Note: Claims are shown in the official language in which they were submitted.
1334
WHAT IS CLAIMED AS NOVEL AND UNOBVIOUS
IN UNITED STATES LETTERS PATENT IS:
1. A pharmaceutical composition, comprising a nucleic acid which comprises an
oligonucleotide
(oligo) consisting of up to about 15% adenosine (A), and which is effective
for alleviating or .inhibiting
bronchoconstriction, allergy(ies) and/or inflammation, the oligo being anti-
sense to a target selected from the
group consisting of
target genes and their corresponding mRNAs;
genomic and mRNA flanking regions selected from the group consisting of 3' and
5' intron-exon borders
and the juxta-section between coding and non-coding regions; and
all mRNA segments encoding polypeptides associated with a disease(s) or
condition(s) afflicting lung
airways;
combinations thereof;
pharmaceutically acceptable salts thereof; and
mixtures thereof.
2. The composition of claim 1, wherein the oligo consists of up to about 10%
A.
3. The composition of claim 2, wherein the oligo consists of up to about 5% A.
4. The composition of claim 3, wherein the oligo consists of up to about 3% A.
5. The composition of claim 4, wherein the oligo is A-free.
6. The composition of claim 1, wherein the target gene is selected from the
group consisting of
target genes and mRNAs encoding polypeptides 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, defensines,
growth factors, vasoactive peptides and
receptors, and binding proteins; and target genes and mRNAs corresponding to
oncogenes, and flanking regions
and intron and exon borders.
7. The agent of claim 6, wherein the encoded polypeptides are selected from
the group consisting of
NfkB 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.beta.), Interleukin 1.beta. Receptor (IL- 1.beta. R),
Eotaxin, Tryptase, Major Basic Protein, .beta.2-adrenergic Receptor Kinase,
Endothelia Receptor A, Endothelia
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,
Cyclooxygenase (COX), Intracellular
Adhesion Molecule 1 (ICAM-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), NFAT
Transcription Factors, STAT 4,
MIP-l.alpha., MCP-2, MCP-3, MCP-4, Cyclophillins, Phospholipase A2, Basic
Fibroblast Growth Factor, Metalloproteinase,
CSBP/p38 MAP Kinase, Tryptose Receptor, PDG2, Interleukin-3 (IL-3),
Interleukin-1.beta. (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 ICAM3, C5a,
CCR3 (Eotaxin Receptor), CCR1, CCR2, CCR4, CCR5, LTB-4, AP-1 Transcription
Factor, Protein kinase C,
Cysteinyl Leukotriene Receptor, Tachychinnen Receptors (tach R), IkB Kinase 1
& 2, STAT 6, c-mas and
NF-Interleukin-6 (NF-IL-6).
8. The composition of claim 1, wherein at least one A is 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 activity at the adenosine
A1, A2b and A3 receptors, and
heteroaromatic bases which have no activity or have an agonist activity at the
adenosine A2a receptor.
9. The composition of claim 8, wherein all As are substituted by universal
bases 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 activity at the adenosine A1, A2b and
A3 receptors, and heteroaromatic bases
which have no activity or have an agonist activity at the adenosine A2a
receptor.
10. The composition of claim 8, 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,
1335
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.
11. The composition of claim 10, wherein the pyrimidines and purines are
substituted at positions 1,
2,3,4,7and8.
12. The composition of claim 11, wherein 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
Image
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.
13. The composition of claim 12, 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.
14. The composition of claim 1, where a methylated cytocine (mC) is
substituted for at least one CpG
dinucleotide if present in the oligo(s). .
15. The composition of claim 1, wherein at least one nucleotide linking
residue of the anti-sense
oligonucleotide(s) is a residue selected from the group consisting of
methylphosphonate, phosphotriester,
phosphorothioate, phosphorodithioate, boranophosphate, formacetal,
thioformacetal, thioether, carbonate,
carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone, sulfite,
sulfoxide, sulfide, hydroxylamine,
methylene(methyimino), methyleneoxy (methylimino), 2'-O-methyl,
phosphoramidate residues and combinations
thereof.
16. The agent of claim 15, wherein all nucleotide linking residues are
selected from the group
consisting of methylphosphonate, phosphotriester, phosphorothioate,
phosphorodithioate, boranophosphate,
formacetal, thioformacetal, thioether, carbonate, carbamate, sulfate,
sulfonate, sulfamate, sulfonamide, sulfone,
sulfite, sulfoxide, sulfide, hydroxylamine, methylene(methyimino),
methyleneoxy (methylinmino), 2'-O-methyl,
phosphoramidate residues and combinations thereof.
17. The composition of claim 1, wherein the anti-sense oligonucleotide
comprises about 7 to 60
mononucleotides.
18. The composition of claim 1, wherein the anti-sense oligonucleotide
comprises fragments 1 to
1670 (SEQ ID NOS: 11 through 1680).
19. The composition of claim 1, wherein the anti-sense oligonucleotide is
linked to an agent selected
from the group consisting of cell internalized or up-taken agent(s) and cell
targeting agents.
20. The composition of claim 19, wherein the cell internalized or up taken
agent is selected from the
group consisting of transferrin, asialoglycoprotein and streptavidin.
21. The composition of claim 19, wherein the nucleic acid is linked to a
vector.
22. The vector of claim 21, which comprises a prokaryotic or eukaryotic
vector.
23. The composition of claim 1, wherein the oligo is hybridized to a
ribonucleic acid.
24. A cell, comprising the agent of claim 1.
25. The composition of claim 1, further comprising a carrier.
26. The composition of claim 25, wherein the carrier comprises a biologically
acceptable carrier.
27. The composition of claim 26, wherein the carrier comprises a
pharmaceutically or veterinarily
acceptable carrier.
28. The composition of claim 25, wherein the carrier is selected from the
group consisting of
gaseous, liquid, solid carriers and mixtures thereof.
29. The composition of claim 25, further comprising an agent selected from the
group consisting of
other therapeutic agents, surfactants, antioxidants, flavoring and coloring
agents, fillers, volatile oils, buffering
agents, dispersants, RNA inactivating agents, anti-oxidants, flavoring agents,
propellants and preservatives.
30. The composition of claim 29, comprising the nucleic acid, a surfactant and
a carrier.
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31. The composition of claim 29, wherein the surfactant is selected from the
group consisting of
surfactant protein A, surfactant protein B, surfactant protein C, surfactant
protein D and surfactant protein and
active fragments thereof, non-dipalmitoyl disaturated phosphatidylcholine,
dipalmitoylphosphatidylcholine,
phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol,
phosphatidylethanolamine, phosphatidylserine,
phosphatidic acid, ubiquinones, lysophosphatidylethanolamine,
lysophosphatidylcholine,
palmitoyl-lysophosphatidylcholin, dehydroepiandrosterone, dolichols,
sulfatidic acid, glycerol-3-phosphate,
dihydroxyacetone phosphate, glycerol, glycero-3-phosphocholine,
dihydroxyacetone, palmitate, cytidine
diphosphate (CDP) diacylglycerol, CDP choline, choline, choline phosphate,
artificial lamellar bodies vehicles for
surfactant components, omega-3 fatty acids, polyenic acid, polyenoic acid,
lecithin, palmitic acid, non-ionic
ethylene and/or propylene oxide block copolymers, polyoxypropylene,
polyoxyethylene, poly (vinyl amine) with
dextran and/or alkanoyl side chains, Brij 35, Triton X-100, ALEC, Exosurf,
Survant and Atovaquone.
32. The composition of claim 31, wherein the RNA inactivating agent comprises
an enzyme, preferably a
ribozyme.
33. The composition of claim 1, wherein the anti-sense oligonucleotide is
present in an amount of
about 0.01 to about 99.99 w/w of the composition.
34. The composition of claim 33, wherein the anti-sense oligonucleotide is
present in an amount of
about 1 to about 40 w/w of the composition.
35. The composition of claim 34, wherein the anti-sense oligonucleotide is
present in an amount of
about 5 to about 20 w/w of the composition.
36. A formulation, comprising the composition of claim 25, selected from the
group consisting of
systemic and topical formulations.
37. The formulation of claim 36, 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.
38. The formulation of claim 37, which is an oral formulation, wherein the
carrier is selected from the
group consisting of solid and liquid carriers.
39. The oral formulation of claim 38, wherein the liquid carrier is selected
from the group consisting
of solutions, suspensions, and oil-in-water and water-in-oil emulsions.
40. The oral formulation of claim 38, which is selected from the group
consisting of a powder,
dragees, tablets, capsules, sprays, aerosols, solutions, suspensions and
emulsions.
41. The formulation of claim 36, 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.
42. The formulation of claim 36, 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.
43. The formulation of claim 36, which is a rectal formulation in the form of
a suppository.
44. The formulation of claim 36, 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.
45. The transdermal formulation of claim 36, 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.
46. An implantable capsule or cartridge, comprising the formulation of claim
44.
47. The formulation of claim 36, 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.
48. The formulation of claim 36, wherein the carrier comprises a hydrophobic
carrier.
49. The formulation of claim 48, wherein the carrier comprises lipid vesicles
or particles.
50. The formulation of claim 49, wherein the vesicles comprise liposomes, and
the particles comprise
microcrystals.
51. The formulation of claim 50, wherein the vesicles comprise liposomes which
comprise the
anti-sense oligonucleotide.
52. The formulation of claim 49, wherein the vesicles comprise N-(1-[2,3-
dioleoxyloxi] propyl)-
N,N,N-trimethyl-ammonium methylsulfate.
53. The formulation of claim 36, comprising a respirable or inhalable
formulation.
54. The respirable or inhalable formulation of claim 53, comprising an
aerosol.
55. The formulation of claim 36, in single or multiple unit form.
56. The formulation of claim 36, in bulk.
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57. A kit, comprising
a delivery device;
in a separate container, the formulation of claim 36; and
instructions for adding a carrier and for use of the formulation.
58. The kit of claim 57, wherein the delivery device comprises a nebulizer
which delivers single
metered doses of the formulation.
59. The kit of claim 58, wherein
the nebulizer comprises an insufflator; and
the composition is provided in a piercable or operable capsule or cartridge.
60. The kit of claim 58, wherein
the delivery device comprises a pressurized inhaler; and
the composition comprises a suspension, solution or dry formulation of the
agent.
61. The kit of claim 57, 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,
cell-internalized or up-taken agents
and coloring agents.
62. The kit of claim 61, comprising, in separate containers, a nucleic acid, a
surfactant and a carrier.
63. The kit of claim 61, wherein the solvent is selected from the group
consisting of organic solvents
and organic solvents mixed with one or more co-solvents.
64. The kit of claim 57, wherein the composition is provided in a capsule or
cartridge.
65. An in vivo method of delivering a nucleic acid comprising an anti-sense
oligonucleotide (oligo)
to a target polynucleotide associated with a disease(s) or condition(s)
afflicting lung airways, comprising
administering to a subject the composition of claim 1 comprising an amount of
the nucleic acid effective to reach
the target polynucleotide.
66. The method of claim 65, wherein the composition is administered into the
subject's respiratory
system.
67. The method of claim 65, wherein the agent is administered directly into
the subject's lung (s).
68. The method of claim 65, wherein the amount of the agent is effective to
bind to the nucleic acid.
69. The method of claim 65, wherein the agent is 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.
70. The method of claim 65, wherein the agent is administered as a respirable
aerosol.
71. The method of claim 65, wherein the disease or condition is associated
with obstruction of the
subject's airways.
72. The method of claim 71, wherein the disease or condition is associated
with asthma.
73. The method of claim 65, wherein the disease or condition is associated
with inflammation.
74. The method of claim 65, wherein the disease or condition is associated
with an allergy, and the
target is selected from the group consisting of polypeptide and antibody
receptor, genes and monas
encoding them, their genomic and RNA flanking sequences and exon and intro
borders of the gene (s) and
RNA(s).
75. The method of claim 65, wherein the disease or condition is associated
with a malignancy or
cancer, and the mRNA encodes a target selected from the group consisting of
immunoglobulins and antibody
receptors, genomic flanking sequences and genes and mRNAs encoding them and
oncogenes.
76. The method of claim 65, wherein the composition is administered by a
transdermal or systemic
route.
77. The method of claim 76, wherein the composition is administered orally,
intracavitarily,
intranasally, intraanally, intravaginally, intrautecally, intraarticularly,
transdermally, intrabucally, intravenously,
subcutaneously, intramuscularly, intravascularly, intratumorously,
intraglandularly, intraocularly, intracranial, into
an organ, intravascuiarly, intrathecally, intralymphatically, intraotically,
by implantation, by inhalation,
intradermally, intrapulmonarily, intraotically, by slow release, by sustained
release and by a pump.
78. The method of claim 65, wherein the subject is a mammal.
79. The method of claim 78, wherein the mammal is selected from the group
consisting of humans
and animals.
80. The method of claim 79, wherein the mammal is a human.
81. The method of claim 79, wherein the subject is an animal.
82. The method of claim 65, wherein the anti-sense oligonucleotide is
administered in amount of
about 0.005 to about 150 mg/kg body weight.
83. The method of claim 82, wherein the anti-sense oligonucleotide is
administered in an amount of
about 0.01 to about 75 mg/kg body weight.
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84. The method of claim 83, wherein the anti-sense oligonucleotide is
administered in an amount of
about 1 to 50 mg/kg body weight.
85. The method of claim 65, which is a prophylactic method.
86. The method of claim 65, which is a therapeutic method.
87. The method of claim 65, 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%.
88. The method of claim 61, wherein the oligo consists of up to about 10% A.
89. The method of claim 88, wherein the oligo consists of up to about 5% A.
90. The method of claim 88, wherein the oligo consists of up to about 3% A.
91. The method of claim 92, wherein the oligo is A-free.
92. The method of claim 65, wherein the target is selected from the group
consisting of genes
and mRNAs encoding polypeptides 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, defensines, growth factors, vasoactive
peptides, peptide receptors and binding
proteins; and
genes and mRNAs corresponding to oncogenes.
93. The method of claim 65, wherein at least one A is 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 activity at the adenosine A1, A2b
and A3 receptors, and heteroaromatic
bases which have no activity or have an agonist activity at the adenosine A2a,
receptor.
94. The method of claim 93, wehrein all As are substituted by universal bases
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 activity at the adenosine A1, A2b and
A3 receptors, and heteroaromatic bases
which have no activity or have an agonist activity at the adenosine A2a
receptor.
95. The method of claim 95, wherein the heteroaromatic bases are selected from
the group consisting
of pyrimidines and purines, which may be substituted by O, halo, NH2, SH, SO,
502, 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.
96. The method of claim 95, wherein the pyrimidines and purines are
substituted at positions 1, 2, 3,
4, 7 and 8.
97. The method of claim 96, wherein 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
Image
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.
98. The method of claim 97, 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.
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99. The method of claim 65, further comprising methylating at least one
cytocine (mC) if a CpG
dinocleotide if present in the oligo(s).
100. The method of claim 65, further comprising substituting at least one
nucleotide linking residue of
the anti-sense oligonucleotide(s) with a residue selected from the group
consisting of methylphosphonate,
phosphotriester, phosphorothioate, phosphorodithioate, boranophosphate,
formacetal, thioformacetal, thioether,
carbonate, carbamate, sulfate, sulfonate, sulfamate, sulfonamide, sulfone,
sulfite, sulfoxide, sulfide, hydroxylamine,
methylene(methyimino), methyleneoxy (methylimino), 2'-O-methyl,
phosphoramidate residues, and combinations
thereof.
101. The method of claim 100, wherein all nucleotide linking residues of the
oligo are substituted.
102. The method of claim 65, further comprising linking the anti-sense
oligonucleotide to an agent
selected from the group consisting of cell internalized and up-taken agent(s)
and cell targeting agents.
103. The method of claim 102, wherein the cell internalized or up taken agent
is selected from the
group consisting of transferrin, asialoglycoprotein, and streptavidin.
104. The method of claim 102, wherein the cell targeting agent is a vector.
105. The method of claim 104, wherein the vector to which the agent is
operatively linked is a
prokaryotic or eukaryotic vector.
106. A method of identifying segments in a target polynucleotide suitable for
constructing
oligonucleotides which are anti-sense to the target polynucleotide and have an
adenosine (A) content of up to and
including about 15% of all nucleotides, comprising
(a) selecting fragments of a target polynucleotide acid having at least 4
contiguous nucleic acids selected
from the group consisting of G and C; and
(a) obtaining a first oligonucleotide 4 to 60 nucleotides long which comprises
the selected fragment and
has a C and G content of up to and including about 15%.
107. A method of obtaining oligonucleotides which are anti-sense to a target
polynucleotide and have
an adenosine content of 0 to up to and including about 15%, comprising
conducting the method of claim 106;
wherein the first oligonucleotide comprises a sequence which is anti-sense to
the selected fragment and has an A
content of up to and including about 15%.
108. The method of claim 107, further comprising, when the anti-sense fragment
comprises at least
one A, substituting at least one A with a universal base selected from the
group consisting of
heteroaromatic bases which bind to thymidine (T) but have less than about 0.3
of A's adenosine A1, A2b
and A3 receptor agonist activity; and
heteroaromatic bases which have no activity or have adenosine A2a receptor
agonist activity.
109. The method of claim 108, 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.
110. The method of claim 109, wherein the pyrimidines and purines are
substituted at positions 1, 2, 3,
4, 7 and 8.
111. The method of claim 109, wherein 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
Image
wherein R1 and R2 are independently ~, alkyl, alkenyl, or alkynyl 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.
112. The method of claim 108, 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.
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113. A method of heating a disease or condition associated with a target
selected from the group
consisting of proteins, gene (s) and their corresponding mRNA(s) encoding the
proteins, the genes and mRNA
flanking regions and their intron and exon borders, associated with a disease
or condition afflicting lung airways,
comprising administering to a subject afflicted with the disease or condition
the composition of claim 1 comprising
an anti-bronchoconstriction, anti-allergic and/or anti-inflammatory effective
amount of the nucleic acid.
114. The method of claim 113, wehrein the amount of nucleic acid administered
is effective to reduce
the production or availability, or to increase the degradation, of the mRNA,
or to reduce the amount of the
polypeptide present in the lungs.
115. The method of claim 113, wherein the nucleic acid is administered
directly to the lung (s) of the
subject.
116. The method of claim 113, wherein the nucleic acid is administered as a
respirable aerosol.
117. The method of claim 113, wherein the disease or condition is a disease or
condition afflicting the
lung (s).
118. The method of claim.117, wherein the disease or condition is associated
with obstruction of the
subject's airways.
119. The method of claim 117, wherein the disease or condition is associated
with asthma.
120. The method of claim 117, wherein the disease or condition is associated
with inflammation.
121. The method of claim 113, wherein the disease or condition is associated
with allergy (ies), and
the target is selected from the group consisting of immunoglobulins and
antibody receptors, gene(s) and
corresponding mRNA(s) encoding them, the genes and mRNA flanking regions and
intron and exon borders.
122. The method of claim 113, wherein
the disease or condition is associated with a malignancy or cancer; and
the target is selected from the group consisting of immunoglobulins and
antibody receptors, gene(s) and
mRNA(s) encoding them, gene(s) and mRNA(s) associated with oncogenes, genomic
and mRNA flanking regions
and exon and intron borders.
123. The method of claim 113, wherein the composition is administered by a
topical or systemic route.
124. The method of claim 123, wherein the composition is administered orally,
intracavitarily,
intranasally, intraanally, intravaginally, intrauterally, intraarticularly,
intraotically, intralymphatically,
transdermally, intrabucally, intravenously, subcutaneously, intramuscularly,
intratumorously, intraglandularly,
intraocularly, intracranial, into an organ, intravascularly, intrathecally, by
implantation, by inhalation,
intradermally, intrapulmonarily, into the ear, by slow release, by sustained
release and by a pump.
125. The method of claim 124, wherein the subject is a mammal.
126. The method of claim 125, wherein the mammals are selected from the group
consisting of
humans and animals.
127. The method of claim 113, wherein the anti-sense oligonucleotide is
administered in amount of
about 0.005 to about 150 mg/kg body weight.
128. The method of claim 127, wherein the anti-sense oligonucleotide is
administered in an amount of
about 0.01 to about 75 mg/kg body weight.
129. The method of claim 130, wherein the anti-sense oligonucleotide is
administered in an amount of
about 1 to about 50 mg/kg body weight.
130. The method of claim 113, which is a prophylactic method.
131. The method of claim 113, which is a therapeutic method.
132. A method of producing anti-sense oligonucleotide(s) (oligos) consisting
of up to and including
about 15% adenosine (A), comprising
selecting a target from the group consisting of polypeptides associated with a
disease(s) and/or
condition(s) afflicting lung airways, genes and RNAs encoding them, the
genomic and mRNA flanking regions and
the gene(s) and mRNA(s) intron and exon borders;
obtaining the sequence of a mRNA(s) selected from the group consisting of
mRNAs corresponding to the
target gene(s) and mRNAs encoding the target polypeptide(s), genomic and mRNA
flanking regions and the genes
and mRNAs intron and exon borders;
selecting at least one segment of the mRNA(s);
synthesizing one or more oligo anti-sense to the selected mRNA segment(s); and
substituting, if necessary, a universal base(s) for one or more A(s) to reduce
the content of A present in the
oligo to up to about 15% of all nucleotides.
133. The method of claim 132, wherein the universal base is 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 activity at the adenosine A1, A2b and A3 receptors, and
heteroaromatic bases which have no
activity or have an agonist activity at the adenosine A2a, receptor.
134. The method of claim 132, wherein all As are substituted with universal
bases selected from the
group consisting of heteroaromatic bases which bind to a thymidine base but
have antagonist activity and less than
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about 0.3 of the adenosine base agonist activity at the adenosine A1, A2b and
A3 receptors, and heteroaromatic bases
which have no activity or have an agonist activity at the adenosine A2a,
receptor.
135. The method of claim 133, 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.
136. The method of claim 135, wherein the pyrimidines and purines are
substituted at positions 1, 2, 3,
4,7and8.
137. The method of claim 135, wherein 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
Image
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.
138. The method of claim 135, 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.
139. The method of claim 132, wherein the proportion of A in the oligo is
reduced to up to about 10%.
140. The method of claim 139, wherein the proportion of A in the oligo is
reduced to up to about 5%.
141. The method of claim 140, wherein the proportion of A in the oligo is
reduced to up to about 3%.
142. The method of claim 141, wherein the proportion of A in the oligo is
reduced to about 0.
143. The method of claim 139, wherein the selected segment contains loss than
about 15% T.
144. The method of claim 132, further comprising substituting a methylated
cytosine for cytosine in at
least one CpG dinucleotide present in the anti-sense oligo(s).
145. The method of claim 132, wherein the anti-sense oligo(s) are about 7 to
about 60 nucleorides
long.
146. The method of claim 132, wherein the target 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, selectins,
defensines, growth factors, vasoactive peptides,
vasoactive peptide receptors, and binding proteins and oncogenes.
147. The method of claim 146, wherein the target genes are selected from the
group consisting of
oncogenes.
148. An anti-sense oligonucleotide produced by the method of claim 132.
