JP2002515513A - Compositions and methods for pulmonary delivery of nucleic acids - Google Patents

Abstract

  (57) [Summary] The present invention relates to compositions and methods for pulmonary delivery of nucleic acids, particularly oligonucleotides. In one preferred embodiment, the compositions and methods of the invention are used to pulmonarily deliver an antisense oligonucleotide to an animal for modulating gene expression in the animal for research, therapeutic or prophylactic purposes. To achieve.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] This application is part of a sequel to US Patent Application Serial No. 09 / 083,586, filed May 21, 1998, the disclosure of which is hereby incorporated by reference herein in its entirety. Can be

FIELD OF THE INVENTION [0002] The present invention relates to compositions and methods for pulmonary delivery of nucleic acid therapeutics and diagnostics to the lungs of animals, particularly humans. More specifically, the present invention shows compositions and methods for pulmonary delivery of oligonucleotide therapeutics and diagnostics, including antisense oligonucleotides. In some preferred embodiments, the present invention provides methods and compositions for pulmonary delivery of a composition for oligonucleotide therapy comprising a penetration enhancer, a carrier compound and / or a transfection agent.

[0003] Further specific objects and advantages of the present invention will become or become apparent to those skilled in the art during the description of the preferred embodiments of the invention.

BACKGROUND OF THE INVENTION [0004] Advances in the field of biotechnology have resulted in significant advances in the treatment of previously intractable diseases such as cancer, genetic diseases, arthritis and AIDS.
Many such advances involve the administration of oligonucleotides and other nucleic acids to a subject, especially a human subject.

[0005] Oligonucleotides are administered by various routes. For example, oligonucleotides administered by the parenteral route have been shown to be effective in treating diseases and / or disorders. See, for example, US Pat. No. 5,595,978, Jan. 21, 1997 to Draper et al., Which discloses intravitreal injection as a means for direct delivery of antisense oligonucleotides to the vitreous humor of the mammalian eye. Also, Robertson, Nature Biotechnology, 1997, 15: 209 and Anon.
See Genetic Engineering News, 1997, 15: 1, each of which discusses the treatment of Crohn's disease through intravenous infusion of antisense oligonucleotides.

[0006] Administering oligonucleotides via the lungs for the treatment of lung disorders is attractive because the oligonucleotides are delivered directly to the target organ. For an overview, see, for example, Nyce, JW, Exp. Opin. Invest. Drugs (1997) 6 (9): 1149-115.
6; Schreier, H., Advanced Drug Delivery Reviews, 19, (1996) 1-2; Wu-Pong
, S., and Byron, PR, Advanced Drug Delivery Reviews, 19, (1996) 47-7
1; and Phan, SH, Thorax 1995; 50: 415-421. However, most reports focus on the intratracheal delivery of larger nucleic acids, which are antisense constructs than antisense oligonucleotides with smaller molecular weights, rather than inhaled delivery. For example, Georges, RN, et al., Cancer Research 53, 1743-1746 (1993
) (Prevention of growth of orthotopic human lung cancer by intratracheal placement of retroviral antisense K-ras structures); and Yoshimura, K., et al., Nucleic Acids Research,
Vol. 20, No. 12, 3233-3240 (1992) (Expression of transmembrane transduction control genes for human cystic fibrosis in mouse lungs after intratracheal plasmid-mediated gene transfer in vivo). .

[0007] Antisense oligonucleotides have been shown to illustrate antisense effects on cells of various diseases or disorders, including cancer. For example, Dosaka-Akita
See, et al., Cancer Res. 55, 1559-1564 (1995) (L-myc antisense DNA inhibits growth for metastatic initiation sites in human small cell lung cancer).

[0008] Compositions that can be effectively provided for pulmonary delivery of nucleic acids, particularly oligonucleotides, and more particularly oligonucleotides having one or more chemical modifications, include:
A long-felt need, along with methods for using such compositions to deliver such oligonucleotides and nucleic acids to the lungs of animals,
t need). The present invention shows these, as well as other important results.

SUMMARY OF THE INVENTION The present invention relates to compositions and methods for pulmonary delivery of oligonucleotides.

[0010] In some preferred embodiments, the present invention provides a pharmaceutical composition for pulmonary delivery of an oligonucleotide comprising at least one oligonucleotide, wherein at least one sugar moiety of the oligonucleotide is 2 '. Is not a deoxyribofuranosyl sugar moiety, or at least one internucleotide linkage of said oligonucleotide is not a phosphodiester or phosphorothioate linkage.

In accordance with the present invention, the following steps are provided: preparing a nucleic acid therapeutic or diagnostic composition; aerosolizing the nucleic acid composition; introducing the aerosolized nucleic acid composition into the lungs of a mammal. And the aerosolized nucleic acid composition comprises at least one oligonucleotide, wherein the sugar moiety of at least one nucleoside unit of said oligonucleotide is
Not a 2'-deoxyribofuranosyl sugar moiety or at least one internucleotide linkage in said oligonucleotide is not a phosphodiester or phosphorothioate linkage. A method is also provided.

The present invention relates to an animal having or suspected of having a disease or condition treatable by one or more nucleic acids, comprising administering a therapeutically effective amount of an aerosolized nucleic acid composition to the lungs of the animal. Wherein the aerosolized nucleic acid composition comprises at least one oligonucleotide, wherein the sugar moiety of at least one nucleoside unit of the oligonucleotide is not a 2'-deoxyribofuranosyl sugar moiety, or At least one internucleotide linkage of the oligonucleotide is not a phosphodiester or phosphorothioate linkage.

The present invention also provides a method of studying the role of a gene or gene product in a non-human animal, comprising administering a therapeutically effective amount of an aerosolized nucleic acid composition to the animal's lungs, wherein the aerosol is provided. The modified nucleic acid composition comprises at least one oligonucleotide, wherein the sugar moiety of at least one nucleoside unit of the oligonucleotide is not a 2′-deoxyribofuranosyl sugar moiety, or at least one internucleotide linkage in the oligonucleotide is Not a phosphodiester or phosphorothioate linkage.

In some preferred embodiments, there is provided a method for delivering an oligonucleotide therapeutic or diagnostic composition to the lungs of an animal, comprising applying a pharmaceutical composition according to the invention to the lungs of the animal. provide.

[0015] Preferably, the oligonucleotide is delivered into cells of said lung. In some preferred embodiments, the methods of the invention are performed on an animal known or suspected of suffering from a disease or condition.

[0016] In some preferred embodiments, the sugar moiety of at least one nucleoside unit of the oligonucleotide is not a 2'-deoxyribofuranosyl sugar moiety.

[0017] In a further preferred embodiment, the nucleoside unit is a 2'-O-substituted nucleoside sugar moiety.

[0018] In some particularly preferred embodiments, the 2-O-substituent of the 2'-O-substituted nucleoside unit is a 2'-O-alkoxyalkoxy substituent.

[0019] In some particularly preferred embodiments, the 2-O-substituent of the 2'-O-substituted nucleoside unit is a 2'-O-dialkylaminooxyalkyl substituent.

[0020] In some preferred embodiments, at least one internucleotide linkage in the oligonucleotide is not a phosphodiester or phosphorothioate linkage.

In a further preferred embodiment, at least one internucleotide linkage in said oligonucleotide is a 3′-methylene phosphonate, a non-phosphorus containing oligonucleoside linkage, a 2′-5 ′ linkage, or a 3 ′ -Deoxy-3'-amino phosphoramidite linkage.

[0022] In some preferred embodiments, the composition further comprises one or more pharmaceutically acceptable carriers.

[0023] In some preferred embodiments, the composition is an aqueous medium. In another preferred embodiment, the aqueous medium is sterile, pyrogen-free water. In a further preferred embodiment, the aqueous medium is a salt solution. In a further preferred embodiment, the pharmaceutical composition is a powder.

[0024] Preferably, the compositions of the present invention comprise an oligonucleotide that is an antisense oligonucleotide.

[0025] In some preferred embodiments, the antisense compound modulates protein expression or modulates the rate of cell growth. In a further preferred embodiment, the antisense oligonucleotide modulates the expression of a cell adhesion protein.

In a further preferred embodiment, the antisense oligonucleotide is an antisense oligonucleotide against a gene sequence involved in a disease or condition, preferably in asthma, lung cancer, pulmonary fibrosis, rhinovirus, tuberculosis, bronchitis or pneumonia. It is.

[0027] In some preferred embodiments, the antisense oligonucleotide is antisense to a portion of the gene encoding the cytokine. In a further preferred embodiment, the antisense oligonucleotide comprises ICAM-1, EL
AM-1, VCAM-1, B7-1, B7-2, CD40, LFA-3, PECAM-1, ras oncogene, H-ras oncogene, K-ras oncogene, anti-protein against protein kinase C Sense or Mycobacterium tuberculosis, M. bov
is, or antisense to a unique part of the genome of Streptococcus pneumoniae.

[0028] In some preferred embodiments, the pharmaceutical compositions of the invention comprise one or more antisense oligonucleotides.

[0029] In a further preferred embodiment, the oligonucleotide is a ribozyme, an external guide sequence, or an antisense peptide nucleic acid.

In a further preferred embodiment, the oligonucleotide is an aptamer (apta
mer) or molecular decoy.

In a further preferred embodiment, the aqueous medium is a sterile, pyrogen-free buffer solution.

[0032] In some preferred embodiments, the nucleic acid therapeutic composition is an aerosolized solution consisting essentially of the antisense oligonucleotide in a saline solution.

[0033] In another preferred embodiment, the nucleic acid therapeutic composition is an aerosolized solution consisting essentially of the antisense oligonucleotide in a buffer solution.

The present invention also provides a method for regulating the expression of a gene in an animal, comprising administering the pharmaceutical composition of the present invention to the animal.

The present invention also provides a medical device for pulmonary delivery of an aerosol, comprising a pharmaceutical composition according to the present invention. Preferably, the medical device is a nebulizer.

In a further aspect, the present invention provides a compound of the formula:

[0037]

Embedded image

Wherein R ₁ has the formula —OR ₅ —OR ₆ ; wherein R ₅ and R ₆ are independently alkyl having 1 to about 5 carbons; A novel compound comprising at least one moiety of the formula:

In a particularly preferred embodiment, R ₅ is —CH ₂ —CH ₂ — and R ₆ is —CH ₃ .

In a further preferred embodiment, the following formula:

[0041]

Embedded image

Wherein R ₁ has the formula —OR ₅ —OR ₆ ; wherein R ₅ and R ₆ are independently alkyl having 1 to about 5 carbons; is methyl cytosine; M is internucleoside linkage; B is an nucleobases; each R ₂ is H, OH, F or wherein R ₇ - be a (R _{8) V} group; wherein R ₇ is R ₈ is C ₃ -C ₂₀ alkyl, C ₄ -C ₂₀ alkenyl, C ₂ -C ₂₀ alkynyl, C ₁ -C ₂₀ alkoxy, C ₂ -C ₂₀ alkenyloxy, or C ₂ -C ₂₀ alkynyloxy; Hydrogen, amino, halogen, hydroxyl, thiol, keto, carboxyl, nitro, nitroso, nitrile, trifluoromethyl, trifluoromethoxy, O-alkyl, S-alkyl, NH-alkyl, N-dialkyl, O-aryl, S- Aryl, NH-aryl, O-aralkyl, S-aralkyl, NH-aralkyl, amino, N-phthalimide , Imidazole, azide, hydrazino, hydroxylamino, isocyanate, sulfoxide, sulfone, sulfide, disulfide, silyl, aryl, heterocyclic group, carbocyclic group, intercalator, reporter molecule, conjugate, polyamine, polyamide , polyalkylene glycols, polyethers, a group for improving the pharmacokinetic properties of the group to improve the pharmacodynamic properties of an oligonucleotide or oligonucleotide,; R ₃ is H or a hydroxyl protecting group; R ₄ is H OH, an internucleoside linkage, a linker attached to a solid support, or Pr
Is a hydroxyl protecting group; and m and n are each independently 0 to about 50.

In some preferred embodiments, R ₅ is —CH ₂ —CH ₂ — and R ₆ is —CH ₃ . In a further preferred embodiment, each R ₁ is —O—CH ₂ —CH ₂ —O—CH ₃ .

In some particularly preferred embodiments, each R ₂ is —O—CH ₂ —CH ₂ —O—CH ₃ and B is the group consisting of 5-methylcytosine, adenine, guanine, uracil, and thymine Is selected from

In a particularly preferred embodiment, the oligonucleotide provides an oligonucleotide comprising one or more 5-methylcytosine-2′-methoxyethoxy nucleoside moieties.

In a further particularly preferred embodiment, there is provided a pharmaceutical composition comprising a compound of the present invention.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention provides compositions and methods for pulmonary delivery of oligonucleotides and other nucleic acids to animal lungs. In a preferred embodiment, the present invention provides compositions and methods for modulating in vivo expression of an animal gene through pulmonary administration of an antisense oligonucleotide, thereby involving administration by intravenous and other routes. Bypass the possible complications and costs. Enhanced delivery of oligonucleotides and other nucleic acids to animal lungs is achieved through the use of the compositions and methods of the present invention.

Studies suggest that oligonucleotides are rapidly cleared from plasma after intravenous administration and accumulate primarily in the liver and kidneys (Miyao et al., Antisense Res.
Dev., 1995, 5: 115; Takakura et al., Antisense & Nucl. Acid Drug Dev. 1996,
6: 177). The effect of measuring and avoiding "first pass clearance" is an effective agent for treating pulmonary diseases or disorders.
) Is required for development.

One way to improve the first-pass clearance effect is to increase the dose of drug administered, which compensates for the loss of drug due to first-pass clearance. This may be easily achieved by intravenous administration, for example, simply supplying the animal with a larger dose of the drug, but other factors affect bioavailability.

The present invention provides a composition for pulmonary administration of an oligonucleotide comprising a carrier compound, a penetration enhancer, and a transfection agent. However,
The present invention also provides compositions and methods for pulmonary administration of oligonucleotides that are essentially not described herein; the term `` essentially free of carriers or permeation enhancers '' It means that only trace amounts (ie, less than or equal to the perceived effective amount) of the carrier or permeation enhancer may be present in the composition. In particular, these modalities for the present invention are due to compositions comprising such carriers or permeation enhancers (less than 10 mole percent, preferably less than 1 mole percent, and most preferably less than 0.1 mole percent). Is caused.

In some preferred embodiments, the present invention provides a pharmaceutical composition for pulmonary administration of large molecule therapeutics, such as oligonucleotides, comprising the oligonucleotide and the drug. At least one substance that facilitates the transport of pulmonary mucosa to the opposite side of the lung mucosa (the so-called “mucosal penetration enhancer (muco
sal penetrate enhancers) ”,“ absorption enhancers ”
Or simply known as "penetration enhancers"). Muranishi, Crit. Rev. Ther. Drug. Carrier Syst
ems, 1990, 7: 1 and Lee et al., Crit. Rev. Ther. Drug Carrier Systems, 1991.
, 8:91.

The present invention provides compositions and methods for pulmonary delivery of one or more nucleic acids to an animal. For the purposes of the present invention, the term "animal" is meant to include humans and other mammals, as well as reptiles, fish, amphibians and birds. The term "pulmonary delivery" means administration to a portion of the animal lung, either directly or by other means. the term"
"Lung" has the meaning commonly used as the tissue for primary respiration (ie, gas exchange) in animals. As used herein, the term “pulmonary delivery” encompasses the absorption of the delivered component from the lining of the lung to the lung tissue.

[0053] The present invention provides compositions and methods for pulmonary delivery of oligonucleotides. The composition may contain a carrier compound, a permeation enhancer, and / or a transfection agent. As used herein, a "carrier compound" refers to an inert (i.e., having no biological activity per se), eg, degrading a biologically active nucleic acid, or Means nucleic acids or their analogs that are recognized as nucleic acids by in vivo processes that reduce the bioavailability of biologically active nucleic acids by facilitating their removal from the circulation I do. Coadministration of a nucleic acid and a carrier compound (typically in excess of the latter substance) may result in a liver, kidney, or other extracirculatory reservoir, possibly due to competition for a common receptor between the carrier compound and the nucleic acid. A substantial reduction in the amount of nucleic acid recovered in the reservoirs can result. For example, recovery of partially phosphorothioated oligonucleotides in liver tissue can be co-administered with polyinosinic acid, dextran sulfate, polycytidic acid, or 4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonsan. (Miyao et al., Antisense Res. Dev., 1995, 5: 115; Takakura et al., Ant)
isense & Nucl. Acid Drug Dev., 1996, 6: 177).

A “pharmaceutical carrier” or “excipient”, as compared to a carrier compound, is a pharmaceutically acceptable carrier for delivering one or more nucleic acids to an animal. Solvents, suspending agents, or other pharmaceutically inert excipients. The excipients may be liquid or solid, and are intended to be administered in a desired bulk, hardness, etc., when combined with the nucleic acid and other components of the defined formulation mixture. Is selected by type. A typical formulation carrier is
Binders (eg, pre-gelled corn starch, polyvinylpyrrolidone, or hydropropyl methylcellulose, etc.), bulking agents (eg, lactose and other sugars,
Microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylate or calcium hydrogen phosphate, etc.), lubricants (for example, magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metal stearic acid) Including, but not limited to, salts, hydrogenated vegetable oils, corn starch, polyethylene glycol, sodium benzoate, etc., disintegrants (eg, starch, sodium starch glycolate, etc.), or wetting agents (sodium lauryl sulfate, etc.). It is not something to be done.

In some preferred embodiments, the present invention uses oligonucleotides for use in antisense modulation of DNA or messenger RNA (mRNA) function encoding the protein for which modulation is desired, and ultimately uses Regulates the amount of such proteins. Hybridization of an antisense oligonucleotide with its mRNA target disrupts the normal role of mRNA and causes its function in the cell. The function of the mRNA to be prevented is the mRNA for the protein translation site
Translocation, translation of actual protein from RNA, one or more
Includes all biological functions such as RNA splicing to generate mRNA species, turnover or degradation of RNA, and possibly independent catalytic activity that will be mediated by RNA. All of the effects of such interference with mRNA function are the modulation of protein expression, where "modulation" means either an increase (stimulation) or a decrease (inhibition) in protein expression. In the context of the present invention, inhibition is a preferred mode of regulating gene expression.

In the context of the present invention, the term “oligonucleotide” means an oligomer or polymer of ribonucleic acid or deoxyribonucleic acid. The term includes oligonucleotides composed of naturally occurring nucleobases, sugars and covalent bonds between sugars (backbone), as well as oligonucleotides having non-naturally occurring portions that have the same function. Such modified or substituted oligonucleotides can be, for example, enhanced uptake into cells, enhanced binding to targets,
And favorable properties such as increased stability in the presence of nucleases,
Often favored over the natural form.

Oligonucleotides are polymers consisting of repeating units commonly known as nucleotides. Unmodified (naturally occurring) nucleotides
(3) Phosphoric acid, which is a nitrogen-containing base, (2) linked to a 5-carbon cyclic sugar by one of its nitrogen atoms, and ester-bonded to the 5-position carbon of the sugar. Having. When linked in an oligonucleotide chain, the phosphate of the first nucleotide is also esterified to the carbon at position 3 of the sugar of the second adjacent nucleotide.
The "backbone" of the unmodified oligonucleotide is composed of (2) and (3). That is, the sugar is linked by a phosphodiester bond between the 5 (5 ') position of the sugar carbon of the first nucleotide and the 3 (3') position of the carbon of the second adjacent nucleotide. "Nucleosides" are free of the base (1) and the phosphate group (3) (
2) is a sugar (Kornberg, A., DNA Replication, WHFreeman & Co., San Francisco, 1980, pp. 4-7). The backbone of an oligonucleotide is positioned by a series of bases in a specific order, and this specific order notation is conventionally written in the order 5 'to 3' and is known as the nucleotide sequence.

[0058] Oligonucleotides will contain a sufficient nucleotide sequence in identity and number to effect specific hybridization with an individual nucleic acid. Oligonucleotides that specifically hybridize to the sense strand of the gene are commonly referred to as "antisense." In the context of the present invention,
"Hybridization" refers to hydrogen bonding between complementary nucleotides which may be Watson-Crick, Hoogsteen or reverse Hoogsteen. For example, adenine and thymine are complementary nucleobases and are paired by hydrogen bond formation. "Complementary," as used herein, refers to the ability for precise pairing between two nucleotides. For example, if a nucleotide at a given position in an oligonucleotide can hydrogen bond with a nucleotide at the same position in a DNA or RNA molecule, the oligonucleotide and DNA or RNA are considered to be complementary to each other at that position. Oligonucleotides and DNA or RNA are complementary to each other when a sufficient number of corresponding positions in each molecule can be occupied by nucleotides that can hydrogen bond to each other. Thus, "specifically hybridizable" and "complementary" indicate a sufficient degree of complementarity or precise pairing that results in a stable and specific bond between the oligonucleotide and the DNA or RNA target. Is a term used for: It is understood in the art that an oligonucleotide need not be 100% complementary to its target DNA sequence to be specifically hybridizable. An oligonucleotide is specifically hybridizable when binding of the oligonucleotide to the target DNA or RNA molecule interferes with the normal function of the target DNA or RNA by causing loss or loss of function, and Non-specific binding to non-target sequences is performed under conditions where specific binding is desired (ie, in the case of an in vivo assay or therapeutic treatment under physiological conditions, or under conditions in which an in vitro assay is performed). It can be said that there is a sufficient degree of complementarity to avoid in the case of).

[0059] Antisense oligonucleotides are commonly used as research reagents, diagnostic aids, and therapeutic agents. For example, antisense oligonucleotides that can inhibit gene expression with excellent specificity are often used by those skilled in the art to define the function of a particular gene, for example, to distinguish the function of various organs in a biological pathway. Have been. This specific inhibitory effect is therefore exploited by those skilled in the art for research purposes. The specificity and sensitivity of oligonucleotides are also utilized by those skilled in the art for therapeutic purposes. For example, the following U.S. patents demonstrate palliative, therapeutic, and other methods utilizing antisense oligonucleotides. US Patent No. 5135
No. 917 provides an antisense oligonucleotide that inhibits human interleukin-1 receptor expression. US Patent No. 5,098,890 shows antisense oligonucleotides complementary to the c-myb oncogene and antisense oligonucleotide therapy in certain cancer conditions. U.S. Pat. No. 5,087,617 provides a method for treating cancer patients with antisense oligonucleotides. U.S. Patent No. 5,166,195 provides oligonucleotide inhibitors of human immunodeficiency virus (HIV). U.S. Patent No. 5,048,810 provides oligomers capable of hybridizing and inhibiting replication of herpes simplex virus Vmw65 mRNA. US Patent No. 5,194,428 provides antisense oligonucleotides having antiviral activity against influenza virus. U.S. Pat. No. 4,806,463 provides antisense oligonucleotides that inhibit HTLV-III replication and methods for using the same. U.S. Pat.
An oligonucleotide having a nucleotide sequence complementary to a part of an oncogene is provided.
U.S. Patent Nos. 5,276,019 and 5,264,423 show phosphorothioated oligonucleotide analogs used to prevent the replication of exogenous nucleic acids in cells. U.S. Patent No. 4,689,320 shows antisense oligonucleotides as antiviral agents specific for cytomegalovirus (CMV). U.S. Patent No.
No. 5098890 provides oligonucleotides that are complementary to at least a portion of the mRNA transcript of the human c-myb gene. U.S. Patent No. 5,242,906 provides antisense oligonucleotides useful for treating latent Epstein-Barr virus (EBV) infection. Other examples of antisense oligonucleotides are provided herein.

[0060] The oligonucleotide according to the invention preferably comprises about 8 to about 30 nucleotides. More preferably, such oligonucleotides comprise from about 15 to about 25 nucleotides. As is known in the art, a nucleotide is a base-sugar linkage that is suitably linked to the adjacent nucleotide by a phosphodiester, phosphorothioate, or other covalent bond. In the context of the present invention, the term "
"Oligonucleotides" include oligonucleotides composed of naturally occurring nucleobases, sugars and covalent bonds between sugars (the backbone), as well as oligonucleotides having non-naturally occurring portions that are similar in function. Such modified or substituted oligonucleotides are often more naturally than their natural counterparts, for example, due to favorable properties such as enhanced cellular uptake, enhanced binding to targets, and increased stability in the presence of nucleases. Is also preferred.

Oligonucleotides are also useful for determining the properties, functions, and potential relevance of various genetic components of the body to the animal's body or disease state. Traditionally, the function of a gene is to construct a loss-of-function mutation in the gene of the animal (eg, a transgenic mouse) (ie, a “knock-out” mutation).
Has been primarily investigated by Because such tasks are difficult, time-consuming, and the "knock-out" mutation produces a lethal phenotype,
It cannot be performed on essential elements of the gene for animal development.
Furthermore, loss-of-function phenotypes cannot be introduced transiently during certain parts of the animal life cycle or during disease states where "knockout" mutations are always present. "Antisense knockout" is a method of selectively regulating the expression of a gene with an antisense oligonucleotide, rather than directly manipulating the gene.
Overcoming these limitations (eg, Albert et al., Trends in Pharmacological Scie
nces, 1994, 15: 250). In addition, some genes produce multiple mRNA transcripts as a result of processes such as alternative splicing, but `` knock-out '' mutations can affect all types of mRNA transcripts produced from such genes. Are usually removed and cannot be used to investigate the biological role of a particular mRNA transcript.
Simple alimentary delivery of oligonucleotides and other nucleic acids
The present invention overcomes these and other drawbacks by providing compositions and methods for the above.

The present invention further includes a composition using a ribozyme. Synthetic RNA molecules and their derivatives that cause high specific endoribonuclease activity are known as ribozymes. (Generally, US patents issued August 6, 1996 by Haseloff et al.
See US Pat. No. 5,543,508 and U.S. Pat. No. 5,545,729 issued Aug. 13, 1996 by Goodchild et al. ) The splitting reaction is catalyzed by the RNA molecule itself. In naturally occurring RNA molecules, the site of autocatalytic cleavage is located within a highly conserved region of RNA secondary structure (
Natl. Acad. Sci. USA, 1986, 83, 8859; Forester et al., C.
ell, 1987, 50, 9). Naturally occurring autocatalytic RNA molecules have been modified to produce ribozymes that can be targeted with a high degree of specificity to particular cells or pathogenic RNA molecules. Thus, ribozymes are nucleic acids that serve the same general purpose as antisense oligonucleotides (ie, regulate the expression of specific genes) and, as with oligonucleotides, have single-stranded elements as an important part. This means that ribozymes have essentially chemical and functional identity to oligonucleotides and are therefore considered equivalent for the purposes of the present invention.

Other biologically active oligonucleotides will be formulated as the compositions of the present invention and will be used by the methods of the present invention for therapeutic, symptomatic, or prophylactic purposes. Such other biologically active oligonucleotides are, inter alia,
Antisense oligonucleotides, antisense PNAs and ribozymes (described above)
And EGS, as well as aptamers and molecular decoys (described below).

The sequence recruiting (recruiting) RNase P has an external guide sequence (External Guide Seq).
uences), hereinafter abbreviated as “EGS”. EGSs are antisense compounds that direct an endogenous nuclease to the targeted nucleic acid (Fo
rster et al., Science, 1990, 249, 783; Geurrier-Takada et al., Proc. Natl. Acad.
Sci. USA, 1997, 94, 8468).

Instead of relying on the recruitment of endogenous nucleases, the antisense compounds can alternatively or additionally comprise a synthetic moiety having nuclease activity covalently linked to an oligonucleotide having an antisense sequence. . Synthetic moieties having nuclease activity include, but are not limited to, enzymatic RNAs (such as ribozymes), lanthanide ion complexes, and the like (Haseloff et al., Nature, 1988, 334, 585).
Baker et al., J. Am. Chem. Soc., 1997, 119, 8749).

Aptamers are single-stranded oligonucleotides that bind to a specific ligand by a mechanism other than Watson-Crick base pairing. Aptamers generally target, for example, proteins and are not intended to bind to nucleic acids (
Ellington et al., Nature, 1990, 346, 818).

Molecular decoys are short, double-stranded nucleic acids that mimic sites on nucleic acids to which factors such as proteins bind (single-stranded nucleic acids intended to “fold back” on themselves) ). Such decoys are expected to competitively inhibit the factor. That is, by binding the factor molecule to the excess decoy,
The concentration of the factor that binds to the site of the cell decreases in response to the decoy, resulting in a therapeutic, palliative, or prophylactic effect. Methods for identifying and constructing nucleic acid molecule decoys are described, for example, in US Pat. No. 5,716,780 to Edwards et al.

Another type of bioactive oligonucleotide is the gene conversion of endogenous nucleic acids (gene c
onversion) is an RNA-DNA hybrid molecule (Cole-Strauss et al., Sc
ience, 1996, 273, 1386).

It has been discovered by the present invention that pulmonary administration of phosphodiester oligonucleotides is particularly useful. In particular, the nuclease activity level in lung tissue is low enough,
It has been discovered by the present invention that there is more room to preserve the longevity of phosphodiester oligonucleotides in lung tissue than previously believed. Thus, contrary to conventional knowledge in the art (eg, Wu-Pong et al., Adv. Drug Deliv
ery, 1996, 19, 47), phosphodiester antisense oligonucleotides are present in the lungs without degradation for long enough to exert antisense effects.

In a further preferred embodiment, the present invention provides an oligonucleotide having at least one 2′-alkoxy-alkyloxy substituent, preferably 2′-methoxyethoxy, preferably a phosphodiesterized and phosphorothioated oligonucleotide. provide. The presence of such a 2'-alkoxy-alkyloxy substituent has been found to confer nuclease resistance and enhanced binding. Further suitable modifications include 2′-dimethylaminooxyethoxy (ie, O (CH ₂ ) ₂ ON (CH ₃ ) ₂ group, also known as 2′-DMAOE), co-application 1998. No. 09 / 016,520, filed Jan. 30, 1998, the contents of which are incorporated herein by reference. Other suitable modifications are 2'-methoxy (2'-O-
CH ₃ ), 2′-aminopropoxy (2′-OCH ₂ CH ₂ CH ₂ NH ₂ ) and 2′-fluoro (2′-F)
including.

[0071] Chemical modifications of other specific oligonucleotides are described in the following subsections.

It is not necessary that all positions in a given compound be uniformly modified; in fact, one or more of the following modifications may be made to one antisense compound or one residue thereof (eg, (In one nucleoside).

Base Modification: For each nucleoside of the oligonucleotide, the base portion of the nucleoside may be selected from a variety of different base units available. These include "modified" or "natural", including the natural purine bases, adenine (A) and guanine (G), and the natural pyrimidine bases, thymine (T), cytosine (C) and uracil (U),
It may be a "natural" base (also referred to herein as a nucleobase). These may further include modified nucleobases including other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, xanthine,
Hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil And cytosine, 6-azouracil, cytosine and thymine, 5-uracil (
Pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halouracils and cytosines, especially 5-bromo , 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3- Deazaadenine. Additional nucleobases include those disclosed in U.S. Patent No. 3,687,808, Concise Encyclopedia Of P
olymer Science And Engineering, pp. 858-859, supervised by Kroschwitz, JI, Joh
n Disclosed in Wiley and Sons, 1990, Englisch et al., Angewandte Chemie, I.
nternational Edition, 1991, 30, 613, and Sanghvi, YS
Chapter 15, Antisense Research and Applications, pages 289-302, Crooke,
Includes those disclosed in ST and Lebleu, B. Supervision, CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. included. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2 ° C (Sanghvi, YS, Crooke, ST and Lebleu, B. Supervision, Antisense Researc
h and Applications, CRC Press, Bocalayton, 1993, pp. 276-278), and are currently preferred for selection as bases. These are described below, 2'-
It is particularly useful when combined with a methoxyethyl sugar modification.

Further exemplary nucleobases include adenine, guanine, cytosine, uridine,
And other non-naturally occurring and natural nucleobases with thymine, such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 6-azouracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, Includes 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine. Additional natural and non-naturally occurring nucleobases include:
No. 3,687,808 (Merigan et al.), Sanghvi, YS, No. 15
Chapter, Antisense Research and Applications, supervised by ST Crooke and B. Lebleu, disclosed in CRC Press, 1993, Englisch et al., Angewandte Chemie, Inte
rnational Edition, 1991, 30, 613-722 (see especially pages 622 and 623), and Concise Encyclopedia of Polymer Science and
Engineering, Kroschwitz, supervised by JI, John Wiley and Sons, 1990, pp. 858-859, Cook, PD, Anti-Cancer Drug Design, 1991, 6, 585-607. , Fully incorporated herein by reference. The term "nucleosidic base" is further intended to include heterocyclic compounds that can act like nucleosidic bases, and in the most classical sense are not nucleosidic bases, but as nucleosidic bases A specific "universal base" that acts
Is included. Of particular mention as a universal base is 3-nitropyrrole.

Representative US patents that describe the preparation of other modified nucleobases along with certain of the above modified nucleobases include, but are not limited to, the aforementioned US Pat. No. 3,687,808.
No. 4,845,205; 5,130,302; 5,134,066; 5,175,2.
No. 73; No. 5,367,066; No. 5,432,272; No. 5,457,187; No. 5,459,255; No. 5,48
No. 4,908; No. 5,502,177; No. 5,525,711; No. 5,552,540; No. 5,587,469; No. 5
No. 5,594,121; 5,596,091; 5,614,617; and 5,681,941. No. 08 / 762,488, filed Dec. 10, 1996, and granted, which is commonly owned and incorporated herein by reference, is also incorporated herein by reference.

In selecting bases for any particular nucleoside of the oligonucleotide, one first considers the need for the base for certain specificities with respect to hybridization to the opposite strand of the particular target. Thus, if an “A” base is required, adenine may be selected, but other considerations, such as when stronger hybridization is desired (compared to that achieved with adenine), 2,6- Other alternative bases capable of achieving hybridization in a manner that mimics the “A” base, such as diaminopurine, may be selected.

Sugar modification: For each nucleoside of the oligonucleotide, the sugar moiety of the nucleoside may be selected from a variety of different sugars or sugar surrogate units available. These may be modified sugar groups, for example sugars containing one or more substituents. Preferred substituents include at the 2 'position: OH; F; O-, S-, or N-alkyl, O
-, S-, or N- alkenyl or O, S-, or an N- alkynyl, the alkyl, alkenyl and alkynyl, to C ₁₀ alkyl or C ₂ to C ₁ to be replaced by C ₁₀ alkenyl and alkynyl May be substituted or unsubstituted. Particularly _{preferred, O [(CH 2) n} O] m CH 3, O (CH 2) n OCH 3, O (CH 2) n NH 2, O
(CH ₂ ) _n CH ₃ , O (CH ₂ ) _n ONH ₂ , and O (CH ₂ ) _n ON [(CH ₂ ) _n CH ₃ )] _{2 wherein} n and m are 1 to about 10 Things. Other preferred substituents include at the 2 ′ position one of the following: C ₁ to C ₁₀ lower alkyl, substituted lower alkyl, alkaryl,
O- alkaryl or O- _{aralkyl, SH, SCH 3, OCN,} Cl, Br, CN, CF 3, OCF 3, SO
CH ₃ , SO ₂ CH ₃ , ONO ₂ , NO ₂ , N ₃ , NH ₂ , heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator ( intercalator), a group for improving the pharmacokinetic properties of the oligonucleotide, or a group for improving the pharmacodynamic properties of the oligonucleotide, and other substituents having similar properties. Preferred modification includes 2'-methoxyethoxy _{(2'-OCH 2 CH 2 OCH} 3, 2'-O- (2- methoxyethyl) or also known as 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78, 486), ie, including alkoxyalkoxy groups. A further preferred modification is 2'-dimethylaminooxyethoxy, also known as 2'-DMAOE, as described in commonly owned U.S. patent application Ser. No. 09 / 016,520, filed Jan. 30, 1998. O (CH ₂ ) ₂ ON
(CH ₃ ) ₂ groups are included, and the contents of the application are incorporated herein by reference.

Other preferred modifications include 2′-methoxy (2′-O—CH ₃ ), 2′-aminopropoxy (
2′-OCH ₂ CH ₂ CH ₂ NH ₂ ) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on the sugar group, especially at the 3 'position of the sugar on the 3' terminal nucleotide or in the 2'-5 'linked oligonucleotide and the 5' position of the 5 'terminal nucleotide. Good. Oligonucleosides of oligonucleotides may also have sugar mimetics such as cyclobutyl moieties instead of pentofuranosyl sugars.

Representative US patents describing the preparation of such modified sugar structures include, but are not limited to, US Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359.
No. 5,043; No. 5,393,878; No. 5,446,137; No. 5,466,786; No. 5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;
5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873.
No. 5,670,633; and 5,700,920, of which certain are owned by the present application, and each of said patents is incorporated herein by reference.
United States Patent Application No. 08 / 468,037, filed June 5, 2016, is also incorporated herein by reference.

Modified Linkage (Backbone): In addition to phosphodiester linkages, certain examples of some preferred modified oligonucleotides envisioned for the present invention include the moiety “M” in the compounds described herein. And those containing modified internucleoside linkages, represented as These internucleoside linkages are also called linkers, backbones or oligonucleotide backbones. A variety of different internucleoside linkages or backbones are available to form these nucleoside linkages. These include modified oligonucleotide backbones, such as phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, 3'-alkylene phosphonates and chiral phosphonates. Including, including methyl and other alkyl phosphonates, phosphinates, 3'-amino phosphoramidates and aminoalkyl phosphoramidates, phosphoramidates, thiono phosphoramidates, thionoalkyl Phosphonates, thionoalkyl phosphotriesters, and boranophosphates, wherein the modified oligonucleotides have a normal 3'-5 'linkage, their 2'-5' linked analogs, and nucleoside units Adjacent pairs are 3'-5 'to 5'-3'
Or those having the opposite polarity linked from 2'-5 'to 5'-2'. Various salts, mixed salts and free acid forms are also included.

Representative United States patents that describe the preparation of the above phosphorus atoms, including linkages, include, but are not limited to, US Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
No. 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019
No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,399,676; No. 5,405,
No. 939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,5
19,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253;
5,571,799; 5,587,361; 5,625,050; and 5,697,248,
Certain of these are owned by the present application, and each of the foregoing patents is hereby incorporated by reference.

For oligonucleotides that do not contain a phosphorus atom, ie, oligonucleosides, preferred internucleoside linkages are short chain alkyl or cycloalkyl sugar linkages, mixed heteroatoms and alkyl or cycloalkyl sugar linkages, or 1
It has a backbone formed by one or more short chain heteroatoms or heterocyclic sugar linkages. These include morpholino linkages (partially formed from the sugar portion of the nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones;
Formacetyl and thioformacetyl skeleton; methyleneformacetyl and thioformacetyl skeleton; alkene-containing skeleton; sulfamate skeleton; methyleneimino and methylenehydrazino skeleton; sulfonate and sulfonamide skeleton;
Those with an amide skeleton; and others with mixed N, O, S and CH ₂ component moieties are included.

Representative US patents describing the preparation of the above-described oligonucleosides include, but are not limited to, US Pat. Nos. 5,034,506; 5,166,315; 5,185,444;
No. 5,214,134; No. 5,216,141; No. 5,235,033; No. 5,264,562; No. 5,264,564
No. 5,405,938; No. 5,434,257; No. 5,466,677; No. 5,470,967; No. 5,489,
No. 677; No. 5,541,307; No. 5,561,225; No. 5,596,086; No. 5,602,240; No. 5,6
No. 10,289; No. 5,602,240; No. 5,608,046; No. 5,610,289; No. 5,618,704;
5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,
No. 439, of which certain are the same as this application, and each of said patents is incorporated herein by reference.

In other preferred oligonucleotides, ie oligonucleotide mimetics, both the sugar and intersugar linkages of the nucleotide unit, ie the backbone, are replaced with new groups. Base units are maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound is an oligonucleotide mimetic that has been shown to have excellent hybridization properties and is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar-phosphate backbone of the oligonucleotide is replaced with an amide-containing backbone, especially an aminoethylglycine backbone. The nucleobase is retained and attached, directly or indirectly, to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents describing the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Incorporated. A further description of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497.

With respect to internucleoside linkages, the most preferred embodiments of the present invention include oligonucleotides having a phosphorothioate backbone and oligonucleosides having a heteroatom backbone, and especially —CH ₂ —NH— of US Pat. No. 5,489,677, cited above. O-CH _2- , -CH _2-
(Known as a methylene (methylimino) or MMI _{backbone), - N (CH 3)} -O-CH 2
_{-CH 2 -ON (CH 3) -CH} 2 -, - CH 2 -N (CH 3) -N (CH 3) -CH 2 - and -ON (CH ₃₎ -CH ₂ -C
H _2- (the natural phosphodiester backbone is shown as —OPO—CH ₂ —), as well as the amide backbone oligonucleotide of US Pat. No. 5,602,240, cited above. Also preferred are the oligonucleotides having a morpholino backbone structure of US Pat. No. 5,034,506, cited above.

Conjugate: The attachment of an effector group to one or more nucleosides or internucleoside linkages of the oligonucleotide modifies various properties of the oligonucleotide. An “effector group” is a chemical moiety capable of performing a specific chemical or biological function. Examples of such effector groups include, but are not limited to, an RNA cleaving group, a reporter group, an intercalating agent, a group for improving the pharmacokinetic properties of the oligonucleotide, or for improving the pharmacodynamic properties of the oligonucleotide. And other substituents having similar properties. A variety of chemical linkers may be used to attach effector groups to the oligonucleotides of the invention.

The oligonucleotides are modified at the 5 ′ and 3 ′ termini, for example, lipophilic
Part (see paragraph immediately following), intercalating agent (Kuyavin et al., WO 96/32496, 1996).
Nguyen et al., US Pat. No. 4,835,263, issued May 30, 1989)
Alternatively, it may be used as a chemical bonding point of a hydroxyalkyl group (Helene et al., WO 96/34008, published on October 31, 1996).

Other positions within the oligonucleotide of the invention may be used to chemically link one or more effector groups to form an oligonucleotide conjugate. For example, U.S. Pat.No. 5,578,718 to Cook et al. Discloses a method of attaching an alkylthio linker to a ribofuranosyl position, a nucleosidic base position, or on an internucleoside linkage, which may be further derivatized to include additional groups. I do. Additional methods of attaching oligonucleotides to various effector groups are known in the art; for example, Protocols for Oligonucleotide Conjugates (Methods in Mol
ecular Biology, Vol. 26) Supervised by Agrawal, S., Humana Press, Totowa, NJ, 1994.

Another preferred further or alternative modification of the oligonucleotides of the invention is that one or more lipophilic moieties that enhance cellular uptake of the oligonucleotide include:
It involves chemically linking to the oligonucleotide. Such a lipophilic moiety may be linked to the oligonucleotide at several different positions on the oligonucleotide. Some preferred positions include the 3 'position of the sugar at the 3' terminal nucleotide, the 5 'position of the sugar at the 5' terminal nucleotide, and the 2 'position of the sugar at any nucleotide. N ⁶ position of a purine nucleobase may also lipophilic moiety, may be utilized to connect to an oligonucleotide of the present invention (Gebeyehu, G. et al., Nucleic Acids Res, 1987, 15 :. 4513
). Such lipophilic moieties include, but are not limited to, cholesteryl moieties (
Nats. Acad. Sci. USA, 1989, 86: 6553), cholic acid (
Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4: 1053), thioesters such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 19).
92, 660: 306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3: 2765), thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20: 533), aliphatic chains such as dodecane. Diol or undecyl residues (Saison-Behmoaras et al., EM
BO J., 1991, 10: 111; Kabanov et al., FEBS Lett., 1990, 259: 327; Svinarchuk.
Biochimie, 1993, 75:49), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3.
-H-phosphonates (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651; Shea et al., Nucl. Acids Res., 1990, 18: 3777), polyamine or polyethylene glycol chains (Manoharan et al., Nucleosides and Nucleotides, 1995, 14). : 969), or adamantaneacetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36: 3651),
Palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264: 229) or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277: 923). Is included. Oligonucleotides containing lipophilic moieties, and methods for preparing such oligonucleotides are described in U.S. Patent Nos. 5,138,045, 5,218,105 and 5,459,
No. 255, the contents of which are incorporated herein by reference.

Representative US patents describing the preparation of such oligonucleotide conjugates include:
Without limitation, U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,
No. 105; No. 5,525,465; No. 5,541,313; No. 5,545,730; No. 5,552,538; No. 5,5
No. 5,580,731; No. 5,580,731; No. 5,591,584; No. 5,109,124;
5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,02.
No. 5, No. 4,762,779; No. 4,789,737; No. 4,824,941; No. 4,835,263; No. 4,876
No. 4,904,582; No. 4,958,013; No. 5,082,830; No. 5,112,963;
No. 5,082,830; No. 5,112,963; No. 5,214,136; No. 5,245,022;
No. 5,254,469; No. 5,258,506; No. 5,262,536; No. 5,272,250; No. 5,292,873
No. 5,317,098; 5,371,241; 5,391,723; 5,416,203; 5,451,
No. 463; No. 5,510,475; No. 5,512,667; No. 5,514,785; No. 5,565,552; No. 5,5
No. 67,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726;
Nos. 5,597,696; 5,599,923; 5,599,928 and 5,688,941, of which certain are owned by the present application and each of the patents is incorporated herein by reference.

Oligonucleotide Synthesis: Oligonucleotides used in accordance with the present invention can be conveniently and routinely made through the well-known technique of solid phase synthesis. Unsubstituted and substituted phosphodiester oligonucleotides are synthesized on an automated DNA synthesizer (Applied Biosystems model 380B) using standard phosphoramidite chemistry using iodine oxidation.

[0092] Phosphorothioates are used due to the stepwise thiation of the phosphite linkage,
Synthesized similarly to the phosphodiester oligonucleotide, except that the standard oxidation bottle was replaced by a 0.2 M solution of 3H-1,2-benzodithiol-3-one 1,1-dioxide in acetonitrile. The thionation wait step was increased to 68 seconds and the capping step was followed. Cut from the CPG column and in concentrated ammonium hydroxide at 55 ° C (1
After 8 hours), the oligonucleotide was purified by precipitation twice from a 0.5 M NaCl solution with 2.5 volumes of ethanol.

[0093] The phosphinate oligonucleotides are disclosed in US Pat.
Prepared as described in 8,270.

[0094] Alkyl phosphonate oligonucleotides are prepared as described in US Pat. No. 4,469,863, which is incorporated herein.

[0095] 3'-deoxy-3'-methylene phosphonate oligonucleotides are prepared as described in US Patent Nos. 5,610,289 or 5,625,050, which are incorporated herein by reference.

[0096] Phosphoramidite oligonucleotides are described in US Pat.
Prepared as described in 5,256,775 or US Patent 5,366,878.

Alkyl phosphonothioate oligonucleotides are described in published PCT application PCT / US94 /
Prepared as described in 00902 and PCT / US93 / 06976 (published as WO 94/17093 and WO 94/02499, respectively).

[0098] 3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as described in US Pat. No. 5,476,925, which is incorporated herein by reference.

[0099] Phosphotriester oligonucleotides are described in US Pat.
Prepared as described in 5,023,243.

The boranophosphate oligonucleotides are prepared as described in US Pat. Nos. 5,130,302 and 5,177,198, both of which are incorporated herein by reference.

[0101] United States patents, all of which are incorporated herein by reference.
5,378,825; 5,386,023; 5,489,677; 5,602,240 and 5,610,289
Methylene-linked oligonucleosides are also identical, methylene methylamino-linked oligonucleosides, MDH-linked oligonucleosides are also identical, methylene dimethylhydrazo-linked oligonucleosides and amide-3-linked oligonucleosides Also identical are methylene carbonyl amino linked oligonucleosides and amide-4 linked oligonucleosides which are also identical, such as, for example, alternating
) Prepare mixed backbone compounds with MMI and PO or PS bonds.

[0102] Formacetal and thioformacetal-linked oligonucleosides are prepared as described in US Patent Nos. 5,264,562 and 5,264,564, incorporated herein by reference.

[0103] Ethylene oxide linked oligonucleosides are prepared as described in United States Patent No. 5,223,618, incorporated herein by reference.

Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential App
Peptide nucleic acids (PNAs) are prepared according to all of the various procedures referenced in lications, Bioorganic & Medicinal Chemistry, 1996, 4,5. They are also incorporated by reference herein, U.S. Patent Nos. 5,539,082; 5,7
No. 00,922 and 5,719,262.

Chimeric oligonucleotides: It is not necessary to uniformly modify all positions of a given compound. In fact, one or more of the above-described modifications can be incorporated into a single compound, or even a single nucleoside in a single oligonucleotide. The present invention also includes compounds that are chimeric compounds. In the context of the present invention, a “chimeric” compound, or alternatively, a “chimeras”, is each composed of at least one monomer unit, ie one nucleotide in the case of one oligonucleotide compound, Compounds containing two or more chemically distinct regions, especially oligonucleotides. These oligonucleotides provide increased resistance to nuclease degradation, increased cellular uptake, and / or
Alternatively, it typically includes at least one region to confer on the oligonucleotide an increased binding affinity for the target nucleic acid. The additional region of the oligonucleotide may serve as a substrate for an enzyme that can cleave RNA: DNA or RNA: RNA hybrids.

By way of example, RNase H is an intracellular endonuclease that cleaves the RNA strand of an RNA: DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby significantly enhancing the effect of the oligonucleotide on inhibiting gene expression.

Thus, when using chimeric oligonucleotides, comparable results can often be obtained with shorter oligonucleotides as compared to phosphorothioate deoxy oligonucleotides that hybridize to the same target region. RNA
Cleavage of the target can be detected in a routine manner by gel electrophoresis and, if necessary, related nucleic acid hybridization techniques known in the art.

The chimeric antisense compounds of the present invention may comprise a combination of two or more oligonucleotides, modified oligonucleotides, oligonucleosides, and / or oligonucleotide analogs, as described above. Can be formed as a mixed structure. Said compounds are also referred to in the art as "hybrids" or "gapmers". Representative U.S. Patents that teach the preparation of the hybrid structures include, but are not limited to, U.S. Patent Nos. 5,013,830, 5,149,797, 5,220,007, 5
No. 5,256,775, No. 5,366,878, No. 5,403,711, No. 5,491,133, No. 5,565,350,
No. 5,623,065, No. 5,652,355, No. 5,652,356, and No. 5,700,922,
Some of them are commonly owned with the present application, and are commonly owned and licensed U.S. Patents filed June 6, 1995, which is hereby incorporated by reference in its entirety. Along with application serial number 08 / 465,880, each of which is incorporated herein by reference in its entirety.

The chimeric oligonucleotides, oligonucleosides, or mixed oligonucleotides / oligonucleosides of the present invention can be of several different types. These are of the first type, wherein the 'gap' portion of the nucleoside to be linked is located between the 5 'and 3' wing 'portions of the nucleoside to be linked, and the' gap 'portion of the oligomeric compound is Includes a second 'open end' type located at either the 5 'or 3' end. Oligonucleotides of the first type may also be referred to in the art as 'gapmers', or alternatively, gapped.
) Also known as oligonucleotides. The second type of oligonucleotide is also known in the art as 'hemimers' or alternatively, 'wingmers'.

[2'-O-Me]-[2'-deoxy]-[2'-O-Me] chimeric phosphorothioate oligonucleotide: 2'-O-alkyl phosphorothioate and 2'-deoxy phosphorothioate oligonucleotide moieties Chimeric oligonucleotide having 2'-deoxy-5'-dimethoxytrityl-3'-O- phosphoramidite in the DNA portion, and 5'-dimethoxy-trityl in the 5 'and 3' wing portions. It is synthesized using -2'-O-methyl-3'-O-phosphoramidite. The waiting step after the delivery of the tetrazole and base is repeated four times for DNA and twice for 2'-O-methyl, increasing to 600 seconds by standard synthesis. Modify the cycle. The fully protected oligonucleotide is cleaved from the support and the phosphate group is
Deprotect in 3: 1 ammonia / ethanol at room temperature overnight, then solidify by lyophilization. Treatment in methanolic ammonia for 24 hours at room temperature deprotects all bases and solidifies the sample again by lyophilization.

[2'-O- (2-methoxyethyl)]-[2'-deoxy]-[-2'-O- (methoxyethyl)] chimeric phosphorothioate oligonucleotide: [2'-O- ( 2-methoxyethyl))-[2'-deoxy]-[-2'-O- (methoxyethyl)] chimeric phosphorothioate oligonucleotide is 2'-O-methylamidite instead of 2'-O- Prepared using (methoxyethyl) amidite and following the procedure for 2'-O-methyl chimeric oligonucleotides above, substituting 2'-O- (methoxyethyl) amidite for 2'-O-methylamidite I do.

[2'-O- (2-methoxyethyl) phosphodiester]-[2'-deoxyphosphorothioate]-[-2'-O- (methoxyethyl) phosphodiester] chimeric oligonucleotide: [2 ' -O- (2-methoxyethyl) phosphodiester]-[2'-deoxyphosphorothioate]-[-2'-O- (methoxyethyl) phosphodiester] chimeric oligonucleotide is the above 2'-O-methyl Prepared according to the procedure for chimeric oligonucleotides, using 2'-O- (methoxyethyl) amidite instead of 2'-O-methylamidite in the wing part. 3, H-1,2 benzodithiol-3-one 1
Sulfurization using dioxide (Beaucage reagent) is used to create phosphorothioate internucleotide linkages within the wing portion of the chimeric structure. Oxidation with iodine is used to create mid-gap phosphodiester internucleotide linkages.

Other chimeric oligonucleotides, chimeric oligonucleosides, and mixed chimeric oligonucleotides / oligonucleosides are synthesized according to US Pat. No. 5,623,065, which is incorporated herein by reference.

The present invention also includes oligonucleotides that are physically pure in terms of chirality, with a focus on specific portions within the oligonucleotide. Examples of oligonucleotides that are materially pure for chirality include, but are not limited to,
Those having a phosphorothioate bond that is at least 75% Sp or Rp (Co
ok et al., US Pat. No. 5,587,361) and materially including chiralities that have pure (Sp or Rp) alkyl phosphonate, phosphoamidate, or phosphotriester linkages (Cook, US Patent Nos. 5,212,295 and 5,521,302).

Examples of specific oligonucleotides that can be used in the formulations of the invention and the target genes they inhibit include:

[0116]

Embedded image

In the above, (i) each oligo backbone bond is a phosphorothioate bond (ISIS-9
(Except 605 and ISIS-17709), and (ii) each saccharide is 2'-deoxy unless indicated in bold and incorporates a 2'-O-methoxyethyl group in bold. (Iii) The underlined cytosine nucleoside has 5-methyl incorporated in the substituent of the nucleobase. ISIS-9605 incorporates a natural phosphodiester bond in the first five and last five bonds, with the remainder being phosphorothioate bonds. ISIS-17709 incorporates a natural phosphodiester bond in the first four and last four bonds, with the remainder being phosphorothioate bonds.

The formulation of the drug formulation and subsequent administration is considered to be within the skill of those in the art. A special description of the present invention is provided below.

Therapeutic Considerations: In general, in therapeutic applications, a subject (ie, an animal, including a human, having, suspected of, or becoming susceptible to, having a disease or disorder) is administered to a subject. A nucleic acid comprising one or more oligonucleotides according to the invention at a dose ranging from 0.01 μg to 100 g per kg body weight, depending on the age of the subject and the weight of the disease or disease state being treated. Is administered. In addition, the treatment program can be continued for a period that will vary depending on the nature of the particular disease or disorder, its severity, and all the conditions of the subject, and can vary from once per day. It can be extended to once every 20 years. The terms "treatment" or "treatment program" in the context of the present invention are meant to include therapeutic palliative and prophylactic aspects. Following treatment, the subject is monitored for changes in his / her condition and alleviation of symptoms of the disease or condition. The dose of nucleic acid may be such that at the current dose level the subject does not show a significant response,
It may be increased or reduced if symptoms of the disease or condition are seen or if the disease or condition is eliminated.

Dosing will depend on the severity or responsiveness of the disease state being treated and, as the treatment progresses, may last from days to months, or until treatment is achieved or the disease state is alleviated. . Optimal dosing schedules can be calculated from measurements of drug accumulation in the subject. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimal dosages may vary depending on the relative potency of the individual oligonucleotides, and are generally
effect in animal models of ro and in vivo are observed, can be evaluated and EC ₅₀ based. Generally, the dose will be from 0.01 μg to 100 g per kg body weight, and once or more daily, weekly, monthly or yearly, or from 2 to
May be given regularly once every 20 years. Optimal dosage regimens are used to deliver a therapeutically effective amount of the administered nucleic acid in a particular manner of administration.

For the purposes of the present invention, the term “therapeutically effective amount” refers to a compound that is effective to achieve its intended purpose without undesirable side effects (eg, toxic, inflammatory, or allergic reactions). Refers to the amount of nucleic acid contained in the formulation.

While individual needs may vary, determination of optimal ranges for effective amounts of prescription is within the skill of the art. Human dosages can be estimated based on animal studies (Katocs et al., Chapter 27 In: Remington's Pharmaceutical Sciences, 18th.
Ed., Gennaro, ed., Mack Publishing Co., Easton, PA, 1990). Generally, the dosage required to provide an effective amount of the formulation, prepared by one of ordinary skill in the art,
It may vary according to age, health, physical condition, weight, type and extent of the recipient's disease or disorder, frequency of treatment, the nature of the concurrent treatment (if any), and the extent of the desired effect ( Nies et al., Chapter 3 In: Goodman &Gilman's
The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al., Eds.,
McGraw-Hill, New York, NY, 1996).

As used herein, the term “high risk person” refers to the likelihood of the onset or recurrence of a disease or disorder, eg, through an individual or family history, or genetic testing. Means a person who is clearly determined to be more likely than normal. As a technology of a treatment program for high-risk individuals, the individual can be treated prophylactically to prevent the onset or recurrence of the disease or disorder. The term “prophylactically effective amount” is meant to refer to an amount of a formulation that produces an effect that is observed as preventing the onset or recurrence of a disease or disorder. A prophylactically effective amount of a prescription is typically determined by the effect compared to the effect observed when a second preservative lacking the active drug is administered to a person with a similar condition. You.

Following successful treatment, a range of from 0.01 μg to 100 g / kg body weight, once daily or more to once every 20 years for prevention of onset of disease or prevention of relapse. It may be desirable to have the nucleic acid administered at a maintenance dose, undergoing maintenance therapy. For example, for individuals known or suspected of being susceptible to an autoimmune or inflammatory condition, from 0.01 μg to 100 g / kg body weight, once daily or more often once every 20 years By administering a prophylactic amount of a range, a prophylactic effect may be achieved. In the same way, an inflammatory condition that is expected to occur as a result of a treatment, such as a graft versus host disease, resulting from the transplantation of cells, tissues, or organs into a person, can Can be difficult.

The compositions of the present invention may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Pharmaceutical formulations, which can be conveniently presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. The technique involves the step of bringing into association the active ingredient with the drug carrier (s) or excipients (s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or neatly separated solid carriers or both.

[0127] 5. Bioequivalents A. Pharmaceutically acceptable salts: The compositions of the present invention can be administered to any pharmaceutically acceptable salt, ester, or salt of said ester, or to animals, including humans. And any other composition capable of providing (directly or indirectly) a biologically active metabolite or a residue thereof. Thus, for example, the disclosure is also directed to penetration enhancers and the "pharmaceutically acceptable salts" of the nucleic acids of the invention, and prodrugs of the nucleic acids. “Pharmaceutically acceptable salts” are physiologically and pharmaceutically acceptable penetration enhancers and salts of the nucleic acids of the present invention, ie, those that retain the desired biological activity of the parent compound. , Are salts that do not impart undesirable toxic effects to it.

The term “pharmaceutically acceptable salt” refers to physiologically and pharmaceutically acceptable oligonucleotides and salts of nucleic acid compounds (ie, salts) for which the compositions of the present invention are used. A salt which retains the desired biological activity of the compound and does not impart undesired toxic effects to it.

Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium,
Polyamines such as ammonium, spermine and spermidine, and the like. Examples of suitable amines are chloroprocaine, choline, N, N'-dibenzylethylenediamine, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (eg, Berge et al.,
See "Pharmaceutical Salts", J. of Pharma Sci., 1977, 66: 1). The base addition salts of said acidic compounds are prepared in conventional manner by contacting the free acid form with a sufficient amount of the desired base to produce the salt. The free acid form can be reproduced by contacting the salt form with an acid and separating off the free acid in a conventional manner. The free acid forms are slightly different from their respective salts in certain physical properties, such as solubility in polar solvents, but in other respects, salts are different from the respective free acids for purposes of the present invention. Are equivalent.

B. Oligonucleotide Prodrugs: The oligonucleotides of the present invention are additionally or alternatively prepared to be carried in a “prodrug” form. The term “prodrug” refers to an active form (ie, drug) that is prepared in an inactive form and that is activated in the body or its cells, depending on endogenous enzymes or other chemicals and / or conditions. Indicates a therapeutic agent that can be changed to In particular, the prodrug forms of the oligonucleotides of the invention are
As ([S-acetyl-2-thioethyl) phosphate] derivative, it is prepared according to the method disclosed by Gosselin et al. In WO 93/24510 published Dec. 9, 1993.

C. Oligonucleotide Deficient Derivatives: During the course of oligonucleotide synthesis, nucleoside monomers are combined at one time in a repeated sequence, such as ligation, oxidation, capping, and detritylation of nucleoside monomers. Linked to one strand. The stepwise yield of the addition of each nucleoside is greater than 99%. The overall result of incomplete ligation leads to incomplete capping, detritylation, and oxidation, so that less than 1% of the sequence strand fails to add nucleoside monomers at each step (Smith, Anal. Chem., 1988, 60, 381A). (N-1)
, (N-2), etc., up to 1 mer (nucleotide), all shorter oligonucleotides are present as impurities in the n-mer oligonucleotide product. Among the impurities, (n-2) -mer and shorter oligonucleotide impurities are present in very small amounts and can be easily removed by chromatographic purification (Warren et al., Chapter 9 In: Methods in Molecular Biology). , Vol. 26: Protoc
ols for Oligonucleotide Conjugates, Agrawal, S., Ed., 1994, Humana Press
Inc., Totowa, NJ, pp. 233-264). However, due to lack of chromatographic selectivity and product yield, the full length (ie, n-mer) after the purification process
Some (n-1) -mer impurities are still present in the oligonucleotide product of This (n-1) portion consists of a mixture of all possible single nucleotide deletion sequences when compared to the original oligonucleotide of the n-mer. The (n-1) impurities can be classified into terminal-deleted or intermediate-deleted sequences depending on the location of the base to be written (that is, either the 5 'or 3' end or inside). When an oligonucleotide containing a single nucleotide deletion sequence impurity is used as a drug (Crooke, Hematologic Path)
, 1995, 9, 59), a truncated sequence impurity can bind to the same target mRNA as the full-length sequence, albeit with slightly lower affinity. Thus, to a certain extent, the impurities can be considered as part of the activated drug component, and are therefore considered to be bioequivalent for the purposes of the present invention.

6. Typical Uses of the Invention: The compositions and methods of the invention include a wide variety of conditions including asthma, lung cancer, pulmonary fibrosis, and rhinovirus, tuberculosis, bronchitis, and pneumonia. Useful for the treatment of various viral infectious diseases.

[0133] occur at the cellular level, two important events that contribute to asthmatic reaction (1) infiltration of the airway lumen by leukocytes, and (2) T lymphocytes from T _H 0 to T _H 2 state ( T cell)
Activation and subsequent production and release of proinflammatory cytokines by activated T cells. Molecules that mediate one or both of these processes are potential targets for treating asthma.

ICAM-1 has been implicated in the pathogenesis of asthma, and monoclonal antibodies to ICAM-1 attenuate eosinophilia and hyperresponse (Wegner et al., Science, 1990, 247, 4).
56). Antisense compounds targeting ICAM-1 are disclosed in U.S. Patent Nos. 5,514,788 and 5,591,623, and co-pending January 20, 1998 and April 1, 1998, respectively.
U.S. Patent Application Serial Nos. 09 / 009,490 and 09 / 062,416 filed on the 7th
(All by Bennett), each of which is incorporated herein in its entirety.

Adhesion molecule-mediated recruitment of eosinophils and other leukocytes is involved in the mechanism of asthmatic inflammation (Bochner et al., Annu. Rev. Immunol., 1994, 12, 2).
95). In addition to ICAM-1, adhesion molecules of particular interest include ELAM-1 (aka E-selectin) and VCAM-1. Antibodies to ELAM-1 prevent neutrophil accumulation in monkey lung (Gundel et al., J. Clin. Invest., 1991, 88, 1407). Antisense compounds targeting the adhesion molecules ELAM-1 and VCAM-1 are described in U.S. Pat.
No. 8 and 5,591,623.

A novel therapeutic class of anti-inflammatory agents in which inhibitors of ICAM-1, VCAM-1 and ELAM-1 expression have activity against a wide range of inflammatory diseases or diseases in inflammatory components such as asthma It was expected to be able to provide. The use of neutralizing antibodies to ICAM-1 in animal models provides a wealth of evidence that such inhibitors, when identified, may have a therapeutic effect on asthma (Wegner et al. , Science
1990, 247, 456-459).

B7-1 and B7-2 are believed to be the first molecules expressed on professional antigen presenting cells (APC) (Liu and Linsley, Curr. Opin. Immunol.,
1992, 4, 265). The B7 protein provides an essential signal for the differentiation of T cells (TH0 lymphocytes) and is thought to contribute to the activation of memory cells. Antisense compounds targeting the B7 protein were co-pending December 1996
No. 08 / 777,266, filed on March 31, filed by Bennett et al.

Another molecule that is expressed on APCs and stimulates T cell activity is CD40 (for overview,
See Banchereau et al., Annu. Rev. Immunol., 1994, 12, 881). Antisense compounds targeting CD40 are described in co-pending U.S. Patent Application Serial No. 09 / 071,433, filed May 1, 1998 by Bennett et al.

Yet another molecule that is expressed on APCs and stimulates T cell activity is LFA-3 (Liu
and Linsley, Curr. Opin. Immunol., 1992, 4, 265). Antisense compounds targeting LFA-3 are described in co-pending US Patent Application Serial No. 09 / 045,106, filed March 20, 1998 by Bennett et al.

The PECAM-1 protein is a glycoprotein expressed on the surface of various cell types (for overview, see Newman, J. Clin. Invest., 1997, 99, 3 and DeLisser et al.
l., Immunol. Today, 1994, 15, 490). In addition to directly participating in cell-cell interactions, PECAM-1 also apparently regulates the activity and / or expression of other molecules involved in cell interactions (Litwin et al., J. Cell. Biol., 1997
, 139, 219), and are therefore key mediators of some cell: cell interactions. An antisense compound targeting PECAM-1 was announced on March 19, 1998 by Bennet
This is described in co-pending US patent application Ser. No. 09 / 044,506, filed by T et al.

The compositions and methods of the present invention are useful for treating lung cancer. For example, antisense oligonucleotides targeting any of a number of molecular targets associated with tumor development, maintenance of hyperproliferation and metastasis, prevent or inhibit lung cancer,
Alternatively, they can be targeted to prevent their spread to other tissues.

The ras oncogene is a guanine-binding protein, for example due to the fact that activated ras oncogene was found in about 30% of human tumors in general; this figure is 100 in exocrine pancreatic carcinoma. % (For overview, Downward, Tre
nds in Biol. Sci., 1990, 15, 469). In addition, the endotracheal device of the retroviral antisense K-ras construct has been used in orthotopic (orthotopic) animal models.
) Prevents human lung cancer growth, indicating a potential antisense approach to lung cancer (Georges, RN, et al., Cancer Research, 1993, 53, 1743).
. Antisense compounds targeting H-ras and K-ras are disclosed in U.S. Patent Nos. 5,582,972 to Lima et al., 5,582,986 to Monia, and 5,661 to Cook et al.
No. 134, and PCT application WO 94/08003, the disclosures of which are incorporated herein by reference in their entirety.

[0143] Protein kinase C (PKC) proteins are also involved in tumorigenesis. Antisense compounds targeting the protein kinase C (PKC) protein are described in U.S. Patent No. 5,620,963 to Cook et al. And 5,681,747 to Boggs et al.

The compositions and methods of the present invention are useful for treating pulmonary fibrosis. Phan (Thorax,
1995, 50, 415) review the current thinking on pulmonary fibrosis, and potential targets for treatment include cell adhesion and / or T cell stimulating molecules (
For example, ICAM-1, ELAM-1, VCAM-1, B7 protein, CD40, LFA-3, PECAM-1, described above). Antisense oligonucleotides targeting one or more of these proteins are sensitive to using the compositions and methods of the invention.

The compositions and methods of the invention also find use in the treatment and / or prevention of rhinovirus. For example, ICAM-1 is a cell receptor for the major serotype of rhinovirus, which has been proposed to account for more than 50% of common colds (Staunton et al., Cell, 1989, 56, 849, Greve et al.
, Cell, 1989, 56, 839).

The compositions and methods of the present invention also find use in the treatment of tuberculosis. For example, an antisense compound targeting the pathogen Mycobacterium tuberculosis or M. bovis can be administered to a patient according to the methods of the invention.

In cases where acute bronchitis results from an infection, the bronchitis is treated by administering one or more antisense compounds targeting the appropriate pathogen in accordance with the methods of the present invention. be able to.

For the compositions and methods of the present invention, for example, the pathogen Streptococcus pneumo
Administering an antisense compound targeting niae also finds use in treating pneumonia.

In addition to the foregoing, the methods and compositions of the present invention also relate to antisense oligonucleotides that target genes involved in other pulmonary conditions. These include, for example, viruses that infect the lungs (eg, respirat rash virus (respirat
ory syncytial virus), H. influenza, parainfluenza), obstructive pulmonary disorders such as pulmonary embolism or anaphylaxis, chronic obstructive pulmonary disease (COPD),
Includes emphysema, chronic bronchitis, bronchiectasis and cystic fibrosis.

The present invention describes the pulmonary administration of biologically active nucleic acids, such as oligonucleotides, to animals. By “having biological activity”, a nucleic acid functions as a reflection of the absolute function of a gene (eg, ribozyme activity), or by producing a protein encoded by such a gene,
It refers to regulating the expression of one or more genes in an animal. In the context of the present invention, "modulate" means to exert either an increase (stimulation) or a decrease (inhibition) on the expression of a gene. Such regulation includes, for example, transcriptional arrest; effects on RNA processing and transport (capping, polyadenylation, and splicing); enhanced or reduced cellular degradation of target nucleic acids; and translational arrest (Cr
Patents, 1996, 6: 1), by various mechanisms known in the art, including, but not limited to, ooke et al., Exp. Opin. Ther. it can.

In non-human animals, the compositions and methods of the invention can be used to study the function of one or more genes in an animal. For example, to study the function of the N-methyl-D-aspartate receptor in neuronal cell death, antisense oligonucleotides to rats, to mice to study the biological function of protein kinase Ca, to mice, And neuropeptides in anxiety
Rats were administered systemically to examine the function of the Y1 receptor (Wahles
tedt et al., Nature, 1993, 363: 260; Dean et al., Proc. Natl. Acad. Sci.
USA, 1994, 91: 11762; and Wahlestedt et al., Science, 1993, 259: 528
). In the case of studying a complex family of related proteins, "antisense knockout" (ie, inhibition of a gene by systemic administration of an antisense oligonucleotide) represents the most rigorous means to probe for specific members of the family (In general, Albert et al., Trends Pharmacol. S
ci., 1994, 15: 250).

The compositions and methods of the present invention can be performed on animals, including humans, having or suspected of having a disease or condition treatable in whole or in part with one or more nucleic acids. Or therapeutic, ie, to provide therapeutic, palliative, or prophylactic relief. The term "disease or condition" includes (1) abnormal conditions of any of the organisms or parts thereof, especially as a result of infection, genetic defects, environmental stress that impairs normal physiological function; 2) Excluding pregnancy itself, but not autoimmune or other diseases associated with pregnancy; (3) Includes cancer and tumors. “Having or suspected of having or being susceptible to” means that a subject animal is at risk of developing a particular disease or condition as defined herein. Indicates an increased condition has been determined or suspected compared to the general population. For example, a subject animal can have a personal and / or family medical history, including the frequency of occurrence of a particular disease or condition. As another example, a subject animal can have such susceptibility as determined by genetic screening according to techniques known in the art (eg, U.S. Pat.
S. Congress, Office of Technology Assessment, Chapter 5 In: Genetic
Monitoring and Screening in the workplace, OTA-BA-455, US Government P
rinting Office, Washington, DC, 1990, pages 75-99). The term "disease or condition treatable in whole or in part with one or more nucleic acids" refers to the administration, modulation or treatment of (1) And / or (2) Refers to a disease or condition as defined herein that can provide therapeutic, palliative and / or prophylactic relief. In preferred embodiments, such diseases or conditions are wholly or partially treatable with antisense oligonucleotides.

[0153] Preferably, the compounds and methods of the present invention utilize particles containing an oligonucleotide therapeutic or diagnostic agent. The particles may be solid or liquid and have a respirable size: a size small enough to pass through the mouth and larynx and into the bronchi and alveoli of the lungs upon inhalation. It is preferably a particle. In general, particles in the size range of about 5-20 microns are respirable and expected to reach the bronchi (Allen, Secundum Artem, online publication updated May 8, 1998) Vol. 6, No. 3, and http: // w
ww.paddocklabs.com/secundum/secarndx.html). It is desirable to avoid non-respirable sized particles as they tend to accumulate or be swallowed in the pharynx and reduce the amount of oligonucleotide that reaches the lungs.

Liquid pharmaceutical compositions of oligonucleotides can be prepared by combining the oligonucleotide with a suitable vehicle, such as sterile pyrogen-free water or saline. Other therapeutic compounds may optionally be included.

The present invention also contemplates using solid particulate compositions. Such compositions preferably comprise particles of the oligonucleotide of respirable size. Grinding the dried oligonucleotide by conventional means, for example using a motor and pestle, and then separating the resulting powder composition through a 400 mesh screen into large particles or agglomerates, Prepare.
The solid particulate composition comprising the active oligonucleotide can optionally contain a dispersant, such as lactose, which optionally functions to facilitate aerosol formation.

According to the method of the present invention, the oligonucleotide composition is aerosolized. Aerosolization of the liquid particles can be made by any suitable means, such as using a nebulizer. See, for example, U.S. Patent No. 4,501,729. Nebulizers convert a solution or suspension into a therapeutic aerosol, either by accelerating a compressed gas, typically air or oxygen, through a narrow venturi hole or by ultrasonic vibration. , A commercially available device. Suitable nebulizers include Pari LC Plus, Pari Dura-Neb 2000, Pari-Baby Size, LC
Sold by Blairex ^™ under the names PARI PRONEB compressor with PLUS, PARI WALKHALER compressor / nebulizer system, PARI LC PLUS reusable nebulizer, and PARI LC Jet + ^™ nebulizer.

A typical composition for use in a nebulizer consists of the oligonucleotide in a liquid or saline solution, such as, for example, sterile and pyrogen-free water, wherein the oligonucleotide is present in the formulation at about 40% Up to and including. Preferably, the oligonucleotide comprises less than 20% w / w. If desired, further additives such as preservatives (eg, methyl hydroxybenzoate), antioxidants, and flavoring agents can be added to the composition.

[0158] Solid particles comprising oligonucleotides can be aerosolized using any of the aerosol generators known in the art. Such aerosol generators produce respirable particles as described above, and also reproducibly produce metered unit doses of the aerosol. Suitable solid particulate aerosol generators include:
Includes insufflators and metered dose inhalers. Metered dose inhalers suitable for use in the art (brand names, manufacturers and indications for use) and useful in the present invention include the following: Delivery Device Brand Name-Manufacturer -Indications Metered dose inhalers Alupent-Boehringer Ingelheim-beta adrenergic bronchodilator Atrovent-Boehringer Ingelheim-anticholinergic bronchodilator Aerobid, Aerobid-M-Forest-steroidal anti-inflammatory drug Beclovent, Beconase-Glaxo Wellcome- Steroidal anti-inflammatory drug Flovent-Glaxo Wellcome-steroidal anti-inflammatory drug Ventolin-Glaxo Wellcome-β adrenergic bronchodilator Proventil-Key Pharm.-β adrenergic bronchodilator Maxair-3M Pharm.-β adrenergic Bronchodilator Azmacort-Rhone-Poulenc Rarer-steroidal anti-inflammatory drug Tilade-Rhone-Poulenc Rorer-anti-inflammatory drug (inhibits inflammatory mediator) Int al-Rhone-Poulenc Rorer-inhibits mast cell degranulation (asthma) Vanceril-Schering-steroidal anti-inflammatory drug Tornalate-Dura Pharm.-β adrenergic bronchodilator.

Solutions for nebulization Alupent-Boehringer Ingelheim-β-adrenergic bronchodilator Pulmozyme-Genetech-recombinant human deoxynuclease I Ventolin-Glaxo Wellcome-β-adrenergic bronchodilator Tornalate-Dura Pharm.-β-adrenergic Sexual bronchodilator Intal-Rhone-Poulenc Rorer-inhibits mast cell degranulation (asthma).

Capsules for Inhalation (Powder) Ventolin-Glaxo Wellcome (Rotcap for use in the Rotohaler device) -β-adrenergic bronchodilator Powder for inhalation Pulmicort-Astra USA (Tabhaler) Apparatus)-steroidal anti-inflammatory drug.

[0161] Preferably, the liquid or solid aerosol is produced at a rate of about 10-150 liters / minute, more preferably at a rate of about 30-150 liters / minute, and most preferably at a rate of about 60 liters / minute.

As used herein, the term “alkyl” includes, but is not limited to, straight, branched and alicyclic hydrocarbon groups. The alkyl group of the present invention may be substituted. Representative alkyl substituents are disclosed in U.S. Patent No., column 12, lines 41-50.

Further exemplary 2 ′ sugar modifications sensitive to the present invention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, Included are O-alkyl imidazoles, and polyethers having the formula (O-alkyl) _m , wherein m is from 1 to about 10. Among these polyethers, linear and cyclic polyethylene glycols (PEG) and (PEG)
Ouch, which contains the containing groups such as crown ethers and their entirety by reference
i et al. (Drug Design and Discovery 1992, 9, 93), Ravasio et al. (J. Org. Chem. 1
991, 56, 4329) and Delgardo et al. (Critical Reviews in Therapeutic Drug)
Carrier Systems 1992, 9, 249) are preferred. Additional sugar modifications are disclosed in Cook, PD (supra). Fluoro, O-alkyl, O-alkylamino, O-alkylimidazole, O-alkylaminoalkyl,
And alkylamino substitutions were filed on March 6, 1995, and entitled "Oligomer Compounds with Pyrimidine Nucleotides with 2 'and 5'Substitutions" (Oligomeric Compoun
Ds having Pyrimidine Nucleotide (s) with 2 'and 5' Substitutions), which is described in U.S. Patent Application Serial No. 08 / 398,901, which is hereby incorporated by reference in its entirety.

Sugars having O-substitution on the ribosyl ring are susceptible to the present invention. Representative substitutions for rings are also O, S, CH ₂ , CHF, and CF ₂ (eg, Secrist, et al., Abstract 21, Progra, which is incorporated by reference herein in its entirety.
m & Abstracts, Tenth International Roundtable, Nucleosides, Nucleotides
and their Biological Applications, Park City, Utah, Sept. 16-20, 1992).

As used herein, the term “aralkyl” refers to an alkyl group having an aryl group, for example, a benzyl group. The term "alkaryl" refers to an aryl group having an alkyl group, for example, a methylphenyl group. "Aryl group" refers to an aromatic cyclic compound including, but not limited to, phenyl, naphthyl, anthracyl, phenanthryl, pyrenyl, and xylyl.

In general, the term “hetero” refers to atoms other than carbon, especially but not limited to N, O, or S. Thus, the term "heterocycloalkyl" refers to an alkyl ring system having one or more heteroatoms (ie, non-carbon atoms). Preferred heterocycloalkyl groups include, for example, morpholino groups and the like. As used herein, the term "heterocycloalkenyl" refers to a ring system having one or more double bonds and one or more heteroatoms. Preferred heterocycloalkenyl groups include, for example, pyrrolidone groups.

[0167] In some preferred embodiments, the compounds of the invention can include a linker attached to a solid support. A solid support is a substance that can function as a solid phase in a solid phase synthesis method, and is described in Caruthers US Pat.
No. 066; 4,500,707; 4,668,777; 4,973,679; and 5,132,418;
And in Koster U.S. Pat. No. 4,725,677 and Re. 34,069. It is known in the art. Linkers are known in the art as short molecules that function to attach a solid support to a functional group (eg, a hydroxyl group) of a first synthon molecule in solid phase synthesis technology. Suitable linkers are, for example, the “Oligonucleotides And Analogues A Practical Approach” (Ekste
in, F. Ed., IRL Press, NY, 1991, Chapter 1, pages 123), which is incorporated herein by reference in its entirety.

The solid supports according to the invention include, for example, controlled release pore glass (CPG), oxalyl-
Controlled release pore glass (eg, Alul, et al., Incorporated by reference in its entirety).
l., Nucleic Acids Research 1991, 19, 1527), TentaGel support--aminopolyethylene glycol derivative support (see, for example, Wright, et al., Tetrahedron Letters 1993, 34, incorporated by reference in its entirety). 3373) and Poros-
-Including those generally known in the art, suitable for use in the solid phase method, including polystyrene / divinylbenzene copolymers.

[0169] In some preferred embodiments of the present invention, it comprises one or more hydroxyl protecting groups. A wide range of hydroxyl protecting groups can be used in the method of the present invention. Preferably, the protecting groups are stable under basic conditions and can be removed under acidic conditions. In general, protecting groups provide chemical functionality that does not react under certain reaction conditions, and confer such functionality in the molecule without substantially damaging the rest of the molecule and Can be removed. Representative hydroxyl protecting groups are described in Beaucage et al. (Tetrahedron 1992, 48, 2223-2311) and Greene
And Wuts (Protective Groups in organic Synthesis, Chapter 2, 2d ed, Jo
hn Wiley & Sons, New York, 1991), each of which is incorporated by reference in its entirety. Preferred protecting groups used for R2, R3 and R3a include dimethoxytrityl (DMT), monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) and 9- (p-methoxyphenyl) xanthen-9-yl ( Mox)
Is included. The R2 or R3 group can be removed from the oligonucleotide of the invention by techniques well known in the art. For example, a dimethoxytrityl protecting group can be removed by a protic acid such as, for example, formic acid, dichloroacetic acid, trichloroacetic acid, p-toluenesulfonic acid or by a Lewis acid such as zinc bromide (eg, Greene and Wuts, supra). See).

In some preferred embodiments of the present invention, the amino group is, for example, alkyl or 2 ′
-In addition to other groups such as alkoxy groups (for example when R1 is alkoxy).
Such amino groups are also commonly found in naturally occurring and non-naturally occurring nucleobases. Generally, it is preferred that these amino groups be in a protected form during the synthesis of the oligomeric compounds of the present invention. Representative amino protecting groups suitable for these purposes are Greene and Wuts (Protective Groups in o
rganic Synthesis, Chapter 7, 2d ed, John Wiley & Sons, New York, 1991)
Are discussed in In general, as used herein, the term "protected" when used in combination with a molecular moiety such as a "nucleobase" refers to one or more of the molecular moieties protected by a protecting group. It indicates that it contains more functionality.

The oligomeric compounds of the invention can be used as diagnostics, therapeutics and research reagents and as kits. These can be used in pharmaceutical compositions by including suitable pharmaceutically acceptable diluents or carriers. They can further be used to treat organisms with diseases characterized by unwanted production of proteins. The organism should be contacted with an oligonucleotide having a sequence capable of specifically hybridizing with the strand of the nucleic acid encoding the unwanted protein. This treatment can be applied to a variety of organisms ranging from unicellular prokaryotes and eukaryotes to multicellular eukaryotes. DNA-
Any organism that utilizes RNA transcription or RNA-protein translation as a fundamental part of its genetic, metabolic, or cellular regulation is responsive to therapeutic and / or prophylactic treatment according to the present invention. Superficially different organisms can be treated, such as bacteria, yeast, protozoa, algae, higher plants, including all plants and all warm-blooded animals. In addition, each cell of a multicellular eukaryote can be treated because each cell of the multicellular eukaryote contains both DNA-RNA transcription and RNA-protein translation as an integral part thereof. In addition, many organelles of eukaryotic cells (eg, mitochondria and chloroplasts) also contain transcription and translation machinery. Thus, single cells, cell populations, organelles can also be included in the definition of an organism that can be treated with a therapeutic or diagnostic oligonucleotide.

[0172]

【Example】

The following examples illustrate the invention and are not intended to be limited to the same. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific substrates and procedures described herein. Such equivalents are considered to be within the scope of the present invention.

Example 1 Oligonucleotide Preparation A. General Synthetic Technique Oligonucleotides were synthesized on an automated DNA synthesizer using standard phosphoramidite chemistry using iodine-based oxidation. β-cyanoethyldiisopropylphosphoramidite applied to Applied Biosystem
s (Foster City CA). Phosphorothioate
For oligonucleotides, a standard oxidation bottle is used for 0.2M 3H-1,2-benzodithiol-3-one in acetonitrile for gradual thiation of phosphite.
The solution was replaced with a -1,1-oxide solution.

The synthesis of 2′-O-methyl- (also known as 2′-methoxy-) phosphorothioate oligonucleotides was carried out according to the procedure set forth above, using 2′-O-methyl b- instead of the standard phosphoramidite. Cyanoethyldiisopropylphosphoramidite (Chem
genes, Needham, MA) and the waiting cycle after pulse delivery of tetrazole and base is increased up to 360 seconds.

Similarly, 2′-O-propyl- (also known as 2′-propoxy-) phosphorothioate oligonucleotides were prepared by slight modifications of this procedure and were essentially filed on February 3, 1995. No. 08 / 383,666, assigned to the same assignee as the present application, which is hereby incorporated by reference.

The 2′-fluoro-phosphorothioate oligonucleotides of the present invention were synthesized using 5′-dimethoxytrityl-3′-phosphoramidite and are described in US Patent Application Serial No. 08 / 383,666, and issued on October 8, 1996
Prepared as disclosed in US Pat. No. 5,459,255, both of which are assigned the same agent as the present application and are hereby incorporated by reference. 2'-
Fluoro-oligonucleotides are prepared using phosphoramidite chemistry and slight modifications of standard DNA synthesis protocols (ie, deprot
Section) was effective with the use of methanolic ammonia at room temperature).

RNA antisense analogs are essentially those disclosed in US Patent Nos. 5,539,082 and 5,539,083.
, Both of which are (1) issued June 23, 1996, (2) assigned the same agent as the present application, and (3) hereby incorporated by reference.

The oligonucleotide containing 2,6-diaminopurine was published on April 9, 1996
Prepared using the composition described in US Patent No. 5,506,351, which was assigned the same agent as the present application, hereby accepted by reference,
ffney et al. (Tetrahedron, 1984, 40: 3), Chollet et al. (Nucl. Acids Res., 1988, 1
6: 305) and Prosnyak et al. (Genomics, 1994, 21: 490). Oligonucleotides containing 2,6-diaminopurines can also be prepared by enzymatic means (Bailly et al., Proc. Natl. Acad. Sci. USA, 1996,
93: 13623).

The 2′-methoxyethoxy oligonucleotides of the present invention are essentially those of Martin et al. (Hel
v. Chim. Acta, 1995, 78, 486). For ease of synthesis, 2'-methoxyethoxy oligonucleotide 3 'nucleotide deoxynucleotide, and _{2'-OCH 2 CH 2 OCH 3} - cytosine is 5-methylcytosine, they are described below It was synthesized according to the following procedure.

B. Synthesis of 5-Methylcytosine Monomer 1. 2,2′-Anhydro [1- (β-D-arabinofuranosyl) -5-methyluridine]: 5-methyluridine (ribosylthymine, Yamasa, Choshi G, 0.279 M), diphenyl carbonate (90.0 g, 0.420 M) and sodium bicarbonate (2.0 g, 0.024 M) in N, N-dimethylformamide (DMF, 300
mL). The mixture was heated to reflux with stirring and the carbon dioxide gas evolved was released in a controlled manner. After 1 hour, the slightly dark solution was concentrated under reduced pressure. The resulting syrup was poured into diethyl ether (2.5 L) with stirring. The product formed a gum. Ether was gently poured and the residue was dissolved in a minimum amount of methanol (about 400 mL). Add the solution to fresh ether (2.5 L
) Yielded a hard rubbery material. Ether was gently poured and the gum dried in a vacuum oven (1 mm Hg at 60 ° C. for 24 hours) to give a solid which was ground to a light tan powder (57 g, 85% crude yield). Material was used for further reactions.

2. 2′-O-methoxy-5-methyluridine: 2,2′-anhydro-5-methyluridine (195 g, 0.81 M), tris (2-methoxyethyl)
Add borate (231 g, 0.98 M) and 2-methoxyethanol (1.2 L) to 2 L
In addition to a stainless steel pressure vessel, it was placed in an oil bath before heating at 160 ° C. 155-160 ℃
After heating for 48 hours at, open the vessel and evaporate the solution to dryness,
(200 mL). The residue was suspended in hot acetone (1 L). Unfortunate
The soluble salts were filtered, washed with acetone and the filtrate was evaporated. Residue (280 g)
CH_ThreeDissolved in CN (600 mL) and evaporated. Silica gel column (3 kg) 0.5% Et _Three CH containing NH_TwoCl_Two/ Acetone / methanol (20: 5: 3). Residue C
H_TwoCl_Two(250 mL) and absorb on silica (150 g) before loading onto the column.
Was. The product was eluted with the packed solvent to give 160 g (63%) of the product.

3. 2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine: 2′-O-methoxyethyl-5′-methyluridine (160 g, 0.506 M) was added to pyridine (250
mL) and the dried residue was dissolved in pyridine (1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g, 0.278 M) was added and the mixture was stirred for 1 hour at room temperature. Dimethoxytrityl chloride (94.3 g, 0
A second aliquot of .278 M) was added and the reaction was stirred for an additional hour. And
The reaction was stopped by adding methanol (170 mL). High pressure liquid chromatography (HP
LC) indicated the presence of about 70% product. The solvent was evaporated to triturated with CH ₃ CN (200 mL). The residue was dissolved in CHCl ₃ (1.5 L) and extracted with x 500 mL of saturated NaHCO ₃ and 2 x 500 mL of saturated NaCl. The organic layer was dried over Na ₂ SO _4, filtered and evaporated. 275 g of residue were obtained. The residue was purified on a 3.5 kg silica gel column, packed and eluted with EtOAc / hexane / acetone (5: 5: 1) containing 0.5% Et ₃ NH. The purified fraction was evaporated, yielding 164 g of product. An additional about 20 g was obtained from the impure fraction, giving a total yield of 183 g (57%).

4. 3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine: 2'-O-methoxyethyl-5'-O-dimethoxytrityl-5 -Methyluridine (106 g, 0
.167 M), DMF / pyridine (75 mL prepared from 562 mL DMF and 188 mL pyridine)
(0 mL of a 3: 1 mixture) and acetic anhydride (24.38 mL, 0.258 M) were mixed and stirred at room temperature for 24 hours. The reaction was analyzed for additional MeOH using thin layer chromatography (tlc).
Along with the first disappearance of the tlc sample. Upon completion of the reaction, as judged by tlc, MeOH (50 mL) was added and the mixture was evaporated at 35 ° C. The residue was washed with CHCl ₃ (800 mL
) And extracted with 2 × 200 mL of saturated sodium bicarbonate and 2 × 200 mL of saturated NaCl. The aqueous layer was re-extracted with 200 mL CHCl ₃ in. The combined organics were dried using sodium sulfate and evaporated to give 122 g residue (about 90% product). Residue
Purified on a 3.5 kg silica gel column and eluted using EtOAc / hexane (4: 1). The purified product fraction was evaporated to yield 96 g (84%).

5. 3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl
-4-Triazole uridine: The first solution was added to CH ₃ CN in 3′-O-acetyl-2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methyluridine (96 g, 0.144 M). Was prepared by dissolving and placed aside. Triethylamine (189 mL, 144 M) was added to a triazole solution (90 g, 1.3 M) in CH ₃ CN (1 L), cooled to −5 ° C., and stirred for 0.5 hour using an overhead stirrer. POCl ₃ dropwise over 30 minutes, 0-
Added to the stirred solution maintained at 10 ° C. and the resulting mixture was stirred for another 2 hours. The first solution was added dropwise to the later solution over 45 minutes. The resulting reaction mixture was stored in a cold room overnight. The salt was filtered from the reaction mixture and the solution was evaporated.
The residue was dissolved in EtOAc (1 L) and the insoluble solid was removed by filtration. 1x 300 filtrate
Washed with mL of NaHCO ₃ and 2 × 300 mL of saturated NaCl, dried over sodium sulfate and evaporated. The residue was triturated with EtOAc to give the title compound.

[0185] 6. 2'-O- methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine: dioxane (500 mL) and _{NH 4 OH (30 mL) 3'} -O- acetyl -2'-O-methoxyethyl in Solution of -5'-O-dimethoxytrityl-5-methyl-4-triazoleuridine (10
3 g, 0.141 M) was stirred at room temperature for 2 hours. Evaporate the dioxane solution and remove the residue to M
It was azeotropically mixed with eOH (2 × 200 mL). Dissolve the residue in MeOH (300 mL) and add 2
Transferred to 1 liter stainless steel pressure vessel. Methanol (400 mL) saturated with NH ₃ gas was added, and the vessel was heated at 100 ° C. for 2 hours (thin layer chromatography, tlc,
Indicated complete conversion). The contents of the vessel were evaporated to dryness and the residue was
(500 mL) and washed once with saturated NaCl (200 mL). The organics were dried over sodium sulfate and the solvent was evaporated, yielding 85 g (95%) of the title compound.

[0186] 7. N^Four-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl
Cytidine: 2′-O-methoxyethyl-5′-O-dimethoxytrityl-5-methylcytidine (85 g, 0.
134 M) in DMF (800 mL) and stir benzoic anhydride (37.2 g, 0.165 M).
Added. After stirring for 3 hours, tlc indicated that the reaction was approximately 95% complete.
The solvent was evaporated and the residue was azeotroped with MeOH (200 mL). CHCl residue _Three (700 mL) and saturated NaHCO_Three(X 300 mL) and saturated NaCl (x 300 mL)
And extracted with MgSO_FourDrying and evaporation gave a residue (96 g). The residue,
0.5% Et as elution solvent_Three1.5 kg sieve using EtOAc / hexane (1: 1) containing NH
Chromatography was performed on a Rica column. Evaporate the purified product fractions
To give 90 g (90%) of the title compound.

[0187] 8. N ⁴ - Benzoyl -2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-Amidito (amidite): N ⁴ - Benzoyl -2'-O-methoxyethyl-5' O- dimethoxytrityl-5-methylcytidine (74 g, 0.10 M) was dissolved in _{CH 2 Cl 2 (1 L)} . Tetraazole diisopropylamine (7.1 g) and 2-cyanoethoxy-tetra- (isopropyl) phosphoric acid (40.
5 mL, 0.123 M) was added with stirring under a nitrogen atmosphere. Allow the resulting mixture at room temperature for 20
Stirred for hours (tlc indicated that the reaction was 95% complete). The reaction mixture is saturated with NaHC
Extracted with O ₃ (1 × 300 mL) and saturated NaCl (3 × 300 mL). The washed aliquot was re-extracted with CH ₂ Cl ₂ (300 mL), the extracts were combined, dried over MgSO ₄ and concentrated. The residue obtained was chromatographed on a 1.5 kg silica column using EtOAc / hexane (3: 1) as elution solvent. The purified fractions were combined to give 90.6 g (87%) of the title compound.

C. Purification of Oligonucleotides: Controlled Pore Glass (CPG) Columns (Applied Biosystems)
After cleavage from and deblocking in concentrated ammonium hydroxide at 55 ° C. for 18 hours, the oligonucleotide was combined with 2.5 volumes of ethanol in 0.5 M
Purification by precipitation from NaCl followed by reverse phase high pressure liquid chromatography (HPLC
) For further purification. Analytical gel electrophoresis, 20% acrylamide, 8 M
Performed in urea and 45 mM Tris-borate buffer (pH 7).

D. Nucleotide Labeling: The antisense oligonucleotides are labeled to detect and / or determine the presence of that amount in a sample employed during the course of the in vivo pharmacokinetic studies described herein. I do. Although radiolabeling by tritium conversion is one of the preferred means of labeling antisense oligonucleotides for such in vivo studies, various radiological, chemical or enzymatic methods have been incorporated into oligonucleotides and other nucleic acids. Various other means are available for incorporating the sign.

[0190] 1. Tritium Conversion Oligonucleotides were labeled by tritium conversion, essentially using the procedure of Graham et al. (Nucleic Acids Research, 1993, 21: 3737). Specifically, about 24
mg of oligonucleotide in 200 μL of sodium phosphate buffer (pH 7.8), 40
It was dissolved in a mixture of 0 μL of 0.1 mM EDTA (pH 8.3) and 200 μL of deionized water. The pH of the resulting mixture was measured and adjusted to pH 7.8 using 0.095 N NaOH. The mixture was lyophilized overnight in a 1.25 mL gasket-attached polypropylene vial. 8.25μ acting on oligonucleotide as free radical scavenger
L-b-mercaptoethanol (Graham et al., Nucleic Acids Research, 1993, 21:37
37), and dissolved in 400 μL of tritiated H ₂₀ (5 Ci / gram). The tube was capped and placed in a 90 BC oil bath without agitation for 9 hours, and centrifuged briefly to remove any condensate from the inside edge of the tube. (As an optional analysis step, two 10 μL aliquots (one for HPLC analysis and one for PAGE analysis) were removed from the reaction tubes; each aliquot was removed with 490 μL of 50 μM sodium phosphate buffer ( pH 7.8) into a separate 1.5 mL standard microfuge tube.) Then freeze the oligonucleotide mixture in liquid nitrogen and lyophilize under high vacuum typically for 3 hours. Transferred to a freeze-drying apparatus. The material was then resuspended in _double- distilled mL of H2O and converted at room temperature for 1 hour. After the incubation, the mixture was snap frozen again and lyophilized overnight. (As optional analysis step, except the oligonucleotide material about 1 mg for HPLC analysis.) 3 times perform additional lyophilization, each double distilled H ₂ 0 to about 1 mL
Together, it ensured the removal of any residual non-incorporated tritium. The final resuspended oligonucleotide solution is transferred to a clean polypropylene vial and assayed. Store the tritium labeled oligonucleotide at about -70BC.

[0191] 2. Other Means of Nucleic Acid Labeling As is well known to those skilled in the art, various means are available for labeling oligonucleotides and other nucleic acids and for separating non-incorporated labels from labeled nucleic acids. For example, double-stranded nucleic acids can be radiolabeled by nick translation and primer extension, and various nucleic acids, including oligonucleotides, can be radiolabeled at the ends by use of enzymes such as T4 polynucleotide kinase or terminal deoxynucleotidyl transferase ( Generally, Chapter 3 In: Short Protocols in
Molecular Biology, 2d Ed., Ausubel et al., Eds., John Wiley & Sons, New York
, NY, pages 3-11 to 3-38; and Chapter 10 In: Molecular Cloning: A Labo
ratory Manual, 2d Ed., Sambrook et al., eds., pages 10.1 to 10.70). Labeling oligonucleotides and other nucleic acids with non-radioactive labels, such as, for example, enzymes, fluorescent moieties, etc., is also well known to those of skill in the art (eg, Beck, Methods in Enzymology, 1992). , 216: 143; and Ruth, Chapter 6
In: Protocols for Oligonucleotide Conjugates (Methods in Molecular Biolo
gy, Volume 26) Agrawal, S., ed., Humana Press, Totowa, NJ, 1994, page 1
67-185).

Example 2 Inhalation Exposure of Oligonucleotides in Mice 1. Spraying of oligonucleotides An aqueous solution of oligonucleotides was sprayed and the resulting aerosol was delivered to an animal model (male CD-1 mice) via a nose-only inhalation system. Particle size was targeted at 1 to 5 μm to reach the bronchial and alveolar regions of the lung. Following single and multiple exposures, mice were evaluated for signs of toxicity and designated tissues were collected for evaluation of organ-specific effects and oligonucleotide concentrations. Male CD-1 mice were chosen as the animal model for this study because of the considerable scientific data available for that species.

[0193] 2. Oligonucleotides used in animal studies The following compounds were tested in this study: 1) ISIS 2105, targeting HPV, sequence: 5'-TTG-CTT-CCA-TCT-TCC-TCG-TC-3 '(SEQ Phosphorothioate antisense 2'-deoxyribose oligonucleotide with ID NO: 9) 2) ISIS 17009, targeting mouse ICAM-1 with sequence: 5'-GGA-GTC-CAG-CAC-TAG-CAC-TG-3 '(Phosphorothioate antisense 2'-deoxyribose oligonucleotide with (SEQ ID NO: 10) 3) ISIS 15163, sequence: 5'-GGA-GTC-CAG-CAC-TAG-CAC-TG-3' (SEQ ID NO : Ten
), Wherein each C is replaced by 5-methylcytosine (iso sequence derivative of 17009), phosphorothioate antisense 2'-deoxyribose oligonucleotide targeting mouse ICAM-1 for injection A solution of the oligonucleotide was formulated using sterile sodium chloride (brine), and sodium chloride for injection was used as a control.

[0194] 3. Single exposure of Isis 2105 in mice I with a concentration of 10 or 100 mg / ml ether in mice with saline control
A 30-minute nasal-only exposure to SIS-2105 solution was performed. The calculated lung dose (see infra) was 1.2 and 12 mg / kg, respectively. Animals were necropsied at 0 minutes (end of exposure), 2 hours, 8 hours, and 24 hours. Animals were generally assessed for their health, with a more limited assessment being made of lung tolerability. Lung concentrations of oligonucleotides and oligonucleotide metabolites were determined by capillary gel electrophoresis (CGE), and the distribution of oligonucleotides in lung tissue was determined immunohistologically.

Results: General animal health The control and low dose groups each had a 7% respiratory rate
Alternatively, it showed a 13% decrease. The high dose group showed a 28 percent decrease in respiratory rate during exposure. Exposure did not affect body weight or organ weight.

[0196] 2. Histological evaluation of the lung Histological results showed that the low-dose group slightly induced an inflammatory response or resulted in increased macrophages. There was a significant inflammatory response in the high dose group, which revealed increased macrophages and a collapsed alveolar space.

[0197] 3. Lung removal (see Figure 1) Figure 1 shows the removal of oligonucleotides from mouse lung in this study. It can be seen that the elimination seems to be monophasic in the low dose group and biphasic in the high dose group. However, completeness (inte
grity) may have been compromised; high doses may have been overdose to the lungs. 20 for parent compound and full-length oligonucleotide
There was a relatively long half-life for both metabolites, which was more than an hour and more than 40 hours for all oligonucleotides. Metabolism of the parent oligonucleotide in the lung appears to be faster than the rate of clearance from the lung, which is consistent with observations in other organs.

[0198] 4. Pulmonary distribution Oligonucleotides were distributed to all cell types in the lung, including bronchial and alveolar epithelium, endothelial cells, and alveolar macrophages. In addition, significant concentrations of oligonucleotides and metabolites were found in lung tissue (by CGE analysis): 80 percent of the oligonucleotides were found untreated at the end of the exposure, and 8 hours after exposure the remaining untreated Untreated was found at 50 percent, and at 24 hours post-dose, 20 to 30 percent.

Significant concentrations of oligonucleotides and metabolites were found in BAL (bronchoalveolar lavage). These are shown in Table 1 below.

[0200]

[Table 1]

For the 12 mg / kg group, the levels of detectable oligonucleotides and / or metabolites found in plasma were: 0.6 micromolar at 0 hours (52 percent full length) and 0.3 micromolar at 2 hours (38% full length). Also significant concentrations found in the liver; 30 micrograms at 24 hours post-exposure; 12-16 percent of untreated parent compound. From these data, it can be seen that for the high dose group, some of the oligonucleotides delivered to the plasma are removed relatively quickly.

The foregoing data indicate that high concentrations of oligonucleotides may affect respiration rate, or alternatively, by respiratory tract irritation or as a result of the relatively high viscosity of the solution. Importantly, pulmonary delivery of oligonucleotides was distributed to all types of cells in the lung.

Example 3 Single and Multiple Exposure Studies of Oligonucleotides in Mice 1. Exposure System Design and Concept The exposure system used was designed to spray only the test article solution or saline. Exposure atmosphere is PARI LC PLUS nebulizer (PARI Respiratory Equipment, In
c, Richmond, VA). Filtered pressurized air was used as the air supply. The airflow pathway was set and maintained at the level required to ensure consistent aerosol production and maintain animal health. An empty port in the production chamber was positioned to obtain a sample for gravimetric measurements and particle size determination or analysis.

Atmospheric concentrations were determined by both gravimetric (developmental stages) and analytical measurements (animal exposure). Glass chamber filter (Gelman # 66075, Gelman scienc
es, Ann Arbor, MI) was placed in an in-line filter holder. A known amount of test atmosphere was sampled by controlling the airflow path. Immediately after sampling, the filters were collected and the mass concentration was calculated. Then, the filter sample was processed, and the test substance deposited on the filter was extracted and analyzed. Analytical measurements were used to calculate the absorbed dose. Samples were collected during each exposure when the animal was placed in the chamber.

Particle size is determined by the Mercer-type cascade impactor (Chen et al., Fundam. Appl. Toxi
col., 1989, 13, 429). Effective cut-off diameters for impactors ranged from 4.8 microns to 0.30 microns. Following the first and last exposures, the particle size was measured for each oligonucleotide tested. The mass median aerodynamic diameter of the three oligonucleotides (the Mass M
The edian Aerodynamic Diameter (MMAD) ranged from 2.72 to 3.26, and the Geometric Standard Deviation (GSD) ranged from 2.44 to 2.46.

Animals were exposed in a nose-only exposure similar to the design described in Cannon et al. (1983), Amer. Ind. Hyg. Assoc. 44 (12) 923-928. "Open" type suppression tubes were used to facilitate the animal's ability to regulate body temperature and to eliminate excetia. The lung dose was calculated based on the following equation. Lung dose = RMV × concentration × time × sedimentation factor / body weight Where: RMV = assumed respiratory volume per minute, 0.03 l / min for a 30 gram mouse Concentration = chamber concentration based on analytical method Time = min Exposure time in units Deposition factor = fraction remaining in the lungs, 10% at 2-3 micrometer particle size
Assuming body weight = average body weight in grams (using 30 grams as average) Based on this equation and the data obtained from the following filter analysis, the estimated lung dose for the low, medium, and high dose groups was 0.8, 1.5 and 3.2 mg / kg.

[0207] 2. Results: A. Oligonucleotide Spray Figure 1 shows a plot of milligram oligonucleotide collected in the impinger versus time. These data indicate successful spraying of the oligonucleotide; that is, the oligonucleotide was sprayed evenly and the size of the resulting particles did not change over time.

B. Toxicity Data collected for the assessment of potential toxicity include clinical observations, body weight, clinical pathology (hematology and serum chemistry), gross necropsy (observation and organ weight) and selection Microscopic examination of isolated tissues. There were no clinical observations attributable to oligonucleotide administration. Weight gain and clinicopathological parameters were all within the normal range of male CD-1 mice. All mice were alive until each necropsy (following either one or four exposures) and there were no gross observations or changes in organ weight at necropsy.

Microscopic observations showed that 4 out of 5 mice out of 5 mice in the high dose ISIS 2105 group were exposed.
Tail, 4 exposures-2 out of 5 mice in the medium dose ISIS 2105 group and 2 or 5 out of 5 mice in the high dose ISIS 15163 single or multiple exposure groups, respectively
Limited to one tail. These effects in the lungs were described as multifocal inflammatory cell infiltrates that were considered minimal in severity. Similar observations have been shown after intravenous administration of oligonucleotides in mice, and these effects have been attributed to the immunostimulatory aspects that occur in rodents receiving this class of compounds.

[0210] No other changes were shown in the lungs and there were no observations of the effects shown on the other tissues examined (liver, kidney, spleen, and nasal passages).

C. Organ Distribution The concentration of each oligonucleotide and its metabolites was determined in lung, liver, kidney and spleen tissue samples. Tables 1 and 2 show the concentrations of total oligonucleotides (parent oligonucleotides and oligonucleotide metabolites) in lung, liver and kidney. The concentrations observed in the lung were dose-dependent, higher for mice given four exposures versus one exposure. Similar concentrations were observed in lungs of mice exposed to phosphorothioate oligonucleotides, ISIS 2105 and ISIS 17709, while higher concentrations were observed in mice exposed to ISIS 15163, a phosphodiester 2'-methoxyethyl modified oligonucleotide . Minimal concentrations of total oligonucleotides were observed in the liver or kidney of mice exposed to ISIS 2105 or ISIS 17009 and in the liver of mice exposed to ISIS 15163. Slightly higher concentrations were observed in the kidneys of mice exposed to ISIS 15163, and these concentrations were dose and frequency dependent.

[0212]

[Table 2]

[0213]

[Table 3]

As can be seen, nasal-only inhalation exposure of the oligonucleotide was well treated in mice. The effects in the lungs are limited to minimal cellular infiltrates such as are due to the general immunostimulation that occurs in mice that have received this class of compounds. The lung is also the tissue with the highest oligonucleotide concentration. Minimal oligonucleotide concentrations were observed in the other organs evaluated, and no histological changes were observed in these organs. Similar observations, tissue concentration and tissue effects, were shown for phosphorothioate oligonucleotides. 2'-Methoxyethyl modified phosphodiester oligonucleotide (ISIS 15163) was detected at higher concentrations in the lung, but histological changes were limited to one animal in each of the single and multiple exposure groups I was

[0215] Each patent, application, printed publication, and other published document mentioned or referred to in this feature is intended to be hereby incorporated by reference in its entirety.

Those skilled in the art will recognize that many changes and modifications may be made to the preferred embodiments of the invention and that such changes and modifications can be made without departing from the spirit of the invention. . Therefore, the appended claims are intended to cover all such equivalent modifications as would fall within the true spirit and scope of the invention.

[Sequence list]

[Brief description of the drawings]

Many objects and advantages of the present invention can be better understood by those skilled in the art with reference to the accompanying drawings. here,

FIG. 1 shows that the oligonucleotides are nebulized homogeneously and that the size of the resulting particles does not change over time.

FIG. 2 shows a PulmoAide Nebulizer (Apguard Medical, Inc., Woodland)
Hills, CA) by nebulizing oligonucleotides for 20 minutes (ISIS 2503
; 40 mg / mL). The fog from the nebulizer is collected in the impinger,
The oligonucleotide content was analyzed by ultraviolet absorption. The straight line in the graph indicates that the nebulization was homogeneous throughout the course of the experiment.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C12Q 1/68 C12N 15/00 ZNAA (81) Designated country EP (AT, BE, CH, CY, DE, DK) , ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR) , NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU) , TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU , LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Ecker, David Jay, United States 92024, Enxinitas, Saxony Road 1041 (72) Inventor Cook, Philip Dunn, United States 92069, Lake San Marcos, La Casa 1155 F-term (reference) 4B024 AA01 BA80 CA01 CA11 HA12 HA17 4B063 QA01 QA19 QQ01 QQ42 QQ52 QR08 QR42 QR56 QR62 QR63 QR82 QS25 QS34 QS36 QX02 4B064 AF27 DA01 4C084 AA13 MA01 MA13 MA55 NA13 ZA591 ZB111 ZB261 ZB331 ZB351 4C086 AA01 AA02 EA16 MA01 MA04 MA13 MA55 NA13 ZA59 ZB11 ZB26 ZB33 ZB35

Claims (63)

[Claims] 1. A pharmaceutical composition for pulmonary delivery of an oligonucleotide, comprising at least one oligonucleotide, wherein the sugar moiety of at least one nucleoside unit of the oligonucleotide is a 2′-deoxyribofuranosyl sugar moiety. Or the at least one internucleotide linkage in the oligonucleotide is not a phosphodiester or phosphorothioate linkage. 2. The pharmaceutical composition according to claim 1, wherein the sugar moiety of at least one nucleoside unit of the oligonucleotide is not a 2′-deoxyribofuranosyl sugar moiety. 3. The pharmaceutical composition according to claim 2, wherein the nucleoside unit is a 2′-O-substituted nucleoside unit. 4. The method according to claim 2, wherein the 2-0-substituent of the 2′-O-substituted nucleoside unit is 2
4. The pharmaceutical composition according to claim 3, which is a '-0-alkoxyalkoxy substituent. 5. The method of claim 2, wherein the 2-0-substituent of the 2′-O-substituted nucleoside unit is 2
4. The pharmaceutical composition according to claim 3, which is a '-0-dialkylaminooxyalkyl substituent. 6. The method of claim 1, wherein at least one internucleotide linkage in the oligonucleotide is not a phosphodiester or phosphorothioate linkage.
A pharmaceutical composition according to claim 1. 7. At least one internucleotide linkage in the oligonucleotide is a 3'-methylene phosphonate, a non-phosphorus containing an oligonucleoside linkage, a 2'-5 'linkage, or a 3'-deoxy-3 The pharmaceutical composition according to claim 6, which is a '-aminophosphoramide bond. 8. The composition of claim 1, further comprising one or more pharmaceutically acceptable carriers.
The pharmaceutical composition according to claim 1. 9. The pharmaceutical composition according to claim 1, wherein said composition is in an aqueous medium. 10. The pharmaceutical composition according to claim 9, wherein said aqueous medium is sterile, pyrogen-free water. 11. The pharmaceutical composition according to claim 9, wherein said aqueous medium is a saline solution. 12. The pharmaceutical composition according to claim 1, wherein said composition is a powder. 13. The pharmaceutical composition according to claim 1, wherein said oligonucleotide is an antisense oligonucleotide. 14. The antisense compound modulates protein expression,
14. The pharmaceutical composition of claim 13, wherein the composition modulates the rate of cell growth. 15. The pharmaceutical composition according to claim 14, wherein said antisense oligonucleotide regulates the expression of a cell adhesion protein. 16. The pharmaceutical composition according to claim 13, wherein the antisense oligonucleotide is antisense to a gene sequence associated with a disease or condition. 17. The pharmaceutical composition according to claim 13, wherein said disease or condition is asthma, lung cancer, pulmonary fibrosis, rhinovirus, tuberculosis, bronchitis, or pneumonia. 18. The pharmaceutical composition according to claim 13, wherein said antisense oligonucleotide is antisense to a part of a gene encoding a cytokine. 19. The method of claim 19, wherein the antisense oligonucleotide is ICAM-1, ELAM-1.
, VCAM-1, B7-1, B7-2, CD40, LFA-3, PECAM-1, ras oncogene, H-ras oncogene, K-ras oncogene, or antisense to a portion of the gene encoding protein kinase C. 14. The pharmaceutical composition according to claim 13, which is present. 20. The method according to claim 20, wherein the antisense oligonucleotide is a Mycobacterium.
14. The pharmaceutical composition according to claim 13, which is antisense to a unique part of the genome of tuberculosis, M. bovis, or Streptococcus pneumoniae. 21. The pharmaceutical composition of claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding ICAM-1. 22. The pharmaceutical composition of claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding ELAM-1. 23. The pharmaceutical composition of claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding VCAM-1. 24. The pharmaceutical composition of claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding B7-1. 25. The pharmaceutical composition of claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding B7-2. 26. The pharmaceutical composition of claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding CD40. 27. The pharmaceutical composition of claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding LFA-3. 28. The pharmaceutical composition according to claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding PECAM-1. 29. The pharmaceutical composition according to claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding the ras oncogene. 30. The pharmaceutical composition of claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding H-ras oncogene. 31. The pharmaceutical composition according to claim 13, wherein the antisense oligonucleotide is antisense to a portion of a gene encoding a K-ras oncogene. 32. The pharmaceutical composition according to claim 13, wherein said antisense oligonucleotide is antisense to a portion of the gene encoding protein kinase C. 33. The pharmaceutical composition according to claim 13, comprising more than one antisense oligonucleotide. 34. The pharmaceutical composition according to claim 1, wherein said oligonucleotide is a ribozyme, an external guide sequence, or an antisense peptide nucleic acid. 35. The pharmaceutical composition according to claim 1, wherein the oligonucleotide is an aptamer or a molecular decoy. 36. The pharmaceutical composition according to claim 9, wherein said aqueous medium is a sterile, pyrogen-free buffer solution. 37. A nucleic acid therapy comprising: preparing a nucleic acid therapeutic or diagnostic composition; aerosolizing the nucleic acid composition; introducing the aerosolized nucleic acid composition into the lungs of a mammal. A method of administering a composition or a nucleic acid diagnostic composition, wherein the aerosolized nucleic acid composition comprises at least one oligonucleotide, wherein the sugar moiety of at least one nucleoside unit of the oligonucleotide is 2'-deoxyribonucleic acid. Such a method, wherein the method is not a furanosyl sugar moiety or at least one internucleotide linkage in the oligonucleotide is not a phosphodiester or phosphorothioate linkage. 38. The method of claim 37, wherein the sugar moiety of at least one nucleoside unit of the oligonucleotide is not a 2'-deoxyribofuranosyl sugar moiety. 39. The method of claim 38, wherein said nucleoside unit is a 2'-O-substituted nucleoside unit. 40. The method of claim 39, wherein said 2-0-substituent of said 2'-O-substituted nucleoside unit is a 2'-0-alkoxyalkoxy substituent. 41. The method of claim 39, wherein said 2-0-substituent of said 2'-O-substituted nucleoside unit is a 2'-0-dialkylaminooxyalkyl substituent. 42. The at least one internucleotide linkage in the oligonucleotide is a phosphodiester or phosphorothioate linkage.
7. The method according to 7. 43. The at least one internucleotide linkage in the oligonucleotide is a 3'-methylene phosphonate, a non-phosphorus containing an oligonucleoside linkage, a 2'-5 'linkage, or a 3'-deoxy-3 43. The method of claim 42, which is a '-aminophosphoramide bond. 44. The method of claim 37, wherein said pharmaceutical composition further comprises one or more pharmaceutically acceptable carriers. 45. The method of claim 37, wherein said nucleic acid therapeutic or diagnostic composition is in an aqueous medium. 46. The nucleic acid therapeutic composition or the nucleic acid diagnostic composition is sterilized,
38. The method of claim 37, wherein the water is pyrogen-free. 47. The method of claim 37, wherein said nucleic acid therapeutic or diagnostic composition is a saline solution. 48. The nucleic acid therapeutic or diagnostic composition is a powder.
38. The method of claim 37. 49. The method of claim 37, wherein the nucleic acid therapeutic composition contains more than one oligonucleotide. 50. The method of claim 37, wherein the oligonucleotide is an antisense oligonucleotide. 51. The method of claim 37, wherein the nucleic acid therapeutic composition is an aerosolized solution consisting essentially of the antisense oligonucleotide in a saline solution. 52. An animal having or suspected of having a disease or condition treatable with one or more nucleic acids, comprising administering to the lungs of the animal a therapeutically effective amount of an aerosolized nucleic acid composition. The method of treating, wherein the aerosolized nucleic acid composition comprises at least one oligonucleotide, wherein the sugar moiety of at least one nucleoside unit of the oligonucleotide is not a 2′-deoxyribofuranosyl sugar moiety, or in the oligonucleotide. The above method, wherein at least one of the internucleotide linkages is not a phosphodiester or phosphorothioate linkage. 53. A method of studying the function of a gene or gene product in a non-human animal, comprising administering a therapeutically effective amount of the aerosolized nucleic acid composition to the lungs of the non-human animal, the method comprising: The modified nucleic acid composition comprises at least one oligonucleotide, wherein the sugar moiety of at least one nucleoside unit of the oligonucleotide is not a 2'-deoxyribofuranosyl sugar moiety, or at least one internucleotide linkage in the oligonucleotide. Such a method, wherein the method is not a phosphodiester or phosphorothioate linkage. 54. A method of delivering an oligonucleotide therapeutic or diagnostic composition to the lung of an animal, comprising applying the pharmaceutical composition of claim 1 to said lung. 55. The method of claim 54, wherein said oligonucleotide is delivered into cells of said lung. 56. The method of claim 55, wherein said animal is known or suspected of suffering from a disease or condition. 57. The method of claim 56, wherein said disease or condition is asthma, lung cancer, pulmonary fibrosis, rhinovirus, tuberculosis, bronchitis, or pneumonia. 58. The method of claim 37, wherein the nucleic acid therapeutic composition is an aerosolized solution consisting essentially of the antisense oligonucleotide in a buffer solution. 59. A method of regulating gene expression in an animal, comprising administering to the animal the pharmaceutical composition of claim 1. 60. A method of regulating gene expression in an animal, comprising administering the pharmaceutical composition of claim 1 to the animal. 61. A medical device for pulmonary delivery of an aerosol, comprising a pharmaceutical composition according to claim 1. 62. The oligonucleotide may be ISIS-15839, ISIS-13312, IS
IS-9605, ISIS-9606, ISIS-14859, ISIS-17709, ISIS-17044, ISIS-28089 and
2. The pharmaceutical composition according to claim 1, wherein the composition is selected from the group consisting of ISIS-104838. 63. The method according to claim 63, wherein the oligonucleotide is ISIS-15839, ISIS-13312, IS
IS-9605, ISIS-9606, ISIS-14859, ISIS-17709, ISIS-17044, ISIS-28089 and
38. The method of claim 37, wherein the method is selected from the group consisting of ISIS-104838.