WO2021142245A1

WO2021142245A1 - Compounds, pharmaceutical compositions and methods for modulating expression of muc5b in lung cells and tissues

Info

Publication number: WO2021142245A1
Application number: PCT/US2021/012680
Authority: WO
Inventors: Balkrishen Bhat; Shraddha SHARMA; Saswata KARMAKAR; Shrirang KARVE; Rebecca GOLDMAN; Sara J. DUNAJ; John R. ANDROSAVICH; Caroline J. WOO; Brian Bettencourt
Original assignee: Translate Bio, Inc.
Priority date: 2020-01-10
Filing date: 2021-01-08
Publication date: 2021-07-15

Abstract

The present disclosure relates to compounds and pharmaceutical compositions for modulating expression of MUC5B mRNA and/or protein in lung cells or tissues. The present disclosure also relates to a method of delivering a compound or pharmaceutical compositions for modulating expression of MUC5B mRNA and/or protein to the lung cells of a subject, the method comprising administering the compound or pharmaceutical composition to the subject via pulmonary delivery. Such compounds, pharmaceutical compositions and methods are useful for treating, preventing, ameliorating, or slowing progression of a lung disease or disorder, such as idiopathic pulmonary fibrosis.

Description

COMPOUNDS, PHARMACEUTICAL COMPOSITIONS AND METHODS FOR MODULATING EXPRESSION OF MUC5B IN LUNG CELLS AND TISSUES

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority to U.S. Provisional Application Serial No.

62/959,474, filed January 10, 2020, the disclosure of which is hereby incorporated by reference.

FIELD OF THE INVENTION

[0002] The disclosure relates to compounds, pharmaceutical compositions and methods for modulating expression of MUC5B mRNA and/or protein in lung cells or tissues. Such compounds, pharmaceutical compositions and methods are useful for treating, preventing, ameliorating, or slowing progression of a lung disease or disorder, such as idiopathic pulmonary fibrosis.

REFERENCE TO THE SEQUENCE LISTING [0003] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled MRT- 2116USPl_SL.txt created on January 10, 2020 which is 133 kilobytes in size. The information in electronic format of the sequence listing is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

[0004] A considerable portion of human diseases of the lung are associated with aberrant expression of a single gene. Some of these diseases, including idiopathic pulmonary fibrosis (IPF), chronic rhinosinusitis (CRS), chronic obstructive pulmonary disease (COPD), diffuse panbronchiolitis (DPB), and cystic fibrosis (CF) have increased expression of a mutated protein, over-expression of a wild-type protein, or a combination thereof.

[0005] Mucins are heavily glycosylated macromolecular components of mucus produced by epithelial cells and mucin- secreting goblet cells. In the lungs, mucus is responsible for trapping inhaled particles, including bacteria, and transporting them out of the airways by ciliary action and cough-driven forces. Additionally, mucus also helps to remove endogenous debris including dying epithelial cells and leukocytes. Mucins are primarily responsible for giving mucus its viscoelastic properties. Mucins can be membrane bound or secreted and, currently, about 20 mucin genes have been identified. Among these, MUC5B is a secreted mucin expressed throughout the upper and lower respiratory tract. MUC5B is one of the two major mucins of lung mucus. Changes in MUC5B expression are associated with lung diseases and disorders such as IPF, CRS, COPD, DPB, asthma and CF. In certain embodiments, changes in MUC5B expression are associated with lung diseases of the upper airways, for example, COPD, DPB, asthma and CF. Current methods of treating such lung diseases and disorders associated with aberrant MUC5B expression are limited because of the difficulty in delivering oligonucleotide based therapeutics directly to lung cells and tissues. [0006] Examples of possible oligonucleotide based therapeutics include antisense oligonucleotides and small interfering RNAs (siRNAs). siRNAs use the RNA interference (RNAi) pathway. RNAi is a sequence- specific RNA degradation process that provides a direct way to knockdown gene expression. In naturally occurring RNAi, a double- stranded RNA (dsRNA) is cleaved by an RNase III/helicase protein, Dicer, into siRNA molecules, which are dsRNA oligonucleotides, commonly 20-25 nucleosides in length. These siRNAs are incorporated into a multicomponent-ribonuclease called the RNA-induced-silencing- complex (RISC). One strand of the siRNA (the antisense strand or guide strand) remains associated with RISC, and guides the complex towards a cognate RNA that has a complementary sequence. The other strand of the siRNA (the sense strand or passenger strand) is degraded. This siRNA-directed RISC digests the cognate RNA, thereby inactivating it. Studies have shown that the use of chemically synthesized siRNAs exhibit RNAi effects in vivo. However, it is presently not possible to predict with a high degree of confidence which of many possible candidate siRNA sequences potentially targeting a particular nucleic acid sequence will, in fact, exhibit effective RNAi activity. Instead, individual candidate siRNA sequences must be generated and tested to determine whether the intended interference with expression of a target nucleic acid has occurred.

SUMMARY OF THE INVENTION

[0007] The invention relates to compounds, pharmaceutical compositions and methods for reducing expression of MUC5B mRNA in lung cells or tissues in a subject in need thereof. In some embodiments, the subject is a human. As a consequence of reducing expression of MUC5B mRNA, MUC5B protein levels are also reduced. Such reduction typically occurs in a time-dependent and dose-dependent manner.

[0008] One aspect of the invention provides a compound comprising an oligonucleotide comprising an antisense strand consisting of 15-30 linked nucleosides, wherein the nucleobase sequence of the antisense strand has at least 12 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-6.

[0009] Another aspect of the invention provides a pharmaceutical composition comprising i) a compound comprising an oligonucleotide comprising an antisense strand consisting of 15-30 linked nucleosides, wherein the nucleobase sequence of the antisense strand has at least 12 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-6, and ii) a lipid nanoparticle.

[00010] Another aspect of the invention provides a method of delivering a compound to lung cells of a subject in need thereof, wherein the method comprises administering a pharmaceutical composition comprising a lipid nanoparticle comprising the compound to the subject via pulmonary delivery, wherein the compound comprises an oligonucleotide comprising an antisense strand consisting of 15-30 linked nucleosides, wherein the nucleobase sequence of the antisense strand has at least 12 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1- 6. In some embodiments, the subject is suffering or at risk of suffering from a lung disease or disorder. In some embodiments the lung disease or disease or disorder is associated with overexpression of MUC5B. In some embodiments overexpression of MUC5B is associated with reduced mucociliary function, reduced alveolar repair, and/or increased lung fibrosis. In some embodiments, the lung disease or disorder is any one of idiopathic pulmonary fibrosis (IPF), chronic rhinosinusitis (CRS), chronic obstructive pulmonary disease (COPD), diffuse panbronchiolitis (DPB), asthma, and cystic fibrosis (CF). In some embodiments, pulmonary delivery is via nebulization of the compound using a nebulizer, preferably a mesh nebulizer. In some embodiments, the nebulizer delivers the compound to lung cells in the form of an aerosol. In some embodiments, the lung cells are lung epithelial cells.

[00011] Another aspect of the invention provides a pharmaceutical composition comprising a lipid nanoparticle comprising a compound for use in a method of treating, preventing, ameliorating, or slowing progression of a lung disease or disorder in a subject, wherein the compound comprises an oligonucleotide comprising an antisense strand consisting of 15-30 linked nucleosides, wherein the nucleobase sequence of the antisense strand has at least 12 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-6. The invention also provides a method of treatment of a subject with a lung disease or disorder, said method comprising administering a pharmaceutical composition comprising a lipid nanoparticle comprising a compound, wherein administering the pharmaceutical composition treats, prevents, ameliorates, or slows progression of the lung disease or disorder, wherein the compound comprises an oligonucleotide comprising an antisense strand consisting of 15-30 linked nucleosides, wherein the nucleobase sequence of the antisense strand has at least 12 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1- 6. The invention also provides use of a pharmaceutical composition comprising a lipid nanoparticle comprising a compound, for the manufacture of a medicament for treating, preventing, ameliorating, or slowing progression of a lung disease or disorder in a subject, wherein the compound comprises an oligonucleotide comprising an antisense strand consisting of 15-30 linked nucleosides, wherein the nucleobase sequence of the antisense strand has at least 12 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-6.

[00012] In some embodiments, a subject in need of treatment with the compound or pharmaceutical composition of the invention is suffering or at risk of suffering from a lung disease or disorder. In some embodiments the lung disease or disorder is associated with overexpression of MUC5B. In some embodiments overexpression of MUC5B is associated with reduced mucociliary function, reduced alveolar repair, and/or increased lung fibrosis. In some embodiments, the lung disease or disorder to be treated is any one of IPF, CRS, COPD, DPB, asthma and CF. In a particular embodiment, the lung disease or disorder to be treated is IPF. A known risk factor for the development of IPF is the presence of a polymorphism (rs35705950) in the promoter region of MUC5B. The polymorphism is associated with both familial and sporadic forms of IPF. Accordingly, in some embodiments, a subject in need of treatment with the compound or pharmaceutical composition of the invention has been identified to have the rs35705950 polymorphism. [00013] In some embodiments, the pharmaceutical composition is administered or is prepared for administration via pulmonary delivery. In some embodiments, pulmonary delivery is via nebulization of the compound using a nebulizer, preferably a mesh nebulizer. [00014] Another aspect of the invention provides a kit comprising a container housing a compound or pharmaceutical composition described herein. In some embodiments, the container may be specifically adapted for use with a nebulizer, e.g., a mesh nebulizer.

[00015] Another aspect of the invention provides a nebulizing apparatus comprising a compound or pharmaceutical composition described herein. In some embodiments the nebulizing apparatus is a mesh nebulizer.

[00016] In some embodiments, the nucleobase sequence of the antisense strand of the oligonucleotide of the invention has at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29 or 30 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-6. In some embodiments, the nucleobase sequence of the antisense strand of the oligonucleotide of the invention is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to an equal length portion of any one of SEQ ID NOs: 1-6, as measured over the entirety of the antisense strand.

[00017] In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide is double- stranded. In some embodiments the oligonucleotide is a siRNA oligonucleotide further comprising a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region. In some embodiments, the sense strand and/or the antisense strand consists of 15-30 linked nucleosides. In some embodiments, the sense strand and/or the antisense strand consists of 15-25 linked nucleosides. In some embodiments, the sense strand and/or the antisense strand consists of 19 linked nucleosides. In some embodiments, the duplex region is 15-30 nucleosides in length. In some embodiments, the duplex region is 15-25 nucleosides in length. In some embodiments, the duplex region is 19 nucleosides in length. In some embodiments, the nucleotide at the 3’ end of the sense strand is adenine, and the nucleotide at the 5’ end of the antisense strand is uracil. [00018] In some embodiments, the oligonucleotide is a siRNA oligonucleotide further comprising a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, and wherein the sense strand and/or the antisense strand further comprises a single-stranded overhang. In some embodiments, both the sense strand and the antisense strand comprise a single- stranded overhang. In some embodiments, the sense strand and/or the antisense strand comprises a 3’ single-stranded overhang. In some embodiments, the single-stranded overhang is two nucleosides in length. In some embodiments, both the sense strand and the antisense strand comprise a 3’ single- stranded overhang consisting of two deoxythymidines. In some embodiments, the internucleoside linkages of the single-stranded overhang are modified internucleoside linkages, preferably phosphothioester internucleoside linkages.

[00019] In some embodiments, the oligonucleotide is a modified oligonucleotide. In some embodiments, the modified oligonucleotide comprises at least one modification selected from a nucleoside analogue, a modified nucleobase, a modified internucleoside linkage and a modified sugar. In some embodiments, the modified oligonucleotide comprises at least one nucleoside analogue. In some embodiments, the modified oligonucleotide comprises at least one modified nucleobase. In some embodiments the at least one modified nucleobase is 5-methyl-cytosine. In some embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage. In some embodiments, the at least one modified internucleoside linkage is a phosphothioester internucleoside linkage. In some embodiments, the antisense strand and/or the sense strand of the modified oligonucleotide comprise two modified internucleoside linkages at either their 3’ or the 5’ end, preferably at the 5’ end. In some embodiments, each of these two modified internucleoside linkages is a phosphothioester internucleoside linkage. In some embodiments, the modified oligonucleotide comprises at least one modified sugar. In some embodiments, the at least one modified sugar is a bicyclic sugar, such as LNA, ENA or cEt. In some embodiments, the at least one modified sugar comprises a 2’-modified sugar moiety, such as 2’-O-methyl, 2’-F, 2’-O-methylethyl, or 2’-O-methoxyethyl. In some embodiments, the modified oligonucleotide is a nucleic acid analogue, such as a peptide nucleic acid (PNA) or a morpholino. [00020] In some embodiments the compound of the invention comprising the oligonucleotide is encapsulated in a liposomal delivery vehicle. In a particular embodiment, the liposomal delivery vehicle is a lipid nanoparticle. A suitable lipid nanoparticle for the present invention comprises one or more of a cationic lipid, a non-cationic lipid, a cholesterol-based lipid, a PEG-modified lipid, an amphiphilic block copolymer and/or a polymer, or a combination thereof.

[00021] An exemplary lipid nanoparticle may be composed of three lipid components: a cationic lipid ( e.g ., a sterol-based cationic lipid), a non-cationic lipid (e.g., DOPE or DEPE) and a PEG-modified lipid (e.g., DMG-PEG2K). In a particular embodiment, a suitable lipid nanoparticle for use with the invention has the following three lipid components: a cationic lipid, DOPE, and DMG-PEG2K. In another particular embodiment, a suitable lipid nanoparticle for use with the invention has the following three lipid components: a cationic lipid, DEPE, and DMG-PEG2K.

[00022] Alternatively, a lipid nanoparticle for use with the invention may be composed of four lipid components: a cationic lipid (e.g., a sterol-based cationic lipid), a non-cationic lipid (e.g., DOPE or DEPE), a cholesterol-based lipid (e.g., cholesterol) and a PEG-modified lipid (e.g., DMG-PEG2K).

[00023] In some embodiments, the one or more cationic lipids is selected from DOTAP (l,2-dioleyl-3-trimethylammonium propane), DODAP (l,2-dioleyl-3- dimethylammonium propane), DOTMA (N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride), DLinKC2DMA, DLin-KC2-DM, C12-200, cKK-E12 (3,6-bi s(4-(bis(2-hydroxydodecyl)amino)butyl)piperazine-2, 5 -dione), HGT5000, HGT5001, HGT4003, a sterol lipid such as imidazole cholesterol ester (ICE), HGT4001, HGT4002, TL1-01D-DMA, TL1-04D-DMA, TL1-08D-DMA, TL1-10D-DMA, OF-02, and combinations thereof.

[00024] In some embodiments, the one or more non-cationic lipids is selected from DSPC (l,2-distearoyl-sn-glycero-3-phosphocholine), DPPC (l,2-dipalmitoyl-sn-glycero-3- phosphocholine), DOPE (l,2-dioleyl-sn-glycero-3-phosphoethanolamine), DEPE (1,2- dierucoyl- sn-glycero-3 -phosphoethanolamine) , DOPC ( 1 ,2-dioleyl- sn-glycero-3 - phosphotidylcholine), DPPE (l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (l,2-dioleoyl-sn-glycero-3- phospho-(l'-rac-glycerol)), and combinations thereof. Exemplary non-cationic lipids for use with the present invention are DOPE and DEPE.

[00025] In some embodiments, the one or more cholesterol-based lipids is selected from cholesterol, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N- oleylamino-propyl)piperazine, or imidazole cholesterol ester (ICE). Typically, the cholesterol- based lipid is cholesterol.

[00026] In some embodiments, the one or more PEG-modified lipids is a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, the PEG-modified lipid is PEGylated cholesterol or PEG-2K. An exemplary PEG-modified lipid for use with the invention is 1,2-dimyristoyl-rac- glycero-3-methoxypolyethylene glycol-2000 ( DMG-PEG2K).

[00027] In some embodiments, cationic lipids constitute about 30-60% of the lipid nanoparticle by molar ratio, e.g., about 35-40%. In some embodiments, the molar ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) may be between about 30-60:25-35:20-30: 1-15, respectively. In a particular embodiment, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG- modified lipid(s) is approximately 50:25:20:5.

[00028] In some embodiments, a lipid nanoparticle for use with the invention has an average size of less than 150 nm. In some embodiments, a lipid nanoparticle has an average size of less than 120 nm. In some embodiments, a lipid nanoparticle has an average size of less than 100 nm. In some embodiments, a lipid nanoparticle has an average size of less than 90 nm. In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the lipid nanoparticles in a pharmaceutical composition provided by the present invention have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50- 80 nm, about 55-75 nm, about 60-70 nm). In some embodiments, substantially all of the lipid nanoparticles have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm). Compositions with lipid nanoparticles having an average size of about 50-70 nm (e.g., 55-65 nm) are particular suitable for pulmonary delivery via nebulization. BRIEF DESCRIPTION OF THE FIGURES [00029] FIG. 1 illustrates the target regions of siRNA oligonucleotides within the murine MUC5B mRNA sequence (GENBANK accession number NM_028801.2). Regions targeted by the oligonucleotides are underlined. siRNA oligonucleotide complementary sequences are shown in bold.

[00030] FIG. 2 illustrates MUC5B knockdown in human alveolar lung epithelium cells. The expression of MUC5B mRNA in A549 cells (human lung cancer line) was determined in response to treatment with chemically modified siRNAs oligonucleotides #1 through #10 targeting MUC5B at an oligonucleotide concentration of 1.25 nM. Each oligonucleotide was tested in triplicate. The fold change of MUC5B mRNA levels relative to control cells (treated with transfection agent only) is downs. MUC5B expression was normalized to the expression of housekeeping genes GAPDH and HPRT.

[00031] FIG. 3 illustrates MUC5B knockdown in human alveolar lung epithelium cells. The expression of MUC5B mRNA in A549 cells was determined in response to treatment with the cytokine IL6 and siRNA oligonucleotide #4 targeting MUC5B (each bar represent the average of n=3 or n=6 for IL6 treatment). The oligonucleotide concentration was 0.325 nM, 1.25 nM or 12.5 nM.

[00032] FIG. 4 illustrates MUC5B knockdown in vivo in the lung. 3-component or 4- component lipid nanoparticles (LNPs) comprising a cationic lipid (as indicated), a non- cationic lipid (DOPE), a PEG-modified lipid (DMG-PEG2K) and optionally cholesterol and encapsulating siRNA oligonucleotide #3 were administered to wild-type Balb/c mice (n=8 per group) via oral aspiration at a dose of 8 μg or 15 μg siRNA, respectively, as indicated. The mice were terminated and lungs were harvested after 72 hrs following dosing. The fold change in MUC5B mRNA levels in treatment groups is shown relative to the saline control group. MUC5B expression was normalized to the housekeeping genes GAPDH and PPIB. [00033] FIG. 5 illustrates MUC5B knockdown in vivo in the lung. 3-component or 4- component lipid nanoparticles (LNPs) comprising a cationic lipid (as indicated), a non- cationic lipid (DOPE), a PEG-modified lipid (DMG-PEG2K) and optionally cholesterol and encapsulating siRNA oligonucleotide #3 were administered to wild-type C57BL/6 mice (n=6 per group) via oral aspiration at a dose of 10 μg siRNA. The mice were terminated and lungs were harvested after 72 hrs following dosing. The fold change in MUC5B mRNA levels in treatment groups is shown relative to the saline control group. MUC5B expression was normalized to the housekeeping genes GUSB and PPIB.

[00034] FIG. 6 illustrates knockdown in MUC5B protein in vivo in the lung. 3- component or 4-component lipid nanoparticles (LNPs) comprising a cationic lipid (as indicated), a non-cationic lipid (DOPE), a PEG-modified lipid (DMG-PEG2K) and optionally cholesterol and encapsulating siRNA oligonucleotide #3 were administered to wild-type C57BL/6 mice (n=6 per group) via oral aspiration at a dose of 10 μg siRNA. In FIG. 6A, MUC5B protein expression was analysed in total protein extracted from isolated mouse lungs using SDS PAGE and Western Blot. MUC5B protein levels were normalized to Calnexin protein levels. In FIG, 6B, the fold change in MUC5B protein levels in treatment groups is shown relative to the saline control group. Each bar represents an average amount of MUC5B protein derived from lungs from three separate animals.

[00035] FIG. 7 illustrates knockdown MUC5B mRNA levels and MUC5B protein in vivo in the lung. 3 -component or 4-component lipid nanoparticles (LNPs) comprising a cationic lipid (as indicated), a non-cationic lipid (DOPE), a PEG-modified lipid (DMG- PEG2K) and optionally cholesterol and encapsulating siRNA oligonucleotide #3 were administered to wild-type C57BL/6 mice (n=6 per group) via oral aspiration at the indicated siRNA doses. The mice were terminated and lungs were harvested after 72 hrs following dosing. As shown in FIG. 7A, MUC5B mRNA expression levels were determined by qPCR. The fold change in MUC5B mRNA levels in treatment groups is shown relative to the saline control group. MUC5B expression was normalized to the housekeeping genes GAPDH and PPIB. As shown in FIG. 7B, MUC5B protein expression was analysed in total protein extracted from isolated mouse lungs using SDS PAGE and Western Blot. MUC5B protein levels were normalized to Calnexin protein levels. The fold change in MUC5B protein levels in treatment groups is shown relative to the saline control group. Each bar represents an average amount of MUC5B protein derived from lungs from three separate animals.

[00036] FIG. 8 illustrates the correlation between siRNA delivered to the lung tissue and the observed MUC5B knockdown. 3-component or 4-component lipid nanoparticles (LNPs) comprising a cationic lipid (as indicated), a non-cationic lipid (DOPE), a PEG- modified lipid (DMG-PEG2K) and optionally cholesterol and encapsulating siRNA oligonucleotide #3 were administered to wild-type C57BL/6 mice (n=6 per group) via oral aspiration at the indicated siRNA doses. The mice were terminated and lungs were harvested after 72 hrs following dosing. In FIG. 8A, a stem-loop PCR-based (SL-qPCR) method of siRNA quantification was used to measure the amount of antisense strand present in mice lungs. The amount in ng of siRNA per g of lung tissue is show for the treatment groups and the saline control group. In FIG. 8B, the siRNA concentration (ng siRNA/g lung tissue) is plotted on the horizontal axis. The percentage of MUC5B mRNA levels in the treatment groups relative to the saline control group (as determined by qPCR) is plotted on the vertical axis. The concentration of siRNA oligonucleotide # 3 in mice lungs was dose-dependent and showed a statistically significant correlation (p<0.0001, r = 0.65; R²=0.42).

[00037] FIG. 9 is a graph that shows MUC5B mRNA expressions in the left lung of wild-type C57BL/6 mice (n = 8) upon oral aspirations of lipid nanoparticles (LNPs) formulation TBL-0346 encapsulating siRNA#3 or siRNA#4. LNPs formulation TBL-0346 comprises a cationic lipid (cKK-E12), a non-cationic lipid (DOPE), a PEG-modified lipid (DMG-PEG2K) and optionally cholesterol. The mice were terminated 24, 48, and 72 hrs post-administation of the LNPs formulations encapsulating siRNA#3 or siRNA#4, and their lungs were harvested. MUC5B mRNA expression levels were determined using qPCR. MUC5B expression is normalized to housekeeping genes GUSB and HPRT. The fold change in MUC5B mRNA levels in treatment groups is shown relative to the saline control group 72 hrs post-administrations.

DETAILED DESCRIPTION OF THE INVENTION [00038] Further aspects of the disclosure, including a description of defined terms, are provided below. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of "or" means "and/or" unless stated otherwise. Furthermore, all transitional phrases such as “comprising”, “including”, “carrying”, “having”, “containing”, “involving”, “holding” and the like are to be understood to be open-ended, e.g., to mean including but not limited to. Additionally, the indefinite articles “a” and “an”, unless clearly indicated to the contrary, should be understood to mean “at least one”. [00039] Those skilled in the art will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and/or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is/are used. [00040] In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[00041] Compounds of the invention include variations of the disclosed compounds in which one or more hydrogen, carbon, nitrogen, oxygen, or sulfur atoms is replaced with a stable isotope of the same element.

[00042] It is also understood that the sequence set forth in each SEQ ID NO contained herein is independent of any nucleoside analogues or any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, compounds defined by a SEQ ID NO may comprise, independently, one or more nucleoside analogues or modifications to a sugar moiety, an internucleoside linkage, or a nucleoside. Moreover, the nucleotides T and U are used interchangeably in sequence descriptions.

Definitions

[00043] Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis.

[00044] Unless otherwise indicated, the following terms have the following meanings: [00045] “2'-deoxynucleoside" means a nucleoside comprising 2' -H(H) ribosyl sugar moiety, as found in naturally occurring deoxyribonucleic acids (DNA). In some embodiments, a 2'-deoxynucleoside may comprise a modified nucleobase or may comprise an RNA nucleobase (uracil).

[00046] “2’-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2’ position of a furanose ring. A 2’-O-F modified sugar is a modified sugar. [00047] “2’-OMe”, “2’-OCH₃” or “2’-O-methyl” each refers to a sugar comprising an -

OCH3 group at the 2’ position of a furanose ring. A 2’-O-methyl modified sugar is a modified sugar.

[00048] “2’-O-methoxyethyl”, “2’-MOE”, “2’-OCH₂CH₂-OCH₃” or “MOE” each refers to an O-methoxy-ethyl modification of the 2’ position of a furanose ring. A 2’-O- methoxyethyl modified sugar is a modified sugar.

[00049] “2'-substituted sugar moiety” or “2' -modified sugar moiety” or “2’ -modified sugar” means a furanosyl sugar moiety comprising at least one 2'-substituent group other than H or OH.

[00050] “2'-substituted nucleoside" or "2-modified nucleoside" means a nucleoside comprising a 2'-substituted, 2'-modified sugar moiety or 2’ -modified sugar.

[00051] “5-methylcytosine” means a cytosine modified with a methyl group attached to the 5’ position. A 5-methylcytosine is a modified nucleobase.

[00052] “Administration” or “administering” refers to bringing a patient or subject, tissue, organ or cells in contact with a compound or pharmaceutical composition described herein. As used herein, administration can be accomplished in vitro , i.e. in a test tube, or in vivo, i.e. in cells or tissues of living organisms, for example, humans. In some embodiments, administering comprises administering to lung cells. In some embodiments, administration to the subject is via pulmonary delivery.

[00053] “Aerosolization” or “aerosolized” refers to the conversion of a pharmaceutical composition into an aerosolized pharmaceutical composition. In some embodiments, aerosolization is accomplished using a propellant or other suitable energy source ( e.g ., ultrasound energy) to convert liquid or particles into a fine spray or dispersed suspension. In some embodiments, a nebulizer is used to aerosolize a pharmaceutical composition for pulmonary delivery. In some embodiments, the nebulizer uses ultrasound waves to generate an aerolized pharmaceutical composition.

[00054] “Aerosolized pharmaceutical composition” refers to a mixture of liquid (e.g., liquid droplets) or particles and air or other inhalable gas. In some embodiments, an aerosolized pharmaceutical composition comprises a fine spray or mist or a dispersed suspension that can be inhaled. In some embodiments, the liquid droplets or particles have a uniform size. [00055] “Alkynyl” refers to any hydrocarbon chain of either linear or branched configuration, having one or more carbon-carbon triple bonds occurring in any stable point along the chain, e.g. C2-C20 alkynyl refers to an alkynyl group having 2-20 carbons.

Examples of an alkynyl group include prop-2-ynyl, but-2-ynyl, but-3-ynyl, pent-2-ynyl, 3- methylpent-4-ynyl, hex-2-ynyl, hex-5-ynyl, etc. In embodiments, an alkynyl comprises one carbon-carbon triple bond. An alkynyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkynyl group may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’, -CO2H, -CO2R’, -CN, -OH, -OR’, -OCOR’, -OCO2R’, -NH₂, -NHR’, - N(R’)2, -SR’ or-S0₂R’, wherein each instance of R’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’ independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1- C3 alkyl). In embodiments, R’ independently is unsubstituted C1-C3 alkyl. In embodiments, the alkynyl is unsubstituted. In embodiments, the alkynyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein).

[00056] “Alkylene” refers to a saturated divalent straight or branched chain hydrocarbon group and is exemplified by methylene, ethylene, isopropylene and the like. [00057] “Alkenylene” refers to an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain.

[00058] “Alkynylene” refers to an unsaturated divalent straight or branched chain hydrocarbon group having one or more unsaturated carbon-carbon triple bonds that may occur in any stable point along the chain. In some embodiments, an alkylene, alkenylene, or alkynylene group may comprise one or more cyclic aliphatic and/or one or more heteroatoms such as oxygen, nitrogen, or sulfur and may optionally be substituted with one or more substituents such as alkyl, halo, alkoxyl, hydroxy, amino, aryl, ether, ester or amide. For example, an alkylene, alkenylene, or alkynylene may be substituted with one or more (e.g., 1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’, -CO2H, -CO2R’, - CN, -OH, -OR’, -OCOR’, -OCO2R’, -NH2, -NHR’, -N(R’)2, -SR’ or-SO2R’, wherein each instance of R’ independently is C₁-C₂₀ aliphatic (e.g., C₁-C₂₀ alkyl, C₁-C₁₅ alkyl, C₁-C₁₀ alkyl, or C₁-C₃ alkyl). In embodiments, R’ independently is an unsubstituted alkyl (e.g., unsubstituted C₁-C₂₀ alkyl, C₁-C₁₅ alkyl, C₁-C₁₀ alkyl, or C₁-C₃ alkyl). In embodiments, R’ independently is unsubstituted C₁-C₃ alkyl. In some embodiments, an alkylene, alkenylene, or alkynylene is unsubstituted. In some embodiments, an alkylene, alkenylene, or alkynylene does not include any heteroatoms.

[00059] “Alkenyl” refers to any linear or branched hydrocarbon chains having one or more unsaturated carbon-carbon double bonds that may occur in any stable point along the chain, e.g. C2-C20 alkenyl refers to an alkenyl group having 2-20 carbons. For example, an alkenyl group includes prop-2-enyl, but-2-enyl, but-3-enyl, 2-methylprop-2-enyl, hex-2-enyl, hex-5-enyl, 2,3-dimethylbut-2-enyl, and the like. In embodiments, the alkenyl comprises 1,

2, or 3 carbon-carbon double bond. In embodiments, the alkenyl comprises a single carbon- carbon double bond. In embodiments, multiple double bonds (e.g., 2 or 3) are conjugated.

An alkenyl group may be unsubstituted or substituted with one or more substituent groups as described herein. For example, an alkenyl group may be substituted with one or more (e.g.,

1, 2, 3, 4, 5, or 6 independently selected substituents) of halogen, -COR’, -CO2H, -CO2R’, - CN, -OH, -OR’, -OCOR’, -OCO2R’, -NH2, -NHR’, -N(R’)2, -SR’ or-SO2R’, wherein each instance of R’ independently is C1-C20 aliphatic (e.g., C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’ independently is an unsubstituted alkyl (e.g., unsubstituted C1-C20 alkyl, C1-C15 alkyl, C1-C10 alkyl, or C1-C3 alkyl). In embodiments, R’ independently is unsubstituted C1-C3 alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In embodiments, an alkenyl group is substituted with a-OH group and may also be referred to herein as a “hydroxyalkenyl” group, where the prefix denotes the - OH group and “alkenyl” is as described herein.

[00060] “Amelioration” refers to a lessening, slowing, stopping, or reversing of at least one indicator of the severity of a condition or disease. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art. [00061] “Amino” refers to groups of the form — N(R’)2 wherein each R’ is independently selected from hydrogen, alkyl, alkenyl, alkynyl, and aryl as described herein. Alkylamino includes both mono-alkylamino and dialkylamino, unless specified. Mono- alkylamino means an -NH(alkyl) group, in which alkyl is as defined herein. Dialkylamino means an -N(alkyl)2 group, in which each alkyl may be the same or different and are each as defined herein for alkyl. In embodiments, an alkyl group is a C₁-C₆ alkyl group. The group may be a terminal group or a bridging group. If the group is a terminal group it is bonded to the remainder of the molecule through the nitrogen atom.

[00062] “Amine” refers to a group having the amide functional group:

An amide group may have 1, 2, or 3 points of attachment to the molecule. Exemplary amide groups include -C(O)N(R’)₂, -C(O)NHR\ -C(O)NH₂, -C(O)NH-,-C(O)NR’-, -NHC(O)- ,and -NR’C(O)-, wherein each instance of R’ independently is C₁-C₂₀ aliphatic (e.g., C₁-C₂₀ alkyl, C₁-C₁₅ alkyl, ci-Cio alkyl, or C₁-C₃ alkyl), or two R’ can combine to form a 3- to 10- membered nitrogen-containing heterocyclyl. In embodiments, R’ independently is an unsubstituted alkyl (e.g., unsubstituted C₁-C₂₀ alkyl, C₁-C₁₅ alkyl, C₁-C₁₀ alkyl, or C₁-C₃ alkyl). In embodiments, R’ independently is unsubstituted C₁-C₃ alkyl. In embodiments, the alkenyl is unsubstituted. In embodiments, the alkenyl is substituted (e.g., with 1, 2, 3, 4, 5, or 6 substituent groups as described herein). In embodiments, an alkenyl group is substituted with a-OH group and may also be referred to herein as a “hydroxy alkenyl” group, where the prefix denotes the -OH group and “alkenyl” is as described herein.

[00063] “Anhydride linkages” are characterized by two acyl groups joined by an oxygen atom, having the general structure:

[00064] “Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense oligonucleotide complementary to a target nucleic acid, compared to target nucleic acid levels in the absence of the antisense oligonucleotide.

[00065] “Antisense oligonucleotide” or “ASO” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding segment of a target nucleic acid. An ASO may act through RNase H.

[00066] “Antisense strand” means an oligonucleotide strand that has a nucleobase sequence that, when written in the 5’ to 3’ direction, comprises the reverse complement of the portion of a target nucleic acid to which it is targeted. [00067] “Approximately” or “about”, as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[00068] “Base complementarity” refers to the capacity for the precise base pairing of nucleobases of an oligonucleotide strand with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.

[00069] “Bicyclic sugar” means a furanose ring modified by the bridging of two carbon atoms. In some embodiments, the bridge connects the 4’-carbon and the 2’-carbon of the sugar ring. A bicyclic sugar is a modified sugar.

[00070] “Bicyclic nucleoside” means a nucleoside comprising a bicyclic sugar, i.e. a sugar moiety comprising a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In some embodiments, the bridge connects the 4’-carbon and the 2’ -carbon of the sugar ring.

[00071] “Biologically active” refers to a characteristic of any agent that has activity in a biological system, and particularly in an organism. For instance, an agent that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active.

[00072] “Cationic Lipid” refers to any of a number of lipid species that have a net positive charge at a selected pH, typically at a pH below 6.5. Accordingly, a cationic lipid for use with the invention typically has a pKa around 6.5 to 7.0. LNPs incorporating such a cationic lipid typically have a neutral surface charge at a physiological pH or a pH in the range of 7.0 to 7.4, and a high surface charge at endosomal pH or a pH between 5.5 and 6.0. Suitable cationic lipids for use in the compounds, pharmaceutical compositions and methods provided herein include the cationic lipids as described in International Patent Publication WO 2011/068810; United States Provisional Patent Application Serial Number 62/864,818, filed on June 21, 2019; United States Provisional Patent Application Serial Number 62/865,555, filed on June 24, 2019; International Patent Publication WO 2012/170889; International Patent Publications WO 2013/063468 and WO 2016/205691; as well as the cationic lipids described in United States Provisional Patent Application Serial Number 62/758,179, filed on November 9, 2018, and Provisional Patent Application Serial Number 62/871,510, filed on July 8, 2019. The cationic lipids described in United States Provisional Patent Application Serial Number 62/864,818, filed on June 21, 2019, and in International Patent Publication WO 2012/170889 were found to be particularly suitable for preparing lipid nanoparticles suitable for pulmonary delivery via nebulization.

[00073] “Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an oligonucleotide.

[00074] “cEt” or “constrained ethyl” is a bicyclic sugar moiety comprising a bridge connecting the 4’ -carbon and the 2’ -carbon, wherein the bridge has the formula: 4’-CH(CH₃)- O-2’ . A cEt modified sugar is a modified sugar.

[00075] “cEt nucleoside” or “constrained ethyl nucleoside” means a nucleoside comprising a cET, i.e. comprising a bicyclic sugar moiety comprising a 4’-CH(CH₃)-O-2’ bridge.

[00076] “Chemically distinct region” refers to a region of an oligonucleotide that is in some way chemically different from another region of the same oligonucleotide. For example, a region having 2’-O-methoxyethyl nucleosides is chemically distinct from a region having nucleosides without 2’-O-methoxyethyl modifications.

[00077] “Chimeric oligonucleotides” are oligonucleotides that contain two or more chemically distinct regions.

[00078] “Co-administration” means administration of two or more pharmaceutical agents to an individual. The two or more pharmaceutical agents may be in a single pharmaceutical composition, or may be in separate pharmaceutical compositions. Each of the two or more pharmaceutical agents may be administered through the same or different routes of administration. Co-administration encompasses parallel or sequential administration. [00079] “Complementary” refers to the capacity for precise base pairing between two nucleotides. For example, if a nucleotide at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleotide at the same position of a target nucleic acid as described herein, then the oligonucleotide and the target nucleic acid are considered to be complementary to each other at that position. An oligonucleotide and target nucleic acid are complementary to each other when a sufficient number of corresponding positions in each molecule are occupied by nucleotides that can hydrogen bond with each other through their bases. With respect of an oligonucleotide the term complementary may be used to indicate a sufficient degree of complementarity or precise base pairing such that stable and specific binding occurs between the oligonucleotide and a target nucleic acid (e.g., between an antisense strand of an oligonucleotide and an mRNA). Thus, it should be appreciated 100% complementarity is not required for an oligonucleotide to be consider complementary to a target nucleic acid, provided that a sufficient degree of complementarity or precise base pairing exist to achieve stable and specific binding between the oligonucleotide and target nucleic acid. “Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In some embodiments, a first nucleic acid is an oligonucleotide comprising an antisense strand and a second nucleic acid is a target nucleic acid.

[00080] “Conjugate group" means a group of atoms that is directly or indirectly attached to an oligonucleotide. Conjugate groups may include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide.

[00081] "Conjugate linker" means a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.

[00082] "Conjugate moiety" means a group of atoms that is attached to an oligonucleotide via a conjugate linker.

[00083] “Contiguous nucleobases” means nucleobases immediately adjacent to each other.

[00084] “Delivery” encompasses both local and systemic delivery. For example, delivery of a compound comprising an oligonucleotide encompasses situations in which an oligonucleotide is delivered to a target tissue and the encoded protein is expressed and retained within the target tissue (also referred to as “local distribution” or “local delivery”), and situations in which an oligonucleotide is delivered to a target tissue and the encoded protein is expressed and secreted into patient’s circulation system (e.g., serum) and systematically distributed and taken up by other tissues (also referred to as “systemic distribution” or “systemic delivery). In some embodiments, delivery is pulmonary delivery, e.g., comprising nebulization.

[00085] “Designing” or “designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.

[00086] “Diluent” means an ingredient in a composition that lacks pharmacological activity, but is pharmaceutically necessary or desirable. For example, in drugs that are injected, the diluent may be a liquid, e.g., saline solution, such as phosphate buffered saline. [00087] “Dose” means a specified quantity of a pharmaceutical agent provided in a single administration, or in a specified time period. In some embodiments, a dose may be administered in one, two, or more boluses, tablets, or injections. For example, in some embodiments where subcutaneous administration is desired, the desired dose requires a volume not easily accommodated by a single injection, therefore, two or more injections may be used to achieve the desired dose. In some embodiments, the pharmaceutical agent is administered by infusion over an extended period of time or continuously. Doses may be stated as the amount of pharmaceutical agent per hour, day, week, or month.

[00088] “Double-stranded" means an oligonucleotide comprises two oligomeric compounds, such as an antisense strand and a sense strand, that are complementary to each other and form a duplex region.

[00089] “Effective amount” in the context of modulating an activity or of treating or preventing a condition means the administration of that amount of pharmaceutical agent to a subject in need of such modulation, treatment, or prophylaxis, either in a single dose or as part of a series, that is effective for modulation of that effect, or for treatment or prophylaxis or improvement of that condition. The effective amount may vary among individuals depending on the health and physical condition of the individual to be treated, the taxonomic group of the individuals to be treated, the composition of the pharmaceutical composition, assessment of the individual’s medical condition, and other relevant factors.

[00090] “Efficacy” means the ability to produce a desired effect.

[00091] “Ethylene bridged nucleic acid” or “ENA” is a bicyclic sugar moiety comprising a bridge connecting the 4’ -carbon and the 2 ’-carbon, wherein the bridge is an ethylene bridge (4’-CH₂CH₂-O-2’). An ENA modified sugar is a modified sugar. [00092] “ENA nucleoside” means a nucleoside comprising an ENA, i.e. comprising a bicyclic sugar moiety comprising an ethylene bridge.

[00093] “Encapsulation” refers to the process of confining a nucleic acid molecule within a liposomal delivery vehicle, such as a lipid nanoparticle.

[00094] “Ester linkage” refers to — OC(=O ) — or — C(=O )O — ; thioester linkage refers to — SC(=O) — or — C(=O)S — .

[00095] “Expression” includes all the functions by which a gene’s coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation, i.e. mRNA and protein.

[00096] “Halogen” means fluorine, chlorine, bromine, or iodine.

[00097] “Heteroalkyl” refers to a branched or unbranched alkyl, alkenyl, or alkynyl group having from 1 to 14 carbon atoms in addition to 1, 2, 3 or 4 heteroatoms independently selected from the group consisting of N, O, S, and P. Heteroalkyls include tertiary amines, secondary amines, ethers, thioethers, amides, thioamides, carbamates, thiocarbamates, hydrazones, imines, phosphodiesters, phosphoramidates, sulfonamides, and disulfides. A heteroalkyl group may optionally include monocyclic, bicyclic, or tricyclic rings, in which each ring desirably has three to six members. Examples of heteroalkyls include polyethers, such as methoxymethyl and ethoxyethyl.

[00098] “Heteroalkylene” refers to a divalent form of a heteroalkyl group as described herein.

[00099] “Heterocycle, heterocyclyl, heterocyclic radical, and heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic or 7-10-membered bicyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term nitrogen includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4- dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+ (as in N-substituted pyrrolidinyl). [000100] “Hybridization” means the annealing of complementary nucleic acid molecules. In some embodiments, complementary nucleic acid molecules include, but are not limited to, an oligonucleotide comprising an antisense strand and a target nucleic acid. [000101] “Improve”, “increase”, or “reduce” indicate values that are relative to a baseline measurement, such as a measurement in the same individual prior to initiation of the treatment described herein, or a measurement in a control subject (or multiple control subject) in the absence of the treatment described herein. A “control subject” is a subject afflicted with the same form of disease as the subject being treated, who is about the same age as the subject being treated.

[000102] “Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.

[000103] “Individual” means a human or non-human subject selected for treatment or therapy.

[000104] “Inhibiting the expression or activity” refers to a reduction or blockade of the expression, level or activity and does not necessarily indicate a total elimination of expression or activity.

[000105] “Internucleoside linkage” refers to the chemical bond between adjacent nucleosides.

[000106] “Intratracheal administration” or “Intratracheal instillation” is the introduction of a substance directly into the trachea. It can be performed, for example, through inserting a needle or catheter down the mouth and throat, or through surgically exposing the trachea and penetrating it with a needle. Generally, short-acting inhaled anesthetic drugs such as halothane, metaphane, or enflurane are used during the instillation procedure.

[000107] “Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.

[000108] “Lipid nanoparticle” is a type of liposomal delivery vehicle and refers to a discrete object comprised of one or more lipids and possessing at least one dimension that is generally less than or equal to 5 micron in size. Lipid nanoparticles may be a variety of different shapes, including but not limited to spheroidal, cuboidal, pyramidal, oblong, cylindrical, toroidal, and the like. In some pharmaceutical compositions, lipid nanoparticles comprise at least one cationic lipid, at least one non-cationic lipid, and at least one aggregation prevention lipid, e.g., PEG-modified lipid.

[000109] “Liposomal delivery vehicles”, e.g., lipid nanoparticles, are characterized as microscopic vesicles having an interior aqua space sequestered from an outer medium by a membrane of one or more bilayers. Bilayer membranes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends BiotechnoL, 16: 307-321, 1998).

Bilayer membranes can also be formed by amphiphilic polymers and surfactants ( e.g ., polymerosomes, niosomes, etc.). In the context of the present invention, a liposomal delivery vehicle typically serves to transport a desired oligonucleotide to a target cell or tissue. [000110] “Liposome” is a type of liposomal delivery vehicle and refers to any lamellar, multilamellar, or solid nanoparticle vesicle. Typically, a liposome as used herein can be formed by mixing one or more lipids or by mixing one or more lipids and polymer(s). Typically, a lipid nanoparticle for use with the present invention is a liposome. Liposomes suitable for practicing the invention typically comprise 3 or 4 lipid components, at least one of one of which is a cationic lipid component and one of which is a PEG-modified lipid component. Typically, such a liposome also comprises a cholesterol-based lipid component. In 3-component liposomes, the cholesterol-based lipid component and the cationic lipid component are typically the same. 3-component and 4-component liposomes usually also comprise non-cationic lipid component (helper lipid).

[000111] “Locked nucleic acid” or “LNA” is a bicyclic sugar moiety comprising a bridge connecting the 4’ -carbon and the 2’ -carbon. An LNA modified sugar is a modified sugar. Examples of such bicyclic sugar include, but are not limited to a-L-Methyleneoxy (4’- CH2-O-2’) LNA , β-D-Methyleneoxy (4’-CH2-O-2’) LNA, Ethyleneoxy (4’-(CH2)2-O-2’) LNA, Aminooxy (4’-CH2-O-N(R)-2’) LNA and Oxyamino (4’-CH2-N(R)-O-2’) LNA. As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4’ and the 2’ position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from [C(Rl)(R2)]n-, C(R1)=C(R2)-, C(R1)=N-, C(=NR1)-, -C(=O)-, -C(=S)-, -O-, -Si(Rl)2-, - S(=O)x- and N(R1)-; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R1 and R2 is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2- C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5- C20 aryl, substituted C5-C20 aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group. Examples of 4’- 2’ bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: [C(Rl)(R2)]n-, [C(Rl)(R2)]n-O-, C(RlR2)-N(Rl)-O- or -C(RlR2)-O-N(Rl)-.

Furthermore, other bridging groups encompassed with the definition of LNA are 4'-CH2-2', 4'-(CH2)2-2', 4'-(CH2)3-2', 4'-CH2-O-2', 4'-(CH2)2-O-2', 4'-CH2-O-N(Rl)-2' and 4'-CH2- N(Rl)-O-2'- bridges, wherein each R1 and R2 is, independently, H, a protecting group or Cl- C12 alkyl. Also included within the definition of LNA are LNAs in which the 2'-hydroxyl group of the ribosyl sugar ring is connected to the 4' carbon atom of the sugar ring, thereby forming a methyleneoxy (4’-CH2-O-2’) bridge to form the bicyclic sugar moiety. The bridge can also be a methylene (-CH2-) group connecting the 2' oxygen atom and the 4' carbon atom, for which the term methyleneoxy (4’-CH2-O-2’) LNA is used a -L- methyleneoxy (4’-CH2-O-2’), an isomer of methyleneoxy (4’-CH2-O-2’) LNA is also encompassed within the definition of LNA, as used herein.

[000112] “LNA nucleoside” means a nucleoside comprising an LNA, i.e. comprising a bicyclic sugar moiety comprising a bridge connecting the 4’-carbon and the 2’-carbon. [000113] “Lung cells” may refer to any cells that reside in the lungs. In some embodiments, lung cells are cells that carry out functions relevant to lung function, development, regeneration and/or repair. In some embodiments, lung cells arise from cells forming the lung endoderm. Non-limiting examples of lung cells include: epithelial cells, type I alveolar cells, type II alveolar cells, macrophages, fibroblasts, neuroendocrine cells, basal cells, secretory cells, ciliated cells, Clara cells, and endothelial cells. In some embodiments, lung cells are epithelial cells residing in the secondary bronchi, tertiary bronchi, bronchioles, and/or alveoli regions of the lung.

[000114] “microRNA” or “miRNA” refers to a small non-coding RNA that functions in RNA silencing and post-transcriptional regulation of gene expression by hybridizing to complementary mRNA sequences. Artificial miRNA or amiRNA are artificially designed miRNAs based on endogenous miRNAs. [000115] “Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.

[000116] “Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e., a 3’ to 5’ phosphodiester internucleoside bond).

[000117] “Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). [000118] “Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase, or a nucleoside analogue.

[000119] “Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, and/or modified nucleobase, or a nucleotide analogue.

[000120] “Modified oligonucleotide” means an oligonucleotide comprising at least one nucleoside analogue, nucleotide analogue, modified internucleoside linkage, modified sugar, and/or modified nucleobase, or a nucleic acid analogue.

[000121] “Modified sugar” means any substitution and/or any change from a natural sugar moiety.

[000122] “Monomer” means a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.

[000123] “Morpholino” or “phosphorodiamidate Morpholino oligomer (PMO)”, is a nucleic acid analogue, wherein methylenemorpholine rings replace the ribose or deoxyribose sugar moieties and non-ionic phosphorodiamidate linkages replacing the anionic phosphates of DNA and RNA.

[000124] “Motif’ means the pattern of unmodified and modified nucleoside in an oligonucleotide.

[000125] “MUC5B nucleic acid” means any nucleic acid encoding MUC5B. For example, in some embodiments, a MUC5B nucleic acid includes a DNA sequence encoding MUC5B, an RNA sequence transcribed from DNA encoding MUC5B including genomic DNA comprising introns and exons ( i.e ., pre-mRNA), and an mRNA sequence encoding MUC5B. “MUC5B mRNA” means an mRNA encoding a MUC5B protein.

[000126] “N/P ratio” refers to a molar ratio of positively charged molecular units in the cationic lipids in a lipid nanoparticle relative to negatively charged molecular units in the oligonucleotide encapsulated within that lipid nanoparticle. As such, N/P ratio is typically calculated as the ratio of moles of amine groups in cationic lipids in a lipid nanoparticle relative to moles of phosphate groups in oligonucleotide encapsulated within that lipid nanoparticle.

[000127] “Natural sugar moiety” means a sugar moiety found in DNA (2’-H) or RNA (2’-OH).

[000128] “Naturally occurring internucleoside linkage” means a 3' to 5' phosphodiester linkage.

[000129] “Nebulization” refers to delivery of a pharmaceutical composition in a fine spray or dispersed suspension that is inhaled into the lungs, typically by means of a nebulizer. [000130] “Nebulizer” is a device that uses a propellant or other suitable energy source such as oxygen, compressed air, or ultrasound waves to convert liquid or particles into a fine spray or mist or a dispersed suspension, typically in form of an aerosol that can be directly inhaled. In some embodiments, a nebulizer for use with the invention contains a piezoelectric element to generate the vibration of a mesh. The vibration pumps a liquid pharmaceutical composition through the mesh. The liquid is emitted from the mesh in droplets generating the aerosol. Such nebulizers are commonly referred to as (vibrating) mesh nebulizers. In some embodiments, a nebulizer is used to aerosolize a pharmaceutical composition for pulmonary delivery. Inhalation from a nebulizer is through a mouthpiece used by the subject [000131] “Non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH.

[000132] “Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.

[000133] “Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single- stranded nucleic acids, double-stranded nucleic acids, siRNAs, ASOs, shRNAs, miRNAs, and amiRNAs. In some embodiments, the nucleotides T and U are used interchangeably in sequence descriptions.

[000134] “Nucleic acid analogue” means a nucleic acid that is analogous (structurally similar) to a naturally occurring nucleic acid, i.e., a synthetic mimic of an oligonucleotide. In some embodiments, the oligonucleotide is a nucleic acid analogue, such as a peptide nucleic acid (PNA) or a morpholino.

[000135] “Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.

[000136] “Nucleobase complementarity” refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In some embodiments, complementary nucleobase refers to a nucleobase of an oligonucleotide that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an oligonucleotide is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.

[000137] “Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.

[000138] “Nucleoside” means a nucleobase linked to a sugar.

[000139] "Nucleoside analogue" means a nucleoside that is analogous (structurally similar) to a naturally occurring nucleoside. A nucleoside analogue may be used to replace the naturally occurring sugar and base, but not necessarily the internucleoside linkage at one or more positions of an oligomeric compound.

[000140] “Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.

[000141] "Nucleotide analogue" means a nucleotide that is analogous (structurally similar) to a naturally occurring nucleoside. A nucleotide analogue may be used to replace the naturally occurring sugar, base and internucleoside linkage at one or more positions of an oligomeric compound. [000142] “Off-target effect” refers to an unwanted or deleterious biological effect associated with modulation of mRNA or protein expression of a gene other than the intended target nucleic acid.

[000143] “Oligomeric compound” or “oligomer” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule. Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogues, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

[000144] “Oligonucleotide” means a polymer of linked nucleosides, each of which can be modified or unmodified, independent one from another. In some embodiments, the oligonucleotide is single- stranded and comprises an antisense strand or a sense strand. In some embodiments, the oligonucleotide is double- stranded and comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region. In some embodiments, the oligonucleotide is a siRNA. In some embodiments, the oligonucleotide is an ASO.

[000145] “PEG-modified lipid” refers to a lipid comprising one or more polyethylene glycol molecules. In some embodiments, the one or more polyethylene glycol molecules are covalently attached to the lipid.

[000146] “Parenteral administration” means administration through injection ( e.g ., bolus injection) or infusion. Parenteral administration includes subcutaneous administration, intravenous administration, intramuscular administration, intraarterial administration, intraperitoneal administration, or intracranial administration, e.g., intrathecal or intracerebroventricular administration.

[000147] “Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Without limitation, as used herein, peptide refers to polypeptides and proteins. [000148] “Peptide nucleic acid” or “PNA” is a nucleic acid analogue wherein the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, for example an aminoethylglycine backbone, and wherein the nucleobases are retained and are bound directly or indirectly to the aza nitrogen atoms of the polyamide backbone. [000149] “Pharmaceutical agent” means a substance that provides a therapeutic benefit when administered to an individual. For example, in some embodiments, a siRNA targeted to MUC5B is a pharmaceutical agent.

[000150] “Pharmaceutically acceptable derivative” encompasses pharmaceutically acceptable salts, conjugates, prodrugs or isomers of the compounds described herein.

[000151] “Pharmaceutically acceptable salts” means physiologically and pharmaceutically acceptable salts of oligonucleotides, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

[000152] “Phosphothioester linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the bridging oxygen atoms with a sulfur atom. A phosphothioester linkage is a modified internucleoside linkage.

[000153] “Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.

[000154] “Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In some embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In some embodiments, a portion is a defined number of contiguous nucleobases of an oligonucleotide.

[000155] “Prevent” or “preventing” refers to delaying or forestalling the onset or development of a disease, disorder, or condition for a period of time from minutes to days, weeks to months, or indefinitely.

[000156] “Prodrug” means a therapeutic agent that is prepared in an inactive form that is converted to an active form within the body or cells thereof by the action of endogenous enzymes or other chemicals or conditions.

[000157] “Prophylactically effective amount” refers to an amount of a pharmaceutical agent that provides a prophylactic or preventative benefit to a subject.

[000158] “Pulmonary delivery” refers to administering the pharmaceutical composition described herein to lung cells in vivo by delivering the pharmaceutical composition to the lung. Non-limiting methods of pulmonary delivery include: nebulization and intratracheal administration/intratracheal instillation. [000159] “Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.

[000160] “Ribonucleotide” means a nucleotide having a hydroxy at the 2’ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.

[000161] “RNAi compound” means an oligomeric compound that acts, at least in part, through RISC or Ago2 to modulate a target nucleic acid and/or protein encoded by a target nucleic acid. RNAi compounds include, but are not limited to siRNA, single-stranded RNA (ssRNA), shRNAs, and miRNA, including miRNA mimics such as amiRNA. In some embodiments, an RNAi compound modulates the amount, activity, and/or splicing of a target nucleic acid. The term RNAi compound excludes ASOs that act through RNase H.

[000162] “Salts” mean physiologically and pharmaceutically acceptable salts of oligonucleotides, i.e., salts that retain the desired biological activity of the parent oligonucleotide and do not impart undesired toxicological effects thereto.

[000163] “Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.

[000164] “Sense strand” means an oligonucleotide strand that has a nucleobase sequence that, when written in the 5’ to 3’ direction, has the same sequence as a portion of a target nucleic acid.

[000165] “Single- stranded" means an oligonucleotide that comprises one oligomeric compound, such as an antisense strand or a sense strand that is not paired with a complementary second oligomeric compound to form a duplex region.

[000166] “Single- stranded overhang” refers to an oligonucleotide region outside of the duplex region of a double- stranded oligonucleotide that is not complementary to either the sense strand or antisense strand.

[000167] "Self-complementary" means an oligonucleotide that at least partially hybridizes to itself. A compound consisting of one oligomeric compound, wherein the oligonucleotide of the oligomeric compound is self-complementary, is a single-stranded compound. A single-stranded oligomeric compound may be capable of binding to a complementary oligomeric compound to form a duplex, in which case the compound would no longer be single- stranded. [000168] “Short hairpin RNAs” or “shRNAs” are artificial RNA molecules with a tight hairpin turn that operate within the RNAi pathway to inhibit expression of specific genes with complementary nucleotide sequences.

[000169] “siRNA”, “siRNA oligonucleotide”, “siRNA molecule" or “siRNA duplex”, also known as short interfering RNA or silencing RNA, is a short double- stranded RNA molecule, e.g. 20-25 base pairs in length, which operates within the RNAi pathway. It interferes with the expression of specific genes with complementary nucleotide sequences by degrading mRNA after transcription, preventing translation. A siRNA does not act through RNase H.

[000170] “Side effects” mean physiological responses attributable to a treatment other than desired effects. In some embodiments, side effects include, without limitation, injection site reactions, liver function test abnormalities, renal function abnormalities, liver toxicity, renal toxicity, central nervous system abnormalities, and myopathies.

[000171] “Sites” are defined as unique nucleobase positions within a target nucleic acid. [000172] “Slowing progression” means a decrease in the development of the disease. [000173] “Specifically hybridizable” refers to an oligonucleotide having a sufficient degree of complementarity between an antisense strand and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.

[000174] “Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target nucleic acid, but to a minimal number of other nucleic acid sequences.

[000175] “Subject” refers to a human or any non-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine, sheep, horse or primate). A subject can be a patient, which refers to a subject, e.g., a human, presenting to a medical or health care provider for evaluation and/or treatment, including for diagnosis or treatment of a lung disease or disorder. The term “subject” is used herein interchangeably with “individual” or “patient.” A subject can be afflicted with or is susceptible to a disease or disorder, but may or may not display symptoms of the disease or disorder. [000176] “Substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[000177] "Sugar moiety" means an unmodified sugar moiety or a modified sugar moiety. As used herein, "unmodified sugar moiety" means a 2'-OH(H) ribosyl moiety, as found in RNA (an "unmodified RNA sugar moiety"), or a 2' -H(H) moiety, as found in DNA (an "unmodified DNA sugar moiety"). As used herein, "modified sugar moiety" or "modified sugar" means a modified furanosyl sugar moiety. As used herein, modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety. In some embodiments, a modified furanosyl sugar moiety is a 2' -substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars.

[000178] “Systemic distribution” or “systemic delivery” refer to a delivery or distribution mechanism or approach that affect the entire body or an entire organism. Typically, systemic distribution or delivery is accomplished via body’s circulation system, e.g., blood stream.

[000179] “Target” refers to a protein, the modulation of which is desired, such as MUC5B.

[000180] “Target gene” refers to a gene encoding a target, such as MUC5B.

[000181] “Targeting” or “targeted” means the process of design and selection of an oligonucleotide that will specifically hybridize to a target nucleic acid and induce a desired effect.

[000182] “Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by oligonucleotides, such as a MUC5B nucleic acid.

[000183] “Target region” means a portion of a target nucleic acid to which one or more oligonucleotides is targeted. [000184] “Target segment” means the sequence of nucleotides of a target nucleic acid to which an oligonucleotide is targeted. “5’ target site” refers to the 5 ’-most nucleotide of a target segment. “3’ target site” refers to the 3 ’-most nucleotide of a target segment. A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping.

[000185] “Target tissues” refers to any tissue that is affected by a disease to be treated. In some embodiments, target tissues include those tissues that display disease-associated pathology, symptom, or feature.

[000186] "Terminal group" means a chemical group or group of atoms that is covalently linked to a terminus of an oligonucleotide.

[000187] “Therapeutically effective amount” means an amount of a pharmaceutical agent that provides a therapeutic benefit to an individual. It will be appreciated by those of ordinary skill in the art that a therapeutically effective amount is typically administered via a dosing regimen comprising at least one unit dose.

[000188] “Treating” "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of and/or reduce incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. Treatment may be administered to a subject who does not exhibit signs of a disease and/or exhibits only early signs of the disease for the purpose of decreasing the risk of developing pathology associated with the disease.

[000189] “Unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases (T), cytosine (C), and uracil (U).

[000190] “Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In some embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).

Oligonucleotides

[000191] The invention relates to compounds and pharmaceutical compositions comprising oligonucleotides for modulating expression of MUC5B mRNA and/or protein in lung cells or tissues. In some embodiments, the cell or tissue is in a subject. In some embodiments, the subject is a human. In some embodiments, MUC5B mRNA levels are reduced. In some embodiments, MUC5B protein levels are reduced. In some embodiments, MUC5B mRNA and protein levels are reduced. Such reduction can occur in a time- dependent manner or in a dose-dependent manner.

[000192] For example, in some embodiments, a dose dependent MUC5B mRNA knockdown is achieved using siRNA delivered in an LNP formulation described herein. In some embodiments, a dose dependent MUC5B mRNA knockdown is achieved with one or more siRNAs, for example, such as those described herein. In some embodiments, the delivered siRNA in an LNP formulation achieves sustained MUC5B mRNA knockdown. For example, in some embodiments, the delivered siRNA in an LNP formulation results in MUC5B mRNA knockdown for at least 24 hours, 48 hours or 72 hours. Accordingly, in some embodiments, the delivered siRNA in an LNP formulation results in MUC5B mRNA knockdown for at least 24 hours. In some embodiments, the delivered siRNA in an LNP formulation results in MUC5B mRNA knockdown for at least 48 hours. In some embodiments, the delivered siRNA in an LNP formulation results in MUC5B mRNA knockdown for at least 72 hours. In some embodiments, the delivered siRNA in an LNP formulation results in MUC5B mRNA knockdown for more than 72 hours.

[000193] The oligonucleotides described herein are targeted to a MUC5B nucleic acid sequence as set forth in GENBANK accession number NM_002458.3 (incorporated herein as SEQ ID NO: 1), GENBANK accession number NM_028801.2 (incorporated herein as SEQ ID NO: 2) GENBANK accession number XM_006230608.2 (incorporated herein as SEQ ID NO: 3), GENBANK accession number XM_006223574.2 (incorporated herein as SEQ ID NO: 4), GENBANK accession number XM_015435240.1 (incorporated herein as SEQ ID NO: 5), or GENBANK accession number XM_021082487.1 (incorporated herein as SEQ ID NO: 6). In a particular embodiment, the target MUC5B nucleic acid sequence is as set forth in GENBANK accession number NM_002458.3 (incorporated herein as SEQ ID NO: 1). [000194] In particular, the present disclosure provides a compound comprising an oligonucleotide comprising an antisense strand consisting of 15-30 linked nucleosides, wherein the nucleobase sequence of the antisense strand has at least 12 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-6. [000195] The oligonucleotide may be a DNA oligonucleotide or a RNA oligonucleotide. The oligonucleotide may be a siRNA, shRNA, miRNA, amiRNA, or an ASO. In a particular embodiment, the oligonucleotide is a siRNA, i.e. an oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region.

[000196] In some embodiments, the nucleobase sequence of the antisense strand has at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29 or 30 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-6.

[000197] In some embodiments, invention provides an oligonucleotide comprising an antisense strand, wherein the nucleobase sequence of the antisense strand has at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29 or 30 contiguous nucleobases that are complementary to an equal length portion of any one of the target regions within the murine MUC5B mRNA sequence (SEQ ID NO: 2), which are shown as underlined in FIG. 1, or the corresponding target regions within the human, rat, cynomolgus or pig MUC5B mRNA sequence of SEQ ID NOs: 1 and 3-6, respectively. [000198] In some embodiments, the nucleobase sequence of the antisense strand is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to an equal length portion of any one of SEQ ID NOs: 1-6, as measured over the entirety of the antisense strand. The nucleobase sequence of the antisense strand comprised in oligonucleotides of the invention is typically 90%, more typically 94% complementary to an equal length portion of any one of SEQ ID NOs: 1-6.

[000199] In some embodiments, the oligonucleotide is single-stranded. In some embodiments, the oligonucleotide is double- stranded. In some embodiments the oligonucleotide is a siRNA oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region. In some embodiments, the sense strand and/or the antisense strand consists of 15-30 linked nucleosides. In some embodiments, the sense strand and/or the antisense strand consists of 15-25 linked nucleosides. In some embodiments, the sense strand and/or the antisense strand consists of 15-20 linked nucleosides. In some embodiments, the sense strand and/or the antisense strand consists of 18-20 linked nucleosides. In some embodiments, the sense strand and/or the antisense strand consists of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 linked nucleosides, or a range defined by any two of these values. In a particular embodiment, the sense strand and/or the antisense strand consists of 19 linked nucleosides.

[000200] In some embodiments, the oligonucleotide comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, and wherein the duplex region is 15-30 nucleosides in length. In some embodiments, the duplex region is 15-25 nucleosides in length. In some embodiments, the duplex region is 15-20 nucleosides in length. In some embodiments, the duplex region is 18-20 nucleosides in length. In some embodiments, the duplex region is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides long, or a range defined by any two of these values. In a particular embodiment, the duplex region is 19 nucleosides in length.

[000201] In some embodiments, the sense strand and/or the antisense strand of the oligonucleotide comprises a single-stranded overhang. In some embodiments, both the sense strand and the antisense strand comprise a single-stranded overhang. In some embodiments, only the sense strand comprises a single- stranded overhang. In some embodiments, only the antisense strand comprises a single- stranded overhang. In some embodiments, the sense strand and/or the antisense strand comprises a 3’ single-stranded overhang. In some embodiments, the sense strand and/or the antisense strand comprises a 5’ single-stranded overhang. In some embodiments, the sense strand or the antisense strand comprises a 5’ single-stranded overhang and a 3’ single-stranded overhang. In some embodiments, the single-stranded overhang is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 nucleosides in length. In some embodiments, the single-stranded overhang is one nucleoside in length. In some embodiments, the single- stranded overhang is two nucleosides in length. In some embodiments, the single- stranded overhang is five nucleosides in length. In some embodiments, the single- stranded overhang is eight nucleosides in length. In some embodiments, the nucleoside(s) of the single-stranded overhang is deoxythymidine (dT). In some embodiments, the nucleoside(s) of the single- stranded overhang is uridine (U). In some embodiments, both the sense strand and the antisense strand comprise a 3’ single- stranded overhang consisting of one deoxy thymidine. In some embodiments, both the sense strand and the antisense strand comprise a 3’ single- stranded overhang consisting of eight deoxythymidines. In a particular embodiment, both the sense strand and the antisense strand comprise a 3’ single- stranded overhang consisting of two deoxythymidines, and optionally the internucleoside linkages of the single- stranded overhang are modified internucleoside linkages, such as phosphothioester internucleoside linkages.

[000202] In some embodiments, the oligonucleotide comprises one blunt end. In some embodiments, the oligonucleotide comprises a 5’ blunt end. In some embodiments, the oligonucleotide comprises a 3’ blunt end. In some embodiments, the oligonucleotide comprises a 5’ and a 3’ blunt end.

[000203] In some embodiments, the nucleotide at an end of the sense and/or antisense strand is uracil. In some embodiments, the nucleotide at an end of the sense and/or antisense strand is adenine. In some embodiments, the nucleotide at the 3’ end of the sense strand is adenine. In some embodiments, the nucleotide at the 5’ end of the antisense strand is uracil. In a particular embodiment, the nucleotide at the 3 ’ end of the sense strand is adenine, and the nucleotide at the 5’ end of the antisense strand is uracil.

[000204] In some embodiments, an oligonucleotide may have a sequence that does not contain guanosine nucleotide stretches (e.g., 3 or more, 4 or more, 5 or more, 6 or more contiguous guanosine nucleotides). In some embodiments, oligonucleotides having guanosine nucleotide stretches have increased non-specific binding and/or off-target effects, compared with oligonucleotides that do not have guanosine nucleotide stretches. In some embodiments, oligonucleotides do no contains any contiguous runs of three or more guanosine (G) or cytidine (C) nucleosides. An oligonucleotide may have a sequence that has greater than 30% G-C content, greater than 40% G-C content, greater than 50% G-C content, greater than 60% G-C content, greater than 70% G-C content, or greater than 80% G-C content. An oligonucleotide may have a sequence that has up to 100% G-C content, up to 95% G-C content, up to 90% G-C content, or up to 80% G-C content. In some embodiments, the G-C content of an oligonucleotide is preferably between about 30-60%. [000205] In some embodiments, oligonucleotides targeted to a MUC5B nucleic acid have chemically modified subunits arranged in patterns, or motifs, to confer to the oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases. Such chimeric oligonucleotides typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. For example, antisense oligonucleotides having a gapmer motif are considered chimeric oligonucleotides.

[000206] In some embodiments, the oligonucleotide is a chimeric oligonucleotide that contains two or more chemically distinct regions, each made up of at least one nucleotide. These chimeric oligonucleotides typically contain at least one region of modified nucleotides that confers one or more beneficial properties (such as, for example, increased nuclease resistance, increased uptake into cells, increased binding affinity for the target).

[000207] Oligonucleotides can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of the oligonucleotide to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the oligonucleotide from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5'- terminus (5'-cap), or at the 3'-terminus (3'-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3' and 5 '-stabilizing groups that can be used to cap one or both ends of an oligonucleotide to impart nuclease stability include those disclosed in WO 03/004602. [000208] Table 1 provides exemplary MUC5B siRNA duplex oligonucleotide sequences of the invention, and details of their chemical modifications. The invention provides a compound comprising a siRNA oligonucleotide with any one of the pairs of antisense strand and sense strands sequences described in Table 1. The invention also provides a compound comprising any one of the siRNA oligonucleotides as described in Table 1. The invention also provides compounds comprising any selection of any number of the pairs of antisense strand and sense strands sequences described in Table 1. The invention also provides compounds comprising any selection of any number of the siRNA oligonucleotides as described in Table 1. Table 1. MUC5B siRNA duplex sequences

An underlined nucleoside denotes a 2’-O-methyl-ribonucleoside A bold and italicized nucleoside denotes a 2’-F-deoxynucleoside A subscript “s” denotes a phosphothioester internucleoside linkage * all 3’-TT overhangs are deoxythymidines

H= Human, M= Mus musculus, R= Rattus norvegicus, C= Macaca fascicularis (cynomolgus monkey)

[000209] In other embodiments the oligonucleotide comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, and wherein the sense strand has a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or 19 contiguous nucleobases of a nucleobase sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 113; and the antisense strand has a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or 19 contiguous nucleobases of a corresponding nucleobase sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114.

[000210] In other embodiments the oligonucleotide comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, and wherein the sense strand has a nucleobase sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27,

29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 113; and the antisense strand has a corresponding nucleobase sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46,

48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114.

[000211] In a further embodiment, both the sense strand and the antisense strand of the oligonucleotide comprise a 3’ single- stranded overhang consisting of two deoxythymidines, optionally wherein both of the internucleoside linkages of the single- stranded overhangs are modified internucleoside linkages, such as phosphothioester internucleoside linkages. In a further embodiment, each nucleoside of the antisense strand and each nucleoside of the sense strand comprises either a 2’-F or a 2'-O-methyl modified sugar. In a further embodiment, the oligonucleotide comprises one or more additional phosphothioester linkages.

[000212] In a particular embodiment, the invention provides a compound comprising an oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, wherein the sense strand has the nucleobase sequence of SEQ ID NO: 7, and the antisense strand has the nucleobase sequence of SEQ ID NO: 8. In a further embodiment, both the sense strand and the antisense strand comprise a 3’ single- stranded overhang consisting of two deoxythymidines. In a further embodiment, both of the internucleoside linkages of the single-stranded overhangs are phosphothioester internucleoside linkages. In a further embodiment, each nucleoside of the antisense strand and each nucleoside of the sense strand comprises either a 2’ -F or a 2 '-O-methyl modified sugar. In a further embodiment, both the sense strand and the antisense strand of the oligonucleotide comprise two additional phosphothioester linkages at their 5’ ends.

[000213] In a particular embodiment, the invention provides a compound comprising an oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, wherein the sense strand has the nucleobase sequence of SEQ ID NO: 7, and the antisense strand has the nucleobase sequence of SEQ ID NO: 8, wherein both the sense strand and the antisense strand comprise a 3’ single-stranded overhang consisting of two deoxythymidines, wherein both of the internucleoside linkages of the single-stranded overhangs are phosphothioester internucleoside linkages, and wherein each nucleoside of the antisense strand and each nucleoside of the sense strand comprises either a 2’-F or a 2'-O-methyl modified sugar.

[000214] In a particular embodiment, the invention provides a compound comprising an oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, wherein the sense strand has the nucleobase sequence of SEQ ID NO: 7, and the antisense strand has the nucleobase sequence of SEQ ID NO: 8, wherein both the sense strand and the antisense strand comprise a 3’ single-stranded overhang consisting of two deoxythymidines, wherein both of the internucleoside linkages of the single-stranded overhangs are phosphothioester internucleoside linkages, wherein both the sense strand and the antisense strand of the oligonucleotide comprise two additional phosphothioester linkages at their 5’ ends, and wherein each nucleoside of the antisense strand and each nucleoside of the sense strand comprises either a 2’-F or a 2'-O-methyl modified sugar.

[000215] In a particular embodiment, the invention provides a compound comprising an oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, wherein the antisense strand is described by the following chemical notation and the sense strand is described by the

following chemical notation

wherein:

An underlined nucleoside = a 2’-O-methyl-ribonucleoside,

A bold and italicized nucleoside = a 2’-F-deoxynucleoside,

A subscript “s” = a phosphothioester internucleoside linkage, and A T nucleoside = a deoxy thymidine.

[000216] In a particular embodiment, the invention provides a compound comprising an oligonucleotide comprising an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, wherein the antisense strand is described by the following chemical notation and the sense strand is described by the

following chemical notation wherein:

An underlined nucleoside = a 2’-O-methyl-ribonucleoside,

A bold and italicized nucleoside = a 2’-F-deoxynucleoside,

Hybridization

[000217] In some embodiments, hybridization occurs between an oligonucleotide disclosed herein and a target nucleic acid. The most common mechanism of hybridization involves hydrogen bonding ( e.g ., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules. It is understood that for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3- nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T.

[000218] Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.

[000219] Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In some embodiments, the oligonucleotides provided herein are specifically hybridizable with a target nucleic acid.

[000220] Oligonucleotides described herein may hybridize to a MUC5B nucleic acid in any stage of RNA processing. For example, described herein are oligonucleotides that are complementary to a pre-mRNA or a mature mRNA. Additionally, oligonucleotides described herein may hybridize to any element of a MUC5B nucleic acid. For example, described herein are oligonucleotides that are complementary to an exon, an intron, the 5’ UTR, the 3’ UTR, a repeat region, a splice junction, an exon:exon splice junction, an exonic splicing silencer (ESS), or an exonic splicing enhancer (ESE). Target Nucleic Acid

[000221] Target nucleic acid sequences that encode MUC5B include, without limitation, the following: GENBANK accession number NM_002458.3 (incorporated herein as SEQ ID NO: 1), GENBANK accession number NM_028801.2 (incorporated herein as SEQ ID NO: 2) GENBANK accession number XM_006230608.2 (incorporated herein as SEQ ID NO: 3), GENBANK accession number XM_006223574.2 (incorporated herein as SEQ ID NO: 4), GENBANK accession number XM_015435240.1 (incorporated herein as SEQ ID NO: 5), or GENBANK accession number XM_021082487.1 (incorporated herein as SEQ ID NO: 6).

[000222] Compounds and pharmaceutical compositions described herein hybridize to any one of SEQ ID NOs: 1-6. In a particular embodiment, compounds and pharmaceutical compositions described herein hybridize to SEQ ID NO: 1. In some embodiments, compounds and pharmaceutical compositions described herein hybridize to a target region or target segment of any one of SEQ ID NOs: 1-6. In a particular embodiment, a target region within the murine MUC5B mRNA sequence (SEQ ID NO: 2) is one of the regions shown as underlined in FIG. 1, or one of the corresponding regions within the human, rat, cynomolgus or pig MUC5B mRNA sequence of SEQ ID NOs: 1 and 3-6, respectively.

[000223] In some embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3’ UTR, a 5’ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for MUC5B can be obtained by accession number from sequence databases such as NCBI and such information is incorporated herein by reference.

[000224] Targeting includes determination of at least one target segment to which an oligonucleotide hybridizes, such that a desired effect occurs. A target region may contain one or more target segments. Multiple target segments within a target region may be overlapping. Alternatively, they may be non-overlapping. In some embodiments, target segments within a target region are separated by no more than about 300 nucleotides. In some embodiments, target segments within a target region are separated by a number of nucleotides that is no more than 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 nucleotides on the target nucleic acid, or is a range defined by any two of the nreceeding values. In some embodiments, target segments within a target region are separated by no more than five nucleotides on the target nucleic acid. In some embodiments, target segments are contiguous. Contemplated are target regions defined by a range having a starting nucleic acid that is any of the 5’ target sites or 3’ target sites listed herein. In some embodiments, a target region may encompass the sequence from a 5’ target site of one target segment within the target region to a 3’ target site of another target segment within the same target region.

[000225] Suitable target segments may be found within a 5’ UTR, a coding region, a 3’ UTR, an intron, an exon, or an exon/intron junction. Target segments containing a start codon or a stop codon are also suitable target segments. A suitable target segment may specifcally exclude a certain structurally defined region such as the start codon or stop codon [000226] The determination of suitable target segments may include a comparison of the sequence of a target nucleic acid to other sequences throughout the genome. For example, the BLAST algorithm may be used to identify regions of similarity amongst different nucleic acids. This comparison can prevent the selection of antisense strand sequences that may hybridize in a non-specific manner to sequences other than a selected target nucleic acid (i.e., non-target or off-target sequences).

[000227] In some embodiments, the desired effect is a reduction in mRNA target nucleic acid levels. Typically, the reduction in mRNA target nucleic acid levels is accompanied by a reduction of levels of protein encoded by the target nucleic acid. In some embodiments, the desired effect is a phenotypic change associated with the target nucleic acid. For example, improved mucociliary function, improved alveolar repair and reduced lung fibrosis may be indicative of reduced levels of MUC5B.

[000228] There may be variation in reduction activity ( e.g ., as defined by percent reduction of target nucleic acid levels) of the oligonucleotides targeting within a target region. In some embodiments, a reduction in levels of MUC5B mRNA in lung cells or tissues is indicative of inhibition of MUC5B expression. In some embodiments, a reduction in levels of MUC5B protein in lung cells or tissues is indicative of inhibition of MUC5B expression. In some embodiments, the oligonucleotide achieves at least 50% reduction in MUC5B mRNA and/or protein levels. In some embodiments, the oligonucleotide achieves at least 60% reduction in MUC5B mRNA and/or protein levels. In some embodiments, the oligonucleotide achieves at least 70% reduction in MUC5B mRNA and/or protein levels. In some embodiments, the oligonucleotide achieves at least 80% reduction in MUC5B mRNA and/or protein levels. In some embodiments, the oligonucleotide achieves at least 85% reduction in MUC5B mRNA and/or protein levels. In some embodiments, the oligonucleotide achieves at least 90% reduction in MUC5B mRNA and/or protein levels. In some embodiments, the oligonucleotide achieves at least 95% reduction in MUC5B mRNA and/or protein levels. Compounds and pharmaceutical compositions of the invention typically will achieve at least 50% reduction in MUC5B mRNA and/or protein levels, more typically at least 60% reduction in MUC5B mRNA and/or protein levels, and preferably at least 70% reduction in MUC5B mRNA and/or protein levels.

Complementarity

[000229] An antisense strand and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense strand can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur ( e.g ., siRNA inhibition of a target nucleic acid, such as any one of SEQ ID NOs: 1-6). [000230] Non-complementary nucleobases between an antisense strand and a MUC5B nucleic acid may be tolerated provided that the antisense strand remains able to specifically hybridize to the MUC5B nucleic acid. Moreover, an antisense strand may hybridize over one or more segments of a MUC5B nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure). [000231] In some embodiments, the antisense strand, or a specified portion thereof, may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to any one of SEQ ID NOs: 1-6, a target region, target segment, or specified portion thereof. Preferably, the antisense strand, or a specified portion thereof, is at least 90%, or 94% complementary to SEQ ID NO: 1.

[000232] Percentage complementarity is measured across the full length of the antisense strand. For example, an antisense strand in which 18 of 20 nucleobases of the antisense strand are complementary to a target region, and would therefore specifically hybridize, would represent 90% complementarity. In this example, the remaining non-complementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense strand which is 18 nucleobases in length having 4 non-complementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid. Percent complementarity of an oligonucleotide with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art.

[000233] In some embodiments, the antisense strand, or specified portion thereof, is fully complementary (i.e., 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense strand may be fully complementary to a target region, or a target segment or target sequence of SEQ ID NO: 1. As used herein, “fully complementary” means each nucleobase of an antisense strand is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 21 nucleobase antisense strand is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 21 nucleobase portion of the target nucleic acid that is fully complementary to the antisense strand. Fully complementary can also be used in reference to a specified portion of the first and /or the second nucleic acid. For example, a 21 nucleobase portion of a 30 nucleobase antisense strand can be “fully complementary” to a target sequence that is 400 nucleobases long. The 21 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 21 nucleobase portion wherein each nucleobase is complementary to the 21 nucleobase portion of the antisense strand. At the same time, the entire 30 nucleobase antisense strand may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense strand are also complementary to the target sequence.

[000234] The location of a non-complementary nucleobase may be at the 5’ end or 3’ end of the antisense strand. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense strand. When two or more non-complementary nucleobases are present, they may be contiguous (i.e., linked) or non-contiguous.

[000235] In some embodiments, antisense strands that are, or are up to 15, 16, 17, 18,

19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non complementary nucleobase(s) relative to any one of SEQ ID NOs: 1-6, such as a target portion of SEQ ID NO: 1.

[000236] The antisense strands provided herein also include those which are complementary to an equal length portion of a target nucleic acid, namely any one of SEQ ID NOs: 1-6. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an oligonucleotide or an antisense strand. In some embodiments, the antisense strand is complementary to at least a 12 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 13 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 14 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 15 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 16 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 17 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least an 18 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 19 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 20 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 21 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 22 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 23 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 24 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 25 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 26 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 27 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 28 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to at least a 29 nucleobase portion of a target nucleic acid. In some embodiments, the antisense strand is complementary to a 30 nucleobase portion of a target nucleic acid, namely any one of SEQ ID NOs: 1-6. Also contemplated are antisense strands that are complementary to at least a 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 27, 28, 29, or 30 nucleobase portion of a target nucleic acid, namely any one of SEQ ID NOs: 1-6, or a range defined by any two of these values.

[000237] In some embodiments, the antisense strand may have a region of complementarity with a MUC5B nucleic acid that has less than a threshold level of complementarity with every sequence of nucleotides, of equivalent length, of an off-target gene. For example, the antisense strand may be designed to ensure that it does not have a sequence that targets genes in a cell other than a MUC5B nucleic acid. The threshold level of sequence identity may be at least 50%, 60%, 70%, 80%, 85%, 90%, 95%, 99% or 100% sequence identity.

[000238] In some embodiments, the antisense strand may be complementary to target genes encoded by homologues of a gene across different species (e.g., a mouse, rat, rabbit, goat, monkey, etc.) In some embodiments, antisense strands having these characteristics may be tested in vivo or in vitro for efficacy in multiple species (e.g., human and mouse). This approach also facilitates development of clinical candidates for treating human disease by selecting a species in which an appropriate animal exists for the disease.

[000239] In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be replaced with any other nucleotide suitable for base pairing (e.g., via a Watson-Crick base pair) with an adenosine nucleotide. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) or uridine (U) nucleotides (or a modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a different pyrimidine nucleotide or vice versa. In some embodiments, any one or more thymidine (T) nucleotides (or modified nucleotide thereof) in a sequence provided herein, including a sequence provided in the sequence listing, may be suitably replaced with a uridine (U) nucleotide (or a modified nucleotide thereof) or vice versa. Identity

[000240] The antisense strands provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or portion thereof. As used herein, an antisense strand is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense strands described herein as well as compounds having non-identical bases relative to the antisense strands provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense strand. Percent identity of an antisense strand is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.

[000241] In some embodiments the oligonucleotide comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, and wherein: the nucleobase sequence of the sense strand is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a nucleic sequence selected from the group consisting of SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, and 113; and the nucleobase sequence of the antisense strand is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to a corresponding nucleic sequence selected from the group consisting of SEQ ID NOs: 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32,

34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, and 114.

[000242] In a particular embodiment, the oligonucleotide comprises an antisense strand and a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region, and wherein: the nucleobase sequence of the sense strand is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 7, and the nucleobase sequence of the antisense strand is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 8.

[000243] In a further embodiment, both the sense strand and the antisense strand comprise a 3’ single- stranded overhang consisting of two deoxythymidines, wherein both of the internucleoside linkages of the single- stranded overhangs are modified internucleoside linkages, such as phosphothioester internucleoside linkages. In a further embodiment, each nucleoside of the antisense strand and each nucleoside of the sense strand comprises either a 2’-F or a 2'-O-methyl modified sugar. In a further embodiment, both the sense strand and the antisense strand of the oligonucleotide comprise two additional phosphothioester linkages at their 5’ ends.

Modifications

[000244] A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2', 3' or 5' hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.

[000245] Modifications to oligonucleotides encompass substitutions or changes to the internucleoside linkages, sugar moieties, or nucleobases. Typically, oligonucleotides are modified to enhance their stability or reduce their immunogenic properties, in particular when administered to a subject as naked oligonucleotides or in complexed form. Therefore, providing a modified oligonucleotide of the present disclosure may have synergistic effects, resulting in the induction of immune tolerance that exceeds what has been observed with unmodified oligonucleotides. Modifications to oligonucleotides may also be employed to enhance cellular uptake, enhance affinity for a nucleic acid target, increase stability in the presence of nucleases, or increase inhibitory activity.

[000246] In some embodiments, the oligonucleotide is unmodified, i.e. it comprises only naturally-occurring nucleosides (or unmodified nucleosides; i.e., adenosine, guanosine, cytidine, thymidine and uridine) and only naturally occurring internucleoside linkages ( e.g 3’-5’ phosphodiester linkages). In some embodiments, the oligonucleotide is a modified oligonucleotide. In some embodiments, the modified oligonucleotide comprises at least one modification selected from a nucleoside analogue, a modified nucleobase, a modified internucleoside linkage and a modified sugar, or any combination thereof.

[000247] It should be appreciated that an oligonucleotide can have any combination of modifications as described herein, therefore any of the modified chemistries or formats of oligonucleotides, nucleosides, sugars, bases and internucleoside linkages described herein can be combined with each other. Moreover, it is not necessary for all positions in a given oligonucleotide to be uniformly modified, and in fact more than one of the modifications described herein may be incorporated in a single oligonucleotide or even at within a single nucleoside within a modified oligonucleotide.

[000248] In some embodiments, the modified oligonucleotide comprises one or more 2’-F modified sugar(s). In some embodiments, the modified oligonucleotide comprises one or more 2'-O-methyl modified sugar(s). In some embodiments, the modified oligonucleotide comprises one or more 2’-F modified sugar(s) and one or more 2'-O-methyl modified sugar(s). In some embodiments, each nucleoside of the modified oligonucleotide comprises either a 2’-F or a 2'-O-methyl modified sugar. In some embodiments, the modified oligonucleotide comprises one or more modified internucleoside linkage(s), such as a phosphothioester linkage. In some embodiments, the modified oligonucleotide comprises one or more 2’-F modified sugar(s), one or more 2'-O-methyl modified sugar(s), and one or more modified internucleoside linkage(s), such as a phosphothioester linkage. In some embodiments, each nucleoside of the modified oligonucleotide comprises either a 2’-F or a 2'-O-methyl modified sugar, and the modified oligonucleotide comprises one or more modified internucleoside linkage(s), such as a phosphothioester linkage.

Nucleoside Analogues

[000249] In some embodiments, the modified oligonucleotide comprises at least one nucleoside analogue. A nucleoside analogue may be used to replace the naturally occurring sugar and base, but not necessarily the internucleoside linkage at one or more positions of an oligomeric compound. In some embodiments, the presence of at least one nucleoside analogue may render a modified oligonucleotide more stable and/or less immunogenic than a control oligonucleotide with the same sequence, but containing only naturally-occurring nucleosides.

[000250] In some embodiments, the nucleoside analogue is selected from 2- aminoadenosine, 3-methyl adenosine, 7-deazaadenosine, 8-oxoadenosine, 8-azaadenosine, 8- azidoadenosine, Nl-methyladenosine, N6-methyladenosine, 2-thiocytidine, 5-methylcytidine, 5-propynyl-cytidine, 5-methylcytidine, 2-thiocytidine, 5-aminoallylcytidine, 5- bromocytidine, 5-iodocytidine, 6-azacytidine, 7-deazaguanosine, Nl-methylguanosine, 06- methylguanosine, 8-oxoguanosine, 2-thiothymidine, 5 propynyl-uridine, 5-bromouridine, 5- fluorouridine, 5-iodouridine, 5-propynyl-uridine, pseudouridine (e.g., N-l-methyl- pseudouridine), 2-thiouridine, 4-thiouridine, 5-aminoallyluridine, 5-methyluridine, 6- azauridine, queosine, beta-D-mannosyl-queosine, wybutoxosine, inosine, 1-methyl-inosine, puromycin and xanthosine. See, e.g., U.S. Patent No. 8,278,036 or WO 2011/012316 for a discussion of 5-methyl-cytidine, pseudouridine, and 2-thio-uridine and their incorporation into mRNA, the disclosures of which are incorporated by reference in their entirety.

[000251] A modified oligonucleotide may have at least one nucleoside analogue that results in an increase in T_m of the oligonucleotide in a range of 1°C, 2 °C, 3°C, 4 °C, or 5°C compared with an oligonucleotide that does not have the at least one nucleoside analogue. A modified oligonucleotide may have a plurality of nucleoside analogues that result in a total increase in T_m of the oligonucleotide in a range of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C or more compared with an oligonucleotide that does not have the nucleoside analogue.

[000252] The sense strand and/or antisense strand may consist of 15-30 linked nucleosides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleosides of the modified oligonucleotide are nucleoside analogues.

Modified Nucleobases

[000253] In some embodiments, the modified oligonucleotide comprises at least one modified nucleobase. A modified nucleobase is any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil, for example a chemically modified base, a biologically modified base (e.g., a methylated base), or an intercalated base.

[000254] In some embodiments, the modified nucleobase is selected from 1 -methyl- adenine, 2-methyl-adenine, 2-methylthio-N-6-isonentenyl -adenine, 2-aminoadenine, 2- (methylamino)adenine, 2-(imidazolylalkyl)adenine, 2-(aminoalklyamino)adenine, 2-propyl- adenine, 3-deazaadenine, 6-methyl-adenine, N6(6-aminohexyl)adenine, 6-isopentenyl- adenine, 7-methyl-adenine, 7-deazaadenine, 8-azaadenine, 8-halo-adenine, 8-amino-adenine, 8-thiol-adenine, 8-thioalkyl-adenine, 8-hydroxyl-adenine, 2-thio-cytosine, 3 -methyl-cytosine, 4-acetyl-cytosine, 5-methyl-cytosine, 5-hydroxymethylcytosine (HMC), glycosyl HMC, gentobiosyl HMC, 5-halo-cytosine, 5-propynyl-cytosine, 5-bromo-cytosine, 5- trifluoromethyl-cytosine, 6-azo-cytosine, isocytosine, pseudoisocytosine, 1 -methyl-guanine, 2-methyl-guanine, 2-propyl-guanine, 2,2-dimethyl-guanine, 3-deazaguanine, 6-methyl- guanine, 0(6)-methylguanine, 7-methyl-guanine, 7-deazaguanine, 8-azaguanine, 8-halo- guanine, 8-amino-guanine, 8-thiol-guanine, 8-thioalkyl-guanine, 8-hydroxyl-guanine, 2- thiothymine, 6-azo-thymine, 1 -methyl-pseudouracil, 2-thio-uracil, 4-thio-uracil, 5- carboxymethylaminomethyl-2-thio-uracil, 5-halo-uracil, 5-(carboxyhydroxymethyl)-uracil, 5- propynyl-uracil, 5-hydroxymethyluracil, 5-fluoro-uracil, 5-bromo-uracil, 5- carboxymethylaminomethyl-uracil, 5-trifluoromethyl-uracil, 5-methyl-2-thio-uracil, 5- methyl-uracil, N-uracil-5-oxyacetic acid methyl ester, 5-methylaminomethyl-uracil, 5- methoxyaminomethyl-2-thio-uracil, 5'-methoxycarbonylmethyl-uracil, 5-methoxy- uracil, uracil- 5 -oxy acetic acid methyl ester, uracil- 5 -oxy acetic acid (v), 6-azo-uracil, pseudouracil (5-uracil), dihydro-uracil, 2, 6-chloropurine, 2,6-diaminopurine, 6-aminopurine, 2- aminopurine, 2-chloro-6-aminopurine, 2,6-diaminopurine, pyrrolo-pyrimidine, 5-Me pyrimidines, xanthine, hypoxanthine and benzimidazole. The preparation of such modified nucleobases is known to a person skilled in the art e.g. from the U.S. Pat. Nos. 4,373,071, 4,401,796, 4,415,732, 4,458,066, 4,500,707, 4,668,777, 4,973,679, 5,047,524, 5,132,418, 5,153,319, 5,262,530, and 5,700,642. Modified nucleobases are described in US patent Nos. 3,687,808, 4,845,205, 5,130,302, 5,134,066, 5,175, 273, 5, 367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,596,091, 5,614,617,

5,750,692, and 5,681,941, as well as Komberg, “DNA Replication,” W. H. Freeman & Co., San Francisco, 1980, pp75-77; and Gebeyehu, G., et al. Nucl. Acids Res., 15:4513 (1987)), the disclosures of which are incorporated by reference in their entirety. Further modified nucleobases comprise those disclosed in United States Patent No. 3,687,808, those disclosed in “The Concise Encyclopedia of Polymer Science And Engineering”, pages 858-859, Kroschwitz, ed. John Wiley & Sons, 1990;, those disclosed by Englisch et al., Angewandle Chemie, International Edition, 1991, 30, page 613, and those disclosed by Sanghvi, Chapter 15, Antisense Research and Applications,” pages 289- 302, Crooke, and Lebleu, eds., CRC Press, 1993, which are incorporated by reference in their entirety.

[000255] Certain modified nucleobases are particularly useful for increasing the binding affinity of the modified oligonucleotides described herein, for example 5-substituted pyrimidine, 6-azapyrimidine, N-2, N-6 and 0-6 substituted purine, 2-aminopropyladenine, 5- propynyluracil and 5- propynylcytosine. In some embodiments, the modified oligonucleotide comprises at least one modified nucleobase, wherein the modified nucleobase is 5-methyl- cytosine. 5-methyl-cytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C. (Sanghvi, in Crooke, and Lebleu, eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).

Modified Internucleoside Linkages

[000256] In some embodiments, the modified oligonucleotide comprises at least one modified internucleoside linkage. A modified internucleoside linkage is any substitution or change from a naturally occurring 3' to 5' phosphodiester internucleoside linkage.

[000257] In some embodiments, the modified oligonucleotide comprises two, three, or four modified internucleoside linkages. In some embodiments, the antisense strand and/or sense strand comprise a single modified internucleoside linkage at the 5’ and/or 3’ end. In some embodiments, the antisense strand and/or sense strand comprise two modified internucleoside linkages at the 5’ and/or 3’ end. In a particular embodiment, the antisense strand and the sense strand comprise two modified internucleoside linkages at their 5’ ends.

In a specific embodiment, each of the modified internucleoside linkages is a phosphothioester internucleoside linkage.

[000258] In some embodiments, the at least one modified internucleoside linkage is a phosphorus containing internucleoside linkage. Phosphorus-containing linkages include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphothioesters, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates comprising 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates comprising 3 '-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3'-5' linkages, 2'-5' linked analogues of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments the at least one modified internucleoside linkage comprises a methylphosphonate, methylphosphoramidate, phosphoramidate, phosphorothioate, phosphothioester, boranophosphate, phosphotriester or positively charged guanidinium group. In some embodiments, the at least one modified internucleoside linkage is a phosphorothioate internucleoside linkage, such as cytidine 5'-O-(l-thiophosphate). In some embodiments, the at least one modified internucleoside linkage is a short chain alkyl linkage, cycloalkyl intersugar linkage, short chain heteroatomic linkage or heterocyclic intersugar linkage. In a particular embodiment, the at least one modified internucleoside linkage is a phosphothioester linkage.

[000259] In some embodiments, each internucleoside linkage of the modified oligonucleotide is a modified internucleoside linkage. For example, each internucleoside linkage of the modified oligonucleotide may be a phosphothioester linkage. Alternatively, the modified oligonucleotide may comprise a heteroatom backbone ( e.g ., a methylene(methylimino) or MMI backbone) or an amide backbone (i.e., a PNA backbone). [000260] Incorporation of modified internucleoside linkages can stabilize the modified oligonucleotide against nucleolytic degradation, enhance cellular uptake, or enhance affinity for target nucleic acids.

Modified Sugars

[000261] In some embodiments, the modified oligonucleotide comprises at least one modified sugar. In some embodiments, the at least one modified sugar is a bicyclic sugar, wherein the furanose ring is modified by the bridging of two carbon atoms. In some embodiments, the modified oligonucleotide comprises at least one bicyclic nucleoside, wherein a bicyclic nucleoside comprises a bicyclic sugar. In some embodiments, the bicyclic sugar has a bridge connecting the 4’ -carbon and the 2’ -carbon of the sugar ring. Examples of bicyclic sugars include, without limitation, LNA, cEt and ENA.

[000262] In some embodiments, the modified oligonucleotide consists entirely of bicyclic nucleosides (e.g., LNA nucleosides, cEt nucleosides, or ENA nucleosides). In some embodiments, the modified oligonucleotide comprises alternating deoxyribonucleosides and bicyclic nucleosides, such as ENA nucleosides, LNA nucleosides or cEt nucleosides. In some embodiments, the modified oligonucleotide comprises alternating ribonucleosides and bicyclic nucleosides, such as ENA nucleosides, LNA nucleosides or cEt nucleosides. In some embodiments, the modified oligonucleotide comprises alternating LNA nucleosides and nucleosides with 2'-O-methyl modified sugars. In some embodiments, the modified oligonucleotide has a 5' nucleotide that is a deoxyribonucleotide. In some embodiments, the modified oligonucleotide has a 5' nucleotide that is a bicyclic nucleotide (e.g., a LNA nucleotide, cEt nucleotide, ENA nucleotide). In some embodiments, the modified oligonucleotide comprises deoxyribonucleosides flanked by at least one bicyclic nucleoside (e.g., a LNA nucleoside, cEt nucleoside, or ENA nucleoside) on each of the 5' and 3' ends of the deoxyribonucleotides. In some embodiments, the modified oligonucleotide comprises deoxyribonucleosides flanked by 1, 2, 3, 4, 5, 6, 7, 8 or more bicyclic nucleosides (e.g., LNA nucleosides, cEt nucleosides, or ENA nucleosides) on each of the 5' and 3' ends of the deoxyribonucleotides. In some embodiments, the modified oligonucleotide has a 3' nucleotide that comprises a 3' hydroxyl group. In some embodiments, the modified oligonucleotide has a 3' nucleotide that comprises a 3' thiophosphate.

[000263] In some embodiments, the at least one modified sugar is a non-bicyclic sugar, such as a 2’ -modified sugar. In some embodiments, the at least one modified sugar is a 2’- modified sugar. In some embodiments, the 2’-modified sugar is a 2'-O-alkyl, 2'-O-alkyl-O- alkyl, 2’-amino, or 2'-F modified sugar. In some embodiments, the 2’-modified sugar is selected from a 2’-O-methyl, 2’-F, 2’-O-methylethyl, 2’-O-methoxyethyl (2’-O-MOE), 2'-O- aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), and 2'-O-N-methylacetamido (2'-O-NMA) modified sugar. In particular embodiments, the 2’-modified sugar is selected from a 2’-F and a 2'-O-methyl modified sugar.

[000264] In some embodiments, either the antisense strand or the sense strand comprise at least one modified sugar wherein the modified sugar is a 2 '-O-methyl modified sugar. In some embodiments, both the antisense strand and the sense strand comprise at least one modified sugar wherein the modified sugar is a 2 '-O-methyl modified sugar. In some embodiments, the sense strand comprises more 2 '-O-methyl modified sugars than the antisense strand. In some embodiments, the sense strand comprises at least one more 2'-O- methyl modified sugar than the antisense strand. In some embodiments, the sense strand comprises 1-10 more 2'-O-methyl modified sugars than the antisense strand. In some embodiments, the sense strand comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more 2'-O-methyl modified sugars than the antisense strand.

[000265] In some embodiments, either the antisense strand or the sense strand comprise at least one modified sugar wherein the modified sugar is a 2'-F modified sugar. In some embodiments, both the antisense strand and the sense strand comprise at least one modified sugar wherein the modified sugar is a 2'-F modified sugar. In some embodiments, either the antisense strand or the sense strand comprise at least one 2'-F modified sugar and at least one 2'-O-methyl modified sugar. In some embodiments, both the antisense strand and the sense strand comprise at least one 2'-F modified sugar and at least one 2 '-O-methyl modified sugar. [000266] In some embodiments, each nucleoside of the antisense strand comprises a modified sugar. In some embodiments, each nucleoside of the sense strand comprises a modified sugar. In some embodiments, each nucleoside of the antisense strand and each nucleoside of the sense strand comprises a modified sugar. In a particular embodiment, each nucleoside of the antisense strand comprises either a 2’-F or a 2'-O-methyl modified sugar.

In a particular embodiment, each nucleoside of the sense strand comprises either a 2’-F or a 2'-O-methyl modified sugar. In a particular embodiment, each nucleoside of the antisense strand and each nucleoside of the sense strand comprises either a 2’-F or a 2'-O-methyl modified sugar.

[000267] In some embodiments, either the antisense strand or the sense strand comprises alternating modified ribonucleosides and modified deoxyribonucleosides. In a particular embodiment, either the antisense strand or the sense strand comprises alternating 2'-O-methyl-ribonucleosides and 2'-F-deoxyribonucleosides. In a particular embodiment, both of the antisense strand and the sense strand comprise alternating modified ribonucleosides and modified deoxyribonucleosides. In a particular embodiment, both of the antisense strand and the sense strand comprise alternating 2'-O-methyl-ribonucleosides and 2'-F-deoxyribonucleosides.

[000268] Such 2’ sugar modifications are routinely incorporated into oligonucleotides and these modified oligonucleotides have been shown to have a higher Tm ( e.g ., higher target binding affinity) than 2'-deoxyoligonucleotides against a given target. Such 2’ sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the modified oligonucleotide. Nucleic Acid Analogues

[000269] In some embodiments, the modified oligonucleotide comprises a nucleic acid analogue. Modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These comprise those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. In some embodiments, the nucleic acid analogue is a morpholino. In some embodiments, the nucleic acid analogue is a PNA.

[000270] Modified oligonucleotides are also known that include oligonucleotides that are based on or constructed from arabinonucleotide or modified arabinonucleotide residues.

In some embodiments, the nucleic acid analogue is formed of arabinonucleosides. Arabinonucleosides are stereoisomers of ribonucleosides, differing only in the configuration at the 2'-position of the sugar ring. In some embodiments, a 2'-arabino modification is 2'-F arabino. In some embodiments, the modified oligonucleotide is 2'-fluoro-D-arabinonucleic acid (FANA) (as described in, for example, Lon et al., Biochem., 41:3457-3467, 2002 and Min et al., Bioorg. Med. Chem. Lett., 12:2651-2654, 2002; the disclosures of which are incorporated herein by reference in their entireties). Similar modifications can also be made at other positions on the sugar, particularly the 3' position of the sugar on a 3' terminal nucleoside or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide.

PCT Publication No. WO 99/67378 discloses arabinonucleic acids (ANA) oligomers and their analogues for improved sequence specific inhibition of expression via association to complementary mRNA.

Conjugates

[000271] Modifed oligonucleotides may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting oligonucleotide. Modified oligonucleotides may be covalently linked to one or more moieties or conjugates at the 5' or 3' end of the oligonucleotide. In some embodiments, the 3' end of the modified oligonucleotide comprises a hydroxyl group or a thiophosphate.

[000272] For example, one or more oligonucleotides, of the same or different types, can be conjugated to each other; or oligonucleotides can be conjugated to targeting moieties with enhanced specificity for a cell type or tissue type. Such conjugate moieties include cholesterol moieties and lipid moieties. Such moieties include, but are not limited to cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S- tritylthiol (Manoharan et al, Ann. N. Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie,

1993, 75, 49- 54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2- di-O-hexadecyl- rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995,

36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Mancharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-t oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937). Additional conjugate moieties include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, fluorophores and dyes. In some embodiments, an oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide. In some embodiments, an oligonucleotide comprises a biotin moiety conjugated to the 5' nucleotide. [000273] Conjugate moieties also include labels. For example, oligonucleotides may be covalently linked to a biotin moiety, cholesterol, Vitamin A, folate, sigma receptor ligands, aptamers, peptides, such as CPP, hydrophobic molecules, such as lipids, ligands of the asialoglycoprotein receptor (ASGPR), such as GalNac, or dynamic polyconjugates and variants thereof. [000274] Conjugate groups may include a conjugate moiety and a conjugate linker that attaches the conjugate moiety to the oligonucleotide. A conjugate linker is a group of atoms comprising at least one bond that connects a conjugate moiety to an oligonucleotide.

Delivery Vehicles

[000275] According to the present invention, compounds as described herein may be delivered as naked oligonucleotides (unpackaged) or via delivery vehicles. As used herein, the terms “delivery vehicle,” “transfer vehicle,” “nanoparticle” or grammatical equivalents, are used interchangeably.

[000276] Delivery vehicles can be formulated in combination with one or more additional nucleic acids, carriers, targeting ligands or stabilizing reagents, or in pharmacological compositions where it is mixed with suitable excipients. Techniques for composition and administration of drugs may be found in “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa., latest edition. A particular delivery vehicle is selected based upon its ability to facilitate the transfection of a nucleic acid to a target cell [000277] In some embodiments, oligonucleotides may be delivered via a single delivery vehicle. In some embodiments, oligonucleotides may be delivered via one or more delivery vehicles, each of a different composition. For example, in some embodiments, oligonucleotides are encapsulated within the same lipid nanoparticles; and in some embodiments, the oligonucleotides are encapsulated within separate lipid nanoparticles.

[000278] According to various embodiments, suitable delivery vehicles include, but are not limited to polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles, liposomes, nanoliposomes, ceramide-containing nanoliposomes, proteoliposomes, both natural and synthetically-derived exosomes, natural, synthetic and semi- synthetic lamellar bodies, nanoparticulates, calcium phosphor-silicate nanoparticulates, calcium phosphate nanoparticulates, silicon dioxide nanoparticulates, nanocrystalline particulates, semiconductor nanoparticulates, poly(D-arginine), sol-gels, nanodendrimers, starch-based delivery systems, micelles, emulsions, niosomes, multi-domain-block polymers (vinyl polymers, polypropyl acrylic acid polymers, dynamic polyconjugates), dry powder compositions, plasmids, viruses, calcium phosphate nucleotides, aptamers, peptides and other vectorial tags. Also contemplated is the use of bionanocapsules and other viral capsid proteins assemblies as a suitable transfer vehicle. (Hum. Gene Ther. 2008 September; 19(9):887-95). In particular embodiments, the delivery vehicle is selected from the group consisting of liposomes, lipid nanoparticles, solid-lipid nanoparticles, polymers, viruses, sol- gels, and nanogels.

Lipid Nanoparticles

[000279] In some embodiments, a suitable delivery vehicle is a lipid nanoparticle. In some embodiments, a lipid nanoparticle comprises one or more cationic lipids. In some embodiments, a lipid nanoparticle comprises one or more cationic lipids, one or more non- cationic lipids, one or more cholesterol-based lipids and one or more PEG-modified lipids. In some embodiments, a lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG-modified lipids. In some embodiments, a lipid nanoparticle comprises no more than four distinct lipid components. A typical lipid nanoparticle for use with the invention is composed of four lipid components: a cationic lipid ( e.g ., a sterol-based cationic lipid), a non-cationic lipid (e.g., DOPE or DEPE), a cholesterol- based lipid (e.g., cholesterol) and a PEG-modified lipid (e.g., DMG-PEG2K). In some embodiments, a lipid nanoparticle comprises no more than three distinct lipid components.

An exemplary lipid nanoparticle is composed of three lipid components: a cationic lipid (e.g., a sterol-based cationic lipid), a non-cationic lipid (e.g., DOPE or DEPE) and a PEG-modified lipid (e.g., DMG-PEG2K).

Formation of Lipid Nanoparticles Encapsulating Oligonucleotides

[000280] The liposomal delivery vehicles for use in the pharmaceutical compositions of the invention can be prepared by various techniques which are presently known in the art.

For example, multilamellar vesicles (MLV) may be prepared according to conventional techniques, such as by depositing a selected lipid on the inside wall of a suitable container or vessel by dissolving the lipid in an appropriate solvent, and then evaporating the solvent to leave a thin film on the inside of the vessel or by spray drying. An aqueous phase may then be added to the vessel with a vortexing motion which results in the formation of MLVs. Unilamellar vesicles (ULV) can then be formed by homogenization, sonication or extrusion of the multilamellar vesicles. In addition, unilamellar vesicles can be formed by detergent removal techniques.

[000281] Various methods are described in published U.S. Application No.

US 2011/0244026, published U.S. Application No. US 2016/0038432, published U.S. Application No. US 2018/0153822, published U.S. Application No. US 2018/0125989 and U.S. Provisional Application No. 62/877,597, filed July 23, 2019 and can be used to practice the present invention, all of which are incorporated herein by reference. As used herein, Process A refers to a conventional method of encapsulating oligonucleotides by mixing oligonucleotides with a mixture of lipids, without first pre-forming the lipids into lipid nanoparticles, as described in US 2016/0038432. As used herein, Process B refers to a process of encapsulating oligonucleotides by mixing pre-formed lipid nanoparticles with the oligonucleotides, as described in US 2018/0153822.

[000282] Briefly, the process of preparing oligonucleotide-loaded lipid nanoparticles includes a step of heating one or more of the solutions (i.e., applying heat from a heat source to the solution) to a temperature (or to maintain at a temperature) greater than ambient temperature, the one or more solutions being the solution comprising the pre-formed lipid nanoparticles, the solution comprising the oligonucleotides and the mixed solution comprising the lipid nanoparticle encapsulated oligonucleotides. In some embodiments, the process includes the step of heating one or both of the oligonucleotide solution and the pre formed lipid nanoparticle solution, prior to the mixing step. In some embodiments, the process includes heating one or more one or more of the solution comprising the pre-formed lipid nanoparticles, the solution comprising the oligonucleotides and the solution comprising the lipid nanoparticle encapsulated oligonucleotides, during the mixing step. In some embodiments, the process includes the step of heating the lipid nanoparticle encapsulated oligonucleotides, after the mixing step. In some embodiments, the temperature to which one or more of the solutions is heated (or at which one or more of the solutions is maintained) is or is greater than about 30 °C, 37 °C, 40 °C, 45 °C, 50 °C, 55 °C, 60 °C, 65 °C, or 70 °C. In some embodiments, the temperature to which one or more of the solutions is heated ranges from about 25-70 °C, about 30-70 °C, about 35-70 °C, about 40-70 °C, about 45-70 °C, about 50-70 °C, or about 60-70 °C. In some embodiments, the temperature greater than ambient temperature to which one or more of the solutions is heated is about 65 °C. [000283] Various methods may be used to prepare an oligonucleotide solution suitable for the present invention. In some embodiments, oligonucleotides may be directly dissolved in a buffer solution described herein. In some embodiments, an oligonucleotide solution may be generated by mixing an oligonucleotide stock solution with a buffer solution prior to mixing with a lipid solution for encapsulation. In some embodiments, an oligonucleotide solution may be generated by mixing an oligonucleotide stock solution with a buffer solution immediately before mixing with a lipid solution for encapsulation. In some embodiments, a suitable oligonucleotide stock solution may contain oligonucleotides in water at a concentration at or greater than about 0.2 mg/ml, 0.4 mg/ml, 0.5 mg/ml, 0.6 mg/ml, 0.8 mg/ml, 1.0 mg/ml, 1.2 mg/ml, 1.4 mg/ml, 1.5 mg/ml, or 1.6 mg/ml, 2.0 mg/ml, 2.5 mg/ml, 3.0 mg/ml, 3.5 mg/ml, 4.0 mg/ml, 4.5 mg/ml, or 5.0 mg/ml.

[000284] In some embodiments, an oligonucleotide stock solution is mixed with a buffer solution using a pump. Exemplary pumps include but are not limited to gear pumps, peristaltic pumps and centrifugal pumps.

[000285] Typically, the buffer solution is mixed at a rate greater than that of the oligonucleotide stock solution. For example, the buffer solution may be mixed at a rate at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x, 15x, or 20x greater than the rate of the oligonucleotide stock solution. In some embodiments, a buffer solution is mixed at a flow rate ranging between about 100-6000 ml/minute ( e.g ., about 100-300 ml/minute, 300-600 ml/minute, 600-1200 ml/minute, 1200-2400 ml/minute, 2400-3600 ml/minute, 3600-4800 ml/minute, 4800-6000 ml/minute, or 60-420 ml/minute). In some embodiments, a buffer solution is mixed at a flow rate of or greater than about 60 ml/minute, 100 ml/minute, 140 ml/minute, 180 ml/minute, 220 ml/minute, 260 ml/minute, 300 ml/minute, 340 ml/minute, 380 ml/minute, 420 ml/minute, 480 ml/minute, 540 ml/minute, 600 ml/minute, 1200 ml/minute, 2400 ml/minute, 3600 ml/minute, 4800 ml/minute, or 6000 ml/minute.

[000286] In some embodiments, an oligonucleotide stock solution is mixed at a flow rate ranging between about 10-600 ml/minute (e.g., about 5-50 ml/minute, about 10-30 ml/minute, about 30-60 ml/minute, about 60-120 ml/minute, about 120-240 ml/minute, about 240-360 ml/minute, about 360-480 ml/minute, or about 480-600 ml/minute). In some embodiments, an oligonucleotide stock solution is mixed at a flow rate of or greater than about 5 ml/minute, 10 ml/minute, 15 ml/minute, 20 ml/minute, 25 ml/minute, 30 ml/minute, 35 ml/minute, 40 ml/minute, 45 ml/minute, 50 ml/minute, 60 ml/minute, 80 ml/minute, 100 ml/minute, 200 ml/minute, 300 ml/minute, 400 ml/minute, 500 ml/minute, or 600 ml/minute. [000287] According to the present invention, a lipid solution contains a mixture of lipids suitable to form lipid nanoparticles for encapsulation of oligonucleotides. In some embodiments, a suitable lipid solution is ethanol based. For example, a suitable lipid solution may contain a mixture of desired lipids dissolved in pure ethanol (i.e., 100% ethanol). In another embodiment, a suitable lipid solution is isopropyl alcohol based. In another embodiment, a suitable lipid solution is dimethylsulfoxide-based. In another embodiment, a suitable lipid solution is a mixture of suitable solvents including, but not limited to, ethanol, isopropyl alcohol and dimethylsulfoxide.

[000288] A suitable lipid solution may contain a mixture of desired lipids at various concentrations. For example, a suitable lipid solution may contain a mixture of desired lipids at a total concentration of or greater than about 0.1 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0 mg/ml, 3.0 mg/ml, 4.0 mg/ml, 5.0 mg/ml, 6.0 mg/ml, 7.0 mg/ml, 8.0 mg/ml, 9.0 mg/ml, 10 mg/ml,

15 mg/ml, 20 mg/ml, 30 mg/ml, 40 mg/ml, 50 mg/ml, or 100 mg/ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration ranging from about 0.1-100 mg/ml, 0.5-90 mg/ml, 1.0-80 mg/ml, 1.0-70 mg/ml, 1.0-60 mg/ml, 1.0-50 mg/ml, 1.0-40 mg/ml, 1.0-30 mg/ml, 1.0-20 mg/ml, 1.0-15 mg/ml, 1.0-10 mg/ml, 1.0-9 mg/ml, 1.0-8 mg/ml, 1.0-7 mg/ml, 1.0-6 mg/ml, or 1.0-5 mg/ml. In some embodiments, a suitable lipid solution may contain a mixture of desired lipids at a total concentration up to about 100 mg/ml, 90 mg/ml, 80 mg/ml, 70 mg/ml, 60 mg/ml, 50 mg/ml, 40 mg/ml, 30 mg/ml, 20 mg/ml, or 10 mg/ml.

[000289] Any desired lipids may be mixed at any ratios suitable for encapsulating oligonucleotides. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including cationic lipids, non-cationic lipids, cholesterol-based lipids, amphiphilic block copolymers (e.g. poloxamers) and/or PEG-modified lipids. In some embodiments, a suitable lipid solution contains a mixture of desired lipids including one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and/or one or more PEG-modified lipids.

[000290] In some embodiments, provided pharmaceutical compositions comprise a lipid nanoparticle wherein the oligonucleotides are associated on both the surface of the lipid nanoparticle and encapsulated within the same lipid nanoparticle. For example, during preparation of the pharmaceutical compositions of the present invention, cationic lipid nanoparticles may associate with the oligonucleotide through electrostatic interactions. [000291] In some embodiments, the compounds, pharmaceutical compositions and methods of the invention comprise oligonucleotides encapsulated in a lipid nanoparticle. In some embodiments, the oligonucleotides may be encapsulated in the same lipid nanoparticle. In some embodiments, the oligonucleotides may be encapsulated in different lipid nanoparticles. In some embodiments, the oligonucleotide is encapsulated in one or more lipid nanoparticles, which differ in their lipid composition, molar ratio of lipid components, size, charge (zeta potential), targeting ligands and/or combinations thereof. In some embodiments, the one or more lipid nanoparticles may have a different composition of sterol- based cationic lipids, neutral lipids, PEG-modified lipids and/or combinations thereof. In some embodiments the one or more lipid nanoparticles may have a different molar ratio of cholesterol-based lipids, cationic lipids, neutral lipids, and PEG-modified lipids used to create the lipid nanoparticles.

[000292] The process of incorporation of a desired oligonucleotide into a lipid nanoparticle is often referred to as “loading”. Exemplary methods are described in Lasic, et al. FEBS Lett., 312: 255-258, 1992, which is incorporated herein by reference. The lipid nanoparticle-incorporated nucleic acids may be completely or partially located in the interior space of the lipid nanoparticle, within the bilayer membrane of the lipid nanoparticle, or associated with the exterior surface of the lipid nanoparticle membrane. The incorporation of an oligonucleotide into lipid nanoparticles is also referred to herein as “encapsulation” wherein the nucleic acid is entirely contained within the interior space of the lipid nanoparticle. The purpose of incorporating an oligonucleotide into a delivery vehicle, such as a lipid nanoparticle, is often to protect the oligonucleotide from an environment which may contain enzymes or chemicals that degrade oligonucleotides and/or systems or receptors that cause the rapid excretion of the oligonucleotides. Accordingly, in some embodiments, a suitable delivery vehicle is capable of enhancing the stability of the oligonucleotides contained therein and/or facilitate the delivery of an oligonucleotide (e.g., siRNA) to the target cell or tissue. [000293] Suitable lipid nanoparticles in accordance with the present invention may be made in various sizes. In some embodiments, provided lipid nanoparticles may be made smaller than previously known lipid nanoparticles. In some embodiments, decreased size of lipid nanoparticles is associated with more efficient delivery of an oligonucleotides ( e.g ., siRNA). Selection of an appropriate lipid nanoparticle size may take into consideration the site of the target cell or tissue and to some extent the application for which the lipid nanoparticle is being made.

[000294] In some embodiments, an appropriate size of lipid nanoparticle is selected to facilitate systemic distribution of the oligonucleotide. Alternatively or additionally, a lipid nanoparticle may be sized such that the dimensions of the lipid nanoparticle are of a sufficient diameter to limit or expressly avoid distribution into certain cells or tissues. [000295] A variety of alternative methods known in the art are available for sizing of a population of lipid nanoparticles. One such sizing method is described in U.S. Pat. No. 4,737,323, incorporated herein by reference. Sonicating a lipid nanoparticles suspension either by bath or probe sonication produces a progressive size reduction down to small ULV less than about 0.05 microns in diameter. Homogenization is another method that relies on shearing energy to fragment large lipid nanoparticles into smaller ones. In a typical homogenization procedure, MLV are recirculated through a standard emulsion homogenizer until selected lipid nanoparticle sizes, typically between about 0.1 and 0.5 microns, are observed. The size of the lipid nanoparticles may be determined by quasi-electric light scattering (QELS) as described in Bloomfield, Ann. Rev. Biophys. Bioeng., 10:421-450 (1981), incorporated herein by reference. Average lipid nanoparticle diameter may be reduced by sonication of formed lipid nanoparticles. Intermittent sonication cycles may be alternated with QELS assessment to guide efficient lipid nanoparticle synthesis.

Provided Lipid Nanoparticles Encapsulating Oligonucleotides

[000296] In some embodiments, the majority of purified lipid nanoparticles in a pharmaceutical composition, i.e., greater than about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the lipid nanoparticles, have a size of about 150 nm (e.g., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm). In some embodiments, substantially all of the purified lipid nanoparticles have a size of about 150 nm ( e.g ., about 145 nm, about 140 nm, about 135 nm, about 130 nm, about 125 nm, about 120 nm, about 115 nm, about 110 nm, about 105 nm, about 100 nm, about 95 nm, about 90 nm, about 85 nm, or about 80 nm).

[000297] In some embodiments, a lipid nanoparticle has an average size of less than 150 nm. In some embodiments, a lipid nanoparticle has an average size of less than 120 nm. In some embodiments, a lipid nanoparticle has an average size of less than 100 nm. In some embodiments, a lipid nanoparticle has an average size of less than 90 nm. In some embodiments, a lipid nanoparticle has an average size of less than 80 nm. In some embodiments, a lipid nanoparticle has an average size of less than 70 nm. In some embodiments, a lipid nanoparticle has an average size of less than 60 nm. In some embodiments, a lipid nanoparticle has an average size of less than 50 nm. In some embodiments, a lipid nanoparticle has an average size of less than 30 nm. In some embodiments, a lipid nanoparticle has an average size of less than 20 nm.

[000298] In some embodiments, greater than about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% of the lipid nanoparticles in a pharmaceutical composition provided by the present invention have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50-80 nm, about 55-75 nm, about 60-70 nm). In some embodiments, substantially all of the lipid nanoparticles have a size ranging from about 40-90 nm (e.g., about 45-85 nm, about 50- 80 nm, about 55-75 nm, about 60-70 nm). Compositions with lipid nanoparticles having an average size of about 50-70 nm (e.g., 55-65 nm) are particular suitable for pulmonary delivery via nebulization.

[000299] In some embodiments, the dispersity, or measure of heterogeneity in size of molecules (PD I), of lipid nanoparticles in a pharmaceutical composition provided by the present invention is less than about 0.5. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.5. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.4. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.3. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.28. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.25. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.23. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.20. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.18. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.16. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.14. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.12. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.10. In some embodiments, a lipid nanoparticle has a PDI of less than about 0.08.

[000300] In some embodiments, greater than about 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the purified lipid nanoparticles in a pharmaceutical composition provided by the present invention encapsulate an oligonucleotide within each individual particle. In some embodiments, substantially all of the purified lipid nanoparticles in a pharmaceutical composition encapsulate an oligonucleotide within each individual particle.

In some embodiments, a lipid nanoparticle has an encapsulation efficiency of between 50% and 99%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 60%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 65%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 70%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 75%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 80%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 85%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 90%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 92%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 95%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 98%. In some embodiments, a lipid nanoparticle has an encapsulation efficiency of greater than about 99%. Typically, lipid nanoparticles for use with the invention have an encapsulation efficiency of at least 90%-95%.

[000301] In some embodiments, a lipid nanoparticle has a N/P ratio of between 1 and 10. In some embodiments, a lipid nanoparticle has a N/P ratio above 1. In some embodiments, a lipid nanoparticle has a N/P ratio of about 1. In some embodiments, a lipid nanoparticle has a N/P ratio of about 2. In some embodiments, a lipid nanoparticle has a N/P ratio of about 3. In some embodiments, a lipid nanoparticle has a N/P ratio of about 4. In some embodiments, a lipid nanoparticle has a N/P ratio of about 5. In some embodiments, a lipid nanoparticle has a N/P ratio of about 6. In some embodiments, a lipid nanoparticle has a N/P ratio of about 7. In some embodiments, a lipid nanoparticle has a N/P ratio of about 8. A typical lipid nanoparticle for use with the invention has an N/P ratio of about 4.

[000302] In some embodiments, a pharmaceutical composition according to the present invention contains at least about 0.5 mg, 1 mg, 5 mg, 10 mg, 100 mg, 500 mg, or 1000 mg of encapsulated oligonucleotides. In some embodiments, a pharmaceutical composition contains about 0.1 mg to 1000 mg of encapsulated oligonucleotides. In some embodiments, a pharmaceutical composition contains at least about 0.5 mg of encapsulated oligonucleotides. In some embodiments, a pharmaceutical composition contains at least about 0.8 mg of encapsulated oligonucleotides. In some embodiments, a pharmaceutical composition contains at least about 1 mg of encapsulated oligonucleotides. In some embodiments, a pharmaceutical composition contains at least about 5 mg of encapsulated oligonucleotides.

In some embodiments, a pharmaceutical composition contains at least about 8 mg of encapsulated oligonucleotides. In some embodiments, a pharmaceutical composition contains at least about 10 mg of encapsulated oligonucleotides. In some embodiments, a pharmaceutical composition contains at least about 50 mg of encapsulated oligonucleotides.

In some embodiments, a pharmaceutical composition contains at least about 100 mg of encapsulated oligonucleotides. In some embodiments, a pharmaceutical composition contains at least about 500 mg of encapsulated oligonucleotides. In some embodiments, a pharmaceutical composition contains at least about 1000 mg of encapsulated oligonucleotides.

Lipid Species

Cationic Lipids

[000303] Suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2010/144740, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid, (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino) butanoate, having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000304] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the present invention include ionizable cationic lipids as described in International Patent Publication WO 2013/149140, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid of one of the following formulas:

or a pharmaceutically acceptable salt thereof, wherein Ri and R2 are each independently selected from the group consisting of hydrogen, an optionally substituted, variably saturated or unsaturated C1-C20 alkyl and an optionally substituted, variably saturated or unsaturated C6-C20 acyl; wherein Li and L2 are each independently selected from the group consisting of hydrogen, an optionally substituted C1-C30 alkyl, an optionally substituted variably unsaturated C1-C30 alkenyl, and an optionally substituted C1-C30 alkynyl; wherein m and o are each independently selected from the group consisting of zero and any positive integer (e.g., where m is three); and wherein n is zero or any positive integer (e.g., where n is one). In some embodiments, the pharmaceutical compositions and methods of the present invention include the cationic lipid (15Z, 18Z)-N,N-dimethyl-6-(9Z,12Z)-octadeca-9,12-dien-l-yl) tetracosa-15,18-dien-l-amine (“HGT5000”), having a compound structure of:

and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include the cationic lipid (15Z, 18Z)-N,N- dimethyl-6-((9Z, 12Z)-octadeca-9, 12-dien- 1-yl) tetracosa-4, 15, 18-trien-l -amine

(“HGT5001”), having a compound structure of:

and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include the cationic lipid and (15Z,18Z)- N,N-dimethyl-6-((9Z,12Z)-octadeca-9,12-dien-l-yl) tetracosa-5,15,18-trien- 1 -amine (“HGT5002”), having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000305] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include cationic lipids described as aminoalcohol lipidoids in International Patent Publication WO 2010/053572, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000306] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016/118725, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000307] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016/118724, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000308] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include a cationic lipid having the formula of 14,25-ditridecyl 15,18,21,24-tetraaza-octatriacontane, and pharmaceutically acceptable salts thereof.

[000309] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publications WO 2013/063468 and WO 2016/205691, each of which are incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid of the following formula:

or pharmaceutically acceptable salts thereof, wherein each instance of R^L is independently optionally substituted C6-C40 alkenyl. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000310] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2015/184256, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid of the following formula:

or a pharmaceutically acceptable salt thereof, wherein each X independently is O or S; each Y independently is O or S; each m independently is 0 to 20; each n independently is 1 to 6; each R_A is independently hydrogen, optionally substituted Cl-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen; and each R_B is independently hydrogen, optionally substituted Cl-50 alkyl, optionally substituted C2-50 alkenyl, optionally substituted C2-50 alkynyl, optionally substituted C3-10 carbocyclyl, optionally substituted 3-14 membered heterocyclyl, optionally substituted C6-14 aryl, optionally substituted 5-14 membered heteroaryl or halogen. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid, “Target 23”, having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000311] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2016/004202, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having the compound structure:

or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having the compound structure:

or a pharmaceutically acceptable salt thereof.

[000312] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the present invention include cationic lipids as described in United States Provisional Patent Application Serial Number 62/758,179, filed on November 9, 2018, and Provisional Patent Application Serial Number 62/871,510, filed on July 8, 2019, which are incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid of the following formula:

or a pharmaceutically acceptable salt thereof, wherein each R¹ and R² is independently H or C1-C6 aliphatic; each m is independently an integer having a value of 1 to 4; each A is independently a covalent bond or arylene; each L¹ is independently an ester, thioester, disulfide, or anhydride group; each L² is independently C2-C10 aliphatic; each X¹ is independently H or OH; and each R³ is independently C6-C20 aliphatic. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid of the following formula:

or a pharmaceutically acceptable salt thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid of the following formula:

or a pharmaceutically acceptable salt thereof.

[000313] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the present invention include the cationic lipids as described in J. McClellan, M. C. King, Cell 2010, 141, 210-217 and in Whitehead et al. , Nature Communications (2014) 5:4277, which is incorporated herein by reference. In some embodiments, the cationic lipids of the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000314] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2015/199952, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having the compound structure:

and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having the compound structure:

and pharmaceutically acceptable salts thereof.

[000315] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/004143, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having the compound structure:

and pharmaceutically acceptable salts thereof.

[000316] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/075531, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid of the following formula:

or a pharmaceutically acceptable salt thereof, wherein one of L¹ or L² is -O(C=O)-, -(C=O)O- , -C(=O)-, -O-, -S(O)_x, -S-S-, -C(=O)S-, -SC(=O)-, -NR^aC(=O)-, -C(=O)NR^a-, NR^aC(=O)NR^a-, -OC(=O)NR^a-, or -NR^aC(=O)O-; and the other of L¹ or L² is -O(C=O)-, - (C=O)O-, -C(=O)-, -O-, -S(O)_x, -S-S-, -C(=O)S-, SC(=O)-, -NR^aC(=O)-, -C(=O)NR^a-, ,NR^aC(=O)NR^a-, -OC(=O)NR^a- or -NR^aC(=O)O- or a direct bond; G¹ and G² are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene; G³ is C1-C24 alkylene, Ci- C₂₄ alkenylene, C₃-C₈ cycloalkylene, C₃-C₈ cycloalkenylene; R^a is H or C₁-C₁₂ alkyl; R¹ and R² are each independently C₆-C₂₄ alkyl or C₆-C₂₄ alkenyl; R³ is H, OR⁵, CN, -C(=O)OR⁴, - 0C(=O)R⁴ or -NR⁵ C(=O)R⁴; R⁴ is C₁-C₁₂ alkyl; R⁵ is H or C₁-C₆ alkyl; and x is 0, 1 or 2. [000317] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/117528, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having the compound structure:

and pharmaceutically acceptable salts thereof. [000318] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/049245, which is incorporated herein by reference. In some embodiments, the cationic lipids of the pharmaceutical compositions and methods of the present invention include a compound of one of the following formulas:

and pharmaceutically acceptable salts thereof. For any one of these four formulas, R4 is independently selected from -(CH₂)_nQ and -(CH₂) _nCHQR; Q is selected from the group consisting of -OR, -OH, -O(CH₂)_nN(R)₂, -OC(O)R, -CX3, -CN, -N(R)C(O)R, -N(H)C(O)R, - N(R)S(O)₂R, -N(H)S(O)₂R, -N(R)C(O)N(R)₂, -N(H)C(O)N(R)₂, -N(H)C(O)N(H)(R), - N(R)C(S)N(R)₂, -N(H)C(S)N(R)₂, -N(H)C(S)N(H)(R), and a heterocycle; and n is 1, 2, or 3. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000319] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the invention include the cationic lipids as described in International Patent Publication WO 2017/173054 and WO 2015/095340, each of which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000320] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the present invention include cationic lipids as described in United States Provisional Patent Application Serial Number 62/865,555, filed on June 24, 2019, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000321] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the present invention include cationic lipids as described in United States Provisional Patent Application Serial Number 62/864,818, filed on June 21, 2019, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure according to the following formula:

₉ or a pharmaceutically acceptable salt thereof, wherein each of R², R³, and R⁴ is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; L¹ is C1-C30 alkylene; C2-C30 alkenylene; or C_2-C₃₀ alkynylene and B¹ is an ionizable nitrogen-containing group. In embodiments, L¹ is C_1-C₁₀ alkylene. In embodiments, L¹ is unsubstituted C_1-C₁₀ alkylene. In embodiments, L¹ is (CH₂)₂, (CH₂)₃, (CH₂)4, or (CH₂)5. In embodiments, L¹ is (CH₂), (CH₂)₆, (CH₂)₇, (CH₂)₈, (CH₂)₉, or (CH₂)I_O. In embodiments, B¹ is independently NH₂, guanidine, amidine, a mono- or dialkylamine, 5- to 6-membered nitrogen-containing heterocycloalkyl, or 5- to 6- membered nitrogen-containing heteroaryl. In embodiments, B¹ is

In embodiments, B¹ is

In embodiments, B¹ is In embodiments, each

of R², R³, and R⁴ is independently unsubstituted linear C₆-C₂₂ alkyl, unsubstituted linear C_6- C₂₂ alkenyl, unsubstituted linear C₆-C₂₂ alkynyl, unsubstituted branched C₆-C₂₂ alkyl, unsubstituted branched C₆-C₂₂ alkenyl, or unsubstituted branched C₆-C₂₂ alkynyl. In embodiments, each of R², R³, and R⁴ is unsubstituted C₆-C₂₂ alkyl. In embodiments, each of R², R³, and R⁴ is -C₆H₁ , -C7H15, -C8H17, -C9H19, -C10H21, -C11H23, -C12H25, -C13H27, -C14H29, -C15H31, -C16H33, -C17H35, -C18H37, -C19H39, -C20H41, -C21H43, -C22H45, -C23H47, -C24H49, or - C₂₅H₅₁. In embodiments, each of R², R³, and R⁴ is independently C₆-C₁₂ alkyl substituted by -0(C0)R⁵ or -C(O)0R⁵, wherein R⁵ is unsubstituted C₆-C₁₄ alkyl. In embodiments, each of R², R³, and R⁴ is unsubstituted C₆-C₂₂ alkenyl. In embodiments, each of R², R³, and R⁴ is - (CH₂) CH=CH₂, -(CH₂)₅CH=CH2, -(CH₂)₆CH=CH₂, -(CH₂)7CH=CH₂, -(CH₂)₈CH=CH₂, - (CH₂)9CH=CH₂, -(CH₂) I_OCH=CH₂, -(CH₂)HCH=CH₂, -(CH₂)I₂CH=CH₂, -(CH₂)I₃CH=CH₂, - (CH₂)I₄CH=CH₂, -(CH₂)I₅CH=CH₂, -(CH₂)I₆CH=CH₂, -(CH₂)I₇CH=CH₂, -(CH₂)I₈CH=CH₂, -(CH₂)₇CH=CH(CH₂)₃CH₃, -(CH₂)₇CH=CH(CH₂)₅CH₃, -(CH₂)₄CH=CH(CH₂)₈CH₃, - (CH₂)₇CH=CH(CH₂)₇CH₃, -(CH₂)₆CH=CHCH₂CH=CH(CH₂)₄CH₃, - (CH₂)₇CH=CHCH₂CH=CH(CH₂)₄CH₃, (CH₂)₇CH=CHCH₂CH=CHCH₂CH=CHCH₂CH₃, - (CH₂)₃CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CH(CH₂)₄CH₃, (CH₂)₃CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH₃, -(CH₂)IICH=CH(CH₂)₇CH₃, or

(CH₂)₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH=CHCH₂CH₃.

In embodiments, said C₆-C₂₂ alkenyl is a monoalkenyl, a dienyl, or a trienyl. In embodiments, each of R², R³, and R⁴ is

In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000322] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the present invention include cleavable cationic lipids as described in International Patent Publication WO 2012/170889, which is incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid of the following formula:

wherein Ri is selected from the group consisting of imidazole, guanidinium, amino, imine, enamine, an optionally-substituted alkyl amino ( e.g ., an alkyl amino such as dimethylamino) and pyridyl; wherein R2 is selected from the group consisting of one of the following two formulas:

and wherein R3 and R4 are each independently selected from the group consisting of an optionally substituted, variably saturated or unsaturated C6-C20 alkyl and an optionally substituted, variably saturated or unsaturated C6-C20 acyl; and wherein n is zero or any positive integer (e.g., one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty or more). In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid, “HGT4001”, having a compound structure of:

and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid, “HGT4002”, having a compound structure of:

and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid, “HGT4003,” having a compound structure of:

and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid, “HGT4004,” having a compound structure of:

and pharmaceutically acceptable salts thereof. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid “HGT4005,” having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000323] Other suitable cationic lipids for use in the pharmaceutical compositions and methods of the present invention include cleavable cationic lipids as described in International Patent Publication WO 2019/222424, and incorporated herein by reference. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid that is any of general formulas or any of structures (la)-(21a) and (lb) - (21b) and (22)-(237) described in International Patent Publication WO 2019/222424.

In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid that has a structure according to Formula (I'),

wherein:

R^x is independently -H, -L'-R¹, or -L^5A-L^5B-B’; each of L¹, L², and L³ is independently a covalent bond, -C(O)-, -C(O)O-, -C(O)S-, or - C(O)NR^L-; each L^4A and L^5A is independently -C(O)-, -C(O)O-, or -C(O)NR^L-; each L^4B and L^5B is independently C1-C20 alkylene; C2-C20 alkenylene; or C2-C20 alkynylene; each B and B’ is NR⁴R⁵ or a 5- to 10-membered nitrogen-containing heteroaryl; each R¹, R², and R³ is independently C6-C30 alkyl, C6-C30 alkenyl, or C6-C30 alkynyl; each R⁴ and R⁵ is independently hydrogen, C1-C10 alkyl; C2-C10 alkenyl; or C2-C10 alkynyl; and each R^L is independently hydrogen, C1-C20 alkyl, C2-C20 alkenyl, or C2-C20 alkynyl. In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid that is Compound (139) of International Application No. PCT/US2019/032522, having a compound structure of:

[000324] In some embodiments, the pharmaceutical compositions and methods of the present invention include a cationic lipid that is TBL-0070 (RL3-DMA-07D) having a compound structure of:

and pharmaceutically acceptable salts thereof.

[000325] In some embodiments, the pharmaceutical compositions and methods of the present invention include the cationic lipid, N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (“DOTMA”). (Feigner el al. (Proc. Nat’l Acad. Sci. 84, 7413 (1987); U.S. Pat. No. 4,897,355, which is incorporated herein by reference). Other cationic lipids suitable for the pharmaceutical compositions and methods of the present invention include, for example, 5-carboxyspermylglycinedioctadecylamide (“DOGS”); 2,3-dioleyloxy- N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (“DOSPA”) (Behr et al. Proc. Nat.’l Acad. Sci. 86, 6982 (1989), U.S. Pat. No. 5,171,678; U.S. Pat. No. 5,334,761); l,2-Dioleoyl-3-Dimethylammonium-Propane (“DODAP”); l,2-Dioleoyl-3- Trimethylammonium- Propane (“DOTAP”).

[000326] Additional exemplary cationic lipids suitable for the pharmaceutical compositions and methods of the present invention also include: l,2-distearyloxy-N,N- dimethyl-3-aminopropane ( “DSDMA”); l,2-dioleyloxy-N,N-dimethyl-3-aminopropane (“DODMA”); 1 ,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (“DLinDMA”); 1,2- dilinolenyloxy-N,N-dimethyl-3-aminopropane (“DLenDMA”); N-dioleyl-N,N- dimethylammonium chloride (“DODAC”); N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”); N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (“DMRIE”); 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis- 9,12-octadecadienoxy)propane (“CLinDMA”); 2-[5’-(cholest-5-en-3-beta-oxy)-3’- oxapentoxy)-3-dimethy l-l-(cis,cis-9’, l-2’-octadecadienoxy)propane (“CpLinDMA”); N,N- dimethyl-3,4-dioleyloxybenzylamine (“DMOBA”); 1 ,2-N,N’-dioleylcarbamyl-3- dimethylaminopropane (“DOcarbDAP”); 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (“DLinDAP”); l,2-N,N’-Dilinoleylcarbamyl-3-dimethylaminopropane (“DLincarbDAP”); 1 ,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (“DLinCDAP”); 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (“DLin-K-DMA”); 2-((8-[(3P)-cholest-5-en-3- yloxy]octyl)oxy)-N, N-dimethyl-3-[(9Z, 12Z)-octadeca-9, 12-dien-l -yloxy]propane-l -amine (“Octyl-CLinDMA”); (2R)-2-((8-[(3beta)-cholest-5-en-3-yloxy]octyl)oxy)-N, N-dimethyl-3- [(9Z, 12Z)-octadeca-9, 12-dien-l-yloxy]propan-l -amine (“Octyl-CLinDMA (2R)”); (2S)-2- ((8-[(3P)-cholest-5-en-3-yloxy]octyl)oxy)-N, fsl-dimethyh3-[(9Z, 12Z)-octadeca-9, 12-dien-l -yloxy]propan-l -amine (“Octyl-CLinDMA (2S)”); 2,2-dilinoleyl-4-dimethylaminoethyl- [l,3]-dioxolane (“DLin-K-XTC2-DMA”); and 2-(2,2-di((9Z,12Z)-octadeca-9,l 2-dien- l-yl)-l ,3-dioxolan-4-yl)-N,N-dimethylethan amine (“DLin-KC2-DMA”) (see, WO 2010/042877, which is incorporated herein by reference; Semple et al. , Nature Biotech. 28: 172-176 (2010)). (Heyes, J., et al. , J Controlled Release 107: 276-287 (2005); Morrissey, DV., et al. , Nat. Biotechnol. 23(8): 1003-1007 (2005); International Patent Publication WO 2005/121348). In some embodiments, one or more of the cationic lipids comprise at least one of an imidazole, dialkylamino, or guanidinium moiety.

[000327] In some embodiments, one or more cationic lipids suitable for the pharmaceutical compositions and methods of the present invention include 2,2-Dilinoleyl-4- dimethylaminoethyl-[l,3]-dk>xolane (“XTC”); (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d] [1 ,3]dioxol-5-amine (“ALNY-100”) and/or 4,7 , 13 -tris(3 -oxo-3 -(undecylamino)propyl)-N 1 ,N 16-diundecyl-4,7 ,10,13- tetraazahexadecane- 1,16-diamide (“NC98-5”).

[000328] In some embodiments, the pharmaceutical compositions of the present invention include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured by weight, of the total lipid content in the pharmaceutical composition, e.g., a lipid nanoparticle. In some embodiments, the pharmaceutical compositions of the present invention include one or more cationic lipids that constitute at least about 5%, 10%, 20%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%, measured as a mol %, of the total lipid content in the pharmaceutical composition, e.g., a lipid nanoparticle. In some embodiments, the pharmaceutical compositions of the present invention include one or more cationic lipids that constitute about 30-70 % (e.g., about 30- 65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35- 50%, about 35-45%, or about 35-40%), measured by weight, of the total lipid content in the pharmaceutical composition, e.g., a lipid nanoparticle. In some embodiments, the pharmaceutical compositions of the present invention include one or more cationic lipids that constitute about 30-70 % (e.g., about 30-65%, about 30-60%, about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%), measured as mol %, of the total lipid content in the pharmaceutical composition, e.g., a lipid nanoparticle.

Non-Cationic Lipids

[000329] In some embodiments, the lipid nanoparticles contain one or more non- cationic lipids. As used herein, the phrase “non-cationic lipid” refers to any neutral, zwitterionic or anionic lipid. As used herein, the phrase “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l- carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), l,2-dierucoyl-sn-glycero-3-phosphoethanolamine (DEPE), phosphatidylserine, sphingolipids, cerebrosides, gangliosides, 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1- stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), or a mixture thereof. In some embodiments, lipid nanoparticles suitable for use with the invention include DOPE as the non-cationic lipid component. In other embodiments, lipid nanoparticles suitable for use with the invention include DEPE as the non-cationic lipid component.

[000330] In some embodiments, a non-cationic lipid is a neutral lipid, i.e., a lipid that does not carry a net charge in the conditions under which the pharmaceutical composition is formulated and/or administered.

[000331] In some embodiments, such non-cationic lipids may be used alone, but are preferably used in combination with other lipids, for example, cationic lipids.

[000332] In some embodiments, a non-cationic lipid may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a pharmaceutical composition. In some embodiments, total non-cationic lipids may be present in a molar ratio (mol%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a pharmaceutical composition. In some embodiments, the percentage of non-cationic lipid in a lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage total non- cationic lipids in a lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of non-cationic lipid in a lipid nanoparticle is no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%. In some embodiments, the percentage total non-cationic lipids in a lipid nanoparticle may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[000333] In some embodiments, a non-cationic lipid may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a pharmaceutical composition. In some embodiments, total non-cationic lipids may be present in a weight ratio (wt%) of about 5% to about 90%, about 5% to about 70%, about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 10 % to about 70%, about 10% to about 50%, or about 10% to about 40% of the total lipids present in a pharmaceutical composition. In some embodiments, the percentage of non-cationic lipid in a lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in a lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of non-cationic lipid in a lipid nanoparticle is no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%. In some embodiments, the percentage total non-cationic lipids in a lipid nanoparticle may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.

Cholesterol-Based Lipids

[000334] In some embodiments, the lipid nanoparticles comprise one or more cholesterol-based lipids. For example, suitable cholesterol-based cationic lipids include, for example, DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino- propyl)piperazine (Gao, et al. Biochem. Biophys. Res. Comm. 179, 280 (1991); Wolf et al. BioTechniques 23, 139 (1997); U.S. Pat. No. 5,744,335), or imidazole cholesterol ester (ICE), as disclosed in International Patent Publication WO 2011/068810, which has the following structure:

[000335] In embodiments, a cholesterol-based lipid is cholesterol.

[000336] In some embodiments, the cholesterol-based lipid may comprise a molar ratio (mol%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a lipid nanoparticle. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 mol%, greater than about 10 mol%, greater than about 20 mol%, greater than about 30 mol%, or greater than about 40 mol%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 mol%, no more than about 10 mol%, no more than about 20 mol%, no more than about 30 mol%, or no more than about 40 mol%.

[000337] In some embodiments, a cholesterol-based lipid may be present in a weight ratio (wt%) of about 1% to about 30%, or about 5% to about 20% of the total lipids present in a lipid nanoparticle. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be greater than about 5 wt%, greater than about 10 wt%, greater than about 20 wt%, greater than about 30 wt%, or greater than about 40 wt%. In some embodiments, the percentage of cholesterol-based lipid in the lipid nanoparticle may be no more than about 5 wt%, no more than about 10 wt%, no more than about 20 wt%, no more than about 30 wt%, or no more than about 40 wt%.

PEG-Modified Lipids

[000338] In some embodiments, the lipid nanoparticle comprises one or more PEGylated lipids.

[000339] For example, the use of polyethylene glycol (PEG)-modified phospholipids and derivatized lipids such as derivatized ceramides (PEG-CER), including N-Octanoyl- Sphingosine-l-[Succinyl(Methoxy Polyethylene Glycol)-2000] (C8 PEG-2000 ceramide) is also contemplated by the present invention, either alone or preferably in combination with other lipid pharmaceutical compositions together which comprise the transfer vehicle ( e.g ., a lipid nanoparticle).

[000340] Contemplated PEG-modified lipids include, but are not limited to, a polyethylene glycol chain of up to 5 kDa in length covalently attached to a lipid with alkyl chain(s) of C6-C20 length. In some embodiments, a PEG-modified or PEGylated lipid is PEGylated cholesterol or PEG-2K. The addition of such components may prevent complex aggregation and may also provide a means for increasing circulation lifetime and increasing the delivery of the lipid-nucleic acid pharmaceutical composition to the target tissues, (Klibanov et al. (1990) FEBS Letters, 268 (1): 235-237), or they may be selected to rapidly exchange out of the pharmaceutical composition in vivo (see U.S. Pat. No. 5,885,613). Particularly useful exchangeable lipids are PEG-ceramides having shorter acyl chains (e.g., C14 or C18). Lipid nanoparticles suitable for use with the invention typically include a PEG- modified lipid such as l,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2K).

[000341] The PEG-modified phospholipid and derivatized lipids of the present invention may comprise a molar ratio from about 0% to about 20%, about 0.5% to about 20%, about 1% to about 15%, about 4% to about 10%, or about 2% of the total lipid present in the liposomal transfer vehicle. In some embodiments, one or more PEG-modified lipids constitute about 4% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids constitute about 5% of the total lipids by molar ratio. In some embodiments, one or more PEG-modified lipids constitute about 6% of the total lipids by molar ratio. For certain applications, such as pulmonary delivery, lipid nanoparticles in which the PEG-modified lipid component constitutes about 5% of the total lipids by molar ratio have been found to be particularly suitable.

Amphiphilic block copolymers

[000342] In some embodiments, a suitable delivery vehicle contains amphiphilic block copolymers (e.g., poloxamers).

[000343] Various amphiphilic block copolymers may be used to practice the present invention. In some embodiments, an amphiphilic block copolymer is also referred to as a surfactant or a non-ionic surfactant. [000344] In some embodiments, an amphiphilic polymer suitable for the invention is selected from poloxamers (Pluronic®), poloxamines (Tetronic®), polyoxyethylene glycol sorbitan alkyl esters (polysorbates) and polyvinyl pyrrolidones (PVPs).

Poloxamers

[000345] In some embodiments, a suitable amphiphilic polymer is a poloxamer. For example, a suitable poloxamer is of the following structure:

wherein a is an integer between 10 and 150 and b is an integer between 20 and 60. For example, a is about 12 and b is about 20, or a is about 80 and b is about 27, or a is about 64 and b is about 37, or a is about 141 and b is about 44, or a is about 101 and b is about 56.

[000346] In some embodiments, a poloxamer suitable for the invention has ethylene oxide units from about 10 to about 150. In some embodiments, a poloxamer has ethylene oxide units from about 10 to about 100.

[000347] In some embodiments, a suitable poloxamer is poloxamer 84. In some embodiments, a suitable poloxamer is poloxamer 101. In some embodiments, a suitable poloxamer is poloxamer 105. In some embodiments, a suitable poloxamer is poloxamer 108. In some embodiments, a suitable poloxamer is poloxamer 122. In some embodiments, t a suitable poloxamer is poloxamer 123. In some embodiments, a suitable poloxamer is poloxamer 124. In some embodiments, a suitable poloxamer is poloxamer 181. In some embodiments, a suitable poloxamer is poloxamer 182. In some embodiments, a suitable poloxamer is poloxamer 183. In some embodiments, a suitable poloxamer is poloxamer 184. In some embodiments, a suitable poloxamer is poloxamer 185. In some embodiments, a suitable poloxamer is poloxamer 188. In some embodiments, a suitable poloxamer is poloxamer 212. In some embodiments, a suitable poloxamer is poloxamer 215. In some embodiments, a suitable poloxamer is poloxamer 217. In some embodiments, a suitable poloxamer is poloxamer 231. In some embodiments, a suitable poloxamer is poloxamer 234. In some embodiments, a suitable poloxamer is poloxamer 235. In some embodiments, a suitable poloxamer is poloxamer 237. In some embodiments, a suitable poloxamer is poloxamer 238. In some embodiments, a suitable poloxamer is poloxamer 282. In some embodiments, a suitable poloxamer is poloxamer 284. In some embodiments, a suitable poloxamer is poloxamer 288. In some embodiments, a suitable poloxamer is poloxamer 304. In some embodiments, a suitable poloxamer is poloxamer 331. In some embodiments, a suitable poloxamer is poloxamer 333. In some embodiments, a suitable poloxamer is poloxamer 334. In some embodiments, a suitable poloxamer is poloxamer 335. In some embodiments, a suitable poloxamer is poloxamer 338. In some embodiments, a suitable poloxamer is poloxamer 401. In some embodiments, a suitable poloxamer is poloxamer 402. In some embodiments, a suitable poloxamer is poloxamer 403. In some embodiments, a suitable poloxamer is poloxamer 407. In some embodiments, a suitable poloxamer is a combination thereof.

[000348] In some embodiments, a suitable poloxamer has an average molecular weight of about 4,000 g/mol to about 20,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 1,000 g/mol to about 50,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 1,000 g/mol.

In some embodiments, a suitable poloxamer has an average molecular weight of about 2,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 3,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 4,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 5,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 6,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 7,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 8,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 9,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 10,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 20,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 25,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 30,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 40,000 g/mol. In some embodiments, a suitable poloxamer has an average molecular weight of about 50,000 g/mol. Other amphiphilic polymers

[000349] In some embodiments, an amphiphilic polymer is a poloxamine, e.g., tetronic

304 or tetronic 904.

[000350] In some embodiments, an amphiphilic polymer is a polyvinylpyrrolidone (PVP), such as PVP with molecular weight of 3 kDa, 10 kDa, or 29 kDa.

[000351] In some embodiments, an amphiphilic polymer is a polyethylene glycol ether (Brij), polysorbate, sorbitan, and derivatives thereof. In some embodiments, an amphiphilic polymer is a polysorbate, such as PS 20.

[000352] In some embodiments, an amphiphilic polymer is polyethylene glycol ether (Brij), poloxamer, polysorbate, sorbitan, or derivatives thereof.

[000353] In some embodiments, an amphiphilic polymer is a polyethylene glycol ether. In some embodiments, a suitable polyethylene glycol ether is a compound of Formula (S-l):

or a salt or isomer thereof, wherein: t is an integer between 1 and 100;

R^IBRU independently is C_10-40 alkyl, C_10-40 alkenyl, or C_10-40 alkynyl; and optionally one or more methylene groups of R^5PEG are independently replaced with C3-10 carbocyclylene, 4 to 10 membered heterocyclylene, C6-10 arylene, 4 to 10 membered heteroarylene, -N(R^N)-, -O-, -S-, -C(O)-, -C(O)N(R^N)-, -NR^NC(O)-, -NR C(O)N(R )-, -C(O)O- -OC(O)-, -OC(O)O- - OC(O)N(R^N)-, -NR^NC(O)O- -C(O)S- -SC(O)-, -C(=NR^N)-,—

C(=NR )N(R )— , - NRNC(=NR^N)- -NR^NC(=NR^N)N(R^N)-, -CCS)-, -C(S)N(R^N)-, -NR^NC(S)-, -NR^NC(S)N(R^N)-, -SCO)-, -OS(O)-, -S(O)O- -OS(O)O- -OS(O)₂- -S(O)₂O- -OS(O)₂O- - N(R^N)S(O)-, - S(O)N(R^N)- -N(R^N)S(O)N(R^N)- -OS(O)N(R^N)- -N(R^N)S(O)0- -S(O)₂- - N(R^N)S(O)₂- - S(O)₂N(R^N)-, -N(R^N)S(O)₂N(R^N)- -OS(O)₂N(R^N)- or -N(R^N)S(O)₂O-; and each instance of R^N is independently hydrogen, C_1-6 alkyl, or a nitrogen protecting group.

[000354] In some embodiment, R^1BRU is C is alkyl. For example, the polyethylene glycol ether is a compound of Formula (S-la):

or a salt or isomer thereof, wherein s is an integer between 1 and 100.

[000355] In some embodiments, R^1BRU is C is alkenyl. For example, a suitable polyethylene glycol ether is a compound of Formula (S-lb):

or a salt or isomer thereof, wherein s is an integer between 1 and 100.

[000356] Typically, an amphiphilic polymer ( e.g ., a poloxamer) is present in a pharmaceutical composition at an amount lower than its critical micelle concentration (CMC). In some embodiments, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture at an amount about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% lower than its CMC. In some embodiments, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture at an amount about 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1% lower than its CMC. In some embodiments, an amphiphilic polymer (e.g., a poloxamer) is present in the mixture at an amount about 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% lower than its CMC.

[000357] In some embodiments, less than about 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% of the original amount of the amphiphilic polymer (e.g., the poloxamer) present in the pharmaceutical composition remains upon removal. In some embodiments, a residual amount of the amphiphilic polymer (e.g., the poloxamer) remains in a pharmaceutical composition upon removal. As used herein, a residual amount means a remaining amount after substantially all of the substance (an amphiphilic polymer described herein such as a poloxamer) in a pharmaceutical composition is removed. A residual amount may be detectable using a known technique qualitatively or quantitatively.

A residual amount may not be detectable using a known technique.

[000358] In some embodiments, a suitable delivery vehicle comprises less than 5% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable delivery vehicle comprises less than 3% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable delivery vehicle comprises less than 2.5% amphiphilic block copolymers ( e.g ., poloxamers). In some embodiments, suitable delivery vehicle comprises less than 2% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable delivery vehicle comprises less than 1.5% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable delivery vehicle comprises less than 1% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable delivery vehicle comprises less than 0.5% (e.g., less than 0.4%, 0.3%, 0.2%, 0.1%) amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable delivery vehicle comprises less than 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, or 0.01% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable delivery vehicle comprises less than 0.01% amphiphilic block copolymers (e.g., poloxamers). In some embodiments, a suitable delivery vehicle contains a residual amount of amphiphilic polymers (e.g., poloxamers). As used herein, a residual amount means a remaining amount after substantially all of the substance (an amphiphilic polymer described herein such as a poloxamer) in a pharmaceutical composition is removed. A residual amount may be detectable using a known technique qualitatively or quantitatively. A residual amount may not be detectable using a known technique.

Polymers

[000359] In some embodiments, a suitable delivery vehicle is formulated using a polymer as a carrier, alone or in combination with other carriers including various lipids described herein. Thus, in some embodiments, liposomal delivery vehicles, as used herein, also encompass nanoparticles comprising polymers. Suitable polymers may include, for example, polyacrylates, polyalkycyanoacrylates, polylactide, polylactide-polyglycolide copolymers, polycaprolactones, dextran, albumin, gelatin, alginate, collagen, chitosan, cyclodextrins, protamine, PEGylated protamine, PLL, PEGylated PLL and polyethylenimine (PEI). When PEI is present, it may be branched PEI of a molecular weight ranging from 10 to 40 kDa, e.g., 25 kDa branched PEI (Sigma #408727).

[000360] According to various embodiments, the selection of cationic lipids, non- cationic lipids, PEG-modified lipids, cholesterol-based lipids, and/or amphiphilic block copolymers which comprise the lipid nanoparticle, as well as the relative molar ratio of such components (lipids) to each other, is based upon characteristics of the selected lipid(s), the nature of the intended target cells, the characteristics of the nucleic acid to be delivered. Additional considerations include, for example, the saturation of the alkyl chain, as well as the size, charge, pH, pKa, fusogenicity and toxicity of the selected lipid(s). Thus the molar ratios may be adjusted accordingly.

Ratio of Distinct Lipid Components

[000361] A suitable lipid nanoparticle for the present invention may include one or more of any of the cationic lipids, non-cationic lipids, cholesterol lipids, PEG-modified lipids, amphiphilic block copolymers and/or polymers described herein at various ratios. In some embodiments, a lipid nanoparticle comprises five and no more than five distinct components of nanoparticle. In some embodiments, a lipid nanoparticle comprises four and no more than four distinct components of nanoparticle. In some embodiments, a lipid nanoparticle comprises three and no more than three distinct components of nanoparticle. As non-limiting examples, a suitable lipid nanoparticle pharmaceutical composition may include a combination selected from cKK-E12, DOPE, cholesterol and DMG-PEG2K; C 12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol and DMG-PEG2K; HGT4001, DOPE, cholesterol and DMG- PEG2K; HGT4002, DOPE, cholesterol and DMG-PEG2K; TL1-01D-DMA, DOPE, cholesterol and DMG-PEG2K; TL1-04D-DMA, DOPE, cholesterol and DMG-PEG2K; TL1- 08D-DMA, DOPE, cholesterol and DMG-PEG2K; TL1-10D-DMA, DOPE, cholesterol and DMG-PEG2K; ICE, DOPE and DMG-PEG2K; HGT4001, DOPE and DMG-PEG2K; or HGT4002, DOPE and DMG-PEG2K.

[000362] In various embodiments, cationic lipids ( e.g ., cKK-E12, C12-200, TL1-01D- DMA, TL1-04D-DMA, TL1-08D-DMA, TL1-10D-DMA, ICE, HGT4001, HGT4002 and/or HGT4003) constitute about 30-60 % (e.g., about 30-55%, about 30-50%, about 30-45%, about 30-40%, about 35-50%, about 35-45%, or about 35-40%) of the lipid nanoparticle by molar ratio. In some embodiments, the percentage of cationic lipids (e.g., cKK-E12, 02- 200, TL1-01D-DMA, TL1-04D-DMA, TL1-08D-DMA, TL1-10D-DMA, ICE, HGT4001, HGT4002 and/or HGT4003) is or greater than about 30%, about 35%, about 40 %, about 45%, about 50%, about 55%, or about 60% of the lipid nanoparticle by molar ratio.

[000363] In some embodiments, the molar ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) may be between about 30- 60:25-35:20-30:1-15, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:20:10, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:30:25:5, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 40:32:25:3, respectively. In some embodiments, the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol- based lipid(s) to PEG-modified lipid(s) is approximately 50:25:20:5.

[000364] In embodiments where a lipid nanoparticle comprises three and no more than three distinct components of lipids, the ratio of total lipid content (i.e., the ratio of lipid component (l):lipid component (2):lipid component (3)) can be represented as x:y:z, wherein

(y + z) = 100 - x.

[000365] In some embodiments, each of “x,” “y,” and “z” represents molar percentages of the three distinct components of lipids, and the ratio is a molar ratio.

[000366] In some embodiments, each of “x,” “y,” and “z” represents weight percentages of the three distinct components of lipids, and the ratio is a weight ratio.

[000367] In some embodiments, lipid component (1), represented by variable “x,” is a sterol-based cationic lipid.

[000368] In some embodiments, lipid component (2), represented by variable “y,” is a non-cationic lipid.

[000369] In some embodiments, lipid component (3), represented by variable “z” is a PEG lipid.

[000370] In some embodiments, variable “x,” representing the molar percentage of lipid component (1) ( e.g ., a sterol-based cationic lipid), is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

[000371] In some embodiments, variable “x,” representing the molar percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is no more than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20%, or about 10%. In embodiments, variable “x” is no more than about 65%, about 60%, about 55%, about 50%, about 40%. [000372] In some embodiments, variable “x,” representing the molar percentage of lipid component (1) ( e.g ., a sterol-based cationic lipid), is: at least about 50% but less than about 95%; at least about 50% but less than about 90%; at least about 50% but less than about 85%; at least about 50% but less than about 80%; at least about 50% but less than about 75%; at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%. In embodiments, variable “x” is at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.

[000373] In some embodiments, variable “x,” representing the weight percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is at least about 10%, about 20%, about 30%, about 40%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%.

[000374] In some embodiments, variable “x,” representing the weight percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is no more than about 95%, about 90%, about 85%, about 80%, about 75%, about 70%, about 65%, about 60%, about 55%, about 50%, about 40%, about 30%, about 20%, or about 10%. In embodiments, variable “x” is no more than about 65%, about 60%, about 55%, about 50%, about 40%.

[000375] In some embodiments, variable “x,” representing the weight percentage of lipid component (1) (e.g., a sterol-based cationic lipid), is: at least about 50% but less than about 95%; at least about 50% but less than about 90%; at least about 50% but less than about 85%; at least about 50% but less than about 80%; at least about 50% but less than about 75%; at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%. In embodiments, variable “x” is at least about 50% but less than about 70%; at least about 50% but less than about 65%; or at least about 50% but less than about 60%.

[000376] In some embodiments, variable “z,” representing the molar percentage of lipid component (3) (e.g., a PEG lipid) is no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In embodiments, variable “z,” representing the molar percentage of lipid component (3) (e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In embodiments, variable “z,” representing the molar percentage of lipid component (3) (e.g., a PEG lipid) is about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 1% to about 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 7.5%, or about 5% to about 10%.

[000377] In some embodiments, variable “z,” representing the weight percentage of lipid component (3) ( e.g ., a PEG lipid) is no more than about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, or 25%. In embodiments, variable “z,” representing the weight percentage of lipid component (3) (e.g., a PEG lipid) is about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%. In embodiments, variable “z,” representing the weight percentage of lipid component (3) (e.g., a PEG lipid) is about 1% to about 10%, about 2% to about 10%, about 3% to about 10%, about 4% to about 10%, about 1% to about 7.5%, about 2.5% to about 10%, about 2.5% to about 7.5%, about 2.5% to about 5%, about 5% to about 7.5%, or about 5% to about 10%.

[000378] For pharmaceutical compositions having three and only three distinct lipid components, variables “x,” “y,” and “z” may be in any combination so long as the total of the three variables sums to 100% of the total lipid content. For example, in typical three- component lipid nanoparticles suitable for use with the invention, the molar ratio of cationic lipid to non-cationic lipid to PEG-modified lipid may be between about 55-65:30-40:1-15, respectively. In some embodiments, a molar ratio of cationic lipid (e.g., a sterol-based lipid) to non-cationic lipid (e.g., DOPE or DEPE) to PEG-modified lipid (e.g., DMG-PEG2K) of 60:35:5 is particularly suitable, e.g., for pulmonary delivery of lipid nanoparticles via nebulization.

Pulmonary Delivery

[000379] Compounds may be formulated for delivery via different administration routes including, but not limited to, oral, rectal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intradermal, transdermal (topical), intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, and/or intranasal administration.

[000380] In accordance with the present invention, the pharmaceutical composition is formulated for pulmonary delivery. As used herein, pulmonary delivery refers to delivery to lung via, e.g., nasal cavity, trachea, bronchi, bronchioles, and/or other pulmonary system. In particular embodiments, a pharmaceutical composition is formulated for nebulization. In these embodiments, the delivery vehicle may be in an aerosolized composition which can be inhaled. In some embodiments, pulmonary delivery involves inhalation ( e.g ., for nasal, tracheal, or bronchial delivery). In some embodiments, the pharmaceutical composition nebulized prior to inhalation.

Nebulization

[000381] The efficacy of nebulizing a pharmaceutical composition for pulmonary delivery depends on the size of the small aerosol droplets. Generally, the smaller the droplet size, the greater its chance of penetration into and retention in the lung. Large droplets (> 10 μm in diameter) are most likely to deposit in the mouth and throat, medium droplets (5 - 10 μm in diameter) are most likely to deposit between the mouth and airway, and small droplets (< 5 μm in diameter) are most likely to deposit and be retained in the lung.

[000382] Inhaled aerosol droplets of a particle size of 1-5 μm can penetrate into the narrow branches of the lower airways. Aerosol droplets with a larger diameter are typically absorbed by the epithelia cells lining the oral cavity, and are unlikely to reach the lower airway epithelium and the deep alveolar lung tissue.

[000383] Particle size in an aerosol is commonly described in reference to the Mass Median Aerodynamic Diameter (MMAD). MMAD, together with the geometric standard deviation (GSD), describes the particle size distribution of any aerosol statistically, based on the weight and size of the particles. Means of calculating the MMAD of an aerosol are well known in the art.

[000384] A specific method of calculating the MMAD using a cascade impactor was first described in 1959 by Mitchell et al. The cascade impactor for measuring particle sizes is constructed of a succession of jets, each followed by an impaction slide, and is based on the principle that particles in a moving air stream impact on a slide placed in their path, if their momentum is sufficient to overcome the drag exerted by the air stream as it moves around the slide. As each jet is smaller than the preceding one, the velocity of the air stream and therefore that of the dispersed particles are increased as the aerosol advances through the impactor. Consequently, smaller particles eventually acquire enough momentum to impact on a slide, and a complete particle size classification of the aerosol is achieved. The improved Next Generation Impactor, used herein to measure the MMAD of the pharmaceutical composition of the invention, was first described by Marple el al. in 2003 and has been widely used in the pharmacopoeia since.

[000385] Another parameter to describe particle size in an aerosol is the Volume Median Diameter (VMD). VMD also describes the particle size distribution of an aerosol based on the volume of the particles. Means of calculating the VMD of an aerosol are well known in the art. A specific method used for determining the VMD is laser diffraction, which is used herein to measure the VMD of the pharmaceutical composition of the invention (see, e.g., Clark, 1995, Int J Pharm. 115:69-78).

[000386] In some embodiments, the mean particle size of the nebulized pharmaceutical composition is between about 4 μm and 6 μm, e.g., about 4 μm, about 4.5 μm, about 5 μm, about 5.5 μm, or about 6 μm.

[000387] The Fine Particle Fraction (FPF) is defined as the proportion of particles in an aerosol which have an MMAD or a VMD smaller than a specified value. In some embodiments, the FPF of the nebulized pharmaceutical composition with a particle size <5 μm is at least about 30%, more typically at least about 40%, e.g., at least about 50%, more typically at least about 60%.

[000388] In some embodiments, nebulization is performed in such a manner that the mean respirable emitted dose (i.e., the percentage of FPF with a particle size < 5 μm; e.g., as determined by next generation impactor with 15 L/min extraction) is at least about 30% of the emitted dose, e.g., at least about 31%, at least about 32%, at least about 33%, at least about 34%, or at least about 35% the emitted dose. In some embodiments, nebulization is performed in such a manner that the mean respirable delivered dose (i.e., the percentage of FPF with a particle size < 5 μm; e.g., as determined by next generation impactor with 15 L/min extraction) is at least about 15% of the emitted dose, e.g., at least 16% or 16.5% of the emitted dose.

Nebulizer

[000389] Nebulization can be achieved by any nebulizer known in the art. A nebulizer transforms a liquid to a mist so that it can be inhaled more easily into the lungs. Nebulizers are effective for infants, children and adults. Nebulizers are able to nebulize large doses of inhaled medications. Typically, a nebulizer for use with the invention comprises a mouthpiece that is detachable. This is important because only clean mouthpieces that are RNase free should be used when administering the pharmaceutical composition of the invention.

[000390] In some embodiments, the reservoir volume of the nebulizer ranges from about 5.0 mL to about 8.0 mL. In some embodiments, the reservoir volume of the nebulizer is about 5.0 mL. In some embodiments, the reservoir volume of the nebulizer is about 6.0 mL. In some embodiments, the reservoir volume of the nebulizer is about 7.0 mL. In some embodiments, the reservoir volume of the nebulizer is about 8.0 mL.

[000391] One type of nebulizer is a jet nebulizer, which comprises tubing connected to a compressor, which causes compressed air or oxygen to flow at a high velocity through a liquid medicine to turn it into an aerosol, which is then inhaled by the patient.

[000392] Another type of nebulizer is the ultrasonic wave nebulizer, which comprises an electronic oscillator that generates a high frequency ultrasonic wave, which causes the mechanical vibration of a piezoelectric element, which is in contact with a liquid reservoir. The high frequency vibration of the liquid is sufficient to produce a vapor mist. Exemplary ultrasonic wave nebulizers are the Omron NE-U17 and the Beurer Nebulizer IH30.

[000393] A third type of nebulizer is a mesh nebulizer such as a vibrating mesh nebulizer comprising vibrating mesh technology (VMT). A VMT nebulizer typically comprises a mesh/membrane with 1000-7000 holes that vibrates at the top of a liquid reservoir and thereby pressures out a mist of very fine aerosol droplets through the holes in the mesh/membrane. VMT nebulizers suitable for delivery of the pharmaceutical composition of the invention include any of the following: eFlow (PARI Medical Ltd.), i-Neb (Respironics Respiratory Drug Delivery Ltd), Nebulizer IH50 (Beurer Ltd.), AeroNeb Go (Aerogen Ltd.), InnoSpire Go (Respironics Respiratory Drug Delivery Ltd), Mesh Nebulizer (Shenzhen Homed Medical Device Co, Ltd.), Portable Nebulizer (Microbase Technology Corporation) and Airworks (Convexity Scientific LLC). In some embodiments, the mesh or membrane of the VMT nebulizer is made to vibrate by a piezoelectric element. In some embodiments, the mesh or membrane of the VMT nebulizer is made to vibrate by ultrasound. [000394] VMT nebulizers have been found to be particularly suitable for practicing the invention because they do not affect the integrity of the oligonucleotide in the pharmaceutical composition of the invention. Typically, at least about 50%, e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, least about 80%, least about 90%, or least about 95% of the oligonucleotide in the pharmaceutical composition of the invention maintains its integrity after nebulization.

[000395] In some embodiments, nebulization is continuous during inhalation and exhalation. More typically, nebulization is breath-actuated. Suitable nebulizers for use with the invention have nebulization rate of >0.2 mL/min. In some embodiments, the nebulization rate is >0.25 mL/min. In other embodiment, the nebulization rate is >0.3 mL/min. In some embodiments, the nebulization rate is >0.45 mL/min. In a typical embodiment, the nebulization rate ranges between 0.2 mL/minute and 0.5 mL/minute.

[000396] A human subject may display adverse effects during treatment, when the nebulization volume exceeds 10 mL. In particular, such adverse effects may be more common when volumes greater than 20 mL are administered. In some embodiments, the nebulization volume does not exceed 20 mL.

[000397] In some embodiments, a single dose of the pharmaceutical composition of the invention can be administered with only a one or two refills per nebulization treatment. For example, if the total volume of the pharmaceutical composition that is to be administered to the patient is 13 mL, then only a single refill is required to administer the entire volume when using a nebulizer with an 8 mL reservoir, but two refills are required to administer the same volume when using a nebulizer with a 5 mL reservoir. In another embodiment, at least three refills are required per nebulization treatment, e.g., to administer a volume of 26 mL, at least three refills are required when using a nebulizer with an 8 mL reservoir. In yet a further embodiment, at least four refills are required. For example, to deliver 42 mL with a nebulizer having a 5 mL reservoir, at least eight refills are required. Typically, no more than 1-3 refills will be required to administer the pharmaceutical composition of the invention.

[000398] The pharmaceutical composition of the invention is typically nebulized at a rate ranging from 0.2 mL/minute to 0.5 mL/minute. A concentration of 0.5 mg/ml to 0.8 mg/ml of the oligonucleotide (e.g. about 0.6 mg/ml) has been found to be particularly suitable, in particular when administered with a vibrating mesh nebulizer.

[000399] In some embodiments, the number of nebulizers used during a single nebulization session ranges from 2-8. In some embodiments, 1 nebulizer is used during a single nebulization session. In some embodiments, 2 nebulizers are used during a single nebulization session. In some embodiments, 3 nebulizers are used during a single nebulization session In some embodiments, 4 nebulizers are used during a single nebulization session In some embodiments, 5 nebulizers are used during a single nebulization session In some embodiments, 6 nebulizers are used during a single nebulization session In some embodiments, 7 nebulizers are used during a single nebulization session In some embodiments, 8 nebulizers are used during a single nebulization session.

Pharmaceutical Compositions

[000400] Compounds of the invention may be admixed with pharmaceutically acceptable active or inert substances. For example, compounds may be admixed with a suitable pharmaceutically acceptable diluent or carrier, such as an aqueous solution. Such solutions may include a buffer, e.g., phosphate -buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Accordingly, in one embodiment, pharmaceutical composition further comprise a pharmaceutically acceptable diluent. In some embodiments, the pharmaceutically acceptable diluent is PBS [000401] Alternatively, an aqueous solution may contain other excepients, either in addition or as an alternative to a buffer. Such excepients may include sucrose or trehalose. For example, a trehalose-based solution has been found to be effective for pulmonary delivery of liposomal compositions, in particular via nebulization. A suitable trehalose concentration is between about 5% and about 15% (w/v), e.g., about 10% (w/v).

[000402] Pharmaceutical compositions of the invention also encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other oligonucleotide which, upon administration to a subject, including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to pharmaceutically acceptable salts of antisense strands, prodmgs, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents. Suitable pharmaceutically acceptable salts include, but are not limited to, sodium and potassium salts.

[000403] Also contemplated herein are lyophilized pharmaceutical compositions comprising one or more of the lipid nanoparticles disclosed herein and related methods for the use of such pharmaceutical compositions as disclosed for example, in United States Provisional Application No. 61/494,882, filed June 8, 2011, the teachings of which are incorporated herein by reference in their entirety.

Therapeutic Use of Pharmaceutical Compositions

[000404] Pharmaceutical compositions of the invention may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject’s age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art. The “effective amount” for the purposes herein may be determined by such relevant considerations as are known to those of ordinary skill in experimental clinical research, pharmacological, clinical, and medical arts. In some embodiments, the amount administered is effective to achieve at least some stabilization, improvement or elimination of symptoms and other indicators as are selected as appropriate measures of disease progress, regression or improvement by those of skill in the art.

[000405] The choice of administration route depends on the target cell or tissues. Pulmonary delivery is commonly used to target the lung epithelium. Accordingly, in some embodiments, oligonucleotide-loaded lipid nanoparticles of the invention are administered by pulmonary delivery via nebulization, typically involving a suitable nebulizing apparatus (e.g., a mesh nebulizer). Additional teaching of pulmonary delivery and nebulization are described in published U.S. Application No. US 2018/0125989 and published U.S. Application No. US 2018/0333457, each of which is incorporated by reference in its entirety.

[000406] Provided methods of the present invention contemplate single as well as multiple administrations of a therapeutically effective amount of the pharmaceutical compositions described herein. Pharmaceutical compositions can be administered at regular intervals, depending on the nature, severity and extent of the subject’s condition. In some embodiments, a therapeutically effective amount of the pharmaceutical composition of the present invention may be administered periodically at regular intervals (e.g., bimonthly (once every two-months), monthly (once every month), biweekly (once every two-weeks), twice a month, once every 30-days, once every 28-days, once every 14-days, once every 10-days, once every 7-days, weekly, twice a week, or daily). Treatment

[000407] Without wishing to be bound by any particular theory, the inventors believe that excessive mucus formation may be caused by the overexpression of MUC5B. Such overexpression may either cause or exacerbate the symptoms associated with common lung diseases or disorders. For example, the presence of a polymorphism (rs35705950) in the promoter region of MUC5B is associated with both familial and sporadic forms of IPF. Overexpression of MUC5B can result in one or more of the following outcomes: reduced mucociliary function, reduced alveolar repair, and increased lung fibrosis.

[000408] Accordingly, in some embodiments, provided herein are methods of treating a subject in need thereof comprising administering a compound or pharmaceutical composition of the invention. In some embodiments, the compounds and pharmaceutical compositions described herein are administered to a subject to treat, prevent, ameliorate or slow progression of a lung disease or disorder. In some embodiments, the subject has a lung disease or disorder. In some embodiments, the individual is at risk for developing a lung disease or disorder. In some embodiments the lung disease or disorder is associated with overexpression of MUC5B. In some embodiments, overexpression of MUC5B is associated with reduced mucociliary function, reduced alveolar repair, and/or increased lung fibrosis. [000409] Accordingly, in some embodiments, provided herein are methods for reducing MUC5B mRNA and/or protein levels in a subject. Lung diseases or disorders that may be treatable with the compounds, pharmaceutical compositions or methods of the invention are commonly associated with reduced mucociliary function, reduced alveolar repair, and/or increased lung fibrosis. In some embodiments, a subject treatable with the compounds, pharmaceutical compositions or methods of the invention may have a polymorphism ( e.g ., rs35705950) in the promoter region of MUC5B that results in overexpression of MUC5B. [000410] Some embodiments include treating a subject in need thereof by administering to the subject a therapeutically effective amount of an oligonucleotide targeted to a MUC5B nucleic acid. In one embodiment, administration of a therapeutically effective amount of an oligonucleotide targeted to a MUC5B nucleic acid is accompanied by monitoring of MUC5B levels in the subject, to determine an individual’s response to administration of the oligonucleotide. A subject’s response to administration of the oligonucleotide may be used by a physician to determine the amount and duration of therapeutic intervention. [000411] In some embodiments, administration of an oligonucleotide targeted to a MUC5B nucleic acid results in reduction of MUC5B mRNA and/or protein levels by at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.

In some embodiments, administration of an oligonucleotide targeted to a MUC5B nucleic acid results in improved mucociliary function, improved alveolar repair and/or reduced lung fibrosis in a subject. In some embodiments, administration of a MUC5B oligonucleotide improves improved mucociliary function, improved alveolar repair and/or reduced lung fibrosis by at least 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 99%, or a range defined by any two of these values.

[000412] It is understood that any reference to uses of compounds, pharmaceutical compositions, oligonucleotides or other molecules throughout the description contemplates their use in the preparation of a pharmaceutical composition or medicament for use in the treatment of a lung disease or disorder associated with overexpression of a MUC5B nucleic acid.

Exemplary Indications

[000413] Outcomes associated with overexpression of MUC5B include reduced mucociliary function, reduced alveolar repair, and increased lung fibrosis. Without wishing to be bound by any particular theory, the inventors believe that MUC5B overexpression may play a role in the pathogenesis of a diverse group of lung disease or disorder including idiopathic pulmonary fibrosis (IPF), chronic rhinosinusitis (CRS), chronic obstructive pulmonary disease (COPD), diffuse panbronchiolitis (DPB), asthma, and cystic fibrosis (CF). Accordingly, in some embodiments, the lung disease or disorder to be treated with the compounds or compositions of the invention are selected from idiopathic pulmonary fibrosis (IPF), chronic rhinosinusitis (CRS), chronic obstructive pulmonary disease (COPD), diffuse panbronchiolitis (DPB), asthma, and cystic fibrosis (CF). In some embodiments, treatment with the compounds or pharmaceutical compositions of the invention is combined with other therapies, including the treatment with a corticosteroid, a bronchodilator (in particular a long- acting bronchodilator), or a combination of a corticosteroid and a bronchodilator. These therapies may be administered are typically admininstered by inhalation and therefore may be administered concomitantly or in combination with the compounds or pharmaceutical compositions of the invention.

[000414] In some embodiments of the present disclosure, the subject is suffering from idiopathic pulmonary fibrosis (IPF). IPF is a disorder that occurs when lung tissue becomes damaged and scarred. This damaged, scarred tissue makes it difficult to breathe. In some cases, IPF can be rapidly progressive. For example, it may be characterized by sequential acute lung injury with subsequent scarring and end-stage lung disease. Common IPF symptoms may include one or more of the following: shortness of breath, radiographically evident diffuse pulmonary infiltrates, varying degrees of pulmonary inflammation and fibrosis, fatigue, unexplained weight loss, aching muscles and joints, and widening and rounding of the tips of the fingers or toes. A risk factor for the development of IPF is the presence of a polymorphism (rs35705950) in the promoter region of MUC5B. The presence of this polymorphism is associated with both familial and sporadic forms of IPF. Accordingly, in some embodiments, a subject to be treated with the compounds or pharmaceutical compositions of the invention is a carrier of the rs35705950 polymorphism. [000415] In some embodiments of the present disclosure, the subject has chronic rhinosinusitis (CRS). Rhinosinusitis is characterized by inflammation of the mucosal linings of the nasal passage and paranasal sinuses. Rhinosinusitis is characterized as chronic when symptoms last for longer than 12 weeks. Symptoms of CRS include facial pain/pressure, hyposmia/anosmia, nasal drainage, and nasal obstruction. Treatment is directed at enhancing mucociliary clearance, improving sinus drainage/outflow, eradicating local infection and inflammation, and improving access for topical medications. Accordingly, in some embodiments, a subject suffering from CRS is treated with a compound or pharmaceutical compositions of the invention in combination with topical medications commonly used for treating CRS, such as a topical corticosteroid.

[000416] In some embodiments, the subject has chronic obstructive pulmonary disease (COPD). Symptoms of COPD include one or more of the following: irreversible airflow obstruction due to chronic bronchitis, emphysema, and/or small airways disease. Airflow obstruction is typically determined by reductions in quantitative spirometric indices, including, but not limited to: forced expiratory volume at 1 second (FEV1) and the ratio of FEV1 to forced vital capacity (FVC). COPD symptoms may additionally include: cough with phlegm, frequent respiratory infections, shortness of breath, wheezing, fatigue, inability to exercise, and chest pressure. In some embodiments, treatment with the compounds or pharmaceutical compositions of the invention is combined with the standard COPD therapies, including treatment with short-acting bronchodilators (e.g., albuterol, levalbuterol, ipratropium, or a combination of albuterol and ipratropium ), long-acting bronchodilators (e.g., aclidinium, arformoterol, formoterol, glycopyrrolate, indacaterol, olodaterol, salmeterol, tiotropium, umeclidinium or combinations thereof), methylxanthines and corticosteroids (e.g., fluticasone, budesonide, or prednisolone, alone or in combination with long-acting bronchodilator).

[000417] In some embodiments of the present disclosure, the subject has diffuse panbronchiolitis (DPB). DPB is an inflammatory lung disease of unknown cause. It is a severe, progressive form of bronchiolitis. Symptoms include nodule-like lesions that appear throughout both lungs, particularly in the terminal bronchioles, severe inflammation, chronic sinusitis, and intense coughing with large amounts of sputum production. If left untreated, DPB progresses to bronchiectasis, an irreversible lung condition that involves enlargement of the bronchioles, and pooling of mucus in the bronchiolar passages. The highest incidence occurs among individuals of East Asian descent, particularly individuals of Japanese descent, followed by individuals of Korean descent. Therapy of DPB may include treatment with a bronchodilator, in addition to treatment with the compounds or pharmaceutical compositions of the invention.

[000418] In some embodiments of the present disclosure, the subject has asthma. Asthma is a condition in which the airways become inflamed, narrow and swell, and produce excess mucus, all of which makes it difficult for a person suffering from asthma to breathe. Common asthma symptoms include one or more of the following: difficulty breathing, chest pain, cough, wheezing, breathing through the mouth, fast breathing, frequent respiratory infections, shortness of breath at night, chest pressure, flare, anxiety, early awakening, fast heart rate, and throat irritation. In some embodiments, treatment with the compounds and pharmaceutical compositions of the invention is combined with standard inhaler therapies used for the treatment of asthma. These include combination of a corticosteroid and a bronchodilator, e.g. fluticasone and salmeterol, budesonide and formoterol, mometasone and formoterol, or fluticasone and vilanterol. [000419] In some embodiments of the present disclosure, the subject has cystic fibrosis (CF). CF is an autosomal inherited disorder resulting from mutation of the CFTR gene, which encodes a chloride ion channel believed to be involved in regulation of multiple other ion channels and transport systems in epithelial cells. Loss of function of CFTR results in chronic lung disease, aberrant mucus production, and dramatically reduced life expectancy. See generally Rowe et al., New Engl. J. Med. 352, 1992-2001 (2005). In some embodiments, treatment with the compounds and pharmaceutical compositions of the invention is combined with other therapies, including treatment with CFTR mRNA therapy. In some embodiments, CFTR mRNA therapy comprises administering a composition comprising an in vitro transcribed mRNA molecule comprising a coding sequence, a 5’-UTR, and a 3’-UTR, wherein the coding sequence encodes the amino acid sequence of the cystic fibrosis transmembrane regulator (CFTR). In some embodiments the transcribed mRNA molecule further comprises a nucleotide sequence encoding a signal peptide.

Dosing

[000420] In one aspect, the disclosure features a method of administering a pharmaceutical composition comprising an oligonucleotide and a lipid nanoparticle to a subject ( e.g ., a human subject). In one embodiment, the unit dose ranges from about 0.001 mg/kg body weight to 500 mg/kg body weight.

[000421] The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with low levels of a MUC5B mRNA or protein; or a disease or disorder associated with expression of a mutant protein.

[000422] In one embodiment, a subject is administered an initial dose and one or more maintenance doses of a pharmaceutical composition comprising a stabilizing oligonucleotide and a particle. The maintenance doses may be administered no more than once every 1, 5,

10, or 30 days.

[000423] Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the pharmaceutical composition may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed. [000424] The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances.

[000425] In some cases, a patient is treated with a pharmaceutical composition comprising an oligonucleotide and a lipid nanoparticle in conjunction with other therapeutic modalities.

Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an oligonucleotide in a pharmaceutical composition can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of an oligonucleotide in a pharmaceutical composition used for treatment may increase or decrease over the course of a particular treatment. For example, the subject can be monitored after administering the pharmaceutical composition. Based on information from the monitoring, an additional amount of a pharmaceutical composition comprising a stabilizing oligonucleotide and a particle can be administered.

In vitro testing of oligonucleotides

[000426] The effects of oligonucleotides on the level, activity or expression of MUC5B nucleic acids can be tested in vitro in a variety of cell types. Cell types used for such analyses are available from commerical vendors (e.g. American Type Culture Collection, Manassus, VA; Zen-Bio, Inc., Research Triangle Park, NC; Clonetics Corporation, Walkersville, MD) and are cultured according to the vendor’s instructions using commercially available reagents (e.g. Invitrogen Life Technologies, Carlsbad, CA). Illustrative cell types include, but are not limited to, A549 cells and primary lung epithelial cells. In general, cells are treated with oligonucleotides when the cells reach approximately 60-80% confluency in culture.

[000427] Cells are treated with oligonucleotides by routine methods. Cells are typically harvested 16-24 hours after oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein.

[000428] The concentration of oligonucleotide used varies from cell line to cell line. Methods to determine the optimal oligonucleotide concentration for a particular cell line are well known in the art. Oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with lipid based reagents. Oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.

RNA Isolation

[000429] RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, CA).

Analysis of inhibition of target levels or expression

[000430] Inhibition of levels or expression of MUC5B mRNA can be assayed in a variety of ways known in the art. For example, target nucleic acid levels can be quantitated by, e.g., Northern blot analysis, competitive polymerase chain reaction (PCR), or quantitaive real-time PCR. RNA analysis can be performed on total cellular RNA or poly(A)+ mRNA. Methods of RNA isolation are well known in the art. Northern blot analysis is also routine in the art. Quantitative real-time PCR can be conveniently accomplished using the commercially available ABI PRISM 7600, 7700, or 7900 Sequence Detection System, available from PE- Applied Biosystems, Foster City, CA and used according to manufacturer’s instructions.

Quantitative Real-Time PCR Analysis of Target RNA Levels

[000431] Quantitation of target RNA levels may be accomplished by quantitative real time PCR using the ABI PRISM 7600, 7700, or 7900 Sequence Detection System (PE- Applied Biosystems, Foster City, CA) according to manufacturer’s instructions. Methods of quantitative real-time PCR are well known in the art.

[000432] Prior to real-time PCR, the isolated RNA is subjected to a reverse transcriptase (RT) reaction, which produces complementary DNA (cDNA) that is then used as the substrate for the real-time PCR amplification. The RT and real-time PCR reactions are performed sequentially in the same sample well. RT and real-time PCR reagents are obtained from Invitrogen (Carlsbad, CA). RT real-time-PCR reactions are carried out by methods well known to those skilled in the art.

[000433] Gene (or RNA) target quantities obtained by real time PCR are normalized using either the expression level of a gene whose expression is constant, such as cyclophilin A, or by quantifying total RNA using RIBOGREEN (Invitrogen, Inc. Carlsbad, CA). Cyclophilin A expression is quantified by real time PCR, by being run simultaneously with the target, multiplexing, or separately. Total RNA is quantified using RIBOGREEN RNA quantification reagent (Invetrogen, Inc. Eugene, OR). Methods of RNA quantification by RIBOGREEN are taught in Jones, L.J., et al, (Analytical Biochemistry, 1998, 265, 368-374). A CYTOFLUOR 4000 instrument (PE Applied Biosystems) is used to measure RIBOGREEN fluorescence.

[000434] Probes and primers are designed to hybridize to a MUC5B mRNA. Methods for designing real-time PCR probes and primers are well known in the art, and may include the use of software such as PRIMER EXPRESS Software (Applied Biosystems, Foster City, CA).

Analysis of Protein Levels

[000435] siRNA inhibition of MUC5B mRNA can be assessed by measuring MUC5B protein levels. Protein levels of MUC5B can be evaluated or quantitated in a variety of ways well known in the art, such as immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), quantitative protein assays, protein activity assays (for example, caspase activity assays), immunohistochemistry, immunocytochemistry or fluorescence-activated cell sorting (FACS). Antibodies directed to a target can be identified and obtained from a variety of sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, MI), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Antibodies useful for the detection of MUC5B are commercially available.

In vivo testing of oligonucleotides

[000436] Oligonucleotides are tested in animals to assess their ability to reduce MUC5B mRNA and/or protein levels and produce phenotypic changes, such as, improved mucociliary function, improved alveolar repair and reduced lung fibrosis. Testing may be performed in normal animals, or in experimental disease models. Calculation of oligonucleotide dosage and dosing frequency is within the abilities of those skilled in the art, and depends upon factors such as route of administration and animal body weight. Following a period of treatment with oligonucleotides, RNA and/or protein is isolated from lung tissue and changes in MUC5B mRNA or protein levels are measured. Kits

[000437] In certain aspects of the disclosure, kits are provided comprising a container housing a compound or a pharmaceutical composition as described herein. In some embodiments, the individual components of the pharmaceutical composition may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical composition separately in two or more containers, e.g., one container for the oligonucleotide and the lipid nanoparticle, and another container with a diluent or buffer (e.g., for reconstitution of a lyophilized pharmaceutical composition).

[000438] The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

EXAMPLES

Example 1. Design of siRNA duplex oligonucleotides targeting MUC5B mRNA

[000439] siRNAs were designed to knockdown MUC5B expression by targeting its mRNA for degradation.

Transcript selection

[000440] Human, cynomolgus monkey (“cyno”), mouse and rat MUC5B transcripts were obtained from the NCBI RefSeq database. Experimentally validated “NM” transcripts were chosen that contained the maximum number of internal exons: for human, for cyno, for mouse, and for rat. Off-target analysis (as described below) also utilized the NCBI RefSeq database.

Selection of oligonucleotide sequences

[000441] All 18mer sub-sequences and complementary antisense sequences that matched the MUC5B transcripts in human, cyno, mouse and rat were generated. The target regions within the murine MUC5B mRNA sequence are shown as underlined in FIG. 1. [000442] siRNA oligonucleotides specific for these target sequences were designed as follows: A single A nucleotide was added to the 3’ end of the sense strand, with a complementary U at the 5’ end of the antisense strand, to yield a 19mer duplex. This UA pair was utilized since the antisense (“guide”) strand’s 5’-most nucleotide is not exposed to and does not bind target mRNAs when loaded in the RISC complex, and the AGO protein subunit prefers 5’ U nucleotides (Noland and Doudna 2013, Nakanishi 2016). Candidate 19mer duplexes were further evaluated for efficacy characteristics and off-target specificity.

Specificity and efficacy selection

[000443] The specificity of the candidate duplexes was evaluated via alignment of both strands to all human and mouse RefSeq transcripts, using the FASTA algorithm with an E value cutoff of 1000. The counts of mismatches between each strand and each transcript (per species) were tabulated. Duplexes were chosen that had at least one 8mer seed (positions 2-9) mismatch on both strands to any human or cyno transcript other than those encoded by the MUC5B gene, and at least one mismatch at any position on both strands to any mouse or rat transcript other than those encoded by the MUC5B gene. Seed mismatches are particularly critical for specificity of siRNA activity (Boudreau et al. 2011). Duplexes were further selected for GC content and thermal asymmetry according to the following guidelines: GC content < 55%, >3 Us or As in the antisense 8mer seed, and a G or C at the antisense 19th position. GC content and asymmetry are two important predictors of siRNA efficacy (Akinc, Bettencourt, and Maier 2015). Any duplexes (strands) with homopolymers of 5 or more nucleotides were excluded. Selection according to these parameters yielded 10 duplexes, which were synthesized and screened.

Example 2: MUC5B siRNA duplexes are active in vitro

[000444] This examples demonstrates that siRNA oligonucleotides are effective in reducing MUC5B mRNA levels in a cell line derived from the human alveolar lung ephithelium.

[000445] In order to determine the efficacy of siRNA duplexes generated in Example 1, 10 chemically modified siRNA duplexes targeting MUC5B were screened in vitro. A549 (lung cancer) cells were seeded at a density of 12,500 cells/well in a 96 well plate. MUC5B siRNA oligonucleotides #1-10 (see Table 1) were added to the cells, at 1.25nM concentration, in the presence of transfection reagent (Dharmafect) to transfect the cells. [000446] After 48 hours, the cells were collected and lysed. Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) was performed in duplicate or triplicate reactions per sample to measure MUC5B gene expression. As shown in FIG. 2, both siRNA oligonucleotides #3 and #4 demonstrated consistent cellular activity ranging from 50-80% knockdown, normalized to a control (transfection reagent only).

Example 3: MUC5B siRNA duplexes are active in vitro in the presence of cytokine IL6

[000447] This examples demonstrates that an siRNA oligonucleotide is effective in reducing MUC5B mRNA levels in the presence of the proinflammatory cytokine IL6, which induces expression of the MUC5B gene.

[000448] In order to determine the efficacy of the siRNA duplexes generated in Example 1 in the presence of IL6, siRNA #4 knockdown of MUC5B gene expression was analyzed. A549 (lung cancer) cells were seeded at a density of 12,500 cells/well in a 96 well plate. siRNA oligonucleotide #4, ranging from 0.312 nM to 12.5 nM, was added to the cells in the presence of transfection reagent (Dharmafect) to transfect the cells. IL6 was added to the cells in a concentration of 0, 50 or 100 ng/ml.

[000449] After 48 hours, the cells were collected and lysed. Reverse transcriptase quantitative polymerase chain reaction (RTqPCR) was performed in duplicate or triplicate reactions per sample to measure MUC5B gene expression. As shown in FIG. 3, in the presence of IL6, transfection of siRNA #4 resulted in a dose dependent decrease in MUC5B mRNA. At a concentration of 12.5 nM, siRNA #4 reduced MUC5B mRNA levels by -80% relative to an untreated control.

Example 4: MUC5B siRNA duplexes are active in vivo

[000450] This example demonstrates successful delivery of an siRNA oligonucleotide to the lungs of mice in vivo using 3-component or 4-component lipid nanoparticles (LNP) as delivery vehicles. [000451] LNPs were prepared using either Process A or Process B described above using 3 or 4 lipid components: a cationic lipid, a non-cationic lipid (DOPE), optionally cholesterol, and a PEG-modified lipid (DMG-PEG2K). The cationic lipid component was varied to identify LNPs that were effective in delivering siRNAs to the lung. The ability of MUC5B siRNAs encapsulated in various LNPs to knockdown MUC5B gene expression in the lung was examined in vivo in wild-type Balb/c and C57BL/6 mice via oro-pharyngeal aspiration. Mice were dosed at 8 μg or 15 μg siRNA oligonucleotide #3 per animal in a first lipid screen (FIG. 4) and 10 μg siRNA oligonucleotide #3 per animal in a second lipid screen (FIG. 5). The animals were terminated 72 hours following dosing with the siRNA. MUC5B mRNA expression was measured by RT-qPCR and was normalized to the housekeeping genes GAPDH or GUSB and PPIB. The results in FIGs. 4 and 5 are shown as relative fold change in MUC5B mRNA levels (normalized to MUC5B mRNA in saline treated animals).

[000452] As shown in FIGs. 4 and 5, LNPs encapsulating siRNA #3 knocked down MUC5B mRNA expression at 72 hours post administration. Up to 75% knockdown of MUC5B mRNA was observed with TBL-0346 (cKK-E12) and TBL-0279 (HGT4002). The tested lipids were generally well tolerated. Less than 10% loss in body weight was observed with the vast majority of the lipids tested.

[000453] To measure MUC5B protein expression at 72 hours post administration, western blot was performed with lung samples obtained from groups of three mice treated with saline, TBL-0346 (cKK-E12) and TBL-0246 (TL1-10D-DMA), respectively. Decrease in MUC5B protein was observed in siRNA treated lung samples (FIG. 6). Further reduction in MUC5B protein is expected at later timepoints.

Example 5: MUC5B siRNA duplexes show dose-dependent knockdown of MUC5B

[000454] This example demonstrates that the knockdown of MUC5B gene expression is dose-dependent and that the dose of administered siRNA oligonucleotide corresponds to the amount of antisense strand detected in the lung.

[000455] The effect of various doses of siRNA on MUC5B knockdown was examined in vivo in C57BL/6 mice. Three different LNPs were selected for testing on the basis that they were found to be well-tolerated and effective at delivering siRNA to the lungs of mice in the experiments described in Example 4. Mice were dosed with siRNA oligonucleotide #3 at 8 μg or 15 μg (TBL-0346) and at 5 μg, 10 μg or 20 μg (TBL-0246 and TBL-0279) per animal via oro-pharyngeal aspiration. The animals were terminated 72 hours following administration. MUC5B mRNA expression was measured by RT-qPCR and normalized to the housekeeping genes GAPDH and PPIB. The results are summarized in FIGs. 7 and 8, which show the relative fold change in MUC5B mRNA levels (normalized to MUC5B mRNA in saline treated animals).

[000456] A dose-dependent knockdown in MUC5B mRNA expression was observed. A -75% knockdown was observed in animals treated with TBL-0346 (cKK-E12) and TBL-0279 (HGT4002) (FIG. 7A). Measurement of MUC5B protein expression by western blot showed a corresponding reduction in protein expression. Typically, a higher reduction in MUC5B protein was observed at the 20 μg dose as compared with 5 μg or 10 μg dose (FIG. 7B).

[000457] The amount of siRNA, specifically the antisense strand of the siRNA oligonucleotide #3, was measured by a stem- loop based qPCR method. The amount of antisense strand detected in the lungs showed a correlation with the dose of siRNA oligonucleotide administered (FIG. 8 A) and with the observed MUC5B knockdown (FIG. 8B).

Example 6: MUC5B siRNA duplexes provide sustainedknockdown of MUC5B

[000458] This example demonstrates sustained knockdown of MUC5B gene expression over a period of 72 hours. To this end, a time-course pharmacodynamics (PD) study was performed in mouse models to assess the ability of MUC5B siRNA to knockdown MUC5B mRNA over a defined time course. The data obtained from these study showed that MUC5B siRNA knocks down MUC5B mRNA over the studied time periods of 24 hours, 48 hours, and 72 hours.

[000459] The effect of exposure time of siRNA on MUC5B knockdown was examined in vivo in C57BL/6 mice. Two different siRNAs - siRNA#3 and siRNA#4 were selected for testing in this example. LNPs formulation TBL-0346 encapsulating either siRNA#3 or siRNA#4 were orally aspirated to the lung of the mice. The mice were terminated 24, 48, and 72 hrs post-administation of the LNPs formulations, and their lungs were harvested. MUC5B mRNA expression levels were determined using qPCR. MUC5B expression was normalized to housekeeping genes GUSB and HPRT. MUC5B mRNA expressions in the left lung of mice upon oral aspirations of lipid nanoparticles (LNPs) formulation were determined. The fold change in MUC5B mRNA levels in treatment groups is shown relative to the saline control group 72 hrs post-administrations (FIG. 9).

[000460] Significant MUC5B knockdown was observed at all time points with siRNAs formulated with TBL-0346. About 60% MUC5B knockdown was observed with siRNA#3 (RN-15838/48) while about 80% MUC5B knockdown was observed with siRNA#4 (RN- 15839/49) at 72 hr time point (FIG. 9).

Claims

1. A compound comprising an oligonucleotide comprising an antisense strand consisting of 15-30 linked nucleosides, wherein the nucleobase sequence of the antisense strand has at least 12 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-6.

2. The compound of claim 1, wherein the nucleobase sequence of the antisense strand has at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or 30 contiguous nucleobases that are complementary to an equal length portion of any one of SEQ ID NOs: 1-6.

3. The compound of claim 1 or claim 2, wherein the nucleobase sequence of the antisense strand is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, or at least 94% complementary to an equal length portion of any one of SEQ ID NOs: 1-6, as measured over the entirety of the antisense strand.

4. The compound of any one of claims 1-3, wherein the nucleobase sequence of the antisense strand is complementary to an equal length portion of SEQ ID NO: 1.

5. The compound of any one of claims 1-4, wherein the oligonucleotide further comprises a sense strand, wherein the antisense strand and the sense strand are complementary to each other and form a duplex region.

6. The compound of claim 5, wherein the sense strand has a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or 19 contiguous nucleobases of SEQ ID NO: 7, and the antisense strand has a nucleobase sequence comprising at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or 19 contiguous nucleobases of SEQ ID NO: 8.

7. The compound of claim 5, wherein the sense strand has the nucleobase sequence of SEQ ID NO: 7, and the antisense strand has the nucleobase sequence of SEQ ID NO: 8

8. The compound of any one of claims 1-6, wherein the antisense strand and/or the sense strand consists of 15-30 linked nucleosides, 15-25 linked nucleosides, 15-20 linked nucleosides, or 18-20 linked nucleosides.

9. The compound of any one of claims 1-6, wherein the antisense strand and the sense strand consist of 19 linked nucleosides.

10. The compound of any one of claims 5-6, wherein the duplex region is 15-30 linked nucleosides, 15-25 linked nucleosides, 15-20 linked nucleosides, or 18-20 linked nucleosides in length.

11. The compound of any one of claims 5-6, wherein duplex region is 19 linked nucleosides in length.

12. The compound of any one of claims 1-11, wherein the oligonucleotide further comprises a single-stranded overhang on the sense and/or antisense strand either 1 or 2 nucleotides in length.

13. The compound of any one of claims 5-12, wherein the sense strand and the antisense strand have a 3’ single- stranded overhang comprising two deoxythymidines, optionally wherein the internucleoside linkages of both of the single- stranded overhangs are phosphothioester internucleoside linkages.

14. The compound of any one of claims 5-13, wherein the nucleotide at the 3’ end of the sense strand is adenine, and the nucleoside at the 5’ end of the antisense strand is uracil.

15. The compound of any one of claims 1-14, wherein the oligonucleotide is a modified oligonucleotide.

16. The compound of claim 15, wherein the modified oligonucleotide comprises at least one nucleoside analogue, modified sugar, modified internucleoside linkage, or modified nucleobase.

17. The compound of claim 16, wherein the modified oligonucleotide comprises at least one modified sugar.

18. The compound of claim 17, wherein the at least one modified sugar is: a. a bicyclic sugar, preferably LNA, ENA or cEt; or b. a 2’-modified sugar comprising one of 2’-O-methyl, 2’-F, 2’-O-methylethyl, and 2’- O-methoxyethyl.

19. The compound of claim 17, wherein the modified oligonucleotide comprises at least one 2’-O-methyl modified sugar and at least one 2’-F modified sugar.

20. The compound of claim 19, wherein each nucleoside of the antisense strand and each nucleoside of the sense strand comprises either a 2’-F or a 2'-O-methyl modified sugar.

21. The compound of claim 16, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage, optionally wherein the at least one modified internucleoside linkage is a phosphothioester internucleoside linkage.

22. The compound of claim 21, wherein the modified oligonucleotide comprises two, three, or four modified internucleoside linkages, optionally wherein each of the modified internucleoside linkages is a phosphothioester internucleoside linkage.

23. The compound of claim 22, wherein the antisense strand and/or the sense strand of the modified oligonucleotide comprise two phosphothioester internucleoside linkages at their 5’ end.

24. A pharmaceutical composition comprising i) a compound of any of claims 1-23 and ii) a lipid nanoparticle.

25. The pharmaceutical composition of claim 24, wherein the compound is encapsulated in the lipid nanoparticle.

26. The pharmaceutical composition of claim 24 or claim 25, wherein the lipid nanoparticle comprises one or more of a cationic lipid, a non-cationic lipid, a cholesterol- based lipid, a PEG-modified lipid, an amphiphilic block copolymer and/or a polymer, or a combination thereof.

27. The pharmaceutical composition of claim 26, wherein the lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, one or more cholesterol-based lipids, and one or more PEG-modified lipids.

28. The pharmaceutical composition of claim 26, wherein the lipid nanoparticle comprises one or more cationic lipids, one or more non-cationic lipids, and one or more PEG- modified lipids.

29. The pharmaceutical composition of any one of claims 24-28, wherein the lipid nanoparticle includes a combination selected from cKK-E12, DOPE, cholesterol and DMG- PEG2K; C 12-200, DOPE, cholesterol and DMG-PEG2K; HGT4003, DOPE, cholesterol and DMG-PEG2K; ICE, DOPE, cholesterol and DMG-PEG2K; HGT4001, DOPE, cholesterol and DMG-PEG2K; HGT4002, DOPE, cholesterol and DMG-PEG2K; TL1-01D-DMA, DOPE, cholesterol and DMG-PEG2K; TL1-04D-DMA, DOPE, cholesterol and DMG- PEG2K; TL1-08D-DMA, DOPE, cholesterol and DMG-PEG2K; TL1-10D-DMA, DOPE, cholesterol and DMG-PEG2K; ICE, DOPE and DMG-PEG2K; HGT4001, DOPE and DMG- PEG2K; or HGT4002, DOPE and DMG-PEG2K.

30. The pharmaceutical composition of any one of claims 24-28, wherein: a. the lipid nanoparticle comprises a cationic lipid selected from DOTAP (l,2-dioleyl-3- trimethylammonium propane), DODAP (l,2-dioleyl-3-dimethylammonium propane), DOTMA (N-[l-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride), DLinKC2DMA, DLin-KC2-DM, C12-200, cKK-E12 (3,6-bi s(4-(bis(2- hydroxydodecyl)amino)butyl)piperazine-2, 5 -dione), HGT5000, HGT5001 , HGT4003, ICE, HGT4001, HGT4002, TL1-01D-DMA, TL1-04D-DMA, TL1-08D-DMA, TL1-10D-DMA, OF-02, and combinations thereof; b. the lipid nanoparticle comprises a non-cationic lipid selected from DSPC (1,2- distearoyl-sn-glycero-3-phosphocholine), DPPC (l,2-dipalmitoyl-sn-glycero-3- phosphocholine), DOPE (l,2-dioleyl-sn-glycero-3-phosphoethanolamine), DEPE 1,2- dierucoyl-sn-glycero-3-phosphoethanolamine, DOPC (1 ,2-diolcyl-sn-glycero-3- phosphotidylcholine), DPPE (l,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine), DMPE (l,2-dimyristoyl-sn-glycero-3-phosphoethanolamine), DOPG (l,2-dioleoyl-sn-glycero-3- phospho-(l'-rac-glycerol)), and combinations thereof; c. the lipid nanoparticle comprises a cholesterol-based lipid selected from DC-Choi (N,N-dimethyl-N-ethylcarboxamidocholesterol), l,4-bis(3-N-oleylamino-propyl)piperazine, or imidazole cholesterol ester (ICE); and/or d. the lipid nanoparticle comprises a PEG-modified lipid selected from PEGylated cholesterol and PEG-2K.

31. The pharmaceutical composition of any one of claims 24-30, wherein cationic lipids constitute about 30-60% of the lipid nanoparticle by molar ratio, preferably about 35-40%.

32. The pharmaceutical composition of any one of claims 24-31, wherein the ratio of cationic lipid(s) to non-cationic lipid(s) to cholesterol-based lipid(s) to PEG-modified lipid(s) is approximately 30-60:25-35:20-30: l-15by molar ratio or wherein the ratio of cationic lipid(s) to non-cationic lipid(s) to PEG-modified lipid(s) is approximately 55-65:30-40:1-15.

33. The pharmaceutical composition of any one of claims 24-32, wherein the lipid nanoparticle has an average size of less than 150 nm.

34. The pharmaceutical composition of claims 33, wherein the lipid nanoparticle has an average size of about 50-70 nm, preferably about 55-65 nm.

35. A method of delivering the compound of any one of claims 1-23 or the pharmaceutical composition of any one of claims 24-34 to lung cells of a subject in need thereof, wherein the method comprises administering the compound or pharmaceutical composition to the subject via pulmonary delivery.

36. The method of claim 35, wherein the subject is suffering or at risk of suffering from a lung disease or disorder.

37. The method of claim 36, wherein the lung disease or disease or disorder is associated with overexpression of MUC5B, optionally wherein overexpression of MUC5B is associated with reduced mucociliary function, reduced alveolar repair, and/or increased lung fibrosis.

38. The method of claim 37, wherein the lung disease or disease or disorder is selected from idiopathic pulmonary fibrosis (IPF), chronic rhinosinusitis (CRS), chronic obstructive pulmonary disease (COPD), diffuse panbronchiolitis (DPB), asthma, and cystic fibrosis (CF).

39. The method of any one of claims 35-38, wherein pulmonary delivery is via nebulization of the compound using a nebulizer, preferably a mesh nebulizer.

40. The method of claims 39, wherein the nebulizer delivers the compound to lung cells in the form of an aerosol.

41. The method of any one of claims 35-40, wherein the lung cells are lung epithelial cells.

42. The compound of any one of claims 1-23 or the pharmaceutical composition of any one of claims 24-34 for use in a method of treating, preventing, ameliorating, or slowing progression of a lung disease or disorder in a subject.

43. The pharmaceutical composition for use according to claim 42, wherein the lung disease or disorder is associated with overexpression of MUC5B, optionally wherein overexpression of MUC5B is associated with reduced mucociliary function, reduced alveolar repair, and/or increased lung fibrosis.

44. The pharmaceutical composition for use according to claim 43, wherein the lung disease or disorder is selected from idiopathic pulmonary fibrosis (IPF), chronic rhinosinusitis (CRS), chronic obstructive pulmonary disease (COPD), diffuse panbronchiolitis (DPB), asthma, and cystic fibrosis (CF).

45. The pharmaceutical composition for use according to any one of claims 42-44, wherein the pharmaceutical composition is administered via pulmonary delivery.

46. The pharmaceutical composition for use according to claim 45, wherein pulmonary delivery is via nebulization of the compound using a nebulizer, preferably a mesh nebulizer.

47. A kit comprising a container housing a compound of any one of claims 1-23 or a pharmaceutical compositions of any one of claims 24-34.

48. A nebulizing apparatus comprising a compound of any one of claim 1-23 or a pharmaceutical composition of any one of claims 24-34.

49. The nebulizing apparatus according to claim 48, wherein the nebulizer is a mesh nebulizer.