CA2330022A1 - Low adenosine anti-sense oligonucleotide, compositions, kit and method for treatment of airway disorders associated with bronchoconstriction, lung inflammation, allergy(ies) and surfactant depletion - Google Patents

Abstract

An in vivo method of selectively delivering a nucleic acid to a target gene or mRNA, comprises the topical administration, e.g. to the respiratory system, of a subject of a therapeutic amount of an oligonucleotide (oligo) that is anti - sense to the initiation codon region, the coding region, the 5' or 3' inton- exon junctions or regions within 2 to 10 nucleotides of the junctions of the gene or antisense to a mRNA complementary to the gene in an amount effective to reach the target polynucleotide and reducing or inhibiting expression. In addition a method of treating and adenosine mediated effect, comprises topically administering to a subject an anti-sense oligo in an amount effective to treat the respiratory, pulmonary, or airway disease. In order t o minimize triggering adenosine receptors by their metabolism, the administere d oligos have a low content of or are essentially free of adenosine. A pharmaceutical composition and formulations comprise the oligo anti-sense to an adenosine receptor, genes and mRNAs encoding them, genomic and mRNA flanking regions, intron and exon borders and all regulatory and functionall y related segments of the genes and mRNAs encoding the polypeptides, their sal ts and mixtures. Various formulations contain a requisite carrier, and optional ly other additives and biologically active agents. The low adenosine or adenosi ne free (des-A) agent for practicing the method of the invention may be prepare d by selecting a target gene(s), genomic flanking region(s), RNA(s) and/or polypeptide(s) associated with a disease(s) or condition(s) afflicting lung airways, obtaining the sequence of the mRNA(s) corresponding to the target gene(s) and/or genomic flanking region(s), and/or RNAs encoding the target polypeptide(s), selecting at least one segment of the mRNA which may be up t o 60 % free of thymidine (T) and synthesizing one or more anti-sense oligonucleotide(s) to the mRNA segments which are free of adenosine (A) by substituting a universal base for A when present in the oligonucleotide. The agent may be prepared by selection of target nucleic acid sequences with GC running stretches, which have low T content, and by optionally replacing A i n the anti-sense oligonucleotides with a "Universal or alternative base". The agent, composition and formulations are used for prophylactic, preventive an d therapeutic treatment of ailments associated with impaired respiration, lung allergy(ies) and/or inflammation and depletion lung surfactant or surfactant hypoproduction, such as pulmonary vasoconstriction, inflammation, allergies, allergic rhynitis, asthma, impeded respiration, lung pain, cystic fibrosis, bronchoconstriction. The present treatment is suitable for administration in combination with other treatments, e.g. before, during and after other treatments, including radiation, chemotherapy, antibody therapy and surgery, among others. Alternatively, the present agent is effectively administered prophylactically or therapeutically by itself for conditions without known therapies or as a substitute for therapies exhibiting undesirable side effects. The treatment of this invention may be administered directly into t he respiratory system of a subject so that the agent has direct access to the lungs, or by other effective routes of administration, e.g. topically, transdermally, by implantation, etc., in an amount effective to reduce or inhibit the symptoms of the ailment.

Description

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WO OOI62'I36 PCT/US00/08020 LOW ADENOSINE ANTI~ENSE OLIGONUCLEOTIDE, COMPOSITIONS, KIT
& METHOD FOR TREATMENT OF AIRWAY DISORDERS ASSOCIATED
WITH BRONCHOCONSTRICTION, LUNG INFLAMMATION, ALLERGY(llES) & SURFACTANT DEPLETION
BACKGROUND OF THE INVENTION
Field of the Invention This patent relates to a composition comprising oligonucleotides (oligos) that are anti-sense to adenine receptors, and contain low amounts of or no adenosine (A). These agents are suitable for the treatment, among others, of pulmonary diseases associated with inflammation, impaired airways, including lung disease and diseases whose secondary effects afflict the lungs of a subject. Examples of these diseases are allergies, asthma, impeded respiration, allergic rhynitis, pain, cystic fibrosis, and cancers such as leukemias, e.g. colon cancer, and the like. The present agent may be administered prophylactically or therapeutically in conjunction with other therapies, or may be utilized as a substitute for therapies that have significant, negative side effects.
Backstround o~'the Invention Respiratory ailments, associated with a variety of diseases and conditions, are extremely common in the general population, and more so in certain ethnic groups, such as African Americans.
In some cases they are accompanied by inflammation, which aggravates the condition of the lungs.
Asthma, for example, is one of the most common diseases in industrialized countries. In the United States it accounts for about I% of all health care costs. An alarming increase in both the prevalence and mortality of asthma over the past decade has been reported, and asthma is predicted to be the preeminent occupational lung disease in the next decade. While the increasing mortality of asthma in industrialized countries could be attributable to the depletion reliance upon beta agonises in the treatment of this disease, the underlying causes of asthma romain poorly understood.
Adenosine may constitute an important mediator in the lung for various diseases, including bronchial asiltma. Its potential role was suggested by the finding that asthmatics respond favorably to aerosolized adenosine with marked bronchoconstriction whereas normal individuals do not. An asthmatic rabbit animal model, the dust mite allergic rabbit model for human asthma, responded in a similar fashion to aerosolized adenosine with marked bronchoconstriction whereas non-asthmatic rabbits showed no response. More recent work with this animal model suggested that adenosine-induced bronchoconstriction and bronchial hyperresponsiveness in asthma may be mediated primarily through the stimulation of adenosine receptors. Adenosine has also been shown to cause adverse effects, including death, when administered therapeutically for other diseases and conditions in subjects with previously undiagnosed hyper reactive airways.
A handful of medicaments have been available for the treatment of respiratory diseases and conditions, although in general they all have limitations. Theophyliine, an important drug in the treatment of asthma, is a known adenosine receptor antagonist which was reported to eliminate adenosine-mediated bronchoconstriction in asthmatic rabbits. A selective adenosine A, receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX) was also reported to inhibit adenosine-mediated bronchoconstriction and bronchial hyperresponsiveness in allergic rabbits. The therapeutic and preventative applications of currently available adenosine A, receptor-specific antagonists are, nevertheless, limited by their toxicity. Theophylline, for example, has been widely used in the treatment of asthma, but is associated with frequent, significant toxicity resulting from its narrow therapeutic dose range. DPCPX is fat too toxic to be useful clinically, The fact that, despite decades of extensive research, no specific adenosine receptor antagonist is available for clinical use attests to the general toxicity of these agents. Anti-sense oligonucleotides have received considerable theoretical consideration as potential useful pharmacological agents in human disease.
Their practical application in actual models of human disease, however, has been somewhat elusive. One important impediment to their effective application has been a difficulty in finding an appropriate route of administration to 2 PCT/US00!(1R(120 deliver them to their site of action. Many in vivo experiments were conducted by administering anti-sense oligonucleotides directly to specific regions of the brain. These applications, however, necessarily have limited clinical utility due to their invasive nature.
Although anti-sense oligonucleotides have received considerable theoretical consideration for their potential use as pharmacological agents in human disease, fording practical and effective applications for these agents in actual models of human disease, however, have been few and far between, particularly because they had to be administered in large doses. Another important consideration in the phartnacoiogic application of these molecules is their route of administration. Many in vivo applications have involved the direct administration of anti-sense oligonucleotides to limited regions of the brain. Such applications, however, have limited clinical utility due to their invasive nature. The systemic administration of anti-sense oligonucleotides as pharmacological agents has been found to have also significant problems, not the least of which being an inherent difficulty in targeting disease-involved nssues. That is, the necessary dilution of the anti-sense oligonucleotide in the circulatory system makes extremely difficult to attain a therapeutic dose at the target tissue by intravenous or oral administration.
The bioavailabiiity of orally administered anti-sense oligonucleotides is very low, of the order of less than about 5%. Anti-sense oligonueleotides have been used in therapy by many, including the present inventor, who in his previous work successfully treated various diseases and conditions by direct administration of these agents to the lung. In many instances, other workers have had to face the difficulties associated with the delivery of DNA molecules to a desired target. Thus, the route of administration may be of extreme importance for treating generalized diseases and conditions as well as those which are localized. In contrast, up to the present time, the delivery of anti-sense agents to the lung has been relatively undeveloped. As described by the present inventor in more detail below, the lung is an excellent target for the direct administration of anti-sense oligonucleotides and provides a non-invasive and a tissue-specific route.
Clearly, there exist presently no effective therapies for treating these ailments, or at least no therapies which are effective and devoid of significant detrimental side effects. Accordingly, there is still a need for an agent for the treatment of adenosine mediated ailments afflicting the pulmonary and respiratory ailments affecting the lung airways, including respiratory problems, bronchoconstriction, inflammation, allergy(ies), depletion or hyposecretion of surfactant, ere., which is highly effective and sufficiently selective to avoid detrimental side effects produced by other therapies. In addition, there is a definite need for making available a delivery method that will require low amounts of therapeutic agents and will be effective for the rapid and targeted access of tissue genes of tnRNAs and the reversal of untoward effects afflicting a subject.
SiJN>1~IARY OF THIS INVENTION
The present invention generally relates to a pharmaceutical or veterinary composition, comprising an anti-sense oligonueleotide(s) (oligo(s)) which is (are) effective for alleviating bronchoconstriction and/or lung inflammation, allergy(ies), and\or surfactant depletion andlor hyposecretion, when administered to a mammal, the oligo containing about 0 to about 15% adenosine (A) and being anti-sense to a target selected from the group consisting of the initiation codon, the coding region, the 5'-end and the 3'-end genomic flanking regions, the 5' and 3' intron-exon junctions, and regions within 2 to 10 nucleotides of the junctions of a gene encoding a target polypeptide associated with lung airway dysfunction or anti-sense to the polypeptide mRNA;
combinations of the oligos; and mixtures of the oligos; and a pharmaceutically or vcterinarily acceptable carrier or diluent.
The targets are typically molecules associated with airway disease, cancer, etc., such as transcription factors, stimulating and activating peptide factors, cytokines, cytokine receptors, chemokines, chemokine receptors, adenosine receptors, bradykinin receptors, endogenously produced specific and non-specific enzymes, immunoglobulins and antibodies, antibody receptors, central nervous system (CNS) and peripheral nervous and non-nervous system receptors, CNS and peripheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensins, growth factors, vasoactive peptides and receptors, binding proteins, and malignancy associated proteins, among others. Examples are oligo(s) targeted to adenosine receptors) and it(they) are typically present in the composition in an amount effective to reduce adenosine mediated effect(s), such as airway obstruction, inflammation, allergy(ies), and sufactant depletion, among others. The adenosine receptor is preferably selected from the group consisting of the adenosine A,, Air, and A~ receptors, and in some instances even adenosine S AZ, receptors. The oligo of the invention may be applied to the preparation of a medicament for (a) reducing adenosine-mediated bronchoconstriction, impeded respiration, inflammation, allergy(ies), depletion production of surfactant, and other detrimental pulmonary effects in a subject in need of treatment, and/or for (b) treating specific diseases and conditions such as asthma, cystic fibrosis, allergic rhynitis, COPD, etc. For the fast time this invention provides the targeted administration of l0 one or mote oligonucleotides directly into the repiratory system. The oligos tray be directed to any target and are intended for fast delivery through the mucosal tissue of the lungs for hybridization to a desired target polynucleotide, e, g. mRNA, to prevent gene transcription and translation, such that protein expression will be reduced, hampered, or completely stopped. Thus, this invention also provides a more general method for administering oligonucleotides that are anti-sense to targeted genes IS and mRNAs associated with any type of diseases, by direct administration into the respiratory system, e. g. by inhalation, by introduction of a solution or aerosol into the respiratory airways, and/or directly into the lung.
The present oligos, moreover, are suitable for reducing effects mediated by a variety of target proteins and genes, for example adenosine-mediated effects, including pulmonary, respiratory, and 20 other associated effects, e. g, bronchoconstnction, inflammation, immune mediated reactions, allergy(ies) and other airway problems, which may be caused by different conditions, including cancer.
Examples of diseases and conditions, which may be treated preventatively, prophylactically and therapeutically with the agent of this invention, are pulmonary vasoconstriction, inf<ammation, allergies, asthma, impeded respiration, respiratory distress syndrome, pain, cystic fibrosis, allergic 25 rhynitis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), bronchitis, and cancers such as leukemias, lymphomas, carcinomas, and the like, e.g. colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellutar carcinoma, kidney cancer, melanoma, hepatic metastases, etc., as well as all types of cancers which may metastasize or have metastasized to the tung(s), including breast and prostate cancer. The present 30 agents are also suitable for administration before, during and after other treatments, including radiation, chemotherapy, antibody therapy, phototherapy and cancer, and other types of surgery. 'the present agent is effectively administered prophylactically and therapeurically in conjunction with other therapies, or by itself for conditions without known therapies or as a substitute for therapies that have significant negative side effects. The oligo(s) may be administered by any means known to a subject, 35 e. g. to the lungs of the subject, more generally through any and all systemic and topical routes. This oligonucleotide(s) (oligo(s)) employed are anti-sense to to a target DNA or RNA, e. g. an adenosine receptor DNA or RNA, and preferably consist essentially of up to about I 5%
adenosine (A), and more preferably contain no adenosine. The oligos are provided in the form of specific compositions and formulations, with a carrier or diluent, and optionally with other therapeutic agents and additives which 40 are used for administration by specific routes, e.g. into the respiratory system, topically, transdermally, parenterally, by implantation, and the Like. The oligo is also provided as a capsule or carnidge, and in the form of a kit. The oligos of the invention may be produced by selection of speciGe targeted segments of the gene or ntRNA encoding the adenosine receptor as described below. In one preferred embodiment, the selection is made to obtain oligos that consisting essentially of less than about i5%
45 adenosine (A). This may be done by selecting the target as done above, which includes genes, genomic flanking regions, RNAs and polypeptide associated with an ailment afflicting the lung airways, obtaining the sequence of a mRNA(s) corresponding to the target genes) and/or their genomic flanking regions) and/or the juxta-membrane regions thereof, and mRNA(s) encoding the target polypeptide(s), selecting at least one segment of the mRNA(s), and synthesizing one or more anti-sense 50 oligonucleotide(s) to the selected mRNA segment(s), and subsfituting, if necessary, an alternative, e. g.

WO 00/62736 PCTlUS001118020 a universal bases) or other bases) for one or more A to reduce the proportion of A present in the oligonucleotide to less than about 1S%, and down to no adenosine. Similarly, alternative and/or universal bases may be substituted for adenosine, e. g. specific adenosine A1, A2b and A3 receptor antagonists or A2a receptor agonists, theophilline, enprophylline, and many other adenosine receptor S antagonists known in the an as well as agonises with significantly reduced agonise activity with respect to adenosine, e. g. less than 0.5%, less than 0.3%, and the like.
The invention will now be described in general in conceptual and experimental terms, with reference to specific examples. Other obaects, advantages and features of the present invention will become apparent to those skilled in the an from the description that follows.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
This invention arose from a desire by the inventor to improve an prior art treatments for pulmonary and other diseases, which technology is generally frought with detrimental side effects and by the need of administering high doses of therapeutical agents. The present invention arises from the inventor's own discovery that adenosine receptor targeted anti-sense oligonucleotides (oligos) may be IS utilized therapeutically in the treatment of diseases or conditions which impair respiration, cause inflammation and/or allergy(ies), constrict bronchial tissue, obstruct the lung airways, depletion surfactant secretion, or otherwise impede normal breathing. In general, many diseases and conditions are associated with or cause inflammation, constrict bronchial tissue or the lung airways, depletion secretion of surfactant, augment allergy(ies), or otherwise impede normal breathing. 'This treatment is selective for specific targets associated with or mediating these symptoms, and the agents are administered in up to 1000-fold lower doses than those seen in the art. The inventor, in addition, wanted to provide a treatment which would improve the outcome and life style of patients undergoing other procedures or being administered other therapies, including antibody therapy, chemotherapy, radiation, phototherapy, and surgery e.g. cancer surgery, and that could be effectively administered preventatively, prophylacticaliy or therapeutically- He reasoned that he could further improve on this discovery by selecting oligos of reduced adenosine content, or reducing the adenosine content of otherwise targeted anti-sense oligos corresponding to endogenous polynucleotide sequences: The present invention is premised on the discovery by the inventor that oligonucleotides are metabolized in vivo to their mononucleotides. Adenosine (A)-containing ohgonucleotides break down and release adenosine which, in turn, activates adenosine receptors, thereby causing bronchoconstriction, inflatnmation, surfactant depletion, allergy(ies), and the like. He, thus, conceived of employing low adenosine-free adenosine oligos to avoid these side effects upon their administration. He succeeded in this endeavor and is providing in this patent novel and improved compositions, formulations and methods which afford greatly improved results when compared with previously known treatments for 3S preventing and alleviating bronchoconstriction, allergy(ies), inflammation, breathing difficulties, surfactant depletion and blockage of airways, as well as for other conditions which affect the lung directly or indirectly. In different embodiments, one or more nucleic acids of the invention may be formulated atone, and/or with one or more surfactant components and/or with a carrier, andlor with other therapeutic agents and/or formulation agents known in the art. The compositions of this invention, thus, may be incorporated into a variety of formulations for systemic and topical administration. Moreover, the inventor also provides a broad method for delivery of anti-sense oligonucleotides (oligos) through the respiratory system, as a fast means of starting treatment to address acute attacks of asthma and other diseases and conditions that have a rapid onset. In addition, the present agents have long halflives and may be administered at very low doses. This makes them 4S ideal for once a week type therapies. In the past, anti-sense oligonueleotides received considerable theoretical consideration as being potentially useful as pharmacologic agents for the treatment of human disease. Wagner, R., Nature 372: 333-335 ( 1994) However, it has been difficult to actually apply these molecules to allemating and curing human diseases. One important consideration in the phacmacologic application of these molecules has been the failure of various routes of administration to deliver the compounds to its target while avoiding invading the circulation and, therefore, other WO 00/62736 PCT/US(1(1/OR1120 untargeted tissues which, thus, produces a plethora of side effects. Most in vivo experiments utilizing anti-sense oligonucleotides involved a direct application of the oligo to limited regions of the brain.
See, Wahlestedt, C., Trends in Pharmacol. Sri. I5: 42-46 (1994); Lai, J. et al., Neuroreport S: 1049-1052 ( t 994); Standifer, K., et al., Neuron 12: 805-810 ( 1994}; Akabayashi, A., et al,, Brain Res. 21:

5 55-61 (1994). Others applied them into the spinal fluid. See, e.g, Tseng, 1.., et al., European J.
Pharmacol. 258: RI-3 (1994); Raffa, R., et al., European J. Phatmtacol. 258:
R5-7 (1994); Gillardon, F., et al., European J. Neurosci. 6: 880-884 ( 1994). Such applications, clearly, have no practical clinical utility due to their invasive nature. Thus, the systemic administration of anti-sense oligonucleotides poses significant problems with respect to their pharmacologic application, not the least of which is the difficulty in selectively targeting disease-involved tissues. The systemic administration of anti-sense oligonucleotides also poses significant problems with respect to their pharmacologic application, not the least of which is the difficulty in selectively targeting disease-involved tissues.
The respiratory system, and in particular the lung, as the ultimate port of entry into the I S organism, however, is an excellent route of administration for anti-sense oligonucleotides. This is so not only for the treatment of lung disease, but also when utilizing the lung as a means for delivery, particularly because of its non-invasive and tissue-specific nature. Thus, local delivery of antisense oligonucleotides directly to the target tissue enables the therapeutic use of these compounds.
Fomivirsen (ISIS 2302) is an example of a local drug delivery into the eye to treat cytomegalovirus (CMV) retinitis, for which a new drug application has been filed by ISIS. The administration of a drug through the Iang offers the further advantage that inhalation is non-invasive whereas direct injection in to the vitreous of the eye is invasive. The composition and formulations of this invention are highly efficacious for preventing and treating diseases and conditions associated with bronchoconstriction, difficult breathing, impeded and obstructed lung ainrvays, allergy(ies), inflammation and surfactant depletion, among others. Examples of diseases and conditions which are suitably treated by the present method are diseases and conditions, including Acute Respiratory Distress Syndrome CARDS), asthma, adenosine administration e.g. in the treatment of SupraVentricular Tachycardia (SVT) and other arrhythmias, and in stress tests to hyper-sensitized individuals, ischemia, renal damage or failure induced by certain drugs, infantile respiratory distress syndrome, pain, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), lung transplantation rejection, pulmonary infections, and cancers such as leukemias, lymphomas, carcinomas, and the like, including colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatoceilular carcinoma, kidney cancer, melanoma, hepatic metastases, ere., as well as all types of cancers which rnay metastasize or have metastasized to the lung(s), including breast and prostate cancer. The invention will be described with respect to the adenosine receptors as targets, but is similarly applicable to any other target with respect to the pulmonary administration of anti-sense oligos. The examples provided below show a complete inhibition of such adenosine receptor associated symptoms in a rabbit model for human bronchoeonstriction, allergy(ies) and inflammation as well as the elimination of the ability of the adenosine receptor agonist par excellence, adenosine, to cause bronchoeonstriction in hyper-responsive monkeys, which are animal models for human hyper-responsiveness to adenosine receptor agonists. The pharmaceutical composition and formulations of the invention, therefore, are suitable for preventing and alleviating the symptoms associated with stimulation of adenosine receptors, such as the adenosine A, receptors. The compositions and formulations of this invention, thus, are also suitable for prevent the untoward side effects of adenosine-mediated hyperresponsiveness in certain individuals, which are generally seen in diseases affecting respiratory activity.
The method of the present invention may be used to neat airway diseases and conditions in a subject of any kind and for any reason, with the intention that the adenosine content of anti-sense compounds be minimized, reduced or eliminated so as to prevent its liberation upon anti-sense degradation. Examples of diseases and conditions, which may be treated preventatively, 6 PCTlUS11111t1802(1 prophyiacticaliy and therapeutically with the compositions and formulations of this invention, are pulmonary vasoconstriction, inflammation, allergies, asthma, allergic rhynitis, impeded respiration, Acute Respiratory Distress Syndrome CARDS), renal damage and failure associated with ischemia as well as the administration of certain drugs, side effects associated with adenosine administration e.g. in SupraVentricular Tachycardia (SVT) and in adenosine stress tests, infantile Respiratory Distress Syndrome (infantile RUS), ARDS, pain, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), lung transplantation rjejection, pulmonary infections, and cancers such as leukemias, lymphomas, carcinomas, and the like, e.g. colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney IO cancer, melanoma, metastatic cancer such as hepatic metastases, lung, breast and prostate metastases, among others. The present compositions and formulations are suitable for administration before, during and after other treatments, including radiation, chemotherapy, antibody therapy, phototherapy and cancer, and other types of surgery. The present compositions and formulations tray also be administered effectively as a substitute for therapies that have significant negative side effects.The terms "anti-sense" oligonucleotides generally refers to small, synthetic oligonucleotides, resembling single-stranded DNA, which in this patent are applied to the inhibition of gene expression by inhibition of a target messenger RNA (mRNA). See, Milligan, 1. F. et al., 1.
Med. Chem. 36(14), 1923-1937 (1993), the relevant portion of which is hereby incorporated in its entirety by reference. For consistency=s sake, all RNAs and oligonucleotides are represented in this patent by a single strand in the 5' to 3' direction, when read from left to right, although their complementary sequences) is (are) also encompassed within the four corners of the invention. In addition, all nucleotide bases and amino acids are represented utilizing the recommendations of the CUPAC-IUB
Biochetrueal Nomenclature Commission, or by the known 3-letter code (for amino acids). Nucleotide sequences ate presented herein by single strand only, in the 5' to 3' direction, from left to right.
In addition, nucleotide and amino acids are represented herein in the manner recommended by the IUPAC-IUB
Biochemical Nomenclature Commission, or (for amino acids) by three letter code, in accordance with 37 CFR ' 1.822 and established usage. See, e.g., PatentIn User Manual, 99-102 (Nov.
1990) (U.S. Patent and Trademark Office, Office of the Assistant Commissioner for Patents, Washington, D.C. 20231); U.S.
Patent No. 4,871,670 to Hudson et al. at col. 3, lines 20-43. The present method utilizes anti-sense agents to inhibit or down-regulate gene expression of target genes, including those listed in Tables l and 2 below. This is generally attained by hybridization of the anti-sense oligonucleotides to coding (sense) sequences of a targeted messenger RNA (rnRNA), as is known in the art.
The exogenously administered agents of the invention decrease the levels of mRNA and protein encoded by the target gene and/or cause changes in the growth characteristics or shapes of the thus treated cells. See, Milligan et al. (1993); Helene, C. and Toulme,1. Biochim. Biophys. Acta 1049, 99-125 (1990); Cohen, J. S. D., Ed., Oligodeoxynucleotides as Anti-sense Inhibitors of Gene Expression; CRC Press: Boca Raton, FL (1987), the relevant portion of which is hereby incorporated in its entirety by reference. As used herein, "anti-sense oligonucleotide or asnti-sense oligo" is generally a short sequence of synthetic nucleotide that (1) hybridizes to any segment of a mRNA encoding a targeted protein under appropriate hybridization conditions, and which (2) upon hybridization causes a decrease in gene expression of the targeted protein. The terms "desAdenosine" (desA) and "des-thymidine" (desT) refer to oligonucleotides substantially lacking either adenosine (dcsA) or lhymidine (desT). In some instances, the des A or des T sequences are naturally occurring, and in others they may result from substitution of an undesirable nucleotide (A) by another lacking its undesirable activity, such as acting as an agonist or having a triggering effect at the adenosine A receptor(s). In the present context, the substitution is generally accomplished by substitution of A with a "universal or alternative base", presently known in the art or to be ascertained at a later time. As used herein, the terms "prevent", "preventing", "treat" or "treating" refer to a preventative, prophylactic, maintenance, or therapeutic treatment which decreases the likelihood that the subject adnlinistered such treatment will manifest symptoms associated with adenosine receptor stimulation. The term "down-regulate" refers to inducing WO 00/G273G PCTlUS00/08020 a decrease in production, secretion or availability and, thus, a decrease in concentration, of intracellular target product, be it a receptor e. g. adenosine A" A=b, A" bradykinin 2B, GATA-3, or other receptors, or an increase in concentration of the adenosine Al, receptor. The present technology relies on the design of anti-sense oligos targeted to mRNAs associated with ailments involving lung airway pathology(ies), and on their modification to reduce the occurrence of undesirable side effects caused by their release of adenosine upon breakdown, while preserving their activity and efficacy for their intended purpose. In this manner, the inventor targets a specific gene to design one or more anti-sense oligonucleotide(s) (oligos) that selectively bmd(s) to the corresponding ntRNA, and then reduces, if necessary, their content of adenosine via substitution with an alternative or a universal base, or an adenosine analog incapable of significantly, or having substantially reduced ability for, activating or antagonizing adenosine A,, Alb or A, receptors or which may act as an agonist at the adenosine A,~, receptor. Any number of adenosines present may be substituted by an alternative andlor universal base, such as heteroaromatic bases, which binds to a thymidine base but has less than about 0.3 of the adenosine base agonist or antagonist activity at the adenosine A" Az" Alb and A, receptors. Based on his prior experience in the field, the inventor reasoned that in addition to "downregulating" specific genes, he could increase the effect of the agents) administered by either selecting segments of RNA
that are devoid, or have a low content, of thymidine (T) or, alternatively, substitute one or more adenosine(s) present in the designed oligonucleotide(s) with other nucleotide bases, so called universal bases, which bind to thymidine but lack the ability to activate adenosine receptors and otherwise exercise the constricting effect of adenosine in the lungs, etc. Given that adenosine (A) is a nucleotide base complementary to thymidine (T), when a T appears in the RNA, the anti-sense oligo will have an A at the same position.
In one aspect of this invention, the anti-sense oligonucleotide has a sequence which specifically binds to a portion or segment of a mRNA molecule which encodes a protein associated with impeded breathing, siiergy(ies), lung inflammation, depletion of lung surfactant or lowering of lung surfactant, airway obstruction, bronchitis, and the like. One effect of this binding i5 to reduce or even prevent the translation of the corresponding mRNA and, thereby, reduce the available amount of target protein in the subject=s lung. In one preferred embodiment of this invention, the phosphodiester residues of the anti-sense oligonucleotide are modified or substituted.
Chemical analogs of oligonucleotides with modified or substituted phosphodiester residues, e.g., to the methylphosphonate, the phosphotriester, the phosphorothioate, the phosphorodlthioate, or the phosphoramidate, a=
methoxy ethyl and similar modifications, which increase the in vivo stability of the oligonucleotide are particularly preferred. The naturally occurring phosphodiester linkages of oligonucleotides are susceptible to some degree of degradation by cellular nucleases. Many of the residues proposed herein, on the contrary, are highly resistant to nuclease degradation. See, Milligan et al,; Cohen, J. S. D., supra.
In another preferred embodiment of the invention, the oligonucleotides may be protected from degradation by adding a "3'-end cap" by which nuclease-resistant linkages are substituted for phosphodiester linkages at the 3' end of the oligonucleotide. See, Tidd, D. M.
and Warenius, H.M., Be.
J. Cancer 60: 343-350 (1989); Shaw, J.P et al., Nucleic Acids Res. 19: 747-750 (1991), the relevant section of which are incorporated in their entireties herein by reference.
Phosphoramidates, phosphorothioates, and methylphosphonate linkages all function adequately in this manner for the purposes of this invention, as do a' modifications, such as a' methoxy ethyl, and the like. The more extensive the modification of the phosphodiester backbone the more stable the resulting agent, and in many instances the higher their RNA affinity and cellular petTneation. See, Milligan, et al., supra. In addition, a plurality of substitutions to the carbohydrate ring are also known to improve stability of nucleic acids. Thus, the number of residues which may be modified or substituted will vary depending on the need, target, and route of administranon, and may be from 1 to all the residues, to any number in between. Many different methods for replacing the entire phosphodiester backbone with novel linkages are known, See, Millikan et al, supra. Preferred backbone analogue residues include phosphoramidate, phosphorothioate, methylphosphonate, phosphorotriester, phosphotriester, thiofotmacetal, WO OO1G273G PCT/US00lU802U
phosphorodithioate, phosphoramidate, fortrlacetal, trifortnacetal, thioether, carbamate, boranophosphate, 3'-thioformacetal, 5'-thioether, carbonate, Cs-substituted nucleotides, 5'-N-carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, 2'-O
methyl, sulfoxide, sulfide, hydroxylamine, methylene(methylimino) (MMI), methoxymethyl (MOM), and methoxyethyl(MOE), and methyleneoxy(methylimino) (MOMI) residues, and combinations thereof.
Phosphorothioate and methylphosphonate-modified oligonucleotides are particularly preferred due to their availability through automated oligonucleotide synthesis. See, Millikan et al, supra. Where appropriate, the agent of this invention may be administered in the form of their pharmaceutically acceptable salts, or as a mixture of the anti-sense ohgonucleotide and its salt. In another embodiment of this invention, a mixture of different anti-sense oligonucleohdes or their pharmaceutically acceptable salts is administered. A single agent of this invention has the capacity to attenuate the expression of a target mRNA and/or various agents to enhance or attenuate the activity of a pathway.
By means of example, the present method may be practiced by identifying all possible deoxyribonucleotide segments which ate low in thymidine (T) or deoxynucleotide segments low in adenosine (A) of about 7 or more mononucleotides, preferably up to about 60 mononucleotides, more preferably about 10 to about 36 mononucleotides, and still more preferably about 12 to about 21 mononucleotides, in a target mRNA
or a gene, respectively. This may be attained by searching for mononucleotide segments within a target sequence which are low in, or lack thymidine (RNA), a nucleotide which is complementary to adenosine, or that are low in adenosine (gene), that are 7 or more nucleotides long. In most cases, this search typically results in about 10 to 30 such sequences, i.e. naturally Lacking or having less than about 40% adenosine, anti-sense oligonucleotides of varying lengths for a typical target mRNA of average length, i.e., about 1800 nucleotides long. Those with high content of 1' or A, respectively, may be fixed by substitution of a universal base for one or more As. The agents) of this invention may be of any suitable length, including but not limited to, about 7 to about 60 nucleotides long, preferably about 12 to about 45, more preferably up to about 30 nucleotides long, and still more preferably up to about 21, although they may be of other lengths as well, depending on the particular target and the mode of delivery. The agents) of the invention may be directed to any and all segments of a target RNA. One preferred group of agents) includes those directed to an mRNA region containing a junction between an introit and an exon. Where the agent is directed to an intron/exon junction, it may either entirely overlie the junction or it may be sufficiently close to the junction to inhibit the splicing-out of the intervening exon during processing of precursor mRNA to mature mRNA, e.g. with the 3' or 5' terminus of the anti-sense oligonucleotide being positioned within about, for example, within about 2 to 10, preferably about 3 to 5, nucleotide of the intron/exon junction. Also preferred are anti-sense oligonucleotides which overlap the initiation codon, and those near the 5' and 3' termini of the coding region. The flanking regions of the exons may also be targeted as well as the spliced segments in the precursor mRNAs. The mRNA sequences of the adenosine receptors and of many other targets are derived from the DNA base sequence of the gene expressing either receptors, e.
g, the adenosine receptors, the enzymes, factors, or other targets associated with airway disease. For example, the sequence of the genomic human A, adenosine receptor is known and is disclosed in U.S. Patent No.
5,320,963 to Stiles, G., et al. The A, adenosine receptor has been cloned, sequenced and expressed in rat (see, Zhou, F., et al., P.N.A.S. (USA) 89: 7432 ( 1992)) and human (see, Jacobson, M. A., et al., U.K, Patent Application No. 9304582.1 ( 1993)). The sequence of the adenosine A2h receptor gene is also known. See, Salvatore, C. A., Luneau, C. J., Johnson, R. G. and Jacobson, M., Genomics ( 1995), the relevant portion of which is hereby incorporated in tts entirety by reference. The sequences of many of the remaining exemplary target genes are also known. See, GenBank, NIH. The sequences of those genes whose sequences are not yet available may be obtained by isolating the target segments applying technology known in the art. Once the sequence of the gene, its RNA
andlor the protein are known, an anti-sense oligonucleotides may be produced according to this invention as described above to reduce the production of the targeted protein in accordance u~irh standard techniques. The sequences for the adenosine A" bradykinin, and other genes as well as methods for preparation of WO 00/62736 PCT/lJS00/08020 oligonucleotides are also known as those of many other target genes and mRNAs for which this invention is suitable. Thus, anti-sense oligonucleotides that downregulate the production of target sequences associated with airway disease, including the adenosine A,, A" Aip, A3, bradykinin, GATA-3, COX-2, and many other receptors, may be produced in accordance with standard techniques.
Examples of diseases and conditions which are suitably treated by the present method are diseases and conditions, including Acute Respiratory Distress Syndrome CARDS), asthma, adenosine administration e.g. in the treatment of SupraVentricular Tachycardia (SVT) and other arrhythmias, and in stress tests to hyper-sensitized individuals, ischemia, renal damage or failure induced by certain drugs, infantile respiratory distress syndrome, pain, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, emphysema, chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, and cancers such as leukemias, lymphomas, carcinomas, and the like, including colon cancer, breast cancer, lung cancer, pancreatic cancer, hepatocellular carcinoma, kidney cancer, melanoma, hepatic metastases, etc., as well as all types of cancers which may metastasize or have metastasized to the lung(s), including breast and prostate I S cancer.
The adenosine receptors discussed above are mere examples of the high power of the inventor=s technology. In fact, a large number of genes may be targeted in a similar manner by the present agent(s), to reduce or down-regulate protein expression. By means of example, if the target disease or condition is one associated with impeded or reduced breathing, bronchoconstriction, chronic ZO bronchitis, pulmonary bronchoconstriction and/or hypertension, chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, allergy, asthma, cystic fibrosis, respiratory distress syndrome, cancers, which either directly or by metastasis afflict the lung, the present method may be applied to a list of potential target mRNAs, which includes the targets listed in Table 1 and Table 2 below, among others. The anti-sense agents) of the invention have a low A
25 content to prevent its liberation upon in vivo degradation of the agent(s).
For example, if the system is the pulmonary or respiratory system, a large number of genes is involved in different functions, including those listed in Table 1 below.
Table 1: Pulmonary Disease or Condition Pulmonary and Inflammation Targets Nf6B Transcription Interleukin-8 Receptor (IL-8 Factor R) 30Interleukin-5 ReceptorInterleukin-4 Receptor (IL-4R) (IL-SR) Interleukin-3 ReceptorInterleukin-lp (IL-1(i) (IL-3R) Interieukin-1 p ReceptorEotaxin (IL-1 ~3R) Tryptase Major Basic Protern p2-adrenergic ReceptorEndothelin Receptor A
Kinase 35Endothelin Receptor Preproendothelin B

Bradykinin B2 ReceptorIgE (High Affinity Receptor) (B2BR) Interleukin-1 (IL-1) Interleukin 1 Receptor (IL-1 R) Interleukin-9 (IL-9) Interleukin-9 Receptor (IL-9 R) lnterleukin-11 (IL-11)Interleukin-11 Receptor (IL-11 R) 40Inducible Nitric OxideCyclooxygenase (COX) Synthase Intracellular Adhesion Molecule 1 (ICAM-1) Vascular Cellular Adhesion Molecule Substance P (VCAM) Rantes Endothelial Leukocyte Adhesion Molecule Endothelia ETA Receptor (SLAM-1) 45Cyclooxygenase2 (COX-2)GM-CSF, f?ndothelin-I

Monocyte Activating Neutrophil Chemotactic Factor Factor Neutrophil Elastase Vefensin 1,2,3 '' Muscarinic AcetylcholinePlatelet Activating Factor Receptors Tumor Necrosis Factor S-lipoxygenase a 50Phosphodiesterase IV Substance P

Substance P Receptor Histamine Receptor Chymase CCR-I CC Chemokine Receptor Interleukin-2 (IL-2) Interleukin-4 (IL-4) Interleukin-12 (IL-12)Interleukin-5 (IL-S) lnterleukin-6 (IL-6) lnterleukin-7 (iI_-7) lnterleukin-8 (IL-8) lnterleukin-12 Receptor (IL-12R) Interleukin-7 ReceptorInterleukin-1 (IL-1) (IL-?R) 5 Interleukin-14 ReceptorInterleukin-14 {IL-l4R) CCR-2 CC Chemokine CCR-3 CC Chemokine Receptor Receptor CCR-4 CC Chemokine CCR-5 CC Chemokine Receptor Receptor Prostanoid Receptors GATA-3 Transcription Factor Neutrophil Adherence MAP Kinase Receptor !0lnterleukin-15 (IL-15)Interleukin-15 Receptor (IL-15R) Interleukin-11 (IL-11)Interleukin-I1 Receptor (IL-IIR) NFAT Transcription STAT 4 Factors MiP-I a MCP-2 15Cyclophillin (A, B, Phosphotipase A2 etc.) Basic Fibroblast GrowthMetalloproteinase Factor CSBP/p38 MAP Kinase 'Cryptase Receptor PDG2 lnterleukin-3 (IL-3) lnterleukin-10 (IL-10)Cyclosporin A - Binding Protein 20FK506-Binding Protein a4p1 Selectin Fibronectin a4ji7 Selectin Table 1: Pulmonary Disease or Condition Pulmonary and Inflammation Targets cMad CAM-1 LFA-1 (CDlla/CD18) PECAM-I LFA-1 Selectin 25C3bi PSGL-1 E-Selectin P-Selectin CD-34 L-Selectin p150,95 Mac-1 (CDIIb/CI)18) Fucosyl transferase VLA-4 CD-181CD 11 a CD 11 b/CD 18 1CAM2 and ICAM3 C5a CCR3 (Eotaxin Receptor)CCR1, CCR2, CCR4, CCRS

LTB-4 AP-1 Transcription Factor 35Protein kinase C Cysteinyl Leukotriene Receptor Tachykinnen Receptors 16B Kinase 1 & 2 (tach R) Interleukin-2 Receptor(e.g., Substance P, NK-1 & NK-3 (IL-2R) Receptors) STAT 6 c-mas NF-Interteukin-6 (NF-IL-6)Interteukin-t0 Receptor (IL-lOR) 40Interleukin-3 (IL-3) lnterleukin-2 Receptor (IL-2R) Interleukin-13 (IL-13)Interleukin-12 Receptor (IL-12R) Interleukin-14 (1L-14)lnterleukin-6 Receptor (IL-6R) Interleukin-16 (IL-16)Interleukin-13 Receptor (IL-13R) Medullasin Interleukin-16 Receptor (IL-16R) 45Adenosine A, Receptor Tryptase-I
(A, R) Adenosine AZb ReceptorAdenosine A, Receptor (A, R) (Aib R) (I Tryptase STAT-3 ' Adenosine Ai, ReceptorlgE Receptor p Subunit (IgE R
(A2, R) p) Fc-epsilon receptor IgE Receptor a Subunit (IgE R
CD23 antigen a) 50IgE Receptor Fe Epsilon Receptor (IgERFc l;
R) Substance P Receptor Histidine decarboxylaseTryptase-1 Prostaglandin D SynthaseEosinophil Cationic Protein Eosinophil Derived Eosinophil Peroxidase Neurotoxin Endothelial Nitric Endothelial Monocyte Activating Oxide Synthase Factor 55Neutrophil Oxidase Cathepsin G
Factor Macrophage InflammatoryInterleukin-8 Receptor a Subunit Protein-i- (IL-8 Ra) Aloha/Rantes Receptor Endotllelin Receptor ET-B

These genes, and others, are involved in the normal functioning of respiration as well as in diseases associated with respiratory pathologies, including cystic fibrosis, asthma, pulmonary hypertension and vasoconstriction, chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, chronic bronchitis, respiratory distress syndrome CARDS), allergic rhinitis, lung cancer and lung metastatic cancers and other airway diseases, including those with inflammatory response.
Anti-sense oligos to the target receptors, e. g. the adenosine A" AZ" Aib, and A~ receptors, CCR3 (chemokine receptors), bradykinin 2B, CAM (vascular cell adhesion molecule), and eosinophil receptors, among others, have been shown to be effective m down-regulating the expression of their genes. Some of these act to alleviate the symptoms or reduce respiratory ailments and/or inflammation, for example, by "down regulation" of the adenosine A" A~" AZe, and/or A, receptors and CCR3, bradykinin 2B, VCAM (vascular cell adhesion molecule) and eosinophil receptors. These agents may be utilized by the present method alone or in conjunction with anti-sense oligos targeted to other genes to validate pathway and/or networks in which they are involved. For better results, the oligos are IS preferably administered directly into the respiratory system, e.g., by inhalation or other means, of the experimental animal, so that they may reach the lungs without widespread systemic dissemination.
This permits the use of low agent doses as compared with those administered systemically of by other generalized routes and, consequently, reduces the number and degree of undesirable side effects resulting from the agent=s widespread distribution in the body. 'IZte agents) of this invention has (have) been shown to reduce the amount of receptor protein expressed by the tissue. These agents, thus, rather than merely interacting with their targets, a g. a receptor, lower the number of target proteins that other drugs may interact with. In this manner, the present agents) affords) extremely high efficacy with low toxicity. Anti-sense oligonucleotides to the A, , A~b, A,, bradykinin B2, GATA-3, CAM (vascular cell adhesion molecule), eosinophil receptors, and COX-2 receptors, among others, have been shown to be affective in the down-regulation of the respective receptor proteins in the cell.
One novel feature of this treatment, as compared to traditional treatments for adenosine-mediated bronchoconstriction, is that administration is direct to the lungs, or in situ to other tissues, organs or systems of the body. Additionally, a receptor protein itself is reduced in amount, rather than merely interacting with a drug, and toxicity is reduced. Other proteins that may be targeted with anti-sense agents for the treatment of lung conditions include, but are not Limited to:
CCR3 (chemokine) receptors, human A2, adenosine receptor, human Azb adenosine receptor, human IgE receptor [3, human Fc-epsilon receptor CD23 antigen, human histidine decarboxylase, human beta tryptase, human tryptase-I, human prostaglandin D synthase, human cyclooxigenase-2, human eosinophil cationic protein, human eosinophil derived neurotoxin, human eosinophil peroxidase, human intercellular adhesion molecule-I (ICAM-1), human vascular cell adhesion molecule-1 (VCAM-i), human endothelial leukocyte adhesion molecule-1 (SLAM-1), human P selectin, human endothelial monocyte activating factor, human IL-3, human IL-4, human IL.-5, human IL-6, human IL-8, human monocyte-derived neutrophil chemotactic factor, human neutrophil elastase, human neutrophil oxidase factot, human cathepsin G, human defensin 1, human defensin 3, human macrophage inflammatory protein-1-alpha, human muscarinic acetylcholine receptor HM3, human fibronectin, human GM-CSF, human tumor necrosis factor a, human leukotriene C4 synthase, hunu3n major basic protein, and human endothelia t. Although not intended to be exclusive, a more extensive list of genes is provided below.
Some of these act to alleviate the symptoms or reduce respiratory ailments and/or inflammation, for example, by "down regulation" of the adenosine A,, AI" A2b, and/or A, receptors and CCR3, bradykinin 2B, VCAM (vascular cell adhesion molecule) and eosinophil receptors. These agents are preferably administered directly into the respiratory system, e.g., by inhalation or other means, so that they may reach the lungs without widespread systemic dissemination. 'This permits the use of substantially lower doses of the agent of the invention as compared with those administered by the prior art, systemically or by other generalized routes and, consequently, reduce undesirable side effects SO resulting from the agent=s widespread distribution in the body. The agents) of this invention has (have) been shown to reduce the amount of receptor protein expressed by the tissue. These agents, thus, rather than merely interacting with their targets, e.g. a receptor, lower the number of target proteins that other drugs may interact with. In this manner, the present agents) affords) extremely high efficacy with low toxicity. In these latter targeu, and in target genes in general, it is particularly imperative to eliminate or reduce the adenosine content of the corresponding anti-sense oligonucIeotide to prevent their breakdown products from liberating adenosine.
As used herein, the term "treat" or "treating" asthma refers to a treatment which decreases the likelihood that the subject administered such treatment will manifest symptoms of the lung disease.
The term "dowttregulate" refers to inducing a decrease in production, secretion or availability (and thus a decrease in concentration) of the targeted intracellular protein. The present invention is concerned primarily with the treatment of human subjects. However, the agents and methods disclosed here may also be employed for veterinary purposes, such as is the case in the treatment of other mammals, such as cattle, horses, wild animals, zoo animals, and domestic animals, e. g. dogs and cats. Targeted proteins are preferably mammalian and more preferably of the same species as the subject being treated. In general, "anti-sense" refers to the use of small, synthetic oligonucleotides, resembling single-stranded DNA, to inhibit gene expression by inhibiting the function of the target messenger RNA (mRNA). Milligan, J. F. et al., J. Med. Chem. 36(14), 1923-1937 (1993). 1n the present invention, inhibition of gene expression of the A, or A3 adenosine receptor is desired. Gene expression is inhibited through hybridization to coding (sense) sequences in a specific messenger RNA (mRNA) target by hydrogen bonding according to Watson-Crick base pairing rules. The mechanism of anti-sense inhibition is that the exogenously applied oligonucleotides decrease the mRNA and protein levels of the target gene or cause changes in the growth Characteristics or shapes of the cells. Id. See, also Nelene, C, and Toulme, J., Biochim. Biophys. Acta 1049, 99-125 (1990); Cohen, J. S. D., Ed., Oligodeoxynucleotides as Anti-sense Inhibitors of Gene Expression; CRC Press:
Boca Raton, FL
( 1987). As used herein, "anti-sense oligonucleotide" is defined as a short sequence of synthetic nucleotide that (1) hybridizes to any coding sequence in an mRNA which codes for the targeted protein, according to hybridization conditions described below, and (2) upon hybridization causes a decrease in gene expression of the A, or A, adenosine receptor. The receptors discussed above are mere examples of the high power of the present technology. In fact, a large number of genes may be targeted in a similar manner by practicing the present methods, to significantly down-regulate or obliterate protein expression and observe any changes wrought to one or more functions within a system, e.g. the respiratory system and other lung disease associated targets.
By means of example, in the respiratory system, the targets may be associated With difficulties of breathing, bronchoconstriction, inflammation, allergic rhynitis, chronic bronchitis, surfactant depletion, and others associated with diseases and conditions such as chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, inhalation bums, Acute Respiratory Distress Syndrome (ARDS), cystic fibrosis, pulmonary fibrosis, radiation pulmonitis, tonsilitis, emphysema, dental pain, oral inflammation, joint pain, esophagitis, cancers afflicting the respiratory system either directly such as Lung cancer, esophageal cancer, and the like, or indirectly by means of metastases, among others. These functions are of great interest because of their association with respiratory dysfunction, as is the case in asthma, allergies, allergic rhinitis, pulmonary bronchoconstriction and hypertension, chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, allergy, asthma, cystic fibrosis (CF), Acute Respiratory Distress Syndrome CARDS) as well as infantile and pregnancy-related RDS, cancer, ere., which either directly or by metastasis afflict the lung, the present anti-sense oligonucleotides may be directed to a list of target mRNAs, which includes the targets listed in Table I above, among others.
The oligos of this invention may be obtained by first selecting fragments of a target nucleic acid having at least 4 contiguous nucleic acids selected from the group consisting of G and C and/or having a specific type and/or extent of activity, and then obtaining a first oligonucleotide 4 to 60 nucleotides long which comprises the selected fragment and has a thymidine (T) nucleic acid content WO 00162736 PCTlUS00/0$020 of up to and including about 15%, preferably, about 12%, about 10%, about 7%, about 5%, about 3%, about 1%, and more preferably no thymidine. The latter step may be conducted by obtaining a second oligonucleotide 4 to GO nucleotides long comprising a sequence which is anti-sense to the selected fragment, the second oligonucleotide having an adenosine base content of up to and including about 15%, preferably about 12%, about t0%, about 7%, about 5%, about 3%, about I%, and more preferably no adenosine. When the selected fragment comprises at (east one thymidine base, an adenosine base may be substituted in the corresponding anti-sense nucleotide fragment with a universal base selected from the group consisting of heteroaromatic bases which bind to a thymidine base but have less than about bout 10%, preferably less than about 1%, and more preferably less than about 0.3% of the adenosine base agonist activity at the adenosine A" Aza, A2,, and A, receptors, and heteroaromatic bases which have no activity at the adenosine A2°
receptor, when validating in the respiratory system. Other adenosine activities in other systems may be detetmmed in other systems, as appropriate. The analogue heteroaromatic bases may be selected from all pyrimidines and purines, which may be substituted by O, halo, NH2, SH, SO, SOi, SO,, OOOH and branched and fused primary and secondary amino, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylaIkyl, arytalkenyl, arylalkynyl, arylcycloalkyl, which may be further substituted by O, halo, NH2, primary, secondary and tertiary amine, SH, SO, SO;, SO" cycloalkyl, heterocyeloalkyl and heteroaryl. The pyrimidines and purines may be substituted at all positions as is known in the art, but preferred are those which are substituted at positions I, 2, 3, 4, 7 and/or 8.
More preferred are pyrimidines and purines such as theophylline, caffeine, dyphylline, etophylline, acephylline piperazine, bamifylline, enprofylline and xantine having the chemical formula ~L i~
Ir,N/1 fC/M~ 3 OsC~.7 jC,~l~~
!! N
R
wherein R' and R2 are independently H, alkyl, alkenyl or alkynyl and R' i.s H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cyeloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloalkynyl, NH2-alkylamino-kctoxyalkyloxy-aryl, mono and dialkylaminoalkyl-N-alkylamino-SOIaryI, among others. Similar modifications in the sugar arc also embodiments of this invention.
Reduced adenosine content of the anti-sense oligos corresponding to the thymidines (T') present in the target RNA serves to prevent the breakdown of the oligos into products that free adenosine into thZ
system, e.g. the lung, brain, heart, kidney, ere., tissue environment and, thereby, to prevent any unwanted effects due to it. By means of example, the Nf6B transcription factor may be selected as a target, and its rnRNA or DNA searched for low thymidine (T) or desthymidine (desT) fragments. Only desT segments of the mRNA or DNA are selected which, in rum, will produce desA
anti-sense as their complementary strand. When a number of RNA des'f segments are found, the sequence of the anti.
sense segments may be deduced. Typically, about 10 to 30 and even larger numbers of desA apti-sense sequences may be obtained. These anti-sense sequences may include some or all desA anti-sense oligonucleotide sequences corresponding to desT segments of the mRNA of the target, such as anyone of those shown in Table I above, in Table 2 below, and others associated with functions of the brain, cardiovascular and renal systems, and many others. When this occurs, the anti-sense oligonucleotides found are said to be 100% A-free. For each of the original desA anti-sense oligonucleotide sequences corresponding to the target gene, e_g. the NF6B transcription factor, typically about 10 to 30 sequences may be found within the target gene or RNA which have a low content of thymidine (RNA). In accordance with this invention, the selected fragment sequences may also contain a small number of thymidine (RNA) nucleotides within the secondary or tertiary or quaternary sequences. In some cases, 1.t a large adenosine content may suffice to render the anti-sense oligonucleotide less active or even inactive against the target. In accordance with this invention, these so called "non-fully desA"
sequences may preferably have a content of adenosine of less than about 15%, about 12%, about 10%, about 7%, about 5%, and about 2% adenosine. Most preferred is no adenosine content (0%). In some instances, however, a higher content of adenosine is acceptable and the oligonucleotides still fail to show detrimental "adenosine activity". A particular important embodiment is that where the adenosine nucleotide is "fixed" or replaced by a "Universal or alternative" base that may base-pair with similar or equal affinity to two or more of the four nucleotide present in natural DNA:
A, G, C, and T.
A universal or alternative base is defined in this patent as any compound, more commonly an adenosine analogue, which has substantial capacity to hybridize to thymidine, while at the same time having reduced, or substantially lacking, ability to bind adenosine receptors or other molecules through which adenosine may exert an undesirable side effect in the experimental animal or in a cell system.
Alternatively, adenosine analogs which completely fail to activate, or have significantly reduce ability for activating, adenosine receptors, such as the adenosine A" ,4z6 andlor A, receptors, most preferably A, receptors, and those that may even act as agonists of the adenosine Az"
receptor, may be used. One example of a universal base is a-deoxyribofuranosol-(5-nitroittdole), and an artisan will know how to select others. This "fixing" step generates further novel sequences, different from those anti-sense to the ones found in nature, that permits the anti-sense ohgonucleotide to bind, preferably equally well, with the target RNA. Other examples of universal or alternative bases are 2-deoxyribosyl-(S-ZO nitroindole). Other examples of universal bases are 3 - nitropyrrole - 2' -deoxynucleoside, 5 - nitro-indole, 2 - deoxyribosyl - (5 - nitroindole), 2-deoxyribofuranosyl - (5-nitroindole), 2' - deoxyinosine, 2' -deoxynebularine, 6H, 8H-3,4-dihydropyrimido [ 4, 5 - c] oxazine - 7 - one and 2 - amino - 6 -methoxy aminopurine. In addition to the above, Universal bases which may be substituted for any other base although with somewhat reduced hybridization potential, include 3 -nitropyrrole 2' - deoxynucleoside 2 - deoxyribofuranosyl - (5 - nitroindole), 2' - deoxyinosine and 2' -deoxynebularine (Glen Research, Sterling, VA). More specific mismatch repairs may be made using "P"
nucleotide, 6H, 8H - 3, 4 -dihydropyrimido [4,5 - c] [l, 2] oxazin - 7 - one, which base pairs with either guanine (G) or adenine (A) and "K" nucleotide, 2 - amino - 6 - methoxyaminopurine, which base pairs with either cytidine (C) or thymidine (T), among others. Others which are known in the art or will become available are also suitable. See, for example, Loakes, D. and Brown, D. M., Nucl. Acids Res.
22:4039-4043 (1994);
Ohtsuka, E. et al., 1. Biol. Chem.260(5):2605-2608 (1985); Lin, P.K.T. and Brown, D. M., Nucleic Acids Res. 20(19):5149-5152 (1992; Nichols, R. et al., Nature 369(b480): 492-493 (1994); Rahmon , M. S. and Humayun, N. Z., Mutation Research 377 (2): 263-8 (1997); Amosova, O., et al., Nucleic Acids Res. 25 (!0): 1930-1934 (1997); Loakes D. & Brown, D. M., Nucleic Acids Res. 22 (20): 4039-4043 ( 1994), the entire sections relating to universal bases and their preparation and use in nucleic acid binding being incorporated herein by reference. When non-fully desT sequences are found in the naturally occurring target, they typically are selected so that about 1 to 3 universal base substitutions will suffice to obtain a 100% "desA" anti-sense oligonucleotide. 'thus, the present method provides either anti-sense oligonucleotides to different targets which are low in, or devoid of, A content, as well as anti-sense oligonucleotides where one or more adenosine nucleotides, e. g.
about 1 to 3, or more, may be "fixed" by replacement with a "Universal" or "replacement" base.
Universal bases are known in the art and need not be listed herein. An artisan will know which bases may act as universal bases, and replace them for A. Table 2 below provides a selected number of targets to which the agents of the invention are effectively applied. Others, however, may also be targeted.
Table 2: Cancer Targets Transforming Therapy Onco2enes Targets ras thymidylate synihetase src thymidylate synthetase myc dihydrofolate redurtase bcl-2 thymidine kinase deoxycytidine kinase ribonucleotide reductase Angiogenesis factors Adhesion Molecules 5 Oncogenes Folate Pathway Enzymes DNA repair genes (One Carbon Pool) Telomerase HMG CoA Reductase Farnesyl Transferase 10 Glucose-6-Phosphate Transferase A group of preferred targets for the treatment of cancer are genes associated with any of different types of cancers, or those generally known to be associated with malignancies, whether they are regulatory or involved in the production of RNA and/or proteins. Examples are transforming oncogenes, including, but not limited to, ras, src, myc, and BGL-2, among others. Other targets are 15 those to which present cancer chemotherapeutic agents are directed to, such as various enzymes, primarily, although not exclusively, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, ribonucleotide reductase, and the like. The present technology is particularly useful in the treatment of cancer ailments given that traditional cancer therapies are naught with the unresolved problem of selectively killing cancer cells while preserving normal living cells from the devastating effects of treatments such as chemotherapy, radiotherapy, and the like. The present technology provides the abiiiry of selectively attenuating or enhancing a desired pathway or target.
This approach provides a significant advantage over standard treatments of cancer because it permits the selection of a pathway, including primary, secondary and possibly tertiary targets, which are not generally expressed simultaneously in normal cells. Thus, the present agent may be administered to a subject to cause a selective increase in toxicity within tumor cells that, for instance, express all three targets while normal cells that may expresses only one or two of the targets will be significantly less affected or even spared. A group of preferred targets for the treatment of cancers are genes associated with different types of cancers, or those generally known to be associated with malignancies, whether they are regulatory or involved in the production of RNA and/or proteins.
Examples are transfotining oncogenes, including, but not limited to, ras, src, myc, and BCL-2, among others. Other targets are those to which present cancer chemotherapeutic agents are directed to, such as various enzymes, primarily, although not exclusively, thymidylate synthetase, dihydrofolate reductase, thymidine kinase, deoxycytidine kinase, ribonucleotide reductase, and the like.
In one embodiment, at least one of the mRNAs to which the oligo of the invention is targeted encodes a protein such as transcription factors, stimulating and activating factors, intracellular and extracellular receptors and peptide transmitters in general, interleukins, interleukin receptors, chemokines, chemokine receptors, endogenously produced specific and non-specific enzymes, immunoglobulins, antibody receptors, central nervous system ((.'NS) and peripheral nervous and non nervous system receptors, CNS and peripheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensines, growth factors, vasoactive peptides and receptors, and binding proteins, among others; or the mRNA is corresponding to an oncogene and other genes associated with various diseases or conditions. Examples of target proteins are eotaxin, major basic protein, preproendothelin, eosinophil cationic protein, P-selectin, STAT 4, MIP-la, MCP-2, MCP-3, MCP-4, STAT 6, c-mas, NF-IL-6, cyclophillins, PDG2, cyclosporin A-binding protein, FKS-binding protein, fibronectin, L<:A-1 (CDlla/CD18), PECAM-1, C3bi, PSGL-1,CD-34, substance P, p150,95, Mac-I
(CD1 IblCDlB), VLA-4, CD-18/CDlla, CDI Ib/CD18, CSa, CCRI, CCR2, CCR4, CCRS, and LT'B-4, among others. Others are, however, suitable, as well. In another embodiment, at least one of the mRNAs to which the oligo is targeted encodes intracellular and extracellular receptors and peptide transmitters such as sympathomimetic receptors, parasympathetic receptors, GABA receptors, adenosine receptors, bradykinin receptors, insulin receptors, glucagon receptors, prostaglandin receptors, thyroid receptors, androgen receptors, anabolic receptors, estrogen receptors, progesterone WO OIIJ62?36 PCT/USt111J118020 receptors, receptors associated with the coagulation cascade, adenohypophyseal receptors, adenohypophyseal peptide transmitters, and histamine receptors (HisR), among others. However others are also contemplated. The encoded sympathomimetic receptors and parasympathomimetic receptors include acetytcholinesterase receptors (AcChaseR) acetyleholine receptors (AcChR), atropine S receptors, muscarinic receptors, epinephrine receptors (EpiR), dopamine receptors (DOPAR), and norepinephrine receptors (NEpiR), among others. Further examples of encoded receptors are adenosine A, receptor, adenosine AzB receptor, adenosine A, receptor, endothelia receptor A, endothelia receptor B, IgE high affinity receptor, muscarinic acetylcholine receptors, substance P
receptor, histamine receptor, CCR-1 CC ehemokine receptor, CCR-2 CC chemokine receptor, CCR-3 CC
chemokine receptor (Eotaxin Receptor), interleukin-lp receptor (IL-1(3R), interleukin-I
receptor (IL-1R), interleukin-Iii receptor (IL-l~iR), inttrleukin-3 receptor (IL-3R), CCR-4 CC
chemokine receptor, cysteinyl leukotriene receptors, prostanoid receptors, GATA-3 transcription factor receptor, interleukin-I receptor (IL-1R), interieukin-4 receptor (IL-4R), interleukin-5 receptor (IL-SR), interleukin-8 receptor (IL-8R), interleukin-9 receptor (IL-9R), intetleukin-11 receptor (IL-I1 R), bradykinin B2 receptor, sympathomimetic receptors, parasympathomimetic receptors, GAGA
receptors, adenosine receptors, bradykinin receptors, insulin receptors, giucagon receptors, prostaglandin receptors, thyroid receptors, androgen receptors, anabolic receptors, estrogen receptors, progesterone receptors, receptors associated with the coagulation cascade, adenohypophyseal receptors, and histamine receptors (HisR). Others are also contemplated even though not listed herein.
The encoded enzymes for development of the oligos of the invention include synthetases, kinases, oxidases, phosphatases, reductases, polysaccharide, triglyceride, and protein hydrolases, esterases, elastases, and , polysaccharide, triglyceride, lipid, and protein syathases, among others. Examples of target enzymes are tryptase, inducible nitric oxide synthase, cyclooxygenase (Cox), MAP kinase, eosinophil peroxidase, ~i2-adrenergic receptor kinase, ieukotriene c-4 synthase, S-lipooxygenase, 2S phosphodiesterase IV, metalloproteinase, tryptase, CSHP/p38 MAP kinase, neutrophil elastase, phospholipase A2, cyclooxygenase 2 (Cox-2), fucosyl transferase, chymase, protein kinase C, thynudylate synthetase, dihydrofolate reductase, thymidinc kinase, deoxycytidine kinase, and ribonucleotide reductase, among others. Any enzyme associated with a disease or condition, however, is suitable as a target for this invention. Suitable encoded factors for application of this invention are, among others, Nf6B transcription factor, granulocyte macrophage colony stimulating factor (CiM-CSF), AP-1 transcription factor, GATA-3 transcription factor, monoeyte activating factor, neutrophil chemotactic factor, granulocyte/macrophage colony-stimulating-factor (G-CSF), NEAT transcription factors, platelet activating factor, tumor necrosis factor a (TNF a), and basic fibroblast growth factor (BFGF). Additional factors are also within the invention even though not specifically mentioned.
Suitable adhesion molecules for use with this invention include intracellular adhesion molecules 1 (ICAM-1), 2 (ICAM-2) and 3 (ICAM-3), vascular cellular adhesion molecule (VCAM), endothelial leukocyte adhesion molecule-1 (ELAM-1), neutrophil adherence receptor, mad CAM-1, and the like.
Other known and unknown factors (at this time) may also be targeted herein.
Among the cytokines, lymphokines and chemokines preferred are interleukin-I (IL-I), interleukin-I(i (IL-lei), interleukin-3 (IL-3), interteukin-~t (IL-4), interleukin-5 (IL-S), interleukin-8 (IL-8), interleukin-9 (IL-9), interleukin-I I (IL-Il),CCR-S CC chemokine, and Rantes. Others, however, may also be targeted, as they are known to be involved in specific diseases or conditions to be treated, or for their generic activities, such as inflammation. Examples of defensins for the practice of this invention are defensin 1, defensin 2, and defensin 3, and of selectins are a4(31 selectin, a4p7 selectin, LFA-l selectin, E-selectin, P-selectin, and L-selectin. Examples of oncogenes, although not an all inclusive list, are ras, src, myc, and bcBCL. Others, however, are also suitable for use with this invention.
The agents administered in accordance with this invention are preferably designed to be anti-sense to target genes and/or mRNAs related in origin to the species to which it is to be administered.
When treating humans, the agents are preferably designed to be anti-sense to a human gene or RNA.
The agents of the invention encompass oligonucleotides which arc anti-sense to naturally occurring DNA and/or RNA sequences, fragments thereof of up to a length of one ( 1 ) base less than the targeted sequence, preferably at least about 7 nucleotides long, oligos having only over about 0.02%, more preferably over about 0.1%, still more preferably over about 1°i°, and even more preferably over about 4% adenosine nucleotides, and up to about 30%, more preferably up to about 15%, still more S preferably up to about I0% and even more preferably up to about S%, adenosine nucleotide, or lacking adenosine altogether, and oligos in which one or more of the adenosine nucleotides have been replaced with so-called universal bases, which may pair up with thymidine nucleotides but fail to substantially trigger adenosine receptor activity. Examples of human sequences and fragments, which are not limiting, of anti-sense oligonucleotide of the invention are the following fragments as well as shorter segments of the fragments and of the full gene or mRNA coding sequences, exons and intron-exon junctions encompassing preferably 7, 10, IS, 18 to 21, 24, 27, 30, n-t nucleotides for each sequence, where n is the sequence=s total number of nucleotides. These fragments may be selected from any portion of the longer oligo, for example, from the middle, S'- end, 3'- end or starting at any other site of the original sequence. Of particular importance are fragments of low adenosine nucleotide content, that 1 S is, those fragments containing less than or about 30%, preferably less than or about 1 S%, more preferably less than or about 10%, and even more preferably Less than or about 5%, and most preferably those devoid of adenosine nucleotide, either by choice or by replacement with a universal base in accordance with this invention. The agent of the invention includes as a most preferred group sequences and their fragments where one or more adenosines present in the sequence have been replaced by a universal base (B), as exemplified here. Similarly, also encompassed are all shorter fragments of the B-containing fragments designed by substitution of B(s) for adenosine(s) (A(s)) contained in the sequences, fragments thereof or segments thereof, as described above. A limited list of sequences and fragments is provided below.
Some of the examples of anti-sense oligonucleotide sequence fragments target the initiation codon of the respective gene, and in some cases adenosine is substituted with a universal or alternative base adenosine analogue denoted as "B", which lacks ability to bind to the adenosine A, andlor A, receptors. In fact, such replacement nucleotide acts as a "spacer. Many of the examples shown below provide one such sequence and many fragments overlapping the initiation codon, preferably wherein the number of nucleotides n is about 7, about 10, about 12, about 15, about 18, about 21 and up to about 28, about 3S, about 40, about 50, about 60.

SEQUENCE LISTING
<110> EAST CAROLINA UNIVERSITY
<120> LOW ADENOSINE ANTI-SENSE OLIGONUCLEOTIDE, COMPOSITIONS, KIT & METHOD
FOR TREATMENT OF AIRWAY DISORDERS ASSOCIATED WITH BRONCHOCONSTRICTION, LUNG
INFLAMMATION, ALLERGY(IES) AND SURFACTANT DEPLETION
<130> 08-892198CA
<140> 2,330,022 <141> 2000-03-24 <150> US 60/127,958 <151> 1999-04-06 <160> 3111 <170> PatentIn version 3.0 <210> 1 <211> 21 <212> DNA
<213> mammalian <400> 1 gatggagggc ggcatggcgg g 21 <210> 2 <211> 21 <212> DNA
<213> Artificial <220>
<223> mismatch molecule <400> 2 gtagcaggcg gggatggggg c 21 <210> 3 <211> 18 <212> DNA
<213> mammalian <400> 3 gttgttgggc atcttgcc <210> 4 <211> 18 <212> DNA
<213> Artificial <220>
<223> mismatch molecule <400> 4 gtacttgcgg atctaggc <210> 5 <211> 18 <212> DNA
<213> mammalian <400> 5 gtgggcctag ctctcgcc 1g <210> 6 <211> 18 <212> DNA
<213> mammalian <400> 6 gtcggggtac ctgtcggc 18 <210> 7 <211> 21 <212> DNA
<213> mammalian <400> 7 ctcgtcgccg tcgccggcgg g 21 <210> 8 <211> 20 <212> DNA
<213> mammalian <400> 8 gggtggtgct attgtcgggc <210> 9 <211> 15 <212> DNA
<213> mammalian <400> 9 ggcccagggc cagcc 15 <210> 10 <211> 21 <212> DNA
<213> mammalian <400> 10 ggccgggcca gccgggcccg g 21 <210> 11 <211> 50 <212> DNA
<213> mammalian <400> 11 gcggcctgga aagctgagat ggagggcggc atggcgggca caggctgggc 50 <210> 12 <211> 49 <212> DNA
<213> mammalian <400> 12 cggcctggaa agctgagatg gagggcggca tggcgggcac aggctgggc 49 <210> 13 <211> 48 <212> DNA
<213> mammalian <400> 13 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca ggctgggc 48 <210> 14 <211> 47 <212> DNA
<213> mammalian <400> 14 gcctggaaag ctgagatgga gggcggcatg gcgggcacag gctgggc 47 <210> 15 <211> 46 <212> DNA
<213> mammalian <400> 15 cctggaaagc tgagatggag ggcggcatgg cgggcacagg ctgggc 46 <210> 16 <211> 45 <212> DNA
<213> mammalian <400> 16 ctggaaagct gagatggagg gcggcatggc gggcacaggc tgggc 45 <210> 17 <211> 44 <212> DNA
<213> mammalian <400> 17 tggaaagctg agatggaggg cggcatggcg ggcacaggct gggc 44 <210> 18 <211> 43 <212> DNA
<213> mammalian <400> 18 ggaaagctga gatggagggc ggcatggcgg gcacaggctg ggc 43 <210> 19 <211> 42 <212> DNA
<213> mammalian <400> 19 gaaagctgag atggagggcg gcatggcggg cacaggctgg 9c 42 <210> 20 <211> 41 <212> DNA
<213> mammalian <400> 20 aaagctgaga tggagggcgg catggcgggc acaggctggg c 41 <210> 21 <211> 40 <212> DNA
<213> mammalian <400> 21 aagctgagat ggagggcggc atggcgggca caggctgggc 40 <210> 22 <211> 39 <212> DNA
<213> mammalian <400> 22 agctgagatg gagggcggca tggcgggcac aggctgggc 39 <210> 23 <211> 38 <212> DNA
<213> mammalian <400> 23 gctgagatgg agggcggcat ggcgggcaca ggctgggc 38 <210> 24 <211> 37 <212> DNA
<213> mammalian <400> 24 ctgagatgga gggcggcatg gcgggcacag gctgggc 37 <210> 25 <211> 36 <212> DNA
<213> mammalian <400> 25 tgagatggag ggcggcatgg cgggcacagg ctgggc 36 <210> 26 <211> 35 <212> DNA
<213> mammalian <400> 26 gagatggagg gcggcatggc gggcacaggc tgggc 35 <210> 27 <211> 34 <212> DNA
<213> mammalian <400> 27 agatggaggg cggcatggcg ggcacaggct gggc 34 <210> 28 <211> 33 <212> DNA
<213> mammalian <400> 28 gatggagggc ggcatggcgg gcacaggctg ggc 33 <210> 29 <211> 32 <212> DNA
<213> mammalian <400> 29 atggagggcg gcatggcggg cacaggctgg gc 32 <210> 30 <211> 31 <212> DNA
<213> mammalian <400> 30 tggagggcgg catggcgggc acaggctggg c <210> 31 <211> 30 <212> DNA
<213> mammalian <400> 31 ggagggcggc atggcgggca caggctgggc 30 <210> 32 <211> 29 <212> DNA
<213> mammalian <400> 32 gagggcggca tggcgggcac aggctgggc 29 <210> 33 <211> 28 <212> DNA
<213> mammalian <400> 33 agggcggcat ggcgggcaca ggctgggc 2g <210> 34 <211> 27 <212> DNA
<213> mammalian <400> 34 gggcggcatg gcgggcacag gctgggc 27 <210> 35 <211> 26 <212> DNA
<213> mammalian <400> 35 ggcggcatgg cgggcacagg ctgggc 26 <210> 36 <211> 25 <212> DNA
<213> mammalian <400> 36 gcggcatggc gggcacaggc tgggc 25 <210> 37 <211> 24 <212> DNA
<213> mammalian <400> 37 cggcatggcg ggcacaggct gggc 24 <210> 38 <211> 23 <212> DNA
<213> mammalian <400> 38 ggcatggcgg gcacaggctg ggc 23 <210> 39 <211> 22 <212> DNA
<213> mammalian <400> 39 gcatggcggg cacaggctgg gc 22 <210> 40 <211> 21 <212> DNA
<213> mammalian <400> 40 catggcgggc acaggctggg c 21 <210> 41 <211> 20 <212> DNA
<213> mammalian <400> 41 atggcgggca caggctgggc 20 <210> 42 <211> 19 <212> DNA
<213> mammalian <400> 42 tggcgggcac aggctgggc 19 <210> 43 <211> 18 <212> DNA
<213> mammalian <400> 43 ggcgggcaca ggctgggc <210> 44 <211> 17 <212> DNA
<213> mammalian <400> 44 gcgggcacag gctgggc 17 <210> 45 <211> 16 <212> DNA
<213> mammalian <400> 45 cgggcacagg ctgggc 16 <210> 46 <211> 15 <212> DNA
<213> mammalian <400> 46 gggcacaggc tgggc 15 <210> 47 <211> 14 <212> DNA
<213> mammalian <400> 47 ggcacaggct gggc 14 <210> 48 <211> 13 <212> DNA
<213> mammalian <400> 48 gcacaggctg ggc 13 <210> 49 <211> 12 ' <212> DNA
<213> mammalian <400> 49 cacaggctgg gc 12 <210> 50 <211> 11 <212> DNA
<213> mammalian <400> 50 acaggctggg c 11 <210> 51 <211> 10 <212> DNA
<213> mammalian <400> 51 caggctgggc 10 <210> 52 <211> 9 <212> DNA
<213> mammalian <400> 52 aggctgggc 9 <210> 53 <211> 51 <212> DNA
<213> mammalian <400> 53 ggcggcctgg aaagctgaga tggagggcgg catggcgggc acaggctggg c 51 <210> 54 <211> 50 <212> DNA
<213> mammalian <400> 54 ggcggcctgg aaagctgaga tggagggcgg catggcgggc acaggctggg 50 <210> 55 <211> 49 <212> DNA
<213> mammalian <400> 55 ggcggcctgg aaagctgaga tggagggcgg catggcgggc acaggctgg 49 <210> 56 <211> 48 <212> DNA
<213> mammalian <400> 56 ggcggcctgg aaagctgaga tggagggcgg catggcgggc acaggctg 48 <210> 57 <211> 47 <212> DNA
<213> mammalian <400> 57 ggcggcctgg aaagctgaga tggagggcgg catggcgggc acaggct 47 <210> 58 <211> 46 <212> DNA
<213> mammalian <400> 58 ggcggcctgg aaagctgaga tggagggcgg catggcgggc acaggc 46 <210> 59 <211> 45 <212> DNA
<213> mammalian <400> 59 ggcggcctgg aaagctgaga tggagggcgg catggcgggc acagg 45 <210> 60 <211> 44 <212> DNA
<213> mammalian <400> 60 ggcggcctgg aaagctgaga tggagggcgg catggcgggc acag 44 <210> 61 <211> 43 <212> DNA
<213> mammalian <400> 61 ggcggcctgg aaagctgaga tggagggcgg catggcgggc aca 43 <210> 62 <211> 42 <212> DNA
<213> mammalian <400> 62 ggcggcctgg aaagctgaga tggagggcgg catggcgggc ac 42 <210> 63 <211> 41 <212> DNA
<213> mammalian <400> 63 ggcggcctgg aaagctgaga tggagggcgg catggcgggc a 41 <210> 64 <211> 40 <212> DNA
<213> mammalian <400> 64 ggcggcctgg aaagctgaga tggagggcgg catggcgggc 40 <210> 65 <211> 39 <212> DNA
<213> mammalian <400> 65 ggcggcctgg aaagctgaga tggagggcgg catggcggg 39 <210> 66 <211> 38 <212> DNA
<213> mammalian t I

<400> 66 ggcggcctgg aaagctgaga tggagggcgg catggcgg 38 <210> 67 <211> 37 <212> DNA
<213> mammalian <400> 67 ggcggcctgg aaagctgaga tggagggcgg catggcg 37 <210> 68 <211> 36 <212> DNA
<213> mammalian <400> 68 ggcggcctgg aaagctgaga tggagggcgg catggc 36 <210> 69 <211> 35 <212> DNA
<213> mammalian <400> 69 ggcggcctgg aaagctgaga tggagggcgg catgg 35 <210> 70 <211> 34 <212> DNA
<213> mammalian <400> 70 ggcggcctgg aaagctgaga tggagggcgg catg 34 <210> 71 <211> 33 <212> DNA
<213> mammalian <400> 71 ggcggcctgg aaagctgaga tggagggcgg cat 33 <210> 72 <211> 32 <212> DNA
<213> mammalian <400> 72 ggcggcctgg aaagctgaga tggagggcgg ca 32 <210> 73 <211> 31 <212> DNA
<213> mammalian <400> 73 ggcggcctgg aaagctgaga tggagggcgg c 31 <210> 74 <211> 30 <212> DNA
<213> mammalian <400> 74 ggcggcctgg aaagctgaga tggagggcgg 30 <210> 75 <211> 29 <212> DNA
<213> mammalian <400> 75 ggcggcctgg aaagctgaga tggagggcg 29 <210> 76 <211> 28 <212> DNA
<213> mammalian <400> 76 ggcggcctgg aaagctgaga tggagggc 2g <210> 77 <211> 27 <212> DNA
<213> mammalian <400> 77 ggcggcctgg aaagctgaga tggaggg 27 <210> 78 <211> 26 <212> DNA
<213> mammalian <400> 78 ggcggcctgg aaagctgaga tggagg <210> 79 <211> 25 <212> DNA
<213> mammalian <400> 79 ggcggcctgg aaagctgaga tggag 25 <210> 80 <211> 24 <212> DNA
<213> mammalian <400> 80 ggcggcctgg aaagctgaga tgga 24 <210> 81 <211> 23 <212> DNA
<213> mammalian <400> 81 ggcggcctgg aaagctgaga tgg 23 <210> 82 <211> 22 <212> DNA
<213> mammalian <400> 82 ggcggcctgg aaagctgaga tg 22 <210> 83 <211> 21 <212> DNA
<213> mammalian <400> 83 ggcggcctgg aaagctgaga t 21 <210> 84 <211> 20 <212> DNA
<213> mammalian <400> 84 ggcggcctgg aaagctgaga 20 <210> 85 <211> 19 <212> DNA
<213> mammalian <400> 85 ggcggcctgg aaagctgag 19 <210> 86 <211> 18 <212> DNA
<213> mammalian <400> 86 ggcggcctgg aaagctga 18 <210> 87 <211> 17 <212> DNA
<213> mammalian <400> 87 ggcggcctgg aaagctg 17 <210> 88 <211> 16 <212> DNA
<213> mammalian <400> 88 ggcggcctgg aaagct 16 <210> 89 <211> 15 <212> DNA
<213> mammalian <400> 89 ggcggcctgg aaagc 15 <210> 90 <211> 14 <212> DNA
<213> mammalian <400> 90 ggcggcctgg aaag 14 <210> 91 <211> 13 <212> DNA
<213> mammalian <400> 91 ggcggcctgg aaa <210> 92 <211> 12 <212> DNA
<213> mammalian <400> 92 ggcggcctgg as <210> 93 <211> 11 <212> DNA
<213> mammalian <400> 93 ggcggcc~gg a <210> 94 <211> 10 <212> DNA
<213> mammalian <400> 94 ggcggcctgg 10 <210> 95 <211> 50 <212> DNA
<213> mammalian <400> 95 gcggcctgga aagctgagat ggagggcggc atggcgggca caggctgggc 50 <210> 96 <211> 49 <212> DNA
<213> mammalian <400> 96 gcggcctgga aagctgagat ggagggcggc atggcgggca caggctggg 49 <210> 97 <211> 48 <212> DNA
<213> mammalian <400> 97 gcggcctgga aagctgagat ggagggcggc atggcgggca caggctgg 48 <210> 98 <211> 47 <212> DNA
<213> mammalian <400> 98 gcggcctgga aagctgagat ggagggcggc atggcgggca caggctg 47 <210> 99 <211> 46 <212> DNA
<213> mammalian <400> 99 gcggcctgga aagctgagat ggagggcggc atggcgggca caggct 46 <210> 100 <211> 45 <212> DNA
<213> mammalian <400> 100 gcggcctgga aagctgagat ggagggcggc atggcgggca caggc 45 <210> 101 <211> 44 <212> DNA
<213> mammalian <400> 101 gcggcctgga aagctgagat ggagggcggc atggcgggca cagg 44 <210> 102 <211> 43 <212> DNA
<213> mammalian <400> 102 gcggcctgga aagctgagat ggagggcggc atggcgggca cag 43 <210> 103 <211> 42 <212> DNA
<213> mammalian <400> 103 gcggcctgga aagctgagat ggagggcggc atggcgggca ca 42 <210> 104 <211> 41 <212> DNA
<213> mammalian <400> 104 gcggcctgga aagctgagat ggagggcggc atggcgggca c 41 <210> 105 <211> 40 <212> DNA
<213> mammalian <400> 105 gcggcctgga aagctgagat ggagggcggc atggcgggca 40 <210> 106 <211> 39 <212> DNA
<213> mammalian <400> 106 gcggcctgga aagctgagat ggagggcggc atggcgggc 39 <210> 107 <211> 38 <212> DNA
<213> mammalian <400> 107 gcggcctgga aagctgagat ggagggcggc atggcggg 3g <210> 108 <211> 37 <212> DNA
<213> mammalian <400> 108 gcggcctgga aagctgagat ggagggcggc atggcgg 37 <210> 109 <211> 36 <212> DNA
<213> mammalian <400> 109 gcggcctgga aagctgagat ggagggcggc atggcg <210> 110 <211> 35 <212> DNA
<213> mammalian <400> 110 gcggcctgga aagctgagat ggagggcggc atggc 35 <210> 111 <211> 34 <212> DNA
<213> mammalian <400> 111 gcggcctgga aagctgagat ggagggcggc atgg ~ 34 <210> 112 <211> 33 <212> DNA
<213> mammalian <400> 112 gcggcctgga aagctgagat ggagggcggc atg 33 <210> 113 <211> 32 <212> DNA
<213> mammalian <400> 113 gcggcctgga aagctgagat ggagggcggc at 32 <210> 114 <211> 31 <212> DNA
<213> mammalian <400> 114 gcggcctgga aagctgagat ggagggcggc a 31 <210> 115 <211> 30 <212> DNA
<213> mammalian <400> 115 gcggcctgga aagctgagat ggagggcggc 30 <210> 116 <211> 29 <212> DNA
<213> mammalian <400> 116 gcggcctgga aagctgagat ggagggcgg 29 <210> 117 <211> 28 <212> DNA
<213> mammalian <400> 117 gcggcctgga aagctgagat ggagggcg 2g <210> 118 <211> 27 <212> DNA
<213> mammalian <400> 118 gcggcctgga aagctgagat ggagggc 2~
<210> 119 <211> 26 <212> DNA
<213> mammalian <400> 119 gcggcctgga aagctgagat ggaggg 26 <210> 120 <211> 25 <212> DNA
<213> mammalian <400> 120 gcggcctgga aagctgagat ggagg 25 <210> 121 <211> 24 <212> DNA
<213> mammalian <400> 121 gcggcctgga aagctgagat ggag 24 <210> 122 <211> 23 <212> DNA
<213> mammalian <400> 122 gcggcctgga aagctgagat gga 23 <210> 123 <211> 22 <212> DNA
<213> mammalian <400> 123 gcggcctgga aagctgagat gg 22 <210> 124 <211> 21 <212> DNA
<213> mammalian <400> 124 gcggcctgga aagctgagat g 21 <210> 125 <211> 20 <212> DNA
<213> mammalian <400> 125 gcggcctgga aagctgagat 20 <210> 126 <211> 19 <212> DNA
<213> mammalian <400> 126 gcggcctgga aagctgaga <210> 127 <211> 18 <212> DNA
<213> mammalian <400> 127 gcggcctgga aagctgag 1g <210> 128 <211> 17 <212> DNA
<213> mammalian <400> 128 gcggcctgga aagctga 17 <210> 129 <211> 16 <212> DNA
<213> mammalian <400> 129 gcggcctgga aagctg 16 <210> 130 <211> 15 <212> DNA
<213> mammalian <400> 130 gcggcctgga aagct 15 <210> 131 <211> 14 <212> DNA
<213> mammalian <400> 131 gcggcctgga aagc 14 <210> 132 <211> 13 <212> DNA
<213> mammalian <400> 132 gcggcctgga aag 13 <210> 133 <211> 12 <212> DNA
<213> mammalian <400> 133 gcggcctgga as 12 <210> 134 <211> 11 <212> DNA
<213> mammalian <400> 134 gcggcctgga a 11 <210> 135 <211> 10 <212> DNA
<213> mammalian <400> 135 gcggcctgga 10 <210> 136 <211> 49 <212> DNA
<213> mammalian <400> 136 cggcctggaa agctgagatg gagggcggca tggcgggcac aggctgggc 49 <210> 137 <211> 48 <212> DNA
<213> mammalian <400> 137 cggcctggaa agctgagatg gagggcggca tggcgggcac aggctggg 48 <210> 138 <211> 47 <212> DNA
<213> mammalian <400> 138 cggcctggaa agctgagatg gagggcggca tggcgggcac aggctgg 47 <210> 139 <211> 46 <212> DNA
<213> mammalian <400> 139 cggcctggaa agctgagatg gagggcggca tggcgggcac aggctg 46 <210> 140 <211> 45 <212> DNA
<213> mammalian <400> 140 cggcctggaa agctgagatg gagggcggca tggcgggcac aggct 45 <210> 141 <211> 44 <212> DNA
<213> mammalian <400> 141 cggcctggaa agctgagatg gagggcggca tggcgggcac aggc 44 <210> 142 <211> 43 <212> DNA
<213> mammalian <400> 142 cggcctggaa agctgagatg gagggcggca tggcgggcac agg 43 <210> 143 <211> 42 <212> DNA
<213> mammalian <400> 143 cggcctggaa agctgagatg gagggcggca tggcgggcac ag 42 <210>144 <211>41 <212>DNA

<213>mammalian <400> 144 cggcctggaa agctgagatg gagggcggca tggcgggcac a 41 <210> 145 <211> 40 <212> DNA
<213> mammalian <400> 145 cggcctggaa agctgagatg gagggcggca tggcgggcac 40 <210> 146 <211> 39 <212> DNA
<213> mammalian <400> 146 cggcctggaa agctgagatg gagggcggca tggcgggca 39 <210> 147 <211> 38 <212> DNA
<213> mammalian <400> 147 cggcctggaa agctgagatg gagggcggca tggcgggc 38 <210> 148 <211> 37 <212> DNA
<213> mammalian <400> 148 cggcctggaa agctgagatg gagggcggca tggcggg 37 <210> 149 <211> 36 <212> DNA
<213> mammalian <400> 149 cggcctggaa agctgagatg gagggcggca tggcgg 36 <210> 150 <211> 35 <212> DNA
<213> mammalian <400> 150 cggcctggaa agctgagatg gagggcggca tggcg 35 <210> 151 <211> 34 <212> DNA
<213> mammalian <400> 151 cggcctggaa agctgagatg gagggcggca tggc 34 <210> 152 <211> 33 <212> DNA
<213> mammalian <400> 152 cggcctggaa agctgagatg gagggcggca tgg 33 <210> 153 <211> 32 <212> DNA
<213> mammalian <400> 153 cggcctggaa agctgagatg gagggcggca tg 32 <210> 154 <211> 31 <212> DNA
<213> mammalian <400> 154 cggcctggaa agctgagatg gagggcggca t <210> 155 <211> 30 <212> DNA
<213> mammalian <400> 155 cggcctggaa agctgagatg gagggcggca 30 <210> 156 <211> 29 <212> DNA
<213> mammalian <400> 156 cggcctggaa agctgagatg gagggcggc 29 <210> 157 <211> 28 <212> DNA
<213> mammalian <400> 157 cggcctggaa agctgagatg gagggcgg 2g <210> 158 <2~11> 27 <212> DNA
<213> mammalian <400> 158 cggcctggaa agctgagatg gagggcg 2~
<210> 159 <211> 26 <212> DNA
<213> mammalian <400> 159 cggcctggaa agctgagatg gagggc 26 <210> 160 <211> 25 <212> DNA
<213> mammalian <400> 160 cggcctggaa agctgagatg gaggg 25 <210> 161 <211> 24 <212> DNA
<213> mammalian <400> 161 cggcctggaa agctgagatg gagg 24 <210> 162 <211> 23 <212> DNA
<213> mammalian <400> 162 cggcctggaa agctgagatg gag 23 <210> 163 <211> 22 <212> DNA
<213> mammalian <400> 163 cggcctggaa agctgagatg ga 22 <210> 164 <211> 21 <212> DNA
<213> mammalian <400> 164 cggcctggaa agctgagatg g 21 <210> 165 <211> 20 <212> DNA
<213> mammalian <400> 165 cggcctggaa agctgagatg 20 <210> 166 <211> 19 <212> DNA
<213> mammalian <400> 166 cggcctggaa agctgagat 19 <210> 167 <211> 18 <212> DNA
<213> mammalian <400> 167 cggcctggaa agctgaga 18 <210> 168 <211> 17 <212> DNA
<213> mammalian <400> 168 cggcctggaa agctgag 17 <210> 169 <211> 16 <212> DNA
<213> mammalian <400> 169 cggcctggaa agctga 16 <210> 170 <211> 15 <212> DNA
<213> mammalian <400> 170 cggcctggaa agctg 15 <210> 171 <211> 14 <212> DNA
<213> mammalian <400> 171 cggcctggaa agct 14 <210> 172 <211> 13 <212> DNA
<213> mammalian <400> 172 cggcctggaa agc 13 <210> 173 <211> 12 <212> DNA
<213> mammalian <400> 173 cggcctggaa ag 12 <210> 174 <211> 11 <212> DNA
<213> mammalian <400> 174 cggcctggaa a 11 <210> 175 <211> 10 <212> DNA
<213> mammalian <400> 175 cggcctggaa 10 <210> 176 <211> 48 <212> DNA
<213> mammalian <400> 176 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca ggctgggc 48 <210> 177 <211> 47 <212> DNA
<213> mammalian <400> 177 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca ggctggg 47 <210> 178 <211> 46 <212> DNA
<213> mammalian <400> 178 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca ggctgg 46 <210> 179 <211> 45 <212> DNA
<213> mammalian <400> 179 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca ggctg 45 <210> 180 <211> 44 <212> DNA
<213> mammalian <400> 180 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca ggct 44 <210> 181 <211> 43 <212> DNA
<213> mammalian <400> 181 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca ggc 43 <210> 182 <211> 42 <212> DNA
<213> mammalian <400> 182 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca gg 42 <210> 183 <211> 41 <212> DNA
<213> mammalian <400> 183 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca g 41 <210> 184 <211> 40 <212> DNA
<213> mammalian <400> 184 ggcctggaaa gctgagatgg agggcggcat ggcgggcaca 40 <210> 185 <211> 39 <212> DNA
<213> mammalian <400> 185 ggcctggaaa gctgagatgg agggcggcat ggcgggcac 39 <210> 186 <211> 38 <212> DNA
<213> mammalian <400> 186 ggcctggaaa gctgagatgg agggcggcat ggcgggca 38 <210> 187 <211> 37 <212> DNA
<213> mammalian <400> 187 ggcctggaaa gctgagatgg agggcggcat ggcgggc 37 <210> 188 <211> 36 <212> DNA
<213> mammalian <400> 188 ggcctggaaa gctgagatgg agggcggcat ggcggg 36 <210> 189 <211> 35 <212> DNA
<213> mammalian <400> 189 ggcctggaaa gctgagatgg agggcggcat ggcgg 35 <210> 190 <211> 34 <212> DNA
<213> mammalian <400> 190 ggcctggaaa gctgagatgg agggcggcat ggcg 34 <210> 191 <211> 33 <212> DNA
<213> mammalian <400> 191 ggcctggaaa gctgagatgg agggcggcat ggc 33 <210> 192 <211> 32 <212> DNA
<213> mammalian <400> 192 ggcctggaaa gctgagatgg agggcggcat gg 32 <210> 193 <211> 31 <212> DNA
<213> mammalian <400> 193 ggcctggaaa gctgagatgg agggcggcat g 31 <210> 194 <211> 30 <212> DNA
<213> mammalian <400> 194 ggcctggaaa gctgagatgg agggcggcat 30 <210> 195 <211> 29 <212> DNA
<213> mammalian <400> 195 ggcctggaaa gctgagatgg agggcggca 29 <210> 196 <211> 28 <212> DNA
<213> mammalian <400> 196 ggcctggaaa gctgagatgg agggcggc 2g <210> 197 <211> 27 <212> DNA
<213> mammalian <400> 197 ggcctggaaa gctgagatgg agggcgg 27 <210> 198 <211> 26 <212> DNA
<213> mammalian <400> 198 ggcctggaaa gctgagatgg agggcg 26 <210> 199 <211> 25 <212> DNA
<213> mammalian <400> 199 ggcctggaaa gctgagatgg agggc 25 <210> 200 <211> 24 <212> DNA
<213> mammalian <400> 200 ggcctggaaa gctgagatgg aggg 24 <210> 201 <211> 23 <212> DNA
<213> mammalian <400> 201 ggcctggaaa gctgagatgg agg 23 <210> 202 <211> 22 <212> DNA
<213> mammalian <400> 202 ggcctggaaa gctgagatgg ag 22 <210> 203 <211> 21 <212> DNA
<213> mammalian <400> 203 ggcctggaaa gctgagatgg a 21 <210> 204 <211> 20 <212> DNA
<213> mammalian <400> 204 ggcctggaaa gctgagatgg 20 <210> 205 <211> 19 <212> DNA
<213> mammalian <400> 205 ggcctggaaa gctgagatg 19 <210> 206 <211> 18 <212> DNA
<213> mammalian <400> 206 ggcctggaaa gctgagat 18 <210> 207 <211> 17 <212> DNA
<213> mammalian <400> 207 ggcctggaaa gctgaga 17 <210> 208 <211> 16 <212> DNA
<213> mammalian <400> 208 ggcctggaaa gctgag 16 <210> 209 <211> 15 <212> DNA
<213> mammalian <400> 209 ggcctggaaa gctga 15 <210> 210 <211> 14 <212> DNA
<213> mammalian <400> 210 ggcctggaaa gctg 14 <210> 211 <211> 13 <212> DNA
<213> mammalian <400> 211 ggcctggaaa get 13 <210> 212 <211> 12 <212> DNA
<213> mammalian <400> 212 ggcctggaaa gc 12 <210> 213 <211> 11 <212> DNA
<213> mammalian <400> 213 ggcctggaaa g 11 <210> 214 <211> 10 <212> DNA
<213> mammalian <400> 214 ggcctggaaa <210> 215 <211> 47 <212> DNA
<213> mammalian <400> 215 gcctggaaag ctgagatgga gggcggcatg gcgggcacag gctgggc 47 <210> 216 <211> 46 <212> DNA
<213> mammalian <400> 216 gcctggaaag ctgagatgga gggcggcatg gcgggcacag gctggg 46 <210> 2I7 <211> 45 <212> DNA
<213> mammalian <400> 217 gcctggaaag ctgagatgga gggcggcatg gcgggcacag gctgg 45 <210> 218 <211> 44 <212> DNA
<213> mammalian <400> 218 gcctggaaag ctgagatgga gggcggcatg gcgggcacag gctg 44 <210> 219 <211> 43 <212> DNA
<213> mammalian <400> 219 gcctggaaag ctgagatgga gggcggcatg gcgggcacag get 43 <210> 220 <211> 42 <212> DNA
<213> mammalian <400> 220 gcctggaaag ctgagatgga gggcggcatg gcgggcacag gc 42 <210> 221 <212> 41 <212> DNA
<213> mammalian <400> 221 gcctggaaag ctgagatgga gggcggcatg gcgggcacag g 41 <210> 222 <211> 40 <212> DNA
<213> mammalian <400> 222 gcctggaaag ctgagatgga gggcggcatg gcgggcacag 40 <210> 223 <211> 39 <212> DNA
<213> mammalian <400> 223 gcctggaaag ctgagatgga gggcggcatg gcgggcaca 39 <210> 224 <211> 38 <212> DNA
<213> mammalian <400> 224 gcctggaaag ctgagatgga gggcggcatg gcgggcac 38 <210> 225 <211> 37 <212> DNA
<213> mammalian <400> 225 gcctggaaag ctgagatgga gggcggcatg gcgggca 37 <210> 226 <211> 36 <212> DNA
<223> mammalian <400> 226 gcctggaaag ctgagatgga gggcggcatg gcgggc 36 <210> 227 <211> 35 <212> DNA
<213> mammalian <400> 227 gcctggaaag ctgagatgga gggcggcatg gcggg 35 <210> 228 <211> 34 <212> DNA
<213> mammalian <400> 228 gcctggaaag ctgagatgga gggcggcatg gcgg 34 <210> 229 <211> 33 <212> DNA
<213> mammalian <400> 229 gcctggaaag ctgagatgga gggcggcatg gcg 33 <210> 230 <211> 32 <212> DNA
<213> mammalian <400> 230 gcctggaaag ctgagatgga gggcggcatg gc 32 <210> 231 <211> 31 <212> DNA
<213> mammalian <400> 231 gcctggaaag ctgagatgga gggcggcatg g 31 <210> 232 <211> 30 <212> DNA
<213> mammalian <400> 232 gcctggaaag ctgagatgga gggcggcatg 30 <210> 233 <211> 29 <212> DNA
<213> mammalian <400> 233 gcctggaaag ctgagatgga gggcggcat 29 <210> 234 <211> 28 <212> DNA
<213> mammalian <400> 234 gcctggaaag ctgagatgga gggcggca 28 <210> 235 <211> 27 <212> DNA
<213> mammalian <400> 235 gcctggaaag ctgagatgga gggcggc 27 <210> 236 <211> 26 <212> DNA
<213> mammalian <400> 236 gcctggaaag ctgagatgga gggcgg 26 <210> 237 <211> 25 <212> DNA
<213> mammalian <400> 237 gcctggaaag ctgagatgga gggcg 25 <210> 238 <211> 24 <212> DNA
<213> mammalian <400> 238 gcctggaaag ctgagatgga gggc 24 <210> 239 <211> 23 <212> DNA
<213> mammalian <400> 239 gcctggaaag ctgagatgga ggg 23 <210> 240 <211> 22 <212> DNA
<213> mammalian <400> 240 gcctggaaag ctgagatgga gg 22 <210> 241 <211> 21 <212> DNA
<213> mammalian <400> 241 gcctggaaag ctgagatgga g 21 <210> 242 <211> 20 <212> DNA
<213> mammalian <400> 242 gcctggaaag ctgagatgga 20 <210> 243 <211> 19 <212> DNA
<213> mammalian <400> 243 gcctggaaag ctgagatgg 19 <210> 244 <211> 18 <212> DNA
<213> mammalian <400> 244 gcctggaaag ctgagatg 18 <210> 245 <211> 17 <212> DNA
<213> mammalian <400> 245 gcctggaaag ctgagat 17 <210> 246 <211> 16 <212> DNA
<213> mammalian <400> 246 gcctggaaag ctgaga 16 <210> 247 <211> 15 <212> DNA
<213> mammalian <400> 247 gcctggaaag ctgag 15 <210> 248 <211> 14 <212> DNA
<213> mammalian <400> 248 gcctggaaag ctga 14 <210> 249 <211> 13 <212> DNA
<213> mammalian <400> 249 gcctggaaag ctg 13 <210> 250 <211> 12 <212> DNA
<213> mammalian <400> 250 gcctggaaag ct 12 <210> 251 <211> 11 <212> DNA
<213> mammalian <400> 251 gcctggaaag c 11 <210> 252 <211> 10 <212> DNA
<213> mammalian <400> 252 gcctggaaag 10 <210> 253 <211> 46 <212> DNA
<213> mammalian <400> 253 cctggaaagc tgagatggag ggcggcatgg cgggcacagg ctgggc 46 <210> 254 <211> 45 <212> DNA
<213> mammalian <400> 254 cctggaaagc tgagatggag ggcggcatgg cgggcacagg ctggg 45 <210> 255 <211> 44 <212> DNA
<213> mammalian <400> 255 cctggaaagc tgagatggag ggcggcatgg cgggcacagg ctgg 44 <210> 256 <211> 43 <212> DNA
<213> mammalian <400> 256 cctggaaagc tgagatggag ggcggcatgg cgggcacagg ctg 43 <210> 257 <211> 42 <212> DNA
<213> mammalian <400> 257 cctggaaagc tgagatggag ggcggcatgg cgggcacagg ct 42 <210> 258 <211> 41 <212> DNA
<213> mammalian <400> 258 cctggaaagc tgagatggag ggcggcatgg cgggcacagg c 41 <210> 259 <211> 40 <212> DNA
<213> mammalian <400> 259 cctggaaagc tgagatggag ggcggcatgg cgggcacagg 40 <210> 260 <211> 39 <212> DNA
<213> mammalian <400> 260 cctggaaagc tgagatggag ggcggcatgg cgggcacag 39 <210> 261 <211> 38 <212> DNA
<213> mammalian <400> 261 cctggaaagc tgagatggag ggcggcatgg cgggcaca 38 <210> 262 <211> 37 <212> DNA
<213> mammalian <400> 262 cctggaaagc tgagatggag ggcggcatgg cgggcac 37 <210> 263 <211> 36 <212> DNA
<213> mammalian <400> 263 cctggaaagc tgagatggag ggcggcatgg cgggca 36 <210> 264 <211> 35 <212> DNA
<213> mammalian <400> 264 cctggaaagc tgagatggag ggcggcatgg cgggc 35 <210> 265 <211> 34 <212> DNA
<213> mammalian <400> 265 cctggaaagc tgagatggag ggcggcatgg cggg 34 <210> 266 <211> 33 <212> DNA
<213> mammalian <400> 266 cctggaaagc tgagatggag ggcggcatgg cgg 33 <210> 267 <211> 32 <212> DNA
<213> mammalian <400> 267 cctggaaagc tgagatggag ggcggcatgg cg 32 <210> 268 <211> 31 <212> DNA
<213> mammalian <400> 268 cctggaaagc tgagatggag ggcggcatgg c 31 <210> 269 <211> 30 <212> DNA
<213> mammalian <400> 269 cctggaaagc tgagatggag ggcggcatgg 30 <210> 270 <211> 29 <212> DNA
<213> mammalian <400> 270 cctggaaagc tgagatggag ggcggcatg 29 <210> 271 <211> 28 <212> DNA
<213> mammalian <400> 271 cctggaaagc tgagatggag ggcggcat 28 <210> 272 <211> 27 <212> DNA
<213> mammalian <400> 272 cctggaaagc tgagatggag ggcggca 27 <210> 273 <211> 26 <212> DNA
<213> mammalian <400> 273 cctggaaagc tgagatggag ggcggc 26 <210> 274 <211> 25 <212> DNA
<213> mammalian <400> 274 cctggaaagc tgagatggag ggcgg 25 <210> 275 <211> 24 <212> DNA
<213> mammalian <400> 275 cctggaaagc tgagatggag ggcg 24 <210> 276 <211> 23 <212> DNA
<213> mammalian <400> 276 cctggaaagc tgagatggag ggc 23 <210> 277 <211> 22 <212> DNA
<213> mammalian <400> 277 cctggaaagc tgagatggag gg 22 <210> 278 <211> 21 <212> DNA
<213> mammalian <400> 278 cctggaaagc tgagatggag g 21 <210> 279 <211> 20 <212> DNA
<213> mammalian <400> 279 cctggaaagc tgagatggag 20 <210> 280 <211> 19 <212> DNA
<213> mammalian <400> 280 cctggaaagc tgagatgga 19 <210> 281 <211> 18 <212> DNA
<213> mammalian <400> 281 cctggaaagc tgagatgg 18 <210> 282 <211> 17 <212> DNA
<213> mammalian <400> 282 cctggaaagc tgagatg 1~
<210> 283 <211> 16 <212> DNA
<213> mammalian <400> 283 cctggaaagc tgagat 16 <210> 284 <211> 15 <212> DNA
<213> mammalian <400> 284 cctggaaagc tgaga 15 <210> 285 <211> 14 <212> DNA
<213> mammalian <400> 285 cctggaaagc tgag 14 <210> 286 <211> 13 <212> DNA
<213> mammalian <400> 286 cctggaaagc tga 13 <210> 287 <211> 12 <212> DNA
<213> mammalian <400> 287 cctggaaagc tg 12 <210> 288 <211> 11 <212> DNA
<213> mammalian <400> 288 cctggaaagc t 11 <210> 289 <211> 10 <212> DNA
<213> mammalian <400> 289 cctggaaagc 10 <210> 290 <211> 45 <212> DNA
<213> mammalian <400> 290 ctggaaagct gagatggagg gcggcatggc gggcacaggc tgggc 45 <210> 291 <211> 44 <212> DNA
<213> mammalian <400> 291 ctggaaagct gagatggagg gcggcatggc gggcacaggc tggg 44 <210> 292 <211> 43 <212> DNA
<213> mammalian <400> 292 ctggaaagct gagatggagg gcggcatggc gggcacaggc tgg 43 <210> 293 <211> 42 <212> DNA
<213> mammalian <400> 293 ctggaaagct gagatggagg gcggcatggc gggcacaggc tg 42 <210> 294 <211> 41 <212> DNA
<213> mammalian <400> 294 ctggaaagct gagatggagg gcggcatggc gggcacaggc t 41 <210> 295 <211> 40 <212> DNA
<213> mammalian <400> 295 ctggaaagct gagatggagg gcggcatggc gggcacaggc 40 <210> 296 <211> 39 <212> DNA
<213> mammalian <400> 296 ctggaaagct gagatggagg gcggcatggc gggcacagg 39 <210> 297 <211> 38 <212> DNA
<213> mammalian <400> 297 ctggaaagct gagatggagg gcggcatggc gggcacag 38 <210> 298 <211> 37 <212> DNA
<213> mammalian <400> 298 ctggaaagct gagatggagg gcggcatggc gggcaca 3~
<210> 299 <211> 36 <212> DNA
<213> mammalian <400> 299 ctggaaagct gagatggagg gcggcatggc gggcac 36 <210> 300 <211> 35 <212> DNA
<213> mammalian <400> 300 ctggaaagct gagatggagg gcggcatggc gggca 35 <210> 301 <211> 34 <212> DNA
<213> mammalian <400> 301 ctggaaagct gagatggagg gcggcatggc gggc 34 <210> 302 <211> 33 <212> DNA
<213> mammalian <400> 302 ctggaaagct gagatggagg gcggcatggc ggg 33 <210> 303 <211> 32 <212> DNA
<213> mammalian <400> 303 ctggaaagct gagatggagg gcggcatggc gg 32 <210> 304 <211> 31 <212> DNA
<213> mammalian <400> 304 ctggaaagct gagatggagg gcggcatggc g 31 <210> 305 <211> 30 <212> DNA
<213> mammalian <400> 305 ctggaaagct gagatggagg gcggcatggc 30 <210> 306 <211> 29 <212> DNA
<213> mammalian <400> 306 ctggaaagct gagatggagg gcggcatgg 29 <210> 307 <211> 28 <212> DNA
<213> mammalian <400> 307 ctggaaagct gagatggagg gcggcatg 28 <210> 308 <211> 27 <212> DNA
<213> mammalian <400> 308 ctggaaagct gagatggagg gcggcat 27 <210> 309 <211> 26 <212> DNA
<213> mammalian <400> 309 ctggaaagct gagatggagg gcggca 26 <210> 310 <211> 25 <212> DNA
<213> mammalian <400> 310 ctggaaagct gagatggagg gcggc 25 <210> 311 <211> 24 <212> DNA
<213> mammalian <400> 311 ctggaaagct gagatggagg gcgg 24 <210> 312 <211> 23 <212> DNA
<213> mammalian <400> 312 ctggaaagct gagatggagg gcg 23 <210> 313 <211> 22 <212> DNA
<213> mammalian <400> 313 ctggaaagct gagatggagg gc 22 <210> 314 <211> 21 <212> DNA
<213> mammalian <400> 314 ctggaaagct gagatggagg g 21 <210> 315 <211> 20 <212> DNA
<213> mammalian <400> 315 ctggaaagct gagatggagg 20 <210> 316 <211> 19 <212> DNA
<213> mammalian <400> 316 ctggaaagct gagatggag 19 <210> 317 <211> 18 <212> DNA
<213> mammalian <400> 317 ctggaaagct gagatgga 18 <210> 318 <211> 17 <212> DNA
<213> mammalian <400> 318 ctggaaagct gagatgg 1~
<210> 319 <211> 16 <212> DNA
<213> mammalian <400> 319 ctggaaagct gagatg 16 <210> 320 <211> 15 <212> DNA
<213> mammalian <400> 320 ctggaaagct gagat 15 <210> 321 <211> 14 <212> DNA
<213> mammalian <400> 321 ctggaaagct gaga 14 <210> 322 <211> 13 <212> DNA
<213> mammalian <400> 322 ctggaaagct gag 13 <210> 323 <211> 12 <212> DNA
<213> mammalian <400> 323 ctggaaagct ga 12 <210> 324 <211> 11 <212> DNA
<213> mammalian <400> 324 ctggaaagct g 11 <210> 325 <211> 10 <212> DNA
<213> mammalian <400> 325 ctggaaagct 10 <210> 326 <211> 44 <212> DNA
<213> mammalian <400> 326 tggaaagctg agatggaggg cggcatggcg ggcacaggct gggc 44 <210> 327 <211> 43 <212> DNA
<213> mammalian <400> 327 tggaaagctg agatggaggg cggcatggcg ggcacaggct ggg 43 <210> 328 <211> 42 <212> DNA
<213> mammalian <400> 328 tggaaagctg agatggaggg cggcatggcg ggcacaggct gg 42 <210> 329 <211> 41 <212> DNA
<213> mammalian <400> 329 tggaaagctg agatggaggg cggcatggcg ggcacaggct g 41 <210> 330 <211> 40 <212> DNA
<213> mammalian <400> 330 tggaaagctg agatggaggg cggcatggcg ggcacaggct 40 <210> 331 <211> 39 <212> DNA
<213> mammalian <400> 331 tggaaagctg agatggaggg cggcatggcg ggcacaggc 39 <210> 332 <211> 38 <212> DNA
<213> mammalian <400> 332 tggaaagctg agatggaggg cggcatggcg ggcacagg 38 <210> 333 <211> 37 <212> DNA
<213> mammalian <400> 333 tggaaagctg agatggaggg cggcatggcg ggcacag 37 <210> 334 <211> 36 <212> DNA
<213> mammalian <400> 334 tggaaagctg agatggaggg cggcatggcg ggcaca 36 <210> 335 <211> 35 <212> DNA
<213> mammalian <400> 335 tggaaagctg agatggaggg cggcatggcg ggcac 35 <210> 336 <211> 34 <212> DNA
<213> mammalian <400> 336 tggaaagctg agatggaggg cggcatggcg ggca 34 <210> 337 <221> 33 <212> DNA
<213> mammalian <400> 337 tggaaagctg agatggaggg cggcatggcg ggc 33 <210> 338 <211> 32 <212> DNA
<213> mammalian <400> 338 tggaaagctg agatggaggg cggcatggcg gg 32 <210> 339 <211> 31 <212> DNA
<213> mammalian <400> 339 tggaaagctg agatggaggg cggcatggcg g 31 <210> 340 <211> 30 <212> DNA
<213> mammalian <400> 340 tggaaagctg agatggaggg cggcatggcg 30 <210> 341 <211> 29 <212> DNA
<213> mammalian <400> 341 tggaaagctg agatggaggg cggcatggc 29 <210> 342 <211> 28 <212> DNA
<213> mammalian <400> 342 tggaaagctg agatggaggg cggcatgg 28 <210> 343 <211> 27 <212> DNA
<213> mammalian <400> 343 tggaaagctg agatggaggg cggcatg 27 <210> 344 <211> 26 <212> DNA
<213> mammalian <400> 344 tggaaagctg agatggaggg cggcat 26 <210> 345 <211> 25 <212> DNA
<213> mammalian <400> 345 tggaaagctg agatggaggg cggca 25 <210> 346 <211> 24 <212> DNA
<213> mammalian <400> 346 tggaaagctg agatggaggg cggc 24 <210> 347 <211> 23 <212> DNA
<213> mammalian <400> 347 tggaaagctg agatggaggg cgg 23 <210> 348 <211> 22 <212> DNA
<213> mammalian <400> 348 tggaaagctg agatggaggg cg 22 <210> 349 <211> 21 <212> DNA
<213> mammalian <400> 349 tggaaagctg agatggaggg c 21 <210> 350 <211> 20 <212> DNA
<213> mammalian <400> 350 tggaaagctg agatggaggg 20 <210> 351 <211> 19 <212> DNA
<213> mammalian <400> 351 tggaaagctg agatggagg 19 <210> 352 <211> 18 <212> DNA
<213> mammalian <400> 352 tggaaagctg agatggag 18 <210> 353 <211> 17 <212> DNA
<213> mammalian <400> 353 tggaaagctg agatgga 17 <210> 354 <211> 16 <212> DNA
<213> mammalian <400> 354 tggaaagctg agatgg 16 <210> 355 <211> 15 <212> DNA
<213> mammalian <400> 355 tggaaagctg agatg 15 <210> 356 <211> 14 <212> DNA
<213> mammalian <400> 356 tggaaagctg agat 14 <210> 357 <211> 13 <212> DNA
<213> mammalian <400> 357 tggaaagctg aga 13 <210> 358 <211> 12 <212> DNA
<213> mammalian <400> 358 tggaaagctg ag 12 <210> 359 <211> 11 <212> DNA
<213> mammalian <400> 359 tggaaagctg a 11 <210> 360 <211> 10 <212> DNA
<213> mammalian <400> 360 tggaaagctg 10 <210> 361 <211> 43 <212> DNA
<213> mammalian <400> 361 ggaaagctga gatggagggc ggcatggcgg gcacaggctg ggc 43 <210> 362 <211> 42 <212> DNA
<213> mammalian <400> 362 ggaaagctga gatggagggc ggcatggcgg gcacaggctg gg 42 <210> 363 <211> 41 <212> DNA
<213> mammalian <400> 363 ggaaagctga gatggagggc ggcatggcgg gcacaggctg g 41 <210> 364 <211> 40 <212> DNA
<213> mammalian <400> 364 ggaaagctga gatggagggc ggcatggcgg gcacaggctg 40 <210> 365 <211> 39 <212> DNA
<213> mammalian <400> 365 ggaaagctga gatggagggc ggcatggcgg gcacaggct 39 <210> 366 <211> 38 <212> DNA
<213> mammalian <400> 366 ggaaagctga gatggagggc ggcatggcgg gcacaggc 38 <210> 367 <211> 37 <212> DNA
<213> mammalian <400> 367 ggaaagctga gatggagggc ggcatggcgg gcacagg 37 <210> 368 <211> 36 <212> DNA
<213> mammalian <400> 368 ggaaagctga gatggagggc ggcatggcgg gcacag 36 <210> 369 <211> 35 <212> DNA
<213> mammalian <400> 369 ggaaagctga gatggagggc ggcatggcgg gcaca 35 <210> 370 <211> 34 <212> DNA
<213> mammalian <400> 370 ggaaagctga gatggagggc ggcatggcgg gcac 34 <210> 371 <211> 33 <212> DNA
<213> mammalian <400> 371 ggaaagctga gatggagggc ggcatggcgg gca 33 <210> 372 <211> 32 <212> DNA
<213> mammalian <400> 372 ggaaagctga gatggagggc ggcatggcgg gc 32 <210> 373 <211> 31 <212> DNA
<213> mammalian <400> 373 ggaaagctga gatggagggc ggcatggcgg g 31 <210> 374 <211> 30 <212> DNA
<213> mammalian <400> 374 ggaaagctga gatggagggc ggcatggcgg 30 <210> 375 <211> 29 <212> DNA
<213> mammalian <400> 375 ggaaagctga gatggagggc ggcatggcg 29 <210> 376 <211> 28 <212> DNA
<213> mammalian <400> 376 ggaaagctga gatggagggc ggcatggc 2g <210> 377 <211> 27 <212> DNA
<213> mammalian <400> 377 ggaaagctga gatggagggc ggcatgg 27 <210> 378 <211> 26 <212> DNA
<213> mammalian <400> 378 ggaaagctga gatggagggc ggcatg 26 <210> 379 <211> 25 <212> DNA
<213> mammalian <400> 379 ggaaagctga gatggagggc ggcat 25 <210> 380 <211> 24 <212> DNA
<213> mammalian <400> 380 ggaaagctga gatggagggc ggca 24 <210> 381 <211> 23 <212> DNA
<213> mammalian <400> 381 ggaaagctga gatggagggc ggc 23 <210> 382 <211> 22 <212> DNA
<213> mammalian <400> 382 ggaaagctga gatggagggc gg 22 <210> 383 <211> 21 <212> DNA
<213> mammalian <400> 383 ggaaagctga gatggagggc g 21 <210> 384 <211> 20 <212> DNA
<213> mammalian <400> 384 ggaaagctga gatggagggc 20 <210> 385 <211> 19 <212> DNA
<213> mammalian <400> 385 ggaaagctga gatggaggg 19 <210> 386 <211> 18 <212> DNA
<213> mammalian <400> 386 ggaaagctga gatggagg 18 <210> 387 <211> 17 <212> DNA
<213> mammalian <400> 387 ggaaagctga gatggag 17 <210> 388 <211> 16 <212> DNA
<213> mammalian <400> 388 ggaaagctga gatgga 16 <210> 389 <211> 15 <212> DNA
<213> mammalian <400> 389 ggaaagctga gatgg 15 <210> 390 <211> 14 <212> DNA
<213> mammalian <400> 390 ggaaagctga gatg 14 <210> 391 <211> 13 <212> DNA
<213> mammalian <400> 391 ggaaagctga gat 13 <210> 392 <211> 12 <212> DNA
<213> mammalian <400> 392 ggaaagctga ga 12 <210> 393 <211> 11 <212> DNA
<213> mammalian <400> 393 ggaaagctga g 11 <210> 394 <211> 10 <212> DNA
<213> mammalian <400> 394 ggaaagctga 10 <210> 395 <211> 42 <212> DNA
<213> mammalian <400> 395 gaaagctgag atggagggcg gcatggcggg cacaggctgg gc 42 <210> 396 <211> 41 <212> DNA
<213> mammalian <400> 396 gaaagctgag atggagggcg gcatggcggg cacaggctgg g 41 <210> 397 <211> 40 <212> DNA
<213> mammalian <400> 397 gaaagctgag atggagggcg gcatggcggg cacaggctgg 40 <210> 398 <211> 39 <212> DNA
<213> mammalian <400> 398 gaaagctgag atggagggcg gcatggcggg cacaggctg <210> 399 <211> 38 <212> DNA
<213> mammalian <400> 399 gaaagctgag atggagggcg gcatggcggg cacaggct 3g <210> 400 <211> 37 <212> DNA
<213> mammalian <400> 400 gaaagctgag atggagggcg gcatggcggg cacaggc 37 <210> 401 <211> 36 <212> DNA
<213> mammalian <400> 401 gaaagctgag atggagggcg gcatggcggg cacagg 36 <210> 402 <211> 35 <212> DNA
<213> mammalian <400> 402 gaaagctgag atggagggcg gcatggcggg cacag 35 <210> 403 <211> 34 <212> DNA
<213> mammalian <400> 403 gaaagctgag atggagggcg gcatggcggg caca 34 <210> 404 <211> 33 <212> DNA
<213> mammalian <400> 404 gaaagctgag atggagggcg gcatggcggg cac 33 <210> 405 <211> 32 <212> DNA
<213> mammalian <400> 405 gaaagctgag atggagggcg gcatggcggg ca 32 <210> 406 <211> 31 <212> DNA
<213> mammalian <400> 406 gaaagctgag atggagggcg gcatggcggg c 31 <210> 407 <211> 30 <212> DNA
<213> mammalian <400> 407 gaaagctgag atggagggcg gcatggcggg 30 <210> 408 <211> 29 <212> DNA
<213> mammalian <400> 408 gaaagctgag atggagggcg gcatggcgg 29 <210> 409 <211> 28 <212> DNA
<213> mammalian <400> 409 gaaagctgag atggagggcg gcatggcg 28 <210> 410 <211> 27 <212> DNA
<213> mammalian <400> 410 gaaagctgag atggagggcg gcatggc 27 <210> 411 <211> 26 <212> DNA
<213> mammalian <400> 411 gaaagctgag atggagggcg gcatgg 26 <210> 412 <211> 25 <212> DNA
<213> mammalian <400> 412 gaaagctgag atggagggcg gcatg 25 <210> 413 <211> 24 <212> DNA
<213> mammalian <400> 413 gaaagctgag atggagggcg gcat 24 <210> 414 <211> 23 <212> DNA
<213> mammalian <400> 414 gaaagctgag atggagggcg gca 23 <210> 415 <211> 22 <212> DNA
<213> mammalian <400> 415 gaaagctgag atggagggcg gc 22 <210> 416 <211> 21 <212> DNA
<213> mammalian <400> 416 gaaagctgag atggagggcg g 21 <210> 417 <211> 20 <212> DNA
<213> mammalian <400> 417 gaaagctgag atggagggcg 20 <210> 418 <211> 19 <212> DNA
<213> mammalian <400> 418 gaaagctgag atggagggc 1g <210> 419 <211> 18 <212> DNA
<213> mammalian <400> 419 gaaagctgag atggaggg 1g <210> 420 <211> 17 <212> DNA
<213> mammalian <400> 420 gaaagctgag atggagg 17 <210> 421 <211> 16 <212> DNA
<213> mammalian <400> 421 gaaagctgag atggag 16 <210> 422 <211> 15 <212> DNA
<213> mammalian <400> 422 gaaagctgag atgga 15 <210> 423 <211> 14 <212> DNA
<213> mammalian <400> 423 gaaagctgag atgg 14 <210> 424 <211> 13 <212> DNA
<213> mammalian <400> 424 gaaagctgag atg 13 <210> 425 <211> 12 <212> DNA
<213> mammalian <400> 425 gaaagctgag at 12 <210> 426 <211> 11 <212> DNA
<213>- mammalian <400> 426 gaaagctgag a 11 <210> 427 <211> 10 <212> DNA
<213> mammalian <400> 427 gaaagctgag 10 <210> 428 <211> 41 <212> DNA
<213> mammalian <400> 428 aaagctgaga tggagggcgg catggcgggc acaggctggg c 41 <210> 429 <211> 40 <212> DNA
<213> mammalian <400> 429 aaagctgaga tggagggcgg catggcgggc acaggctggg 40 <210> 430 <211> 39 <212> DNA
<213> mammalian <400> 430 aaagctgaga tggagggcgg catggcgggc acaggctgg 39 <210> 431 <211> 38 <212> DNA
<213> mammalian <400> 431 aaagctgaga tggagggcgg catggcgggc acaggctg 38 <210> 432 <211> 37 <212> DNA
<213> mammalian <400> 432 aaagctgaga tggagggcgg catggcgggc acaggct 37 <210> 433 <211> 36 <212> DNA
<213> mammalian <400> 433 aaagctgaga tggagggcgg catggcgggc acaggc 36 <210> 434 <211> 35 <212> DNA
<213> mammalian <400> 434 aaagctgaga tggagggcgg catggcgggc acagg 35 <210> 435 <211> 34 <212> DNA
<213> mammalian <400> 435 aaagctgaga tggagggcgg catggcgggc acag 34 <210> 436 <211> 33 <212> DNA
<213> mammalian <400> 436 aaagctgaga tggagggcgg catggcgggc aca 33 <210> 437 <211> 32 <212> DNA
<213> mammalian <400> 437 aaagctgaga tggagggcgg catggcgggc ac 32 <210> 438 <211> 31 <212> DNA
<213> mammalian <400> 438 aaagctgaga tggagggcgg catggcgggc a 31 <210> 439 <211> 30 <212> DNA
<213> mammalian <400> 439 aaagctgaga tggagggcgg catggcgggc 30 <210> 440 <211> 29 <212> DNA
<213> mammalian <400> 440 aaagctgaga tggagggcgg catggcggg 29 <210> 441 <211> 28 <212> DNA
<213> mammalian <400> 441 aaagctgaga tggagggcgg catggcgg 2g <210> 442 <211> 27 <212> DNA
<213> mammalian <400> 442 aaagctgaga tggagggcgg catggcg 27 <210> 443 <211> 26 <212> DNA
<213> mammalian <400> 443 aaagctgaga tggagggcgg catggc 26 <210> 444 <211> 25 <212> DNA
<213> mammalian <400> 444 aaagctgaga tggagggcgg catgg 25 <210> 445 <211> 24 <212> DNA
<213> mammalian <400> 445 aaagctgaga tggagggcgg catg 24 <210> 446 <211> 23 <212> DNA
<213> mammalian <400> 446 aaagctgaga tggagggcgg cat 23 <210> 447 <211> 22 <212> DNA
<213> mammalian <400> 447 aaagctgaga tggagggcgg ca 22 <210> 448 <211> 21 <212> DNA
<213> mammalian <400> 448 aaagctgaga tggagggcgg c 21 <210> 449 <211> 20 <212> DNA
<213> mammalian <400> 449 aaagctgaga tggagggcgg 20 <210> 450 <211> 19 <212> DNA
<213> mammalian <400> 450 aaagctgaga tggagggcg 19 <210> 451 <211> 18 <212> DNA
<213> mammalian <400> 451 aaagctgaga tggagggc 18 <210> 452 <211> 17 <212> DNA
<213> mammalian <400> 452 aaagctgaga tggaggg 17 <210> 453 <211> 16 <212> DNA
<213> mammalian <400> 453 aaagctgaga tggagg 16 <210> 454 <211> 15 <212> DNA
<213> mammalian <400> 454 aaagctgaga tggag 15 <210> 455 <211> 14 <212> DNA
<213> mammalian <400> 455 aaagctgaga tgga 14 <210> 456 <211> 13 <212> DNA
<213> mammalian <400> 456 aaagctgaga tgg 13 <210> 457 <211> 12 <212> DNA
<213> mammalian <400> 457 aaagctgaga tg 12 <210> 458 <211> 11 <212> DNA
<213> mammalian <400> 458 aaagctgaga t 11 <210> 459 <211> 10 <212> DNA
<213> mammalian <400> 459 aaagctgaga 10 <210> 460 <211> 40 <212> DNA
<213> mammalian <400> 460 aagctgagat ggagggcggc atggcgggca caggctgggc 40 <210> 461 <211> 39 <212> DNA
<213> mammalian <400> 461 aagctgagat ggagggcggc atggcgggca caggctggg 39 <210> 462 <211> 38 <212> DNA
<213> mammalian <400> 462 aagctgagat ggagggcggc atggcgggca caggctgg 38 <210> 463 <211> 37 <212> DNA
<213> mammalian <400> 463 aagctgagat ggagggcggc atggcgggca caggctg 37 <210> 464 <211> 36 <212> DNA
<213> mammalian <400> 464 aagctgagat ggagggcggc atggcgggca caggct 36 <210> 465 <211> 35 <212> DNA
<213> mammalian <400> 465 aagctgagat ggagggcggc atggcgggca caggc 35 <210> 466 <211> 34 <212> DNA
<213> mammalian <400> 466 aagctgagat ggagggcggc atggcgggca cagg 34 <210> 467 <211> 33 <212> DNA
<213> mammalian <400> 467 aagctgagat ggagggcggc atggcgggca cag 33 <210> 468 <211> 32 <212> DNA
<213> mammalian <400> 468 aagctgagat ggagggcggc atggcgggca ca 32 <210> 469 <211> 31 <212> DNA
<213> mammalian <400> 469 aagctgagat ggagggcggc atggcgggca c 31 <210> 470 <211> 30 <212> DNA
<213> mammalian <400> 470 aagctgagat ggagggcggc atggcgggca 30 <210> 471 <211> 29 <212> DNA
<213> mammalian <400> 471 aagctgagat ggagggcggc atggcgggc 29 <210> 472 <211> 28 <212> DNA
<213> mammalian <400> 472 aagctgagat ggagggcggc atggcggg 2g <210> 473 <211> 27 <212> DNA
<213> mammalian <400> 473 aagctgagat ggagggcggc atggcgg 27 <210> 474 <211> 26 <212> DNA
<213> mammalian <400> 474 aagctgagat ggagggcggc atggcg 26 <210> 475 <211> 25 <212> DNA
<213> mammalian <400> 475 aagctgagat ggagggcggc atggc 25 <210> 476 <211> 24 <212> DNA
<213> mammalian <400> 476 aagctgagat ggagggcggc atgg 24 <210> 477 <211> 23 <212> DNA
<213> mammalian <400> 477 aagctgagat ggagggcggc atg 23 <210> 478 <211> 22 <212> DNA
<213> mammalian <400> 478 aagctgagat ggagggcggc at 22 <210> 479 <211> 21 <212> DNA
<213> mammalian <400> 479 aagctgagat ggagggcggc a 21 <210> 480 <211> 20 <212> DNA
<213> mammalian <400> 480 aagctgagat ggagggcggc 20 <210> 481 <211> 19 <212> DNA
<213> mammalian <400> 481 aagctgagat ggagggcgg 19 <210> 482 <211> 18 <212> DNA
<213> mammalian <400> 482 aagctgagat ggagggcg 1g <210> 483 <211> 17 <212> DNA
<213> mammalian <400> 483 aagctgagat ggagggc 17 <210> 484 <211> 16 <212> DNA
<213> mammalian <400> 484 aagctgagat ggaggg 16 <210> 485 <211> 15 <212> DNA
<213> mammalian <400> 485 aagctgagat ggagg 15 <210> 486 <211> 14 <212> DNA
<213> mammalian <400> 486 aagctgagat ggag 14 <210> 487 <211> 13 <212> DNA
<213> mammalian <400> 487 aagctgagat gga 13 <210> 488 <211> 12 <212> DNA
<213> mammalian <400> 488 aagctgagat gg 12 <210> 489 <211> 11 <212> DNA
<213> mammalian <400> 489 aagctgagat g 11 <210> 490 <211> 10 <212> DNA
<213> mammalian <400> 490 aagctgagat 10 <210> 491 <211> 39 <212> DNA
<213> mammalian <400> 491 agctgagatg gagggcggca tggcgggcac aggctgggc 39 <210>492 <211>38 <212>DNA

<213>mammalian <400> 492 agctgagatg gagggcggca tggcgggcac aggctggg 38 <210> 493 <211> 37 <212> DNA
<213> mammalian <400> 493 agctgagatg gagggcggca tggcgggcac aggctgg 37 <210>494 <211>36 <212>DNA

<213>mammalian <400> 494 agctgagatg gagggcggca tggcgggcac aggctg 36 <210> 495 <211> 35 <212> DNA
<213> mammalian <400> 495 agctgagatg gagggcggca tggcgggcac aggct 35 <210> 496 <211> 34 <212> DNA
<213> mammalian <400> 496 agctgagatg gagggcggca tggcgggcac aggc 34 <210> 497 <211> 33 <212> DNA
<213> mammalian <400> 497 agctgagatg gagggcggca tggcgggcac agg 33 <210> 498 <211> 32 <212> DNA
<213> mammalian <400> 498 agctgagatg gagggcggca tggcgggcac ag 32 <210> 499 <211> 31 <212> DNA
<213> mammalian <400> 499 agctgagatg gagggcggca tggcgggcac a 31 <210> 500 <211> 30 <212> DNA
<213> mammalian <400> 500 agctgagatg gagggcggca tggcgggcac 30 <210> 501 <211> 29 <212> DNA
<213> mammalian <400> 501 agctgagatg gagggcggca tggcgggca 29 <210> 502 <211> 28 <212> DNA
<213> mammalian <400> 502 agctgagatg gagggcggca tggcgggc 28 <210> 503 <211> 27 <212> DNA
<213> mammalian <400> 503 agctgagatg gagggcggca tggcggg 27 <210> 504 <211> 26 <212> DNA
<213> mammalian <400> 504 agctgagatg gagggcggca tggcgg 26 <210> 505 <211> 25 <212> DNA
<213> mammalian <400> 505 agctgagatg gagggcggca tggcg 25 <210> 506 <211> 24 <212> DNA
<213> mammalian <400> 506 agctgagatg gagggcggca tggc 24 <210> 507 <211> 23 <212> DNA
<213> mammalian <400> 507 agctgagatg gagggcggca tgg 23 <210> 508 <211> 22 <212> DNA
<213> mammalian <400> 508 agctgagatg gagggcggca tg 22 <210> 509 <211> 21 <212> DNA
<213> mammalian <400> 509 agctgagatg gagggcggca t 21 <210> 510 <211> 20 <212> DNA
<213> mammalian <400> 510 agctgagatg gagggcggca 20 <210> 511 <211> 19 <212> DNA
<213> mammalian <400> 511 agctgagatg gagggcggc 19 <210> 512 <211> 18 <212> DNA
<213> mammalian <400> 512 agctgagatg gagggcgg 18 <210> 513 <211> 17 <212> DNA
<213> mammalian <400> 513 agctgagatg gagggcg 17 <210> 514 <211> 16 <212> DNA
<213> mammalian <400> 514 agctgagatg gagggc 16 <210> 515 <211> 15 <212> DNA
<213> mammalian <400> 515 agctgagatg gaggg 15 <210> 516 <211> 14 <212> DNA
<213> mammalian <400> 516 agctgagatg gagg 14 <210> 517 <211> 13 <212> DNA
<213> mammalian <400> 517 agctgagatg gag 13 <210> 518 <211> 12 <212> DNA
<213> mammalian <400> 518 agctgagatg ga 12 <210> 519 <211> 11 <212> DNA
<213> mammalian <400> 519 agctgagatg g 11 <210> 520 <211> 10 <212> DNA
<213> mammalian <400> 520 agctgagatg 10 <210> 521 <211> 38 <212> DNA
<213> mammalian <400> 521 gctgagatgg agggcggcat ggcgggcaca ggctgggc 38 <210> 522 <211> 37 <212> DNA
<213> mammalian <400> 522 gctgagatgg agggcggcat ggcgggcaca ggctggg 37 <210> 523 <211> 36 <212> DNA
<213> mammalian <400> 523 gctgagatgg agggcggcat ggcgggcaca ggctgg 36 <210> 524 <211> 35 <212> DNA
<213> mammalian <400> 524 gctgagatgg agggcggcat ggcgggcaca ggctg 35 <210> 525 <211> 34 <212> DNA
<213> mammalian <400> 525 gctgagatgg agggcggcat ggcgggcaca ggct 34 <210> 526 <211> 33 <212> DNA
<213> mammalian <400> 526 gctgagatgg agggcggcat ggcgggcaca ggc 33 <210> 527 <211> 32 <212> DNA
<213> mammalian <400> 527 gctgagatgg agggcggcat ggcgggcaca gg 32 <210> 528 <211> 31 <212> DNA
<213> mammalian <400> 528 gctgagatgg agggcggcat ggcgggcaca g 31 <210> 529 <211> 30 <212> DNA
<213> mammalian <400> 529 gctgagatgg agggcggcat ggcgggcaca 30 <210> 530 <211> 29 <212> DNA
<213> mammalian <400> 530 gctgagatgg agggcggcat ggcgggcac 29 <210> 531 <211> 28 <212> DNA
<213> mammalian <400> 531 gctgagatgg agggcggcat ggcgggca 28 <210> 532 <211> 27 <212> DNA
<213> mammalian <400> 532 gctgagatgg agggcggcat ggcgggc 27 <210> 533 <211> 26 <212> DNA
<213> mammalian <400> 533 gctgagatgg agggcggcat ggcggg 26 <210> 534 <211> 25 <212> DNA
<213> mammalian <400> 534 gctgagatgg agggcggcat ggcgg 25 <210> 535 <211> 24 <212> DNA
<213> mammalian <400> 535 gctgagatgg agggcggcat ggcg 24 <210> 536 <211> 23 <212> DNA
<213> mammalian <400> 536 gctgagatgg agggcggcat ggc 23 <210> 537 <211> 22 <212> DNA
<213> mammalian <400> 537 gctgagatgg agggcggcat gg 22 <210> 538 <211> 21 <212> DNA
<213> mammalian <400> 538 gctgagatgg agggcggcat g 21 <210> 539 <211> 20 <212> DNA
<213> mammalian <400> 539 gctgagatgg agggcggcat 20 <210> 540 <211> 19 <212> DNA
<213> mammalian <400> 540 gctgagatgg agggcggca 19 <210> 541 <211> 18 <212> DNA
<213> mammalian <400> 541 gctgagatgg agggcggc 18 <210> 542 <211> 17 <212> DNA
<213> mammalian <400> 542 gctgagatgg agggcgg 1~
<210> 543 <211> 16 <212> DNA
<213> mammalian <400> 543 gctgagatgg agggcg 16 <210> 544 <211> 15 <212> DNA
<213> mammalian <400> 544 gctgagatgg agggc 15 <210> 545 <211> 14 <212> DNA
<213> mammalian <400> 545 gctgagatgg aggg 14 <210> 546 <211> 13 <212> DNA
<213> mammalian <400> 546 gctgagatgg agg 13 <210> 547 <211> 12 <212> DNA
<213> mammalian <400> 547 gctgagatgg ag 12 <210> 548 <211> 11 <212> DNA
<213> mammalian <400> 548 gctgagatgg a 11 <210> 549 <211> 10 <212> DNA
<213> mammalian <400> 549 gctgagatgg 10 <210> 550 <211> 37 <212> DNA
<213> mammalian <400> 550 ctgagatgga gggcggcatg gcgggcacag gctgggc 37 <210> 551 <211> 36 <212> DNA
<213> mammalian <400> 551 ctgagatgga gggcggcatg gcgggcacag gctggg 36 <210> 552 <211> 35 <212> DNA
<213> mammalian <400> 552 ctgagatgga gggcggcatg gcgggcacag gctgg 35 <210> 553 <211> 34 <212> DNA
<213> mammalian <400> 553 ctgagatgga gggcggcatg gcgggcacag gctg 34 <210> 554 <211> 33 <212> DNA
<213> mammalian <400> 554 ctgagatgga gggcggcatg gcgggcacag get 33 <210> 555 <211> 32 <212> DNA
<213> mammalian <400> 555 ctgagatgga gggcggcatg gcgggcacag gc 32 <210> 556 <211> 31 <212> DNA
<213> mammalian <400> 556 ctgagatgga gggcggcatg gcgggcacag g 31 <210> 557 <211> 30 <212> DNA
<213> mammalian <400> 557 ctgagatgga gggcggcatg gcgggcacag 30 <210> 558 <211> 29 <212> DNA
<213> mammalian <400> 558 ctgagatgga gggcggcatg gcgggcaca 29 <210> 559 <211> 28 <212> DNA
<213> mammalian <400> 559 ctgagatgga gggcggcatg gcgggcac 28 <210> 560 <211> 27 <212> DNA
<213> mammalian <400> 560 ctgagatgga gggcggcatg gcgggca 27 <210> 561 <211> 26 <212> DNA
<213> mammalian <400> 561 ctgagatgga gggcggcatg gcgggc 26 <210> 562 <211> 25 <212> DNA
<213> mammalian <400> 562 ctgagatgga gggcggcatg gcggg 25 <210> 563 <211> 24 <212> DNA
<213> mammalian <400> 563 ctgagatgga gggcggcatg gcgg 24 <210> 564 <211> 23 <212> DNA
<213> mammalian <400> 564 ctgagatgga gggcggcatg gcg 23 <210> 565 <211> 22 <212> DNA
<213> mammalian <400> 565 ctgagatgga gggcggcatg gc 22 <210> 566 <211> 21 <212> DNA
<213> mammalian <400> 566 ctgagatgga gggcggcatg g 21 <210> 567 <211> 20 <212> DNA
<213> mammalian <400> 567 ctgagatgga gggcggcatg 20 <210> 568 <211> 19 <212> DNA
<213> mammalian <400> 568 ctgagatgga gggcggcat 19 <210> 569 <211> 18 <212> DNA
<213> mammalian <400> 569 ctgagatgga gggcggca 18 <210> 570 <211> 17 <212> DNA
<213> mammalian <400> 570 ctgagatgga gggcggc 17 <210> 571 <211> 16 <212> DNA
<213> mammalian <400> 571 ctgagatgga gggcgg 16 <210> 572 <211> 15 <212> DNA
<213> mammalian <400> 572 ctgagatgga gggcg 15 <210> 573 <211> 14 <212> DNA
<213> mammalian <400> 573 ctgagatgga gggc 14 <210> 574 <211> 13 <212> DNA
<213> mammalian <400> 574 ctgagatgga ggg 13 <210> 575 <211> 12 <212> DNA
<213> mammalian <400> 575 ctgagatgga gg 12 <210> 576 <211> 11 <212> DNA
<213> mammalian <400> 576 ctgagatgga g 11 <210> 577 <211> 10 <212> DNA
<213> mammalian <400> 577 ctgagatgga 10 <210> 578 <211> 36 <212> DNA
<213> mammalian <400> 578 tgagatggag ggcggcatgg cgggcacagg ctgggc 36 <210> 579 <211> 35 <212> DNA
<213> mammalian <400> 579 tgagatggag ggcggcatgg cgggcacagg ctggg 35 <210> 580 <211> 34 <212> DNA
<213> mammalian <400> 580 tgagatggag ggcggcatgg cgggcacagg ctgg 34 <210> 581 <211> 33 <212> DNA
<213> mammalian <400> 581 tgagatggag ggcggcatgg cgggcacagg ctg 33 <210> 582 <211> 32 <212> DNA
<213> mammalian <400> 582 tgagatggag ggcggcatgg cgggcacagg ct 32 <210> 583 <211> 31 <212> DNA
<213> mammalian <400> 583 tgagatggag ggcggcatgg cgggcacagg c 31 <210> 584 <211> 30 <212> DNA
<213> mammalian <400> 584 tgagatggag ggcggcatgg cgggcacagg 30 <210> 585 <211> 29 <212> DNA
<213> mammalian <400> 585 tgagatggag ggcggcatgg cgggcacag 29 <210> 586 <211> 28 <212> DNA
<213> mammalian <400> 586 tgagatggag ggcggcatgg cgggcaca 2g <210> 587 <211> 27 <212> DNA
<213> mammalian <400> 587 .
tgagatggag ggcggcatgg cgggcac 27 <210> 588 <211> 26 <212> DNA
<213> mammalian <400> 588 tgagatggag ggcggcatgg cgggca 26 <210> 589 <211> 25 <212> DNA
<213> mammalian <400> 589 tgagatggag ggcggcatgg cgggc 25 <210> 590 <211> 24 <212> DNA
<213> mammalian <400> 590 tgagatggag ggcggcatgg cggg 24 <210> 591 <211> 23 <212> DNA
<213> mammalian <400> 591 tgagatggag ggcggcatgg cgg 23 <210> 592 <211> 22 <212> DNA
<213> mammalian <400> 592 tgagatggag ggcggcatgg cg 22 <210> 593 <211> 21 <212> DNA
<213> mammalian <400> 593 tgagatggag ggcggcatgg c 21 <210> 594 <211> 20 <212> DNA
<213> mammalian <400> 594 tgagatggag ggcggcatgg 20 <210> 595 <211> 19 <212> DNA
<213> mammalian <400> 595 tgagatggag ggcggcatg 19 <210> 596 <211> 18 <212> DNA
<213> mammalian <400> 596 tgagatggag ggcggcat 18 <210> 597 <211> 17 <212> DNA
<213> mammalian <400> 597 tgagatggag ggcggca 17 <210> 598 <211> 16 <212> DNA
<213> mammalian <400> 598 tgagatggag ggcggc 16 <210> 599 <211> 15 <212> DNA
<213> mammalian <400> 599 tgagatggag ggcgg 15 <210> 600 <211> 14 <212> DNA
<213> mammalian <400> 600 tgagatggag ggcg 14 <210> 601 <211> 13 <212> DNA
<213> mammalian <400> 601 tgagatggag ggc 13 <210> 602 <211> 12 <212> DNA
<213> mammalian <400> 602 tgagatggag gg 12 <210> 603 <211> 11 <212> DNA
<213> mammalian <400> 603 tgagatggag g 11 <210> 604 <211> 10 <212> DNA
<213> mammalian <400> 604 tgagatggag 10 <210> 605 <211> 35 <212> DNA
<213> mammalian <400> 605 gagatggagg gcggcatggc gggcacaggc tgggc 35 <210> 606 <211> 34 <212> DNA
<213> mammalian <400> 606 gagatggagg gcggcatggc gggcacaggc tggg 34 <210> 607 <211> 33 <212> DNA
<213> mammalian <400> 607 gagatggagg gcggcatggc gggcacaggc tgg 33 <210> 608 <211> 32 <212> DNA
<213> mammalian <400> 608 gagatggagg gcggcatggc gggcacaggc tg 32 <210> 609 <211> 31 <212> DNA
<213> mammalian <400> 609 gagatggagg gcggcatggc gggcacaggc t 31 <210> 610 <211> 30 <212> DNA
<213> mammalian <400> 610 gagatggagg gcggcatggc gggcacaggc 30 <210> 611 <211> 29 <212> DNA
<213> mammalian <400> 611 gagatggagg gcggcatggc gggcacagg 29 <210> 612 <211> 28 <212> DNA
<213> mammalian <400> 612 gagatggagg gcggcatggc gggcacag 28 <210> 613 <211> 27 <212> DNA
<213> mammalian <400> 613 gagatggagg gcggcatggc gggcaca 27 <210> 614 <211> 26 <212> DNA
<213> mammalian <400> 614 gagatggagg gcggcatggc gggcac 26 <210> 615 <211> 25 <212> DNA
<213> mammalian <400> 615 gagatggagg gcggcatggc gggca 25 <210> 616 <211> 24 <212> DNA
<213> mammalian <400> 616 gagatggagg gcggcatggc gggc 24 <210> 617 <211> 23 <212> DNA
<213> mammalian <400> 617 gagatggagg gcggcatggc ggg 23 <210> 618 <211> 22 <212> DNA
<213> mammalian <400> 618 gagatggagg gcggcatggc gg 22 <210> 619 <211> 21 <212> DNA
<213> mammalian <400> 619 gagatggagg gcggcatggc g 21 <210> 620 <211> 20 <212> DNA
<213> mammalian <400> 620 gagatggagg gcggcatggc 20 <210> 621 <211> 19 <212> DNA
<213> mammalian <400> 621 gagatggagg gcggcatgg 19 <210> 622 <211> 18 <212> DNA
<213> mammalian <400> 622 gagatggagg gcggcatg 18 <210> 623 <211> 17 <212> DNA
<213> mammalian <400> 623 gagatggagg gcggcat 17 <210> 624 <211> 16 <212> DNA
<213> mammalian <400> 624 gagatggagg gcggca 16 <210> 625 <211> 15 <212> DNA
<213> mammalian <400> 625 gagatggagg gcggc 15 <210> 626 <211> 14 <212> DNA
<213> mammalian <400> 626 gagatggagg gcgg 14 <210> 627 <211> 13 <212> DNA
<213> mammalian <400> 627 gagatggagg gcg 13 <210> 628 <211> 12 <212> DNA
<213> mammalian <400> 628 gagatggagg gc 12 <210> 629 <211> 11 <212> DNA
<213> mammalian <400> 629 gagatggagg g 11 <210> 630 <211> 10 <212> DNA
<213> mammalian <400> 630 gagatggagg 10 <210> 631 <211> 34 <212> DNA
<213> mammalian <400> 631 agatggaggg cggcatggcg ggcacaggct gggc 34 <210> 632 <211> 33 <212> DNA
<213> mammalian <400> 632 agatggaggg cggcatggcg ggcacaggct ggg 33 <210> 633 <211> 32 <212> DNA
<213> mammalian <400> 633 agatggaggg cggcatggcg ggcacaggct gg 32 <210> 634 <211> 31 <212> DNA
<213> mammalian <400> 634 agatggaggg cggcatggcg ggcacaggct g 31 <210> 635 <211> 30 <212> DNA
<213> mammalian <400> 635 agatggaggg cggcatggcg ggcacaggct 30 <210> 636 <211> 29 <212> DNA
<213> mammalian <400> 636 agatggaggg cggcatggcg ggcacaggc 29 <210> 637 <211> 28 <212> DNA
<213> mammalian <400> 637 agatggaggg cggcatggcg ggcacagg 28 <210> 638 <211> 27 <212> DNA
<213> mammalian <400> 638 agatggaggg cggcatggcg ggcacag 2~
<210> 639 <211> 26 <212> DNA
<213> mammalian <400> 639 agatggaggg cggcatggcg ggcaca 26 <210> 640 <211> 25 <212> DNA
<213> mammalian <400> 640 agatggaggg cggcatggcg ggcac 25 <210> 641 <211> 24 <212> DNA
<213> mammalian <400> 641 agatggaggg cggcatggcg ggca 24 <210> 642 <211> 23 <212> DNA
<213> mammalian <400> 642 agatggaggg cggcatggcg ggc 23 <210> 643 <211> 22 <212> DNA
<213> mammalian <400> 643 agatggaggg cggcatggcg gg 22 <210> 644 <211> 21 <212> DNA
<213> mammalian <400> 644 agatggaggg cggcatggcg g 21 <210> 645 <211> 20 <212> DNA
<213> mammalian <400> 645 agatggaggg cggcatggcg 20 <210> 646 <211> 19 <212> DNA
<213> mammalian <400> 646 agatggaggg cggcatggc 1g <210> 647 <211> 18 <212> DNA
<213> mammalian <400> 647 agatggaggg cggcatgg 1g <210> 648 <211> 17 <212> DNA
<213> mammalian <400> 648 agatggaggg cggcatg 17 <210> 649 <211> 16 <212> DNA
<213> mammalian <400> 649 agatggaggg cggcat 16 <210> 650 <211> 15 <212> DNA
<213> mammalian <400> 650 agatggaggg cggca 15 <210> 651 <211> 14 <212> DNA
<213> mammalian <400> 651 agatggaggg cggc 14 <210> 652 <211> 13 <212> DNA
<213> mammalian <400> 652 agatggaggg cgg 13 <210> 653 <211> 12 <212> DNA
<213> mammalian <400> 653 agatggaggg cg 12 <210> 654 <211> 11 <212> DNA
<213> mammalian <400> 654 agatggaggg c 11 <210> 655 <211> 10 <212> DNA
<213> mammalian <400> 655 agatggaggg 10 <210> 656 <211> 33 <212> DNA
<213> mammalian <400> 656 gatggagggc ggcatggcgg gcacaggctg ggc 33 <210> 657 <211> 32 <212> DNA
<213> mammalian <400> 657 gatggagggc ggcatggcgg gcacaggctg gg 32 <210> 658 <211> 31 <212> DNA
<213> mammalian <400> 658 gatggagggc ggcatggcgg gcacaggctg g 31 <210> 659 <211> 30 <212> DNA
<213> mammalian <400> 659 gatggagggc ggcatggcgg gcacaggctg 30 <210> 660 <211> 29 <212> DNA
<213> mammalian <400> 660 gatggagggc ggcatggcgg gcacaggct 29 <210> 661 <211> 28 <212> DNA
<213> mammalian <400> 661 gatggagggc ggcatggcgg gcacaggc 28 <210> 662 <211> 27 <212> DNA
<213> mammalian <400> 662 gatggagggc ggcatggcgg gcacagg 27 <210> 663 <211> 26 <212> DNA
<213> mammalian <400> 663 gatggagggc ggcatggcgg gcacag 26 <210> 664 <211> 25 <212> DNA
<213> mammalian <400> 664 gatggagggc ggcatggcgg gcaca 25 <210> 665 <211> 24 <212> DNA
<213> mammalian <400> 665 gatggagggc ggcatggcgg gcac 24 <210> 666 <211> 23 <212> DNA
<213> mammalian <400> 666 gatggagggc ggcatggcgg gca 23 <210> 667 <211> 22 <212> DNA
<213> mammalian <400> 667 gatggagggc ggcatggcgg gc 22 <210> 668 <211> 21 <212> DNA
<213> mammalian <400> 668 gatggagggc ggcatggcgg g 21 <210> 669 <211> 20 <212> DNA
<213> mammalian <400> 669 gatggagggc ggcatggcgg 20 <210> 670 <211> 19 <212> DNA
<213> mammalian <400> 670 gatggagggc ggcatggcg 19 <210> 671 <211> 18 <212> DNA
<213> mammalian <400> 671 gatggagggc ggcatggc 18 <210> 672 <211> 17 <212> DNA
<213> mammalian <400> 672 gatggagggc ggcatgg 17 <210> 673 <211> 16 <212> DNA
<213> mammalian <400> 673 gatggagggc ggcatg 16 <210> 674 <211> 15 <212> DNA
<213> mammalian <400> 674 gatggagggc ggcat 15 <210> 675 <211> 14 <212> DNA
<213> mammalian <400> 675 gatggagggc ggca 14 <210> 676 <211> 13 <212> DNA
<213> mammalian <400> 676 gatggagggc ggc 13 <210> 677 <211> 12 <212> DNA
<213> mammalian <400> 677 gatggagggc gg 12 <210> 678 <211> 11 <212> DNA
<213> mammalian <400> 678 gatggagggc g 11 <210> 679 <211> 10 <212> DNA
<213> mammalian <400> 679 gatggagggc <210> 680 <211> 32 <212> DNA
<213> mammalian <400> 680 atggagggcg gcatggcggg cacaggctgg gc 32 <210> 681 <211> 31 <212> DNA
<213> mammalian <400> 681 atggagggcg gcatggcggg cacaggctgg g 31 <210> 682 <211> 30 <212> DNA
<213> mammalian <400> 682 atggagggcg gcatggcggg cacaggctgg 30 <210> 683 <211> 29 <212> DNA
<213> mammalian <400> 683 atggagggcg gcatggcggg cacaggctg 29 <210> 684 <211> 28 <212> DNA
<213> mammalian <400> 684 atggagggcg gcatggcggg cacaggct 2g <210> 685 <211> 27 <212> DNA
<213> mammalian <400> 685 atggagggcg gcatggcggg cacaggc 27 <210> 686 <211> 26 <212> DNA
<213> mammalian <400> 686 atggagggcg gcatggcggg cacagg 26 <210> 687 <211> 25 <212> DNA
<213> mammalian <400> 687 atggagggcg gcatggcggg cacag 25 <210> 688 <211> 24 <212> DNA
<213> mammalian <400> 688 atggagggcg gcatggcggg caca 24 <210> 689 <211> 23 <212> DNA
<213> mammalian <400> 689 atggagggcg gcatggcggg cac 23 <210> 690 <211> 22 <212> DNA
<213> mammalian <400> 690 atggagggcg gcatggcggg ca 22 <210> 691 <211> 21 <212> DNA
<213> mammalian <400> 691 atggagggcg gcatggcggg c 21 <210> 692 <211> 20 <212> DNA
<213> mammalian <400> 692 atggagggcg gcatggcggg 20 <210> 693 <211> 19 <212> DNA
<213> mammalian <400> 693 atggagggcg gcatggcgg 19 <210> 694 <211> 18 <212> DNA
<213> mammalian <400> 694 atggagggcg gcatggcg 18 <210> 695 <211> 17 <212> DNA
<213> mammalian <400> 695 atggagggcg gcatggc 17 <210> 696 <211> 16 <212> DNA
<213> mammalian <400> 696 atggagggcg gcatgg 16 <210> 697 <211> 15 <212> DNA
<213> mammalian <400> 697 atggagggcg gcatg 15 <210> 698 <211> 14 <212> DNA
<213> mammalian <400> 698 atggagggcg gcat 14 <210> 699 <211> 13 <212> DNA
<213> mammalian <400> 699 atggagggcg gca 13 <210> 700 <211> 12 <212> DNA
<213> mammalian <400> 700 atggagggcg gc 12 <210> 701 <211> 11 <212> DNA
<213> mammalian <400> 701 atggagggcg g 11 <210> 702 <211> 10 <212> DNA
<213> mammalian <400> 702 atggagggcg 10 <210> 703 <211> 31 <212> DNA
<213> mammalian <400> 703 tggagggcgg catggcgggc acaggctggg c 31 <210> 704 <211> 30 <212> DNA
<213> mammalian <400> 704 tggagggcgg catggcgggc acaggctggg 30 <210> 705 <211> 29 <212> DNA
<213> mammalian <400> 705 tggagggcgg catggcgggc acaggctgg 29 <210> 706 <211> 28 <212> DNA
<213> mammalian <400> 706 tggagggcgg catggcgggc acaggctg 2g <210> 707 <211> 27 <212> DNA
<213> mammalian <400> 707 tggagggcgg catggcgggc acaggct 27 <210> 708 <211> 26 <212> DNA
<213> mammalian <400> 708 tggagggcgg catggcgggc acaggc 26 <210> 709 <211> 25 <212> DNA
<213> mammalian <400> 709 tggagggcgg catggcgggc acagg 25 <210> 710 <211> 24 <212> DNA
<213> mammalian <400> 710 tggagggcgg catggcgggc acag 24 <210> 711 <211> 23 <212> DNA
<213> mammalian <400> 711 tggagggcgg catggcgggc aca 23 <210> 712 <211> 22 <212> DNA
<213> mammalian <400> 712 tggagggcgg catggcgggc ac 22 <210> 713 <211> 21 <212> DNA
<213> mammalian <400> 713 tggagggcgg catggcgggc a 21 <210> 714 <211> 20 <212> DNA
<213> mammalian <400> 714 tggagggcgg catggcgggc 20 <210> 715 <211> 19 <212> DNA
<213> mammalian <400> 715 tggagggcgg catggcggg 1g <210> 716 <211> 18 <212> DNA
<213> mammalian <400> 716 tggagggcgg catggcgg 1g <210> 717 <211> 17 <212> DNA
<213> mammalian <400> 717 tggagggcgg catggcg 17 <210> 718 <211> 16 <212> DNA
<213> mammalian <400> 718 tggagggcgg catggc 16 <210> 719 <211> 15 <212> DNA
<213> mammalian <400> 719 tggagggcgg catgg <210> 720 <211> 14 <212> DNA
<213> mammalian <400> 720 tggagggcgg catg 14 <210> 721 <211> 13 <212> DNA
<213> mammalian <400> 721 tggagggcgg cat <210> 722 <211> 12 <212> DNA
<213> mammalian <400> 722 tggagggcgg ca <210> 723 <211> 11 <212> DNA
<213> mammalian <400> 723 tggagggcgg c 11 <210> 724 <211> 10 <212> DNA
<213> mammalian <400> 724 tggagggcgg 10 <210> 725 <211> 30 <212> DNA
<213> mammalian <400> 725 ggagggcggc atggcgggca caggctgggc 30 <210> 726 <211> 29 <212> DNA
<213> mammalian <400> 726 ggagggcggc atggcgggca caggctggg 29 <210> 727 <211> 28 <212> DNA
<213> mammalian <400> 727 ggagggcggc atggcgggca caggctgg 2g <210> 728 <211> 27 <212> DNA
<213> mammalian <400> 728 ggagggcggc atggcgggca caggctg 27 <210> 729 <211> 26 <212> DNA
<213> mammalian <400> 729 ggagggcggc atggcgggca caggct 26 <210> 730 <211> 25 <212> DNA
<213> mammalian <400> 730 ggagggcggc atggcgggca caggc 25 <210> 731 <211> 24 <212> DNA
<213> mammalian <400> 731 ggagggcggc atggcgggca cagg 24 <210> 732 <211> 23 <212> DNA
<213> mammalian <400> 732 ggagggcggc atggcgggca cag 23 <210> 733 <211> 22 <212> DNA
<213> mammalian <400> 733 ggagggcggc atggcgggca ca 22 <210> 734 <211> 21 <212> DNA
<213> mammalian <400> 734 ggagggcggc atggcgggca c 21 <210> 735 <211> 20 <212> DNA
<213> mammalian <400> 735 ggagggcggc atggcgggca 20 <210> 736 <211> 19 <212> DNA
<213> mammalian <400> 736 ggagggcggc atggcgggc 1g <210> 737 <211> 18 <212> DNA
<213> mammalian <400> 737 ggagggcggc atggcggg 1g <210> 738 <211> 17 <212> DNA
<213> mammalian <400> 738 ggagggcggc atggcgg 17 <210> 739 <211> 16 <212> DNA
<213> mammalian <400> 739 ggagggcggc atggcg 16 <210> 740 <211> 15 <212> DNA
<213> mammalian <400> 740 ggagggcggc atggc 15 <210> 741 <211> 14 <212> DNA
<213> mammalian <400> 741 ggagggcggc atgg 14 <210> 742 <211> 13 <212> DNA
<213> mammalian <400> 742 ggagggcggc atg 13 <210> 743 <211> 12 <212> DNA
<213> mammalian <400> 743 ggagggcggc at 12 <210> 744 <211> 11 <212> DNA
<213> mammalian <400> 744 ggagggcggc a 11 <210> 745 <211> 10 <212> DNA
<213> mammalian <400> 745 ggagggcggc 10 <210> 746 <211> 29 <212> DNA
<213> mammalian <400> 746 gagggcggca tggcgggcac aggctgggc 29 <210> 747 <211> 28 <212> DNA
<213> mammalian <400> 747 gagggcggca tggcgggcac aggctggg 28 <210> 748 <211> 27 <212> DNA
<213> mammalian <400> 748 gagggcggca tggcgggcac aggctgg 27 <210> 749 <211> 26 <212> DNA
<213> mammalian <400> 749 gagggcggca tggcgggcac aggctg 26 <210> 750 <211> 25 <212> DNA
<213> mammalian <400> 750 gagggcggca tggcgggcac aggct 25 <210> 751 <211> 24 <212> DNA
<213> mammalian <400> 751 gagggcggca tggcgggcac aggc 24 <210> 752 <211> 23 <212> DNA
<213> mammalian <400> 752 gagggcggca tggcgggcac agg 23 <210> 753 <211> 22 <212> DNA
<213> mammalian <400> 753 gagggcggca tggcgggcac ag 22 <210> 754 <211> 21 <212> DNA
<213> mammalian <400> 754 gagggcggca tggcgggcac a 21 <210> 755 <211> 20 <212> DNA
<213> mammalian <400> 755 gagggcggca tggcgggcac 20 <210> 756 <211> 19 <212> DNA
<213> mammalian <400> 756 gagggcggca tggcgggca 19 <210> 757 <211> 18 <212> DNA
<213> mammalian <400> 757 gagggcggca tggcgggc 1g <210> 758 <211> 17 <212> DNA
<213> mammalian <400> 758 gagggcggca tggcggg 17 <210> 759 <211> 16 <212> DNA
<213> mammalian <400> 759 gagggcggca tggcgg 16 <210> 760 <211> 15 <212> DNA
<213> mammalian <400> 760 gagggcggca tggcg 15 <210> 761 <211> 14 <212> DNA
<213> mammalian <400> 761 gagggcggca tggc 14 <210> 762 <211> 13 <212> DNA
<213> mammalian <400> 762 gagggcggca tgg 13 <210> 763 <211> 12 <212> DNA
<213> mammalian <400> 763 gagggcggca tg 12 <210> 764 <211> 11 <212> DNA
<213> mammalian <400> 764 gagggcggca t 11 <210> 765 <211> 10 <212> DNA
<213> mammalian <400> 765 gagggcggca 10 <210> 766 <211> 28 <212> DNA
<213> mammalian <400> 766 agggcggcat ggcgggcaca ggctgggc 28 <210> 767 <211> 27 <212> DNA
<213> mammalian <400> 767 agggcggcat ggcgggcaca ggctggg 27 <210> 768 <211> 26 <212> DNA
<213> mammalian <400> 768 agggcggcat ggcgggcaca ggctgg 26 <210> 769 <211> 25 <212> DNA
<213> mammalian <400> 769 agggcggcat ggcgggcaca ggctg 25 <210> 770 <211> 24 <212> DNA
<213> mammalian <400> 770 agggcggcat ggcgggcaca ggct 24 <210> 771 <211> 23 <212> DNA
<213> mammalian <400> 771 agggcggcat ggcgggcaca ggc 23 <210> 772 <211> 22 <212> DNA
<213> mammalian <400> 772 agggcggcat ggcgggcaca gg 22 <210> 773 <211> 21 <212> DNA
<213> mammalian <400> 773 agggcggcat ggcgggcaca g 21 <210> 774 <211> 20 <212> DNA
<213> mammalian <400> 774 agggcggcat ggcgggcaca 20 <210> 775 <211> 19 <212> DNA
<213> mammalian <400> 775 agggcggcat ggcgggcac 19 <210> 776 <211> 18 <212> DNA
<213> mammalian <400> 776 agggcggcat ggcgggca 1g <210> 777 <211> 17 <212> DNA
<213> mammalian <400> 777 agggcggcat ggcgggc 17 <210> 778 <211> 16 <212> DNA
<213> mammalian <400> 778 agggcggcat ggcggg 16 <210> 779 <211> 15 <212> DNA
<213> mammalian <400> 779 agggcggcat ggcgg 15 <210> 780 <211> 14 <212> DNA
<213> mammalian <400> 780 agggcggcat ggcg 14 <210> 781 <211> 13 <212> DNA
<213> mammalian <400> 781 agggcggcat ggc 13 <210> 782 <211> 12 <212> DNA
<213> mammalian <400> 782 agggcggcat gg 12 <210> 783 <211> 11 <212> DNA
<213> mammalian <400> 783 agggcggcat g 11 <210> 784 <211> 10 <212> DNA
<213> mammalian <400> 784 agggcggcat 10 <210> 785 <211> 27 <212> DNA
<213> mammalian <400> 785 gggcggcatg gcgggcacag gctgggc 27 <210> 786 <211> 26 <212> DNA
<213> mammalian <400> 786 gggcggcatg gcgggcacag gctggg 26 <210> 787 <211> 25 <212> DNA
<213> mammalian <400> 787 gggcggcatg gcgggcacag gctgg 25 <210> 788 <211> 24 <212> DNA
<213> mammalian <400> 788 gggcggcatg gcgggcacag gctg 24 <210> 789 <211> 23 <212> DNA
<213> mammalian <400> 789 gggcggcatg gcgggcacag get 23 <210> 790 <211> 22 <212> DNA
<213> mammalian <400> 790 gggcggcatg gcgggcacag gc 22 <210> 791 <211> 21 <212> DNA
<213> mammalian <400> 791 gggcggcatg gcgggcacag g 21 <210> 792 <211> 20 <212> DNA
<213> mammalian <400> 792 gggcggcatg gcgggcacag 20 <210> 793 <211> 19 <212> DNA
<213> mammalian <400> 793 gggcggcatg gcgggcaca 19 <210> 794 <211> 18 <212> DNA
<213> mammalian <400> 794 gggcggcatg gcgggcac 18 <210> 795 <211> 17 <212> DNA
<213> mammalian <400> 795 gggcggcatg gcgggca 17 <210> 796 <211> 16 <212> DNA
<213> mammalian <400> 796 gggcggcatg gcgggc 16 <210> 797 <211> 15 <212> DNA
<213> mammalian <400> 797 gggcggcatg gcggg 15 <210> 798 <211> 14 <212> DNA
<213> mammalian <400> 798 gggcggcatg gcgg 14 <210> 799 <211> 13 <212> DNA
<213> mammalian <400> 799 gggcggcatg gcg 13 <210> 800 <211> 12 <212> DNA
<213> mammalian <400> 800 gggcggcatg gc 12 <210> 801 <211> 11 <212> DNA
<213> mammalian <400> 801 gggcggcatg g 11 <210> 802 <211> 10 <212> DNA
<213> mammalian <400> 802 gggcggcatg 10 <210> 803 <211> 26 <212> DNA
<213> mammalian <400> 803 ggcggcatgg cgggcacagg ctgggc 26 <210> 804 <211> 25 <212> DNA
<213> mammalian <400> 804 ggcggcatgg cgggcacagg ctggg 25 <210> 805 <211> 24 <212> DNA
<213> mammalian <400> 805 ggcggcatgg cgggcacagg ctgg 24 <210> 806 <211> 23 <212> DNA
<213> mammalian <400> 806 ggcggcatgg cgggcacagg ctg 23 <210> 807 <211> 22 <212> DNA
<213> mammalian <400> 807 ggcggcatgg cgggcacagg ct 22 <210> 808 <211> 21 <212> DNA
<213> mammalian <400> 808 ggcggcatgg cgggcacagg c 21 <210> 809 <211> 20 <212> DNA
<213> mammalian <400> 809 ggcggcatgg cgggcacagg 20 <210> 810 <211> 19 <212> DNA
<213> mammalian <400> 810 ggcggcatgg cgggcacag 19 <210> 811 <211> 18 <212> DNA
<213> mammalian <400> 811 ggcggcatgg cgggcaca 18 <210> 812 <211> 17 <212> DNA
<213> mammalian <400> 812 ggcggcatgg cgggcac 17 <210> 813 <211> 16 <212> DNA
<213> mammalian <400> 813 ggcggcatgg cgggca 16 <210> 814 <211> 15 <212> DNA
<213> mammalian <400> 814 ggcggcatgg cgggc 15 <210> 815 <211> 14 <212> DNA
<213> mammalian <400> 815 ggcggcatgg cggg 14 <210> 816 <211> 13 <212> DNA
<213> mammalian <400> 816 ggcggcatgg cgg 13 <210> 817 <211> 12 <212> DNA
<213> mammalian <400> 817 ggcggcatgg cg 12 <210> 818 <211> 11 <212> DNA
<213> mammalian <400> 818 ggcggcatgg c 11 <210> 819 <211> 10 <212> DNA
<213> mammalian <400> 819 ggcggcatgg 10 <210> 820 <211> 25 <212> DNA
<213> mammalian <400> 820 gcggcatggc gggcacaggc tgggc 25 <210> 821 <211> 24 <212> DNA
<213> mammalian <400> 821 gcggcatggc gggcacaggc tggg 24 <210> 822 <211> 23 <212> DNA
<213> mammalian <400> 822 gcggcatggc gggcacaggc tgg 23 <210> 823 <211> 22 <212> DNA
<213> mammalian <400> 823 gcggcatggc gggcacaggc tg 22 <210> 824 <211> 21 <212> DNA
<213> mammalian <400> 824 gcggcatggc gggcacaggc t 21 <210> 825 <211> 20 <212> DNA
<213> mammalian <400> 825 gcggcatggc gggcacaggc 20 <210> 826 <211> 19 <212> DNA
<213> mammalian <400> 826 gcggcatggc gggcacagg 19 <210> 827 <211> 18 <212> DNA
<213> mammalian <400> 827 gcggcatggc gggcacag 18 <210> 828 <211> 17 <212> DNA
<213> mammalian <400> 828 gcggcatggc gggcaca 17 <210> 829 <211> 16 <212> DNA
<213> mammalian <400> 829 gcggcatggc gggcac 16 <210> 830 <211> 15 <212> DNA
<213> mammalian <400> 830 gcggcatggc gggca 15 <210> 831 <211> 14 <212> DNA
<213> mammalian <400> 831 gcggcatggc gggc 14 <210> 832 <211> 13 <212> DNA
<213> mammalian <400> 832 gcggcatggc ggg 13 <210> 833 <211> 12 <212> DNA
<213> mammalian <400> 833 gcggcatggc gg 12 <210> 834 <211> 11 <212> DNA
<213> mammalian <400> 834 gcggcatggc g 11 <210> 835 <211> 10 <212> DNA
<213> mammalian <400> 835 gcggcatggc 10 <210> 836 <211> 24 <212> DNA
<213> mammalian <400> 836 cggcatggcg ggcacaggct gggc 24 <210> 837 <211> 23 <212> DNA
<213> mammalian <400> 837 cggcatggcg ggcacaggct ggg 23 <210> 838 <211> 22 <212> DNA
<213> mammalian <400> 838 cggcatggcg ggcacaggct gg 22 <210> 839 <211> 21 <212> DNA
<213> mammalian <400> 839 cggcatggcg ggcacaggct g 21 <210> 840 <211> 20 <212> DNA
<213> mammalian <400> 840 cggcatggcg ggcacaggct 20 <210> 841 <211> 19 <212> DNA
<213> mammalian <400> 841 cggcatggcg ggcacaggc 19 <210> 842 <211> 18 <212> DNA
<213> mammalian <400> 842 cggcatggcg ggcacagg 18 <210> 843 <211> 17 <212> DNA
<213> mammalian <400> 843 cggcatggcg ggcacag 17 <210> 844 <211> 16 <212> DNA
<213> mammalian <400> 844 cggcatggcg ggcaca 16 <210> 845 <211> 15 <212> DNA
<213> mammalian <400> 845 cggcatggcg ggcac 15 <210> 846 <211> 14 <212> DNA
<213> mammalian <400> 846 cggcatggcg ggca 14 <210> 847 <211> 13 <212> DNA
<213> mammalian <400> 847 cggcatggcg ggc 13 <210> 848 <211> 12 <212> DNA
<213> mammalian <400> 848 cggcatggcg gg 12 <210> 849 <211> 11 <212> DNA
<213> mammalian <400> 849 cggcatggcg g 11 <210> 850 <211> 10 <212> DNA
<213> mammalian <400> 850 cggcatggcg 10 <210> 851 <211> 23 <212> DNA
<213> mammalian <400> 851 ggcatggcgg gcacaggctg ggc 23 <210> 852 <211> 22 <212> DNA
<213> mammalian <400> 852 ggcatggcgg gcacaggctg gg 22 <210> 853 <211> 21 <212> DNA
<213> mammalian <400> 853 ggcatggcgg gcacaggctg g 21 <210> 854 <211> 20 <212> DNA
<213> mammalian <400> 854 ggcatggcgg gcacaggctg 20 <210> 855 <211> 19 <212> DNA
<213> mammalian <400> 855 ggcatggcgg gcacaggct 19 <210> 856 <211> 18 <212> DNA
<213> mammalian <400> 856 ggcatggcgg gcacaggc 18 <210> 857 <211> 17 <212> DNA
<213> mammalian <400> 857 ggcatggcgg gcacagg 17 <210> 858 <211> 16 <212> DNA
<213> mammalian <400> 858 ggcatggcgg gcacag 16 <210> 859 <211> 15 <212> DNA
<213> mammalian <400> 859 ggcatggcgg gcaca 15 <210> 860 <211> 14 <212> DNA
<213> mammalian <400> 860 ggcatggcgg gcac 14 <210> 861 <211> 13 <212> DNA
<213> mammalian <400> 861 ggcatggcgg gca 13 <210> 862 <211> 12 <212> DNA
<213> mammalian <400> 862 ggcatggcgg gc 12 <210> 863 <211> 11 <212> DNA
<213> mammalian <400> 863 ggcatggcgg g 11 <210> 864 <211> 10 <212> DNA
<213> mammalian <400> 864 ggcatggcgg 10 <210> 865 <211> 22 <212> DNA
<213> mammalian <400> 865 gcatggcggg cacaggctgg gc 22 <210> 866 <211> 21 <212> DNA
<213> mammalian <400> 866 gcatggcggg cacaggctgg g 21 <210> 867 <211> 20 <212> DNA
<213> mammalian <400> 867 gcatggcggg cacaggctgg 20 <210> 868 <211> 19 <212> DNA
<213> mammalian <400> 868 gcatggcggg cacaggctg 19 <210> 869 <211> 18 <212> DNA
<213> mammalian <400> 869 gcatggcggg cacaggct 1g <210> 870 <211> 17 <212> DNA
<213> mammalian <400> 870 gcatggcggg cacaggc 17 <210> 871 <211> 16 <212> DNA
<213> mammalian <400> 871 gcatggcggg cacagg 16 <210> 872 <211> 15 <212> DNA
<213> mammalian <400> 872 gcatggcggg cacag 15 <210> 873 <211> 14 <212> DNA
<213> mammalian <400> 873 gcatggcggg caca 14 <210> 874 <211> 13 <212> DNA
<213> mammalian <400> 874 gcatggcggg cac 13 <210> 875 <211> 12 <212> DNA
<213> mammalian <400> 875 gcatggcggg ca 12 <210> 876 <211> 11 <212> DNA
<213> mammalian <400> 876 gcatggcggg c 11 <210> 877 <211> 10 <212> DNA
<213> mammalian <400> 877 gcatggcggg 10 <210> 878 <211> 21 <212> DNA
<213> mammalian <400> 878 catggcgggc acaggctggg c 21 <210> 879 <211> 20 <212> DNA
<213> mammalian <400> 879 catggcgggc acaggctggg 20 <210> 880 <211> 19 <212> DNA
<213> mammalian <400> 880 catggcgggc acaggctgg 19 <210> 881 <211> 18 <212> DNA
<213> mammalian <400> 881 catggcgggc acaggctg 18 <210> 882 <211> 17 <212> DNA
<213> mammalian <400> 882 catggcgggc acaggct 17 <210> 883 <211> 16 <212> DNA
<213> mammalian <400> 883 catggcgggc acaggc 16 <210> 884 <211> 15 <212> DNA
<213> mammalian <400> 884 catggcgggc acagg 15 <210> 885 <211> 14 <212> DNA
<213> mammalian <400> 885 catggcgggc acag 14 <210> 886 <211> 13 <212> DNA
<213> mammalian <400> 886 catggcgggc aca 13 <210> 887 <211> 12 <212> DNA
<213> mammalian <400> 887 catggcgggc ac 12 <210> 888 <211> 11 <212> DNA
<213> mammalian <400> 888 catggcgggc a 11 <210> 889 <211> 10 <212> DNA
<213> mammalian <400> 889 catggcgggc 10 <210> 890 <211> 20 <212> DNA
<213> mammalian <400> 890 atggcgggca caggctgggc 20 <210> 891 <211> 19 <212> DNA
<213> mammalian <400> 891 atggcgggca caggctggg 19 <210> 892 <211> 18 <212> DNA
<213> mammalian <400> 892 atggcgggca caggctgg 1g <210> 893 <211> 17 <212> DNA
<213> mammalian <400> 893 atggcgggca caggctg 17 <210> 894 <211> 16 <212> DNA
<213> mammalian <400> 894 atggcgggca caggct 16 <210> 895 <211> 15 <212> DNA
<213> mammalian <400> 895 atggcgggca caggc 15 <210> 896 <211> 14 <212> DNA
<213> mammalian <400> 896 atggcgggca cagg 14 <210> 897 <211> 13 <212> DNA
<213> mammalian <400> 897 atggcgggca cag 13 <210> 898 <211> 12 <212> DNA
<213> mammalian <400> 898 atggcgggca ca 12 <210> 899 <211> 11 <212> DNA
<213> mammalian <400> 899 atggcgggca c <210> 900 <211> 10 <212> DNA
<213> mammalian <400> 900 atggcgggca 10 <210> 901 <211> 19 <212> DNA
<213> mammalian <400> 901 tggcgggcac aggctgggc 19 <210> 902 <211> 18 <212> DNA
<213> mammalian <400> 902 tggcgggcac aggctggg 1g <210> 903 <211> 17 <212> DNA
<213> mammalian <400> 903 tggcgggcac aggctgg 17 <210> 904 <211> 16 <212> DNA
<213> mammalian <400> 904 tggcgggcac aggctg 16 <210> 905 <211> 15 <212> DNA
<213> mammalian <400> 905 tggcgggcac aggct 15 <210> 906 <211> 14 <212> DNA
<213> mammalian <400> 906 tggcgggcac aggc 14 <210> 907 <211> 13 <212> DNA
<213> mammalian <400> 907 tggcgggcac agg 13 <210> 908 <211> 12 <212> DNA
<213> mammalian <400> 908 tggcgggcac ag 12 <210> 909 <211> 11 <212> DNA
<213> mammalian <400> 909 tggcgggcac a <210> 910 <211> 10 <212> DNA
<213> mammalian <400> 910 tggcgggcac <210> 911 <211> 18 <212> DNA
<213> mammalian <400> 911 ggcgggcaca ggctgggc 18 <210> 912 <211> 17 <212> DNA
<213> mammalian <400> 912 ggcgggcaca ggctggg 17 <210> 913 <211> 16 <212> DNA
<213> mammalian <400> 913 ggcgggcaca ggctgg 16 <210> 914 <211> 15 <212> DNA
<213> mammalian <400> 914 ggcgggcaca ggctg <210> 915 <211> 14 <212> DNA
<213> mammalian <400> 915 ggcgggcaca ggct 14 <210> 916 <211> 13 <212> DNA
<213> mammalian <400> 916 ggcgggcaca ggc 13 <210> 917 <211> 12 <212> DNA
<213> mammalian <400> 917 ggcgggcaca gg 12 <210> 918 <211> 11 <212> DNA
<213> mammalian <400> 918 ggcgggcaca g 11 DEMANDES OU BREVETS VOLUMINEUX
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COMPREND PLUS D'UN TOME.

NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
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Claims (91)

WHAT IS CLAIMED AS NOVEL & UNOBVIOUS
IN UNITED STATES LETTERS PATENT IS:

1. A pharmaceutical composition, comprising an oligonucleotide(s) (oligo(s)) which is (are) effective for alleviating bronchoconstriction and/or lung inflammation, allergy(ies), or surfactant depletion or hyposecretion, when administered to a mammal, the oligo containing about 0 to about 15% adenosine (A) and being anti-sense to a target selected from the group consisting of the initiation codon, the coding region, the 5'-end and the 3'-end genomic flanking regions, the 5' and 3' intron-exon junctions, and regions within 2 to 10 nucleotides of the junctions of a gene encoding a target polypeptide associated with lung airway dysfunction or anti-sense to the polypeptide mRNA; combinations of the oligos; and mixtures of the oligos; and a pharmaceutically or veterinarily acceptable carrier or diluent.

2. The composition of claim 1, wherein the oligo is A-free.

3. The composition of claim 1, wherein the target is selected from the group consisting of the initiation codon, the coding region, the 5'-end and the 3'-end genomic flanking regions, the 5' and 3' intron-exon junctions, and regions within 2 to 10 nucleotides of the junctions of an oncogene(s) and a gene(s) encoding a target polypeptide(s) associated with lung airway dysfunction or anti-sense to the oncogene mRNA and the polypeptide mRNA; combinations of the oligos; and mixtures of the oligos; the polypeptides being selected from the group consisting of peptide factors and transmitters, antibodies, cytokines and chemokines, enzymes, binding proteins, adhesion molecules, their receptors, and malignancy associated proteins.

4. The composition of claim 3, wherein the target is selected from the group consisting of the initiation codon, the coding region, the 5'-end and the 3'-end genomic flanking regions, the 5' and 3' intron-exon junctions, and regions within 2 to 10 nucleotides of the junctions of an oncogene(s) and a gene(s) encoding a target polypeptide(s) associated with lung airway dysfunction or anti-sense to the oncogene mRNA and the polypeptide mRNA; combinations of the oligos; and mixtures of the oligos; wherein the polypeptides are selected from the group consisting of transcription factors, stimulating and activating peptide factors, cytokines, cytokine receptors, chemokines, chemokine receptors, adenosine receptors, bradykinin receptors, endogenously produced specific and non-specific enzymes, immunoglobulins and antibodies, antibody receptors, central nervous system (CNS) and peripheral nervous and non-nervous system receptors, CNS and peripheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensins, growth factors, vasoactive peptides and receptors, binding proteins, and malignancy associated proteins.

5. The agent of claim 4, wherein the encoded polypeptide(s) is(are) selected from the group consisting of adenosine receptors A1, A2a, A2b and A3, bradykinin receptors B1 and B2, Nf6B
Transcription Factor, Interleukin-8 Receptor (IL-8 R), Interleukin 5 Receptor (IL-5 R), Interleukin 4 Receptor (IL-4 R), Interleukin 3 Receptor (IL-3 R), Interleukin-1 .beta. (IL-1 (3), Interleukin 1 .beta. Receptor (IL- 1.beta. R), Eotaxin, Tryptase, Major Basic Protein, .beta.2-adrenergic Receptor Kinase, Endothelin Receptor A, Endothelin Receptor B, Preproendothelin, Bradykinin B2 Receptor, IgE High Affinity Receptor, Interleukin 1 (IL-1), Interleukin 1 Receptor (IL-1 R), Interleukin 9 (IL-9), Interleukin-9 Receptor (IL-9 R), Interleukin 11 (IL-11), Interleukin-11 Receptor (IL-11 R), Inducible Nitric Oxide Synthase, Cyclo-oxygenase-1 (COX-1), Cyclo-oxygenase-2 (COX-2), Intracellular Adhesion Molecule 1 (1CAM-1) Vascular Cellular Adhesion Molecule (VCAM), Rantes, Endothelial Leukocyte Adhesion Molecule (ELAM-1), Monocyte Activating Factor, Neutrophil Chemotactic Factor, Neutrophil Elastase, Defensin 1, 2 and 3, Muscarinic Acetylcholine Receptors, Platelet Activating Factor, Tumor Necrosis Factor .alpha., 5-lipoxygenase, Phosphodiesterase IV, Substance P, Substance P Receptor, Histamine Receptor, Chymase, CCR-1 CC Chemokine Receptor, CCR-2 CC Chemokine Receptor, CCR-3 CC Chemokine Receptor, CCR-4 CC Chemokine Receptor, CCR-5 CC Chemokine Receptor, Prostanoid Receptors, GATA-3 Transcription Factor, Neutrophil Adherence Receptor, MAP Kinase, Interleukin-9 (IL-9), NEAT Transcription Factors, STAT 4, MIP-1.alpha., MCP-2, MCP-3, MCP-4, Cyclophillins, Phospholipase A2, Basic Fibroblast Growth Factor, Metalloproteinase, CSBP/p38 MAP

Kinase, Tryptose Receptor, PDG2, Interleukin-1.beta. (IL-3), Interleukin-1 (3 (IL-1.beta.), Cyclosporin A-Binding Protein, FK5-Binding Protein, .alpha.4.beta.1 Selectin, Fibronectin, .alpha.4.beta.7 Selectin, Mad CAM-1, LFA-1 (CD11a/CD18), PECAM-1, LFA-1 Selectin, C3bi, PSGL-1, E-Selectin, P-Selectin, CD-34, L-Selectin, p150,95, Mac-1 (CD11b/CD18), Fucosyl transferase, VLA-4, CD-18/CD11a, CD11b/CD18, ICAM2 and 1CAM3, C5a, CCR3 (Eotaxin Receptor), CCR1, CCR2, CCR4, CCR5, LTB-4, Transcription Factor, Protein kinase C, Cysteinyl Leukotriene Receptor, Tachychinnen Receptors (tach R), 16B Kinase 1 & 2, STAT 6, c-mas and NF-Interleukin-6 (NF-IL-6).

6. The composition of claim 1, wherein one or more As is(are) substituted by a universal base selected from the group consisting of heteroaromatic bases which bind to a thymidine base but have antagonist activity and less than about 0.3 of the adenosine base agonist or antagonist activity at the adenosine A1, A2a A2b and A3 receptors.

7. The composition of claim 6, wherein the heteroaromatic bases are selected from the group consisting of pyrimidines and purines, which may be substituted by O, halo, NH2, SH, SO, SO2, SO3, COOH and branched and fused primary and secondary amino, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, which may be further substituted by O, halo, NH2, primary, secondary and tertiary amine, SH, SO, SO2, SO3, cycloalkyl, heterocycloalkyl and heteroaryl.

8. The composition of claim 7, wherein the pyrimidines and purines are substituted at a position selected from the group consisting of positions 1, 2, 3, 4, 7, and 8, and the pyrimidines and purines are selected from the group consisting of theophylline, caffeine, dyphylline, etophylline, acephyliine piperazine, bamifylline, enprofylline and xantine having the chemical formula wherein R1 and R2 are independently H, alkyl, alkenyl or alkynyl and R3 is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloalkynyl, NH2-alkylamino-ketoxyalkyloxy-aryl and mono and dialkylaminoalkyl-N-alkylamino-SO2 aryl.

9. The composition of claim 8, wherein the universal base is selected from the group consisting of 3-nitropyrrole-2'-deoxynucleoside, 5-vitro-indole, 2-deoxyribosyl-(5-nitroindole), 2-deoxyribofuranosyl-(5-nitroindole), 2'-deoxyinosine, 2'-deoxynebularine, 6H, 8H-3,4-dihydropyrimido [4,5-c] oxazine-7-one or 2-amino-6-methoxyaminopurine.

10. The composition of claim 1, where one or more methylated cytocine(s) (m C) is(are) substituted for a C in one or more CpG dinocleotide(s), if present in the oligo(s).

11. The composition of claim 1, wherein one or more mononucleotide(s) of the oligo(s) is(are) linked or modified by one or more methylphosphonate, 5'-N-carbamate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methyimino) (MMI), methoxymethyl (MOM), methoxyethyl (MOE), methyleneoxy (methylimino) (MOMI), 2'-O-methyl, phosphoramidate, C-5 substituted residues, or combinations thereof.

12. The composition of claim 11, wherein the mononucleotide residues are linked by phosphorothioate residues.

13. The composition of claim 1, wherein the anti-sense oligo comprises about 7 to about 60 mononucleotides.

14. The composition of claim 1, wherein the anti-sense olio comprises fragments 1, 3, 5,7 and 8 to 2313 (SEQ.ID NOS: 1 through 2419).

15. The composition of claim 1, wherein the anti-sense oligo is operatively linked to, or complexed with, an agent selected from the group consisting of cell internalized or up-taken agents and cell targeting agents.

16. The composition of claim 15, wherein the cell internalized or up-taken agent is selected from the group consisting of transferrin, asialoglycoprotein and streptavidin.

17. The composition of claim 1, wherein the oligo is operatively linked to a vector that is a prokaryotic or eukaryotic vector.

18. The composition of claim 1, wherein the oligo(s) is(are) hybridized to a ribonucleic acid.

19. A cell, carrying the oligo of claim 1.

20. The composition of claim 1, wherein the carrier or diluent is selected from the group consisting of gaseous, liquid, and solid carriers or diluents.

21. The composition of claim 20, further comprising an agent selected from the group consisting of other therapeutic agents, surfactants, flavoring and coloring agents, fillers, volatile oils, buffering agents, dispersants, RNA inactivating agents, anti-oxidants, flavoring agents, propellants and preservatives.

22. The composition of claim 21, comprising one or more oligo(s), a surfactant, and a carrier or diluent for the oligo and the surfactant.

23. The composition of claim 21, wherein the the agent is an RNA inactivating agent which comprises an enzyme, optionally an ribozyme.

24. The composition of claim 1, wherein the anti-sense oligo is present in an amount of about 0.01 to about 99.99 w/w of the composition.

25. The composition of claim 1, which is a systemic or topical formulation.

26. The formulation of claim 25, selected from the group consisting of oral, intrabuccal, intrapulmonary, rectal, intrauterine, intratunor, intracranial, nasal, intramuscular, subcutaneous, intravascular, intrathecal, inhalable, transdermal, intradermal, intracavitary, implantable, iontophoretic, ocular, vaginal, intraarticular, otical, intravenous, intramuscular, intraglandular, intraorgan, intralymphatic, implantable, slow release and enteric coating formulations.

27. The formulation of claim 26, which is an oral formulation, wherein the carrier is selected from the group consisting of solid and liquid carriers.

28. The oral formulation of claim 27, which is selected from the group consisting of a powder, dragees, tablets, capsules, sprays, aerosols, solutions, suspensions and emulsions, optionally oil-in-water and water-in-oil emulsions.

29. The formulation of claim 25, which is a topical formulation, wherein the carrier is selected from the group consisting of creams, gels, ointments, sprays, aerosols, patches, solutions, suspensions and emulsions.

30. The formulation of claim 26, which is an injectable formulation, wherein the carrier is selected from the group consisting of aqueous and alcoholic solutions and suspensions, oily solutions and suspensions and oil-in-water and water-in-oil emulsions.

31. The formulation of claim 26, which is a rectal formulation, optionally a suppository.

32. The formulation of claim 26, which is a transdermal formulation, wherein the carrier is selected from the group consisting of aqueous and alcoholic solutions, oily solutions and suspensions and oil-in-water and water-in-oil emulsions.

33. The transdermal formulation of claim 32, which is an iontophoretic transdermal formulation, wherein the carrier is selected from the group consisting of aqueous and alcoholic solutions, oily solutions and suspensions and oil-in-water and water-in-oil emulsions, and wherein the formulation further comprises a transdermal transport promoting agent.

34. The formulation of claim 26, which is provided in an implant, a capsule or a cartridge.

35. The composition of claim 20, wherein the carrier is selected from the group consisting of aqueous and alcoholic solutions and suspensions, oily solutions and suspensions and oil-in-water and water-in-oil emulsions.

36. The formulation of claim 20, wherein the carrier comprises a hydrophobic carrier.

37. The formulation of claim 36, wherein the carrier comprises lipid vesicles, optionally liposomes, or particles, optionally microcrystals.

38. The formulation of claim 37, wherein the carrier comprises liposomes, and the liposomes comprise the anti-sense oligo.

39. The formulation of claim 26, which is a respirable or inhalable formulation, optionally an aerosol.

40. The composition of claim 1, in single or multiple unit form.

41. The composition of claim 1, in bulk.

42. A kit, comprising a delivery device;
in a separate container(s), the oligo(s) of claim 1; and instructions for adding a carrier and for use of the kit.

43. The kit of claim 42, wherein the formulation is a respirable formulation and the delivery device comprises a nebulizer which delivers single metered doses of the formulation.

44. The kit of claim 43, wherein the nebulizer comprises an insufflator and the composition is provided in a piercable or operable capsule or cartridge.

45. The kit of claim 44, wherein the delivery device comprises a pressurized inhaler and the composition comprises a suspension, solution or dry formulation of the oligo.

46. The kit of claim 45, further comprising, in a separate container, an agent selected from the group consisting of other therapeutic agents, surfactants, anti-oxidants, flavoring agents, fillers, volatile oils, dispersants, antioxidants, propellants, preservatives, buffering agents, RNA
inactivating agents, cell-internalized or up-taken agents and coloring agents.

47. The kit of claim 46, comprising, in separate containers, one or more oligos, one or more surfactants, and a carrier or diluent, and optionally other therapeutic agents.

48. The kit of claim 42, wherein the device is a transdermal delivery device, and the kit further comprises a transdermal delivery agent, a transdermal carrier or diluent, and instructions for preparing a transdermal delivery formulation.

49. The kit of claim 42, wherein the device is an iontophoretic delivery device, and the kit further comprises iontophoretic agents and instructions for preparing an iontophoretic formulation.

50. An in vivo method of delivering an anti-sense oligonucleotide(s) (oligo(s)) to one or more target polynucleotide(s), comprising administering into the respiratory system of a subject one or more oligo(s) that are anti-sense to the polynucleotide(s), in an amount effective to reach and hybridize to the target polynucleotide(s), and reduce the production or availability, or to increase the degradation, of the target mRNA, or to reduce the amount of the target polypeptide present in the lungs.

51. An in vivo method of delivering an anti-sense oligonucleotide (oligo) to a target polynucleotide associated with bronchoconstriction and/or lung inflammation, allergy(ies) and/or surfactant hypoproduction, comprising administering to a subject the composition of claim 1, that comprises an amount of the oligo(s) effective to reach and hybridize to the target polynucleotide(s), and reduce or inhibit the polynucleotide(s)' transcription and/or expression and, thereby, alleviating bronchoconstriction and/or lung inflammation, allergy(ies) and/or surfactant hypoproduction.

52. The method of claim 51, wherein the administered composition comprises an amount of the oligo(s) and is administered under conditions effective for alleviating bronchoconstriction and/or lung inflammation, allergy(ies) and/or surfactant depletion or hyposecretion, when administered to a mammal.

53. The method of claim 51, wherein the composition is administered into the subject's respiratory system.

54. The method of claim 53, wherein the composition is administered directly into the subject's lung (s).

55. The method of claim 51, wherein the administered composition comprises an amount of the oligo(s) and is administered under conditions effective to reduce the production or availability, or to increase the degradation, of the target mRNA or to reduce the amount of the target polypeptide present in the lungs.

56. The method of claim 51, wherein the agent is administered as a respirable aerosol.

57. The method of claim 51, wherein the pulmonary obstruction, and/or bronchoconstriction and\or lung inflammation, allergy(ies) and/or surfactant hypoproduction are associated with a disease or condition selected from the group consisting of pulmonary vasoconstriction, inflammation, allergies, asthma, impeded respiration, respiratory distress syndrome (RDS), pain, cystic fibrosis (CF), allergic rhynitis (AR), pulmonary hypertension, emphysema, chronic obstructive pulmonary disease (COPD), pulmonary transplantation rejection, pulmonary infections, bronchitis, and cancer.

58. The method of claim 57, wherein the disease or condition is associated with an allergy(ies), and the oligo is anti-sense to a target selected from the group consisting of the initiation codon,the coding region, the 5'-end and the 3'-end genomic flanking regions, the 5' and 3' intron-exon junctions, and regions within 2 to 10 nucleotides of the junctions of a gene(s) encoding an immunoglobulin(s) and antibody(ies) and immunoglobulin and antibody receptors or are anti-sense to the immunoglobulin(s) and antibody(ies) and immunoglobulin and antibody receptors mRNA;
combinations of the oligo(s); and mixtures of the oligos.

59. The method of claim 57, wherein the disease or condition is associated with a malignancy or cancer, and the oligo is anti-sense to a target selected from the group consisting of the initiation codon,the coding region, the 5'-end and the 3'-end genomic flanking regions, the 5' and 3' intron-exon junctions, and regions within 2 to 10 nucleotides of the junctions of an oncogene(s) and\or encodes a malignancy associated protein, or is(are) anti-sense to the oncogene or malignancy associated protein mRNA; combinations of the oligo(s); and mixtures of the oligos and the oligo(s) is(are) administered in an amount effective to reduce either the level of the protein mRNA or of the malignancy associated protein, or to reduce the growth of or provide beneficial characteristics to malignant cells.

60. The method of claim 51, wherein the composition is administered transdermally or systemically.

61. The method of claim 60, wherein the composition is administered orally, intracavitarily, intranasally, intraanally, intravaginally, intrauterally, intraarticularly, transdermally, intrabucally, intravenously, subcutaneously, intramuscularly, intravascularly, intratumorously, intraglandularly, intraocularly, intracranial, into an organ, intravascularly, intrathecally, intralymphatically, intraotically, by implantation, by inhalation, intradermally, intrapulmonarily, intraotically, by slow release, by sustained release and by a pump.

62. The method of claim 51, wherein the subject is a non-human mammal.

63. The method of claim 51, wherein the mammal is a human.

64. The method of claim 51, wherein the oligo is administered in amount of about 0.005 to about 150 mg/kg body weight.
65. The method of claim 51, wherein the oligo is obtained by (a) selecting fragments of a target nucleic acid having at least 4 contiguous nucleic acids selected from the group consisting of G and C;
(b) obtaining a first oligonucleotide 4 to 60 nucleotides long which comprises the selected fragment and has a C and G nucleic acid content of up to and including about 15%; and (c) obtaining a second oligonucleotide 4 to 60 nucleotides long comprising a sequence which is anti-sense to the selected fragment, the second oligonucleotide having an A base content of up to and including about 15%.

65. The method of claim 64, wherein the oligo is A-free.

66. The method of claim 51, wherein the target is selected from the group consisting of the initiation codon, the coding region, the 5'-end and the 3'-end genomic flanking regions, the 5' and 3' intron-exon junctions, and regions within 2 to 10 nucleotides of the junctions of an oncogene or a gene encoding a target polypeptide associated with lung airway dysfunction or anti-sense to the polypeptide or oncogene mRNA; combinations of the oligo(s); and mixtures of the oligos;
wherein the polypeptide is selected from the group consisting of transcription factors, stimulating and activating factors, interleukins, interleukin receptors, chemokines, chemokine receptors, endogenously produced specific and non-specific enzymes, immunoglobulins, antibody receptors, central nervous system (CNS) and peripheral nervous and non-nervous system receptors, CNS and peripheral nervous and non-nervous system peptide transmitters, adhesion molecules, defensives, growth factors, vasoactive peptides, peptide receptors and binding proteins, and malignancy associated proteins.

67. The method of claim 51, wherein one or more As in the oligo(s) is(are) substituted by a universal base selected from the group consisting of heteroaromatic bases which bind to a thymidine base but have less than about 0.3 of the adenosine base agonist or antagonist activity at an adenosine A1, A2a, A2b and A3 receptors.

68. The method of claim 67, wherein the heteroaromatic bases are selected from the group consisting of pyrimidines and purines, which may be substituted by O, halo, NH2, SH, SO, SO2, SO3, COOH and branched and fused primary and secondary amino, alkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, alkoxy, alkenoxy, acyl, cycloacyl, arylacyl, alkynoxy, cycloalkoxy, aroyl, arylthio, arylsulfoxyl, halocycloalkyl, alkylcycloalkyl, alkenylcycloalkyl, alkynylcycloalkyl, haloaryl, alkylaryl, alkenylaryl, alkynylaryl, arylalkyl, arylalkenyl, arylalkynyl, arylcycloalkyl, which may be further substituted by O, halo, NH2, primary, secondary and tertiary amine, SH, SO, SO2, SO3, cycloalkyl, heterocycloalkyl and heteroaryl.

69. The method of claim 67, wherein the pyrimidines and purines are substituted at positions 1, 2, 3, 4, 7 and 8 and the pyrimidines and purines are selected from the group consisting of theophylline, caffeine, dyphylline, etophylline, acephylline piperazine, bamifylline, enprofylline and xantine having the chemical formula wherein R1 and R2 are independently H, alkyl, alkenyl or alkynyl and R3 is H, aryl, dicycloalkyl, dicycloalkenyl, dicycloalkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, O-cycloalkyl, O-cycloalkenyl, O-cycloalkynyl, NH2-alkylamino-ketoxyalkyloxy-aryl and mono and dialkylaminoalkyl-N-alkylamino-SO2 aryl.

70. The method of claim 69, wherein the universal base is selected from the group consisting of 3-nitropyrrole-2'-deoxynucleoside, 5-nitro-indole, 2-deoxyribosyl-(5-nitroindole), 2-deoxyribofuranosyl-(5-nitroindole), 2'-deoxyinosine, 2'-deoxynebularine, 6H, 8H-3,4-dihydropyrimido [4,5-c] oxazine-7-one or 2-amino-6-methoxyaminopurine.

71. The method of claim 51, further comprising substituting a methylated cytocine ("'C) for a C in one or more CpG dinucleotide(s), if present in the oligo(s).

72. The method of claim 51, further comprising substituting by, or modifying one or more nucleotide residue(s) of the oligo(s) with, methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methyimino) (MMI), methoxymethyl (MOM), methoxyethyl (MOE), methyleneoxy (methylimino) (MOMI), methoxy methyl (MOM), 2'-O-methyl, phosphoramidate, C-5 substituted residues, or combinations thereof.

73. The method of claim 51, further comprising operatively linking to, or complexing the oligo(s) with, an agent selected from the group consisting of cell internalized and uptaken agent(s) and cell targeting agents.

74. The method of claim 73, wherein the cell internalized or up taken agent is selected from the group consisting of transferrin, asialoglycoprotein, and streptavidin.

75. The method of claim 73, wherein the cell targeting agent is a vector, optionally a prokaryotic or eukaryotic vector.

76. A method of treating a disease or condition associated with a target selected associated with a disease or condition afflicting lung airways, comprising conducting the method of claim 56.

77. The method of claim 76, wehrein the amount of oligo(s) administered is (are) effective to reduce the production or availability, or to increase the degradation, of the mRNA, or to reduce the amount of the polypeptide present in the lungs.

78. The method of claim 77, wherein the amount of oligo(s) administered is (are) effective to reduce the production or availability, or to increase the degradation, of the mRNA, or to increase the amount of the surfactant present in the subject's lungs.

79. The composition of claim 4, wherein the oligo(s) is(are) anti-sense to the initiation codon, the coding region, the 5'-end and the 3'-end genomic flanking regions, the 5' and 3' intron-exon junctions, and regions within 2 to 10 nucleotides of the junctions of a gene(s) encoding an adenosine A1, A2a, A2b and\or A3 receptor, or anti-sense to the adenosine A1, A2a, A2b and\or A3 receptor mRNA.

80. The composition of claim 79, wherein all nucleotide linking residues are phosphorothioates.

81. The composition of claim 1, wherein the oligo is a DNA.

82. The composition of claim 1, wherein the oligo is an RNA.

83. The composition of claim 1, wherein the oligo comprises about 7 to up to about 60 mononucleotides.

84. The composition of claim 79, wherein the oligo(s) is selected from the group consisting of fragment(s) SEQ ID NOS: 1, 3, 5, 7, 8, and\or 11 through 2419, optionally wherein at least one mononucleotide residue is substituted or modified by methylphosphonate, phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate, formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methyimino), (MMI), methoxymethyl (MOM), methoxyethyl (MOE), methyleneoxy (methylimino) (MOMA), methoxy methyl (MOM), 2'-O-methyl, phosphoramidate residues and/or combinations thereof.

85. The method of claim 51, wherein the oligo is administered topically to the airway, respiratory or pulmonary epithelium of the subject.

86. The composition of claim 1, wherein the oligo has a particle size of about 5-10 µm or in the range of 10-500 µm.

87. The composition of claim 1, further comprising a propellant.

88. The method of claim 50, wherein the oligo has a particle size of about 5-10 µm or in the range of 10-500 µm.

89. The method of claim 50, further comprising adding to the oligo a propellant.

90. The method of claim 51, wherein the oligo has a particle size of about 5-10 µm or in the range of 10-500 µm.

91. The method of claim 51, further comprising adding to the oligo a propellant.