary activity in response to downregulation of the receptors. But this is not so. Antisense Oligo I (SEQ ID NO: 1) does not cause any harmful intrapulmonary effects and administration of small amounts of this antisense oligo does not produce unexpected or unwanted side effects. This demonstrates that oligo I (SEQ ID NO: 1) is not in significant amounts causing toxic effects when administered directly to the lungs. This is in contrast to conventional adenosine receptor antagonists such as diophylline, which can be released from the lungs and cause toxic and even life-threatening effects.

Example 25 Long-Term Sustained Effect of Oligo I

Oligo I (SEQ ID NO: 1) showed long-lasting effects, as evidenced by the value of PC ₅₀ and resistance obtained by administering oligo I (SEQ ID NO: 1) prior to adenosine treatment. As described above, the persistence of the effect was measured with respect to the value of PC ₅₀ of adenosine antisense oligo I, when the same amount of 5 mg each was administered to the nebulizer each time through an endotracheal tube. The effect of the agent was significant from the first day to the eighth day after administration. When the action of antisense oligo I (SEQ ID NO: 1) disappeared, animals were administered saline (control) and the value of PC ₅₀ adenosine was measured again for all animals. Saline-treated animals showed a baseline PC ₅₀ adenosine value (n = 6).

As described above, airway resistance was measured in 6 allergic rabbits administered with 20 mg of antisense oligo I (SEQ ID NO: 1), measuring the persistence of the effect on resistance and the average calculated duration of effect It was 8.3 days in both PC ₅₀ adenosine (p <0.05) and resistance (p <0.05). These results show that antisense oligo I (SEQ ID NO: 1) has a very long effect period, which is completely unexpected.

Example 26 Antisense Oligo II

It has been found that antisense oligo IIs targeted to other regions of adenosine A ₁ receptor mRNA are very active against adenosine A ₁ -mediated effects. As described above, the effect of administration of antisense oligo II (SEQ ID NO: 7) was measured by compliance and resistance values when 20 mg of antisense oligo II or saline (control) was administered to two groups of allergic rabbits. It was. After administration of adenosine or saline, compliance and resistance values were measured as described in Example 13.

The effect of the antisense oligos of the present invention was statistically significant, with p <0.05 for compliance with the paired t-test and p <0.01 for resistance, which differed from the control.

These results showed that antisense oligo II (SEQ ID NO: 7) targeting the adenosine A ₁ receptor effectively maintains compliance and reduces resistance when administered adenosine.

Example 27 Antisense Oligos III and IV

As described above, antisense oligo III (SEQ ID NO: 8) and antisense oligo IV (SEQ ID NO: 9) are actually specifically targeted to adenosine A3 receptors, thus antisense oligo III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) was shown to reduce the number of inflammatory and inflammatory cells when administered 20 mg in an allergic rabbit. After placing the inflammatory cells in the bronchial lavage solution for 3 hours, the number of inflammatory cells was measured and there was at least 100 viable cells per wash. The effect of antisense oligo III (SEQ ID NO: 8) and IV (SEQ ID NO: 9) on total cells in granulocytes and bronchial liquor was assessed after exposure to dust allergens. These results showed that antisense oligo III (SEQ ID NO: 8) and antisense oligo IV (SEQ ID NO: 9) are very useful as anti-inflammatory agents in asthma lungs after exposure to dust allergens. As is known in the art, when administered antisense oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9), granulocytes, more specifically, eosinopil cells, are the primary inflammatory cells of asthma. ) Decreased by about 40% and 66%, respectively. Moreover, antisense oligos IV (SEQ. ID NO: 9) and III (SEQ. ID NO: 8) also reduced the total number of cells by about 40% and 80% in bronchial lavage, respectively. It is also an important indicator of anti-inflammatory activity by the anti-adenosine A ₃ agonists of the invention. Inflammation is known to underlie bronchial hypersensitivity and allergen-induced bronchial stenosis in asthma. Antisense Oligos III (SEQ ID NO: 8) and IV (SEQ ID NO: 9), which target adenosine A ₃ receptors, are both important new classes of anti-inflammatory agents that can be designed to specifically target lung receptors in species This can be a representative example.

Example 28 Antisense Oligo V

Antisense oligo V (SEQ. ID NO: 10) targeting adenosine A _2b adenosine receptor mRNA is very effective in reducing adenosine A2b-mediated action and reducing the number of adenosine A2b receptors by less than half.

Example 29 Unexpected Excellence of Oligo I-DS Substituted with Phosphodiester-Residue (SEQ. ID NO: 1681)

As described above, oligo I (SEQ. ID NO: 1) and I-DS (SEQ. ID NO: 1681) were separately isolated and administered to allergic rabbits, and then rabbits were administered adenosine. Phosphodiester oligo I-DS (SEQ. ID NO: 1681) is statistically significantly less effective at reducing the effects of adenosine, while oligo I (SEQ. ID NO: 1) is shown in 20 mg of PC ₅₀ adenosine. It has high efficiency.

Example 30 Antisense Oligo VI

For this task, an additional antisense phosphorothioate oligo (Oligo IV) was designed that targets the adenosine A ₁ receptor. As described in the patent application, antisense oligos are designed for the treatment of selected species and are generally specific to that species unless the fraction of adenosine mRNA of the other species selected is of similar sequence. This antisense oligo is prepared as follows and in vivo in a rabbit model for bronchial stenosis, inflammation and pulmonary allergy, with respiratory distress and obstruction as in the case of diseases such as asthma, as described in the same application above. Tested. The added oligos and their effects on rabbit models were studied and the results of the study were recorded and reviewed below. Oligos (antisense oligo VIs) of the present invention were selected in this study to supplement the results of SEQ ID NO: I (oligo I), which is an antisense of adenosine A ₁ receptor mRNA in the above-patent application. This additional oligo is identical to the antisense oligo VI and, unlike oligo I, targets another region of adenosine A ₁ receptor mRNA. The design and synthesis of this antisense oligo was carried out according to the method shown in Example 1 of this patent application.

Antisense Oligo VI is a phosphorothioate designed to target the coding region of rabbit adenosine A ₁ receptor mRNA corresponding to +964 to +984 relative to the start codon (starting point). Oligo VI is prepared as described and has 20 nucleotides in length, as described herein above. Oligo VI leads to the adenosine A ₁ receptor gene and has the sequence 5'-CGC CGG CGG CTG CGG GCC GG-3 '(SEQ. ID NO: _).

Phosphorothioate antisense oligo VIs having the sequences described in (5) above were synthesized in an applied biosystem model oligonucleotide synthesizer and purified using NENSORB chromatography (DuPont, DE). TETD (tetraethylthiuram dicellate) was used as a vulcanizing agent during the synthesis.

Example 31 Preparation of Allergic Rabbit

Newly born New Zealand White Pass ?? la-deficient rabbits are within 10 hours of birth, as described above in 10% kaolin (Metzger, WJ, in Late Phase Allergic Reactions, Dorsch, W., Ed., CRC Handbook, pp.347-362 , CRC Press, Boca Raton (1990); relevant portions of which are incorporated herein by reference in their entirety.) 312 antigen units / ml house dust mite (D. farinae) extract (Berkeley Biologicals, Berkeley, Intraperitoneal immunization with 0.5 ml of CA) (Metzger, WJ, in Late Phase Allergic Reactions, Dorsch, W., Ed., CRC Handbook, pp.347-362, CRC Press, Boca RAton (1990); Ali, S Metzger, WJ and Mustafa, SJ, Am. J. Resp. Crit.Care Med. 149: 908 (1994), all of which are hereby incorporated by reference in their entirety.

Immunized once a week for the first month and once every two weeks until the next four months. These rabbits preferably produce allergen-specific IgE antibodies, typically respond to allergen attacks in both early and late asthma reactions, and bronchial hypersensitivity (BHR) is shown. Allergens (312 unit dust allergens) are administered intraperitoneally each month to continuously stimulate and maintain allergen-specific IgE antibodies and BHR. At the age of four months, the immunized rabbits were prepared for aerosol administration as mentioned by Ali et al. (1994).

Example 32 Preparation of Adenosine Aerosol

Adenosine aerosol (20 mg / ml) was prepared by an ultrasonic nebulizer (Model 646, DeVilbiss, Somerset, PA) producing aerosol droplets. 80% of the aerosol droplets are smaller than 5 μm in diameter. The same amount of aerosol was administered directly to the lungs through the endotracheal tube in all three rabbits.

The animals then received aerosolized adenosine and calculated the amount of adenosine (PC50 adenosine) on the first day that the pretreatment value for responsiveness to adenosine made 50% loss of compliance. The aerosolized antisense (5 mg / 1.0 ml) was then administered to the animal via an endotracheal tube for 2 minutes (total 20 mg) twice a day for two days. Post-treatment PC ₅₀ values were recorded on the third morning (post-treatment dose). The results of these studies are mentioned in (9) below.

Example 33 Antisense Oligo Prescription

Each of the antisense oligos was individually dissolved in an aqueous solution and administered in 5 mg aliquots four times using an atomizer through an endotracheal tube, as described for antisense oligo I in (e) above.

Example 34 Oligo VI Reduces Response to Adenosine Administration More Than Oligo I.

Adenosine VI was tested and prepared in three allergic rabbits, as described in (7) and this patent application. Oligo VI targets the portion of the coding region of oligo I and other A ₁ receptors. All of these target sequences were randomly selected from many possible coding region target sequences. Three rabbits were treated as above Oligo I. Briefly, 5 mg of Oligo VI was sprayed into rabbits for two days at 8 hour intervals twice a day. The PC ₅₀ adenosine study was then compared to pretreatment PC ₅₀ values. The third morning was performed and the procedure was described in more detail in Nyce and Metzger (Nyce & Metzger, Nature 385: 721-725 (1997)).

The results obtained in three rabbits are shown in Table 1 below.

Table 1: PC ₅₀ Adenosine Before and After Aerosolized Adenosine Treatment

Processing time PC ₅₀ Adenosine After treatment 3.0 ± 2.1〉 20.0 * Maximum amount adenosine can be achieved by insolubility in saline

All three animals treated with Oligo VI completely reduced the sensitivity to adenosine up to the measurable level of the agent, as shown in Table 1 above. That is, administration of oligo VI alleviated adenosine-induced bronchial stenosis in three allergic rabbits. Thus, the actual efficacy of oligo IV is greater than can be measured in the experimental system used.

In comparison to the results from oligo I, oligo IV was found to exhibit as much or better effect than oligo I.

Example 34 Conclusion

The results of the work, as will be described in the Examples, show that all antisense oligonucleotides designed in accordance with the above-present application are very effective in neutralizing or reducing the action mediated by the receptors targeted by them. That is, two antisense oligos that target adenosine A ₁ receptor mRNA, one antisense oligo that targets adenosine A _2b receptor mRNA, and two antisense oligos that target A ₃ receptor mRNA are mediated by the specific receptors they target It is possible to alleviate the effects of extracellular administration of adenosine. In addition, the activity of the antisense oligos of the invention is specific to the target and cannot substantially inhibit other targets. In addition, the results show that the administration of the agents of the invention is very low or rarely present with side effects or toxicity. This shows 100% success due to the administration of agents that are highly effective and specific for the treatment of bronchial stenosis and / or inflammatory response. The invention can be broadly applied in the same manner to all genes and corresponding mRNAs encoding proteins associated with airway disease. Compared to the same oligonucleotide type in which phosphodiester bonds and phosphodiester bonds are substituted with phosphorothioester bonds, phosphothioester oligonucleotides have unexpected superiority over phosphodiester antisense oligos.

The above embodiments illustrate the invention, but are not limited thereto. In the same category of claims contained in the invention, the invention can be described by the following claims.

Claims (58)

5 between the target genes, coding or non-coding regions of RNA corresponding to target genes, initiation codons of genes, flanking regions of the genome, intron-exon boundaries, coding and non-coding regions Selected from the group consisting of all RNA fragments encoding proteins associated with '-terminal and 3'-terminal and juxta-compartment and one or more diseases or conditions or mixtures thereof An agent comprising an oligonucleotide that is anti-sense for at least two mRNAs. The method of claim 1, wherein one mRNA is transcriptional, stimulatory and active factors, interleukins, interleukin receptors, chemokines, chemokine receptors, intrinsically produced specific and non-specific Enzymes, immunoglobulins, antibody receptors, central nervous system (CNS) and peripheral and non-neural receptors, CNS and peripheral and non-neural peptide delivery agents, adhesion molecules, diphene An agent characterized in that it corresponds to an oncogene or codes for a protein selected from the group consisting of defensines, growth factors, vascular functional peptides and receptors, and binding proteins. The method of claim 2, wherein the encoded receptors and peptides are sympathetic neurostimulatory receptors, parasympathetic receptors, GABA receptors, adhesion receptors, adenosine receptors, bradykinin receptors, insulin receptors, glucagon (glucagon) receptors, prostaglandin receptors, thyroid receptors, androgen receptors, anabolic receptors, estrogen receptors, progesterone receptors, coagulation cascades and An agent characterized in that it is selected from the group consisting of related receptors, adenohypophyseal receptors, adenohypopisyl peptide transporters, and histamine receptors (HisR). The method of claim 2, wherein the encoded sympathetic receptors and parasympathetic receptors are acetylcholinesterase receptors (AcChaseR), acetylcholine receptors (AcChR), atropine receptors, muscarinic receptors, An agent characterized in that it is selected from the group consisting of epinephrine receptors (EpiR), dopamine receptors (DOPAR), tachychinnen receptors and norepinephrine receptors (NEpiR). The enzymes of claim 2, wherein the encoded enzymes are polymerases, kinases, oxidases, phosphatases, reductases, polysaccharins, triglycerides and proteolytic enzymes, esterases, elastases, and polysaccharides, triglycerides. An agent, characterized in that selected from the group consisting of cerides, lipids and protein polymerases. The enzymes of claim 5, wherein the encoded enzymes are tryptase, inducible nitric oxide polymerase, cyclooxygenase (Cox), MAP kinase, eosinophil peroxidase, β2-adrenergic receptor kinase, leukotriene c -4 polymerase, 5-lipooxygenase, phosphodiesterase IV, metalloprotenase, CSBP / p38 MAP kinase, neutrophyll elastase, phospholipase A2, cyclooxygenase 2 ( Cox-2), fucosyltransferase, IκB kinase 1 and 2, dentifrice, protein kinase C, thymitylate polymerase, tryptase, dihydrofolate reductase, tryptase, thymidine kinase, deoxyciti An agent characterized in that it is selected from the group consisting of Dean kinase, and ribonucleide reductase. The method according to claim 2, wherein the encoded factor is NfκB transcription factor, granulocyte macrophage colony stimulating factor (GM-CSP), AP-1 transcription factor, monocyte activating factor, Neutrophil chemotactic factor, granulocyte An agent characterized in that it is selected from the group consisting of colony-stimulating-factor (G-CSF), NFAT transcription factors, platelet activating factor, tumor necrosis factorα (TNFα), basal fibroblast growth factor (BFGF). The method of claim 2, wherein the encoded adhesion protein comprises intracellular adhesion molecules 1 (ICAM-1), 2 (ICAM-2) and 3 (ICAM-3), vascular cell adhesion molecule (VACM), endothelial leukocyte adhesion molecule. -1 (ELAM-1), GATA transcription factor, Neutrophil Adhesion Receptor, mad CAM-1, characterized in that the agent selected from the group consisting of. The method of claim 2, wherein the encoded cytokines, lymphokines, chemokines are interleukin-1 (IL-1), interleukin-1β (IL-1) interleukin-3 (IL-3), interleukin-4 (IL-). 4), interleukin-5 (IL-5), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-11 (IL-11), CCR-5 CC chemokines and Rantes Agent selected from the group consisting of: The method of claim 2, wherein the encoded receptor is adenosine A ₁ receptor, adenosine A ₂ B receptor, adenosine A ₃ receptor, endothelin receptor A, endothelin receptor B, IgE high-affinity receptor, muscarinic acetylcholine receptors. , Substrate P receptor, histamine receptor, CCR-1 CC chemokine receptor, substrate P, NK-1 and NK-3 receptors, CCR-2CC chemokine receptor, CCR-3 CC chemokine receptor (Eotaxin Receptor), interleukin- 1β receptor (IL-1βR), interleukin-1 receptor (IL-1R), interleukin-1β receptor (IL-1βR), interleukin-3 (IL-3R), CCR-4CC chemokine receptor, cysteine leukotriene receptors, pro Staroid receptors, interleukin-1 receptor (IL-1R), interleukin-4 receptor (IL-4R), interleukin-5 receptor (IL-5R), interleukin-8 receptor (IL-8R), interleukin-9 receptor ( IL-9R), interleukin-11 receptor (IL-11R), bradykinin B2 receptor, sympathetic stimulatory receptors, parasympathetic nervous system receptor, GABA receptor Sieves, adenosine receptors, bradykinin receptors, insulin receptors, glucagon receptors, prostaglandin receptors, thyroid receptors, androgen receptors, anabolic receptors, estrogen receptors, progestron receptors , An adenohypopicil receptors, and histamine receptors (HisR). The method of claim 2, wherein the encoded protein is eotaxin, major basic protein, preproendothelin, eosinophil cationic protein, P-selectin, STAT 4, STAT 6, c-mas, NF-interleukin-6 (NF-IL-6), MIP-1α, MCP-2, MCP-3, MCP-4, cyclophylline, PDG2, cyclosporin A-binding protein, FK5 -Binding protein, fibronectin, LFA-1 (CD11a / CK18), PECAM-1, C3bi, PSGL-1, CD-34, substrate P, p150,95, Mac-1 (CD11b / CD18), VLA-4 , CD-18 / CD11a, CD11b / CD 18, C5a, CCR1, CCR2, CCR4, CCR5, and LTB-4. 3. The agent of claim 2, wherein the encoded defensin is selected from the group consisting of defensin 1, defensin 2, and defensin 3. The agent of claim 2, wherein the encoded selectin is selected from the group consisting of α4β1 selectin, α4β7 selectin, LFA-1 selectin, E-selectin, P-selectin, and L-selectin. The agent according to claim 2, wherein the mRNA corresponds to an oncogene selected from the group consisting of ras, src, myc, and bcl-2. The method of claim 1, wherein at least one mononucleotide-linked phosphodiester moiety of the anti-sense oligonucleotide is methylphosphonate, phosphoester, phosphorothioate, phosphorodithioate, boranophosphate. , Formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene (methimino), methylenoxy (Methyllimino), 2'-0-methyl, phosphoramidate residues, and combinations thereof. The agent of claim 15, wherein the agent is substituted with all phosphodiester residues. The agent of claim 1, wherein the anti-sense oligonucleotide consists of about 7 to 60 mononucleotides. The agent of claim 1, wherein the oligo comprises at least about 15% A. 3. The agent of claim 1, wherein the oligo is free of adenosine. The method of claim 1, wherein the anti-sense oligo is characterized in that selected from the group consisting of oligos consisting of SEQ ID NO-to-, SEQ ID NO-to-, and SEQ ID NO-to-(fragment number-to-). agent. The method of claim 1, wherein at least one A is heteroaromatic bases that bind to thymidine base but have antagonist activity, no more than about 0.3 adenosine bases having agonist activity at adenosine A ₁ , A _2b and A ₃ receptors, And heteroaroma bases having no activity on the adenosine A _2a receptor or having antagonist activity. The method of claim 21, wherein the heteroaromatic base is selected from the group consisting of pyrimidines and purines, O, halo, NH ₂ , SH, SO, SO ₂ , SO ₃ , COOH, and branched and conjugated primary and secondary amino, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl , Heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsuloxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcyclo May be substituted by alkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, Agent which can be further substituted by O, halo, NH, primary, secondary and tertiary amines, SH, SO, SO ₂ , SO ₃ , cycloalkyl, heterocycloalkyl and heteroaryl. The agent of claim 22, wherein the pyrimidines and purines are substituted at positions 1, 2, 3, 4, 7 and 8 24. The pyrimidines and purines of claim 23, wherein the pyrimidines and purines are of the formula-theophylline, caffeine, dyphylline, etophylline, acephylline, piperazine ), Bamifylline, enprofylline and xanthine. [Formula 1] Wherein R ¹ and R ² are independently H, alkyl, alkenyl, or alkynyl, R ³ is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloakenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloalkynyl, NH ₂ -alkylamino-ketoxyalkyloxy-aryl and mono and diaalkylaminoalkyl-N-alkylamino-SO ₂ -aryl. The method of claim 24, wherein the general base is 3-nitropyrrole-2'-dioxynucleotide, 5'-nitro-indole, 2-dioxyribosyl- (5-nitroindole), 2-dioxyribofuranosyl- (5 -Nitroindole), 2'-deoxyinosine, 2'-deoxynebulin, 6H, 8H-3,4-dihydropyrimido [4,5-c] oxazine-7-1 or 2-amino An agent selected from the group consisting of -6-methoxyaminopurine. The agent of claim 1, wherein the methylated cytosine ( ^m C) is substituted with at least one CpG dinucleotide when present as an oligo. The agent of claim 1, wherein the anti-sense oligonucleotide is operably linked to an up-taken agent internalized or uptake into a cell, or to a eukaryotic or prokaryotic vector. 28. The agent of claim 27, wherein the agent that has been refractory to or taken up by the cell is selected from the group consisting of transferrin, aziallolycoprotein, and streptavidin. A composition comprising the agent according to claim 1 and a pharmaceutically or veterinary acceptable carrier. 30. The composition of claim 29, wherein the carrier is selected from the group consisting of gas, liquid and solid carrier. The method of claim 29, wherein the other therapeutic compounds, surfactants, antioxidants, fragrances and colorants, fillers, volatile oils, buffers, dispersants, RNA inerts, antioxidants, fragrances, A composition further comprising an agent selected from the group consisting of propellants and preservatives. 30. The composition of claim 29, wherein the anti-sense oligonucleotide is present in an amount of about 0.01 relative to about 99.99 w / w of the composition. 33. The composition of claim 32 comprising nucleic acids, surfactants and carriers. 34. The surfactant of claim 33, wherein the surfactant is selected from surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant protein and active fragments thereof, non-dipalmitoyl unsaturated phosphatidylcholine, dipalmi Toylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidyl incitol, phosphatidylethanolamine, phosphatidylserine, phosphoric acid, ubiquinones, lysososphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dihydroepiandrosterone, dolly Field, sulfuric acid, glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycerol-3-phosphocholine, dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline , Choline phosphate, artificial lamellar excipients for surfactant components, Omega-3 Sulfated Acids, Polyenic Acid, Polyenoic Acid, Lecithin, Palmitic Acid, Non-Ion Ethylene and / or Propylene Oxide Copolymers, Polyoxypropylene, Polyoxyethylene, Dextran and / or Poly (vinyl amine) with alkanoyl side chains, Brij 35, Triton X-100, ALEC, Exosurf, Survant and Atovaquone. A formulation comprising the composition of claim 26 wherein the carrier is a hydrophobic carrier. 36. The formulation of claim 35, wherein the carrier is lipid particulates or excipient. 37. The formulation of claim 36, wherein the excipients are liposomes and the microparticles are microcrystals. 36. The formulation of claim 35, wherein the lipid excipients consist of N- (1- [2,3-dioleooxyloxy] propyl) -N, N, N-trimethyl-ammonium methylsulfate. 35. A formulation according to claim 34, which contains a formulation suitable for respiration. 35. The formulation of claim 34, which contains an aerosol. 35. A product according to claim 34, wherein the product is in the form of a single or multiple unit. 35. A product according to claim 34, which is in a bulk state. A capsule or cartrige containing the composition of claim 26. A kit comprising a delivery device in a separate container, instructions for composition and use of claim 26. 45. The kit of claim 44, wherein the delivery device comprises a nebulizer for delivering a single measured dose of the formulation. 45. The kit of claim 44, wherein the nebulizer consists of an insufflator and the composition is provided in a pierceable or openable capsule or medicine. 45. The kit of claim 44, wherein the delivery device consists of a compressed inhaler and the composition consists of a suspension or solution phase agent. 45. The method according to claim 44, wherein the other therapeutic compounds, surfactants, antioxidants, fragrances and colorants, fillers, volatile oils, buffers, dispersants, internalized or uptake into cells taken) A kit further comprising an agent selected from the group consisting of agents, RNA inerts, antioxidants, fragrances, propellants and preservatives in a separate container. 49. The surfactant of claim 48, wherein the surfactant is selected from: surfactant protein A, surfactant protein B, surfactant protein C, surfactant protein D and surfactant proteins and active fragments thereof, non-dipalmitoyl unsaturated phosphatidylcholine, dipalmi Toylphosphatidylcholine, phosphatidylcholine, phosphatidylglycerol, phosphatidyl incitol, phosphatidylethanolamine, phosphatidylserine, phosphoric acid, ubiquinones, lysososphatidylethanolamine, lysophosphatidylcholine, palmitoyl-lysophosphatidylcholine, dihydroepiandrosterone, dolly Field, sulfuric acid, glycerol-3-phosphate, dihydroxyacetone phosphate, glycerol, glycerol-3-phosphocholine, dihydroxyacetone, palmitate, cytidine diphosphate (CDP) diacylglycerol, CDP choline, choline , Choline phosphate, artificial lamellar excipients for surfactant components, Omega-3 Sulfated Acids, Polyenic Acid, Polyenoic Acid, Lecithin, Palmitic Acid, Non-Ionic Ethylene and / or Propylene Oxide Block Copolymers, Polyoxypropylene, Polyoxyethylene, Dextran and / or Alkanoyl A kit characterized in that it is selected from the group consisting of poly (vinyl amine) with side chains, Brij 35, Triton X-100, ALEC, Exosurf, Survant and Atovaquone. 45. The kit of claim 44, wherein the composition is provided in capsule or medicinal form. A cell comprising the agent of claim 1. A method of treating a disease or disorder associated with mRNA corresponding to at least one or more target gene (s), flanking regions of the genome, or proteins, Comprising the step of administering the agent of claim 1 to the subject or diseased subject, Wherein said agent is administered by a subject at a dosage effective to reduce productivity or effectiveness or increase degradation of at least one or more target mRNAs. 53. The method of claim 52, wherein said agent is administered in a dosage effective to reduce production or effectiveness or increase degradation of at least two target mRNAs. 53. The method of claim 52, wherein said agent is administered directly to the subject's lungs. 55. The method of claim 54, wherein said agent is administered in an aerosol suitable for respiration. The method of claim 52, wherein the disease or condition is a lung disease or disease, and the at least one target mRNA comprises a protein selected from the group consisting of adenosine A ₁ receptor, adenosine A ₂ B receptor, adenosine A ₃ receptor, and bradykin B2 receptor. Coding. The method of claim 56, wherein the disease or condition is associated with a disorder of the subject's airways. 59. The method of claim 57, wherein the disease or condition is asthma.