RNAi agents for inhibiting expression of matrix metalloproteinase 7 (MMP7), compositions thereof, and methods of use

Abstract

RNAi agents, compositions comprising RNAi agents, and methods for suppressing matrix metallopeptidase 7 (MMP7) gene are described. The MMP7 RNAi agents and RNAi agent conjugates disclosed herein suppress the expression of MMP7 gene. Pharmaceutical compositions comprising one or more MMP7 RNAi agents, optionally with one or more additional therapeutic agents, are also described. In vivo delivery of the described MMP7 RNAi agents to lung cells can result in suppression of MMP7 gene expression and provide therapeutic benefit to subjects, including human subjects, in the treatment of various diseases, including pulmonary inflammatory diseases such as idiopathic pulmonary fibrosis (IPF).

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 63 / 270,849, filed October 22, 2021, U.S. Provisional Patent Application No. 63 / 308,289, filed February 9, 2022, and U.S. Provisional Patent Application No. 63 / 345,654, filed May 25, 2022, the contents of each of which are incorporated by reference in their entirety herein.

[0002] Sequence Listing This application contains a Sequence Listing which has been submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy is named 30673_WO_SequenceListing.xml and is 3,575kb in size.

[0003] The present disclosure relates to RNA interference (RNAi) agents, e.g., double-stranded RNAi agents, compositions comprising MMP7 RNAi agents, and methods of their use, for inhibiting matrix metalloproteinase 7 ("MMP7" or "matrilysin") gene expression. [Background technology]

[0004] Matrix metalloproteinase 7 ("MMP7" or "matrilysin") is the smallest (28 kDa) member of the metalloproteinase (MMP) family of 24 related secreted zinc-dependent endopeptidases with diverse substrates and functions and can degrade components of the extracellular matrix (e.g., elastin, proteoglycans, type IV collagen, fibronectin, entactin / nidogen, and proteoglycan core proteins), in addition to cleaving and regulating active non-extracellular matrix substrates such as cytokines (Fujishima, Shiomi et al., Arch Pathol Lab Med 134(8): 1136-1142 (2010); Craig, Zhang et al., Am JRespir Cell Mol Biol 53(5): 585-600 (2015)). Due to their functional roles in regulating extracellular matrix remodeling and cytokine signaling, members of the MMP family have been linked to common pathogenic mechanisms contributing to cancer, chronic inflammation, and fibrosis. However, despite being attractive as drug targets, the development of highly selective small molecule MMP inhibitors has been challenging due to the shared structural similarity of the zinc-dependent catalytic domains among family members (Vandenbroucke and Libert, Nat Rev Drug Discov 13(12): 904-927 (2014); Fields, Cells 8(9) (2019)).

[0005] MMP7 is constitutively expressed and secreted by epithelial cells throughout the body, including skin and lung, as well as glandular epithelium of the liver, intestine, pancreas, salivary glands, and genital tract, and is involved in epithelial repair (Pilcher, Wang et al., Ann NY Acad Set 878:12-24 (1999)). Increased MMP7 expression has been shown to be associated with increased pulmonary function in the lung (Rosas, Richards et al., PLoSMed 5(4): e93 (2008)), liver (Hung, Chang et al., Hepatology 50(4): 1184-1193 (2009);Roeb, Matrix Biol 68-69: 463-473 (2018);Nomden, Beljaars et al., Front Med (Lausanne) 7:617261 (2020);Irvine, Okano et al., Sci Rep 11(1):2858 (2021)), and kidney (Ke, Fan et al., Front Physiol 8:21 (2017);Zhang, Ren et al., Kidney Blood Press Res 42(3): 541-552 (2017);Tan, Li et al., JCI Insight 4(24) (2019)). MMP7 enzyme levels are associated with pathogenic fibrosis through multiple potential mechanisms including promoting epithelial-mesenchymal transition (EMT), extracellular matrix degradation, abnormal matrix repair, and tissue remodeling. MMP7 promotes fibrosis by cleaving E-cadherin to activate epithelial cells and proteolytically activating heparin-binding epidermal growth factor precursor (pro-HB-EGF) to release active HB-EGF, promoting abnormal epithelial migration and human lung fibroblast proliferation (Zhang, Rice et al., Am J Respir Cell Mol Biol 24(2): 123-131 (2001);McGuire, Li et al., Am J Pathol 162(6): 1831-1843 (2003)).MMP7 is also known to promote fibroblast survival and resistance to apoptosis through cleavage of the osteopontin and mFasL pathways (Agnihotri, Crawford et al., J Biol Chem 276(30):28261-28267 (2001); Mummler, Burgy et al., FASEB J 32(2):703-716 (2018); Nareznoi, Konikov-Rozenman et al., Cells 9(2) (2020)).

[0006] One specific type of fibrosis is idiopathic pulmonary fibrosis (IPF), a chronic lung disease that is often fatal and has a relatively unpredictable clinical course and rate of disease progression (Id.). In patients with IPF, MMP7 expression is known to be increased in peripheral blood, bronchoalveolar lavage fluid, and lung tissue (Zuo, Kaminski et al., Proc Natl Acad Sci USA 99(9): 6292-6297 (2002)). Serum MMP7 expression has been well-validated as a serum biomarker for IPF and correlates with the severity and progression of IPF (Song, Do et al., Chest 143(5): 1422-1429 (2013);Tzouvelekis, Herazo-Maya et al., Respirology 22(3):486-493 (2017)). Consistent with its known mechanisms, MMP7 regulates multiple pathways that contribute to abnormal epithelial cell, fibroblast, and immune cell function in IPF. Importantly, MMP7 knockout mice are protected from bleomycin-mediated lung injury (the standard rodent model of IPF) and exhibit reduced lung inflammation, fibrosis, and mortality, thus suggesting that MMP7 plays a role in IPF (Craig, Zhang et al., Am J Respir Cell Mol Biol 53(5):585-600 (2015)).

[0007] Genome-wide association studies (GWAS) have shown that the gain-of-function MMP7 gene variant rs11568818AA is associated with IPF risk (Richards, Park et al., Am J Physiol Lung Cell Mol Physiol 302(8):L746-754 (2012)). Furthermore, such GWAS studies have described that MMP7 gene variants are associated with multiple cancer diseases and sclerosis (Moreno-Ortiz, Gutierrez-Angulo et al., Genet Mol Res 13(2):3537-3544 (2014);Fu, Chien et al., Anticancer Res 40(2):695-702 (2020)). Summary of the Invention

[0008] There is a need for novel RNA interference (RNAi) agents (also called RNAi agents, RNAi triggers, or triggers), e.g., double-stranded RNAi agents, that can selectively and efficiently suppress expression of the MMP7 gene, including for use as therapeutic agents or medicines. Additionally, there is a need for novel MMP7-specific RNAi agent compositions for the treatment of diseases or disorders associated with pathological inflammation (such as IPF) and / or disorders that may be at least partially mediated by a decrease in MMP7 gene expression.

[0009] The nucleotide sequence and chemical modifications of the MMP7 RNAi agents disclosed herein, and their combination with specific targeting ligands suitable for selectively and efficiently delivering the MMP7 RNAi agents to relevant lung cells in vivo, are different from those previously disclosed or known in the art. The MMP7 RNAi agents disclosed herein highly potently and efficiently suppress the expression of the MMP7 gene.

[0010] In general, the present disclosure features MMP7 gene-specific RNAi agents, compositions comprising MMP7 RNAi agents, and methods of suppressing expression of the MMP7 gene in vitro and / or in vivo using the MMP7 RNAi agents and compositions comprising the MMP7 RNAi agents described herein. The MMP7 RNAi agents described herein can selectively and efficiently reduce expression of the MMP7 gene, thereby reducing the abundance of MMP7, which is believed to be profibrotic, possibly through multiple mechanisms including cleaving E-cadherin to activate epithelial cells, proteolytically activating heparin-binding epidermal growth factor precursor (pro-HB-EGF) to release active HB-EGF, promoting aberrant epithelial migration and human lung fibroblast proliferation, and cleaving and activating other profibrotic substrates such as osteopontin and membrane-bound Fas ligand (mFasL).

[0011] The described MMP7 RNAi agents can be used in methods for therapeutic treatment (including preventative or prophylactic treatment) of conditions and diseases including, but not limited to, idiopathic pulmonary fibrosis (IPF), asthma, various other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), infectious diseases (e.g., SARS-COV-2), acute lung injury (e.g., acute respiratory distress syndrome (ARDS)), pulmonary hypertension, various cancers, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), fatty liver disease, biliary atresia, and chronic kidney disease (CKD).

[0012] In one aspect, the disclosure features an RNAi agent for suppressing expression of the MMP7 gene, the RNAi agent including a sense strand (also referred to as a passenger strand) and an antisense strand (also referred to as a guide strand). The sense strand and the antisense strand can be partially, substantially, or fully complementary to each other. The length of each of the sense strands of the RNAi agents described herein can be 12-49 nucleotides in length. The length of each of the antisense strands of the RNAi agents described herein can be 18-30 nucleotides in length. In some embodiments, the sense strand and the antisense strand are independently 18-26 nucleotides in length. The sense strand and the antisense strand can be either the same length or different lengths. In some embodiments, the sense strand and the antisense strand are independently 21-26 nucleotides in length. In some embodiments, the sense strand and the antisense strand are independently 21-24 nucleotides in length. In some embodiments, the sense strand and the antisense strand are both 21 nucleotides in length. In some embodiments, the antisense strands are independently 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the sense strands are independently 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides in length. In vivo and / or in vitro, the RNAi agents described herein, when delivered to a cell expressing MMP7, such as a lung cell, inhibit the expression of one or more MMP7 gene variants.

[0013] The MMP7 RNAi agents disclosed herein target the human MMP7 gene (see, e.g., SEQ ID NO: 1). In some embodiments, the MMP7 RNAi agents disclosed herein target a portion of the MMP7 gene having any of the sequences disclosed in Table 1.

[0014] In another aspect, the present disclosure features compositions, including pharmaceutical compositions comprising one or more of the disclosed MMP7 RNAi agents, which can selectively and efficiently reduce the expression of the MMP7 gene. Compositions comprising one or more of the MMP7 RNAi agents described herein can be administered to subjects, such as human or animal subjects, for the treatment (including prophylactic treatment or suppression) of conditions and diseases, including, but not limited to, various lung diseases, including idiopathic pulmonary fibrosis (IPF), asthma, various other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), infectious diseases (e.g., SARS-COV-2), acute lung injury (e.g., acute respiratory distress syndrome (ARDS)), pulmonary hypertension, various cancers, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), fatty liver disease, biliary atresia, and chronic kidney disease (CKD).

[0015] Examples of MMP7 RNAi agent sense and antisense strands that can be used in MMP7 RNAi agents are shown in Tables 3, 4, 5, and 6. Examples of MMP7 RNAi agent duplexes are shown in Tables 7A, 7B, 8, 9, and 10. Examples of 19 nucleotide core stretch sequences that may consist of or be included in the sense and antisense strands of certain MMP7 RNAi agents disclosed herein are shown in Table 2.

[0016] In another aspect, the present disclosure features a method for delivering MMP7 RNAi agent to epithelial cells of a subject, such as a mammal, in vivo.Furthermore, described herein is a composition for use in such a method.In some embodiments, disclosed herein is a method for delivering MMP7 RNAi agent to lung cells (epithelial cells, macrophages, smooth muscle, endothelial cells) in vivo to a subject.In some embodiments, the subject is a human subject.

[0017] The methods disclosed herein include administering one or more MMP7 RNAi agents to a subject, e.g., a human or animal subject, by any suitable means known in the art. The pharmaceutical compositions disclosed herein that include one or more MMP7 RNAi agents can be administered in a number of ways, depending on whether local or systemic treatment is desired. Administration can be, for example, but is not limited to, intravenous, intraarterial, subcutaneous, intraperitoneal, subdermal (e.g., via an implant device), and intrapleural. In some embodiments, the pharmaceutical compositions described herein are administered by inhalation (such as dry powder inhalation or aerosol inhalation), intranasal administration, intratracheal administration, or oropharyngeal aspiration administration.

[0018] In some embodiments, the MMP7 RNAi agents described herein inhibit expression of the MMP7 gene in pulmonary epithelium, preferably by administration by inhalation (e.g., by an inhaler device, such as a metered dose inhaler, or a nebulizer, such as a jet or vibrating mesh nebulizer, or a soft mist inhaler).

[0019] One or more MMP7 RNAi agents can be delivered to a target cell or tissue using any oligonucleotide delivery technique known in the art. In some embodiments, the MMP7 RNAi agent is delivered to a cell or tissue by covalently linking the RNAi agent to a targeting group. In some embodiments, the targeting group can include a cell receptor ligand, such as an integrin targeting ligand. Integrins are a family of transmembrane receptors that promote cell-extracellular matrix (ECM) adhesion. In particular, integrin alpha-v-beta-6 (αvβ6) is an epithelial-specific integrin known as a receptor for ECM proteins and TGF-β latency-associated peptide (LAP), and is expressed in a variety of cells and tissues. Integrin αvβ6 is known to be upregulated in injured lung epithelium. In some embodiments, the MMP7 RNAi agent described herein is linked to an integrin targeting ligand that has affinity for integrin αvβ6. As referred to herein, an "αvβ6 integrin targeting ligand" is a compound that has affinity for integrin αvβ6 and can be utilized as a ligand to facilitate targeting and delivery of an RNAi agent to which it binds to a desired cell and / or tissue (i.e., to a cell expressing integrin αvβ6). In some embodiments, multiple αvβ6 integrin targeting ligands or a cluster of αvβ6 integrin targeting ligands are linked to an MMP7 RNAi agent. In some embodiments, the MMP7 RNAi agent-αvβ6 integrin targeting ligand conjugate is selectively internalized by lung epithelial cells, either through receptor-mediated endocytosis or by other means.

[0020] Examples of targeting groups useful for delivering MMP7 RNAi agents, including αvβ6 integrin targeting ligands, are disclosed, for example, in International Patent Application Publication No. WO 2018 / 085415 and International Patent Application Publication No. WO 2019 / 089765, the contents of each of which are incorporated by reference in their entireties.

[0021] The targeting group can be linked to the 3' or 5' end of the sense strand or antisense strand of the MMP7 RNAi agent. In some embodiments, the targeting group is linked to the 3' or 5' end of the sense strand. In some embodiments, the targeting group is linked to the 5' end of the sense strand. In some embodiments, the targeting group is internally linked to a nucleotide of the sense strand and / or antisense strand of the RNAi agent. In some embodiments, one or more targeting ligands are internally linked to one or more nucleotides of the sense strand of the RNAi agent. In some embodiments, the targeting group is linked to the RNAi agent via a linker.

[0022] In another aspect, the disclosure features a composition that includes one or more MMP7 RNAi agents having a double-stranded structure as disclosed in Tables 7A, 7B, 8, 9, and 10.

[0023] The use of MMP7 RNAi agents provides methods for the curative (including preventative) treatment of diseases or disorders in which a reduction in MMP7 may provide a therapeutic benefit. The MMP7 RNAi agents disclosed herein can be used to treat various pulmonary diseases, including idiopathic pulmonary fibrosis (IPF), asthma, various other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), infectious diseases (e.g., SARS-COV-2), acute lung injury (e.g., acute respiratory distress syndrome (ARDS)), pulmonary hypertension, various cancers, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), fatty liver disease, biliary atresia, and chronic kidney disease (CKD). In some embodiments, the MMP7 RNAi agents disclosed herein can be used to treat pulmonary inflammatory diseases or conditions. The MMP7 RNAi agents can be used, for example, to treat IPF or other types of pulmonary fibrosis. Such methods of treatment include administration of the MMP7 RNAi agents to humans or animals in which a reduction in MMP7 levels is desired.

[0024] As used herein, the terms "oligonucleotide" and "polynucleotide" each independently mean a polymer of linked nucleosides, which may be modified or unmodified.

[0025] As used herein, an "RNAi agent" (also referred to as an "RNAi trigger") refers to a composition containing an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that can sequence-specifically reduce or suppress (e.g., reduce or suppress under appropriate conditions) translation of a messenger RNA (mRNA) transcript of a target mRNA. As used herein, an RNAi agent can act through the RNA interference mechanism (i.e., induce RNA interference through interaction with the RNA interference pathway machinery (RNA-induced silencing complex or RISC) of mammalian cells) or by any alternative mechanism(s) or pathway(s). Although it is believed that an RNAi agent acts primarily through the RNA interference mechanism as the term is used herein, the disclosed RNAi agents are not constrained or limited to any particular pathway or mechanism of action. The RNAi agent disclosed herein is composed of a sense strand and an antisense strand, and includes, but is not limited to, short (or small) interfering RNA (siRNA), double-stranded RNA (dsRNA), microRNA (miRNA), short hairpin RNA (shRNA), and dicer substrate. The antisense strand of the RNAi agent described herein is at least partially complementary to the target mRNA (i.e., MMP7 mRNA). The RNAi agent can include one or more modified nucleotides and / or one or more non-phosphodiester bonds.

[0026] As used herein, when referring to the expression of a given gene, the terms "silencing," "reducing," "suppressing," "downregulating," or "knockdown" mean that expression of the gene is reduced in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, as measured at the level of RNA transcribed from the gene or at the level of a polypeptide, protein, or protein subunit translated from the mRNA, when a cell, group of cells, tissue, organ, or subject is administered an RNAi agent as described herein, compared to another cell, group of cells, tissue, organ, or subject that has not received such administration.

[0027] As used herein, the terms "sequence" and "nucleotide sequence" mean a sequence or order of nucleic acid bases or nucleotides designated as a series of letters using standard nomenclature.

[0028] As used herein, "base", "nucleotide base" or "nucleobase" refers to a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, including the major purine bases adenine and guanine, and the major pyrimidine bases cytosine, thymine and uracil. Nucleobases can be further modified to include, but are not limited to, universal bases, hydrophobic bases, mixed bases, size-extended bases, and fluorinated bases (see, for example, Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008). The synthesis of such modified nucleobases (including phosphoramidite compounds that include modified nucleobases) is known in the art.

[0029] As used herein, and unless otherwise indicated, the term "complementary" when used to describe a first nucleobase or nucleotide sequence (e.g., an RNAi agent's sense strand or a targeting mRNA) in the context of a second nucleobase or nucleotide sequence (e.g., an RNAi agent's antisense strand or a single-stranded antisense oligonucleotide) refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize (form base-pair hydrogen bonds under mammalian physiological conditions (or otherwise suitable in vivo or in vitro conditions)) with an oligonucleotide comprising the second nucleotide sequence and form a double-stranded or double-helical structure under certain standard conditions. A person skilled in the art would be able to select the most suitable set of conditions for the hybridization test. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimetics, so long as at least the above hybridization requirements are met. Sequence identity or complementarity is independent of modifications. For example, a and Af, as defined herein, are complementary to U (or T) and identical to A for purposes of determining identity or complementarity.

[0030] As used herein, "perfectly complementary" or "fully complementary" means that in a hybridized pair of nucleic acid base or nucleotide sequence molecules, all (100%) of the bases of a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases of a contiguous sequence of a second oligonucleotide. The contiguous sequence may include all or a portion of the first or second nucleotide sequence.

[0031] As used herein, "partially complementary" means that in a hybridized pair of nucleic acid base or nucleotide sequence molecules, at least 70% (but not all) of the bases of a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases of a contiguous sequence of a second oligonucleotide. The contiguous sequence may include all or a portion of the first or second nucleotide sequence.

[0032] As used herein, "substantially complementary" means that in a hybridized pair of nucleic acid base or nucleotide sequence molecules, at least 85% (but not all) of the bases of a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases of a contiguous sequence of a second oligonucleotide. The contiguous sequence may include all or a portion of the first or second nucleotide sequence.

[0033] As used herein, the terms "complementary," "fully complementary," "partially complementary," and "substantially complementary" are used in reference to nucleobase or nucleotide matches between a sense strand and an antisense strand of an RNAi agent, or between an antisense strand of an RNAi agent and the sequence of an MMP7 mRNA.

[0034] As used herein, the term "substantially identical" or "substantially identical" as applied to a nucleic acid sequence means that a nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% or more sequence identity, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. The percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window. The percentage is calculated by determining the number of positions where the same type of nucleobase occurs in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity. The invention disclosed herein encompasses nucleotide sequences that are substantially identical to those disclosed herein.

[0035] As used herein, the terms "treat", "treatment" and the like refer to methods or steps taken to result in a reduction or alleviation of the number, severity, and / or frequency of one or more symptoms of a disease in a subject. As used herein, "treat" and "treatment" can include prevention, management, prophylactic treatment, and / or suppression or reduction in the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0036] As used herein, the phrase "introducing into cell" refers to delivering RNAi agent into cell so that RNAi agent can function. The phrase "functional delivery" refers to delivering RNAi agent into cell in such a way that RNAi agent can have expected biological activity, for example, sequence-specific suppression of gene expression.

[0037] Unless otherwise noted, symbols used herein [ka] The use of means that any group or groups according to the scope of the invention described herein can be linked thereto.

[0038] As used herein, the term "isomers" means compounds that have identical molecular formulae but differ in the nature or sequence of bonding of their atoms or the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are called "stereoisomers." Stereoisomers that are not mirror images of one another are called "diastereoisomers" and stereoisomers that are non-superimposable mirror images are called "enantiomers" or may also be called optical isomers. A carbon atom bonded to four non-identical substituents is called a "chiral center."

[0039] As used herein, unless otherwise specified in a structure having a particular configuration, for each structure in which asymmetric centers exist and thus give rise to enantiomers, diastereomers, or other stereoisomeric configurations, each structure disclosed herein is intended to represent all such possible isomers, including their optically pure and racemic forms. For example, the structures disclosed herein are intended to cover not only single stereoisomers, but also mixtures of diastereomers.

[0040] As used in the claims herein, the phrase "consisting of" excludes any element, step, or ingredient not specified in the claim. As used in the claims herein, the phrase "consisting essentially of" limits the scope of the claim to those materials or steps specified and that do not materially affect the basic and novel feature(s) of the claimed invention.

[0041] Those skilled in the art will readily understand and appreciate that the compounds and compositions disclosed herein may have certain atoms (e.g., N, O, or S atoms) in a protonated or deprotonated state depending on the environment in which the compound or composition is placed. Thus, as used herein, the structures disclosed herein contemplate that certain functional groups, such as OH, SH, or NH, may be protonated or deprotonated. The disclosures herein are intended to cover the disclosed compounds and compositions regardless of the state of protonation based on the environment (e.g., pH), as would be readily understood by those skilled in the art. Correspondingly, compounds described herein with labile protons or basic atoms should also be understood to represent salt forms of the corresponding compounds. The compounds described herein can be in the form of free acids, free bases, or salts. It should be understood that pharma-ceutically acceptable salts of the compounds described herein are within the scope of the present invention.

[0042] As used herein, when referring to a connection between two compounds or molecules, the term "linked" or "conjugated" means that the two compounds or molecules are joined by a covalent bond. Unless otherwise stated, the terms "linked" and "conjugated" as used herein can refer to a connection between a first compound and a second compound with or without an intervening atom or group of atoms.

[0043] As used herein, the term "including" is used herein to mean, and is used interchangeably with, the phrase "including but not limited to." The term "or" is used herein to mean, and is used interchangeably with, the term "and / or," unless context clearly dictates otherwise.

[0044] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Additionally, the materials, methods, and examples are illustrative only and are not intended to be limiting.

[0045] Other objects, features, aspects, and advantages of the present invention will become apparent from the following detailed description, the accompanying drawings, and the claims. [Brief description of the drawings]

[0046] [Figure 1] FIG. 1 depicts the chemical structure of a tridentate αvβ6 epithelial cell targeting ligand, referred to herein as Tri-SM6.1-αvβ6-(TA14). [Diagram 2] FIG. 2 depicts the chemical structure of the peptide αvβ6 epithelial cell targeting ligand, referred to herein as αvβ6-pep1. [Diagram 3] FIG. 3: MTT colorimetric assay for cell metabolic activity showing little cytotoxic effect of MMP7 RNAi agents, as described in Example 24. [Figure 4A]FIG. 4A is a schematic diagram of the sense and antisense strands of a modified MMP7 RNAi agent conjugate having the structure of AC001651 (see, e.g., Tables 8 and 10) and having a tridentate αvβ6 epithelial cell targeting ligand linked to the 5′ end of the sense strand. The following abbreviations are used in Figures 4A-4F: a, c, g, and u are 2'-O-methyl modified nucleotides; Af, Cf, Gf, and Uf are 2'-fluoro modified nucleotides; o is a phosphodiester bond; s is a phosphorothioate bond; invAb is an inverted abasic residue (see, e.g., Table 11); cPrpu is a 5'-phosphonate cyclopropyl-2'-O-methyluridine modified nucleotide (see, e.g., Table 11); cPrpa is a 5'-phosphonate cyclopropyl-2'-O-methyladenosine modified nucleotide (see, e.g., Table 11); Tri-SM6.1-αvβ6-(TA14) is a tridentate αvβ6 epithelial cell targeting ligand having the structure shown in Figure 1; and (TriAlk14) is a linking group shown in Table 11, suitable for subsequent conjugation to a targeting ligand (see also Example 1 herein). [Figure 4B] FIG. 4B is a schematic diagram of the sense and antisense strands of a modified MMP7 RNAi agent conjugate having the structure of AC001514 (see, e.g., Tables 8 and 10) and having a tridentate αvβ6 epithelial cell targeting ligand linked to the 5′ end of the sense strand. [Figure 4C] FIG. 4C is a schematic diagram of the sense and antisense strands of a modified MMP7 RNAi agent conjugate having the structure of AC002023 (see, e.g., Tables 8 and 10) and having a tridentate αvβ6 epithelial cell targeting ligand linked to the 5′ end of the sense strand. [Figure 4D] FIG. 4D is a schematic of the sense and antisense strands of a modified MMP7 RNAi agent duplex having the structure of AD09887 (see, eg, Tables 8 and 10) and having a (TriAlk14) linker at the 5′ end of the sense strand. [Figure 4E]FIG. 4E is a schematic of the sense and antisense strands of a modified MMP7 RNAi agent duplex having the structure of AD09667 (see, eg, Tables 8 and 10) and having a (TriAlk14) linker at the 5′ end of the sense strand. [Figure 4F] FIG. 4F is a schematic of the sense and antisense strands of a modified MMP7 RNAi agent duplex having the structure of AD10441 (see, eg, Tables 8 and 10) and having a (TriAlk14) linker at the 5′ end of the sense strand. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] RNAi agents Described herein are RNAi agents (herein referred to as MMP7 RNAi agents or MMP7 RNAi triggers) for suppressing expression of the MMP7 gene. Each of the MMP7 RNAi agents disclosed herein includes a sense strand and an antisense strand. The sense strand can be 12-49 nucleotides in length. In some embodiments, the sense strand is 12-49 nucleotides in length. The antisense strand can be 18-49 nucleotides in length. The sense strand and the antisense strand can be the same length, or they can be different lengths. In some embodiments, the sense strand and the antisense strand are each independently 18-27 nucleotides in length. In some embodiments, both the sense strand and the antisense strand are each independently 21-26 nucleotides in length. In some embodiments, the sense strand and the antisense strand are each independently 21-24 nucleotides in length. In some embodiments, the sense strand and the antisense strand are each independently 19-21 nucleotides in length. In some embodiments, the sense strand is about 19 nucleotides in length, while the antisense strand is about 21 nucleotides in length. In some embodiments, the sense strand is about 21 nucleotides long, while the antisense strand is about 23 nucleotides long. In some embodiments, the sense strand is 23 nucleotides long, and the antisense strand is 21 nucleotides long. In some embodiments, both the sense strand and the antisense strand are each 21 nucleotides long. In some embodiments, the sense strand of the RNAi agent is each independently 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, or 49 nucleotides long. In some embodiments, the antisense strands of the RNAi agent are each independently 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length. In some embodiments, the RNAi agent is double-stranded and the length of the duplex is about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 nucleotides in length.In some embodiments, the RNAi agent is double-stranded, and the length of the duplex is 19, 20, 21, 22, or 23 nucleotides in length.

[0048] Exemplary nucleotide sequences used to form MMP7 RNAi agents are shown in Tables 2, 3, 4, 5, 6, and 10. Exemplary RNAi agent duplexes comprising the sense and antisense strand sequences of Tables 2, 3, 4, 5, 6 are shown in Tables 7A, 7B, 8, 9, and 10.

[0049] In some embodiments, the region of complete, substantial, or partial complementarity between the sense strand and the antisense strand is 16 to 26 (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or 26) nucleotides in length and occurs at or near the 5' end of the antisense strand (e.g., this region can be separated from the 5' end of the antisense strand by 0, 1, 2, 3, or 4 nucleotides that are not completely, substantially, or partially complementary).

[0050] The sense strand of the MMP7 RNAi agent described herein comprises at least 12 consecutive nucleotides having at least 85% identity with the core stretch sequence of the same nucleotide number of MMP7 mRNA (also referred to herein as "core stretch" or "core sequence"). In some embodiments, the core stretch sequence of the sense strand is 100% (fully) complementary or at least about 85% (substantially) complementary with the core stretch sequence of the antisense strand, and thus the core stretch sequence of the sense strand is typically fully identical or at least about 85% identical to the nucleotide sequence of the same length present in the MMP7 mRNA target (e.g., sometimes referred to as the target sequence). In some embodiments, the core stretch of the sense strand is 16, 17, 18, 19, 20, 21, 22, or 23 nucleotides in length. In some embodiments, the core stretch of the sense strand is 17 nucleotides in length. In some embodiments, the core stretch of the sense strand is 19 nucleotides in length. In some embodiments, the core stretch of the sense strand is 21 nucleotides in length.

[0051] The antisense strand of the MMP7 RNAi agent described herein comprises at least 15 consecutive nucleotides having at least 85% complementarity with the core stretch of the same number of nucleotides of MMP7 mRNA and the core stretch of the same number of nucleotides of the corresponding sense strand. In some embodiments, the core stretch of the antisense strand is 100% (fully) complementary or at least about 85% (substantially) complementary with the same length of nucleotide sequence present in the MMP7 mRNA target (e.g., target sequence). In some embodiments, the core stretch of the antisense strand is 17, 18, 19, 20, 21, 22, or 23 nucleotides long. In some embodiments, the core stretch of the antisense strand is 19 nucleotides long. In some embodiments, the core stretch of the antisense strand is 17 nucleotides long. The core stretch sequence of the sense strand may be the same length as the corresponding antisense core sequence or may be different in length.

[0052] The sense strand and the antisense strand of the MMP7 RNAi agent anneal to form a duplex. The sense strand and the antisense strand of the MMP7 RNAi agent can be partially, substantially, or completely complementary to each other. Within the complementary duplex region, the core stretch sequence of the sense strand is at least 85% complementary or 100% complementary to the antisense core stretch sequence. In some embodiments, the core stretch sequence of the sense strand contains a sequence of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that are at least 85% or 100% complementary to a corresponding 16, 17, 18, 19, 20, 21, 22, or 23 nucleotide sequence of the core stretch sequence of the antisense strand (i.e. the sense and antisense core stretch sequences of the MMP7 RNAi agent have a region of at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 nucleotides that are at least 85% base paired or 100% base paired).

[0053] In some embodiments, the antisense strand of an MMP7 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 2 or Table 3 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of an MMP7 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 10 by 0, 1, 2, or 3 nucleotides.

[0054] In some embodiments, the sense strand and / or the antisense strand can optionally and independently contain an additional 1, 2, 3, 4, 5, or 6 nucleotides (extensions) at the 3' end, the 5' end, or both the 3' end and the 5' end of the core stretch sequence. If an additional nucleotide of the antisense strand is present, it may or may not be complementary to the corresponding sequence in the MMP7 mRNA. If an additional nucleotide of the sense strand is present, it may or may not be identical to the corresponding sequence in the MMP7 mRNA. If an additional nucleotide of the antisense strand is present, it may or may not be complementary to the corresponding additional nucleotide of the sense strand, if present.

[0055] As used herein, an extension comprises 1, 2, 3, 4, 5, or 6 nucleotides at the 5' and / or 3' end of the core stretch sequence of the sense strand and / or the core stretch sequence of the antisense strand. The extension nucleotides of the sense strand may or may not be complementary to the corresponding nucleotides of the antisense strand (either the core stretch sequence nucleotides or the extension nucleotides). Conversely, the extension nucleotides of the antisense strand may or may not be complementary to the corresponding nucleotides of the sense strand (either the core stretch sequence nucleotides or the extension nucleotides). In some embodiments, both the sense and antisense strands of the RNAi agent contain 3' and 5' extensions. In some embodiments, one or more 3' extension nucleotides of one strand are base-paired with one or more 5' extension nucleotides of the other strand. In other embodiments, one or more 3' extension nucleotides of one strand are not base-paired with one or more 5' extension nucleotides of the other strand. In some embodiments, the MMP7 RNAi agent has an antisense strand with a 3' extension and a sense strand with a 5' extension. In some embodiments, the extension nucleotide(s) is / are unpaired and forms an overhang. As used herein, "overhang" refers to a stretch of one or more unpaired nucleotides located at either end of the sense strand or the antisense strand that does not form part of the hybridized or double-stranded portion of the RNAi agent disclosed herein (see, for example, U.S. Patent No. 8,362,231).

[0056] In some embodiments, the MMP7 RNAi agent comprises an antisense strand having a 3' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In other embodiments, the MMP7 RNAi agent comprises an antisense strand having a 3' extension of 1, 2, or 3 nucleotides in length. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are complementary to the corresponding MMP7 mRNA sequence. In some embodiments, one or more of the antisense strand extension nucleotides comprise nucleotides that are not complementary to the corresponding MMP7 mRNA sequence.

[0057] In some embodiments, the MMP7 RNAi agent comprises a sense strand having a 3' extension of 1, 2, 3, 4, or 5 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprise an adenosine, uracil, or thymidine nucleotide, an AT dinucleotide, or a nucleotide that corresponds to or is identical to a nucleotide in the MMP7 mRNA sequence. In some embodiments, the 3' sense strand extension comprises or consists of, but is not limited to, one of the following sequences: T, UT, TT, UU, UUT, TTT, or TTTT (listed from 5' to 3', respectively).

[0058] The sense strand can have a 3' extension and / or a 5' extension. In some embodiments, the MMP7 RNAi agent comprises a sense strand having a 5' extension of 1, 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, one or more of the sense strand extension nucleotides comprise nucleotides that correspond to or are identical to nucleotides in the MMP7 mRNA sequence.

[0059] Examples of sequences used to form MMP7 RNAi agents are shown in Tables 2, 3, 4, 5, 6, and 10. In some embodiments, the antisense strand of the MMP7 RNAi agent comprises any of the sequences in Tables 2, 3, or 10. In certain embodiments, the antisense strand of the MMP7 RNAi agent comprises or consists of any one of the modified sequences in Table 3. In some embodiments, the antisense strand of the MMP7 RNAi agent comprises nucleotides (5' end to 3' end) 1-17, 2-15, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, or 2-21 of any of the sequences in Tables 2 or 3. In some embodiments, the sense strand of the MMP7 RNAi agent comprises any of the sequences in Tables 2, 4, 5, or 6. In some embodiments, the sense strand of the MMP7 RNAi agent comprises the sequence of nucleotides (5' to 3') 1-18, 1-19, 1-20, 1-21, 2-19, 2-20, 2-21, 3-20, 3-21, or 4-21 of any of the sequences in Table 2, 4, 5, or 6. In certain embodiments, the sense strand of the MMP7 RNAi agent comprises or consists of a modified sequence of any one of the modified sequences in Table 4, 5, 6, or 10.

[0060] In some embodiments, the sense strand and antisense strand of an RNAi agent described herein contain the same number of nucleotides. In some embodiments, the sense strand and antisense strand of an RNAi agent described herein contain a different number of nucleotides. In some embodiments, the sense strand 5' end and the antisense strand 3' end of an RNAi agent form a blunt end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of an RNAi agent form a blunt end. In some embodiments, both ends of an RNAi agent form a blunt end. In some embodiments, neither end of an RNAi agent is blunt. As used herein, "blunt end" refers to an end of a double-stranded RNAi agent where the terminal nucleotides of the two annealed strands are complementary (form complementary base pairs).

[0061] In some embodiments, the sense strand 5' end and the antisense strand 3' end of the RNAi agent form a non-complementary end. In some embodiments, the sense strand 3' end and the antisense strand 5' end of the RNAi agent form a non-complementary end. In some embodiments, both ends of the RNAi agent form non-complementary ends. In some embodiments, neither end of the RNAi agent is a non-complementary end. As used herein, non-complementary ends refers to ends of a double-stranded RNAi agent where the terminal nucleotides of the two annealed strands are base-paired (i.e., do not form an overhang) but are not complementary (i.e., form non-complementary base pairs). In some embodiments, one or more unpaired nucleotides at the end of one strand of the double-stranded RNAi agent form an overhang. The unpaired nucleotides may be in the sense strand or the antisense strand and form either a 3' or 5' overhang. In some embodiments, the RNAi agent contains a blunt end and a non-complementary end, a blunt end and a 5' overhanging end, a blunt end and a 3' overhanging end, a non-complementary end and a 5' overhanging end, a non-complementary end and a 3' overhanging end, two 5' overhanging ends, two 3' overhanging ends, a 5' overhanging end and a 3' overhanging end, two non-complementary ends, or two blunt ends. Typically, the overhang, if present, is located at the 3' end of the sense strand, the antisense strand, or both the sense strand and the antisense strand.

[0062] The MMP7 RNAi agent disclosed herein can also be composed of one or more modified nucleotides. In some embodiments, substantially all of the nucleotides of the sense strand of the MMP7 RNAi agent and substantially all of the nucleotides of the antisense strand are modified nucleotides. The MMP7 RNAi agent disclosed herein can further be composed of one or more modified internucleoside linkages, for example, one or more phosphorothioate linkages. In some embodiments, the MMP7 RNAi agent contains one or more modified nucleotides and one or more modified internucleoside linkages. In some embodiments, 2'-modified nucleotides are combined with modified internucleoside linkages.

[0063] In some embodiments, the MMP7 RNAi agent is prepared or provided as a salt, mixed salt, or free acid. In some embodiments, the MMP7 RNAi agent is prepared as a pharmaceutically acceptable salt. In some embodiments, the MMP7 RNAi agent is prepared as a pharmaceutically acceptable sodium salt. Such forms known in the art are within the scope of the invention disclosed herein.

[0064] Modified Nucleotides Modified nucleotides, when used in various oligonucleotide constructs, can preserve the activity of the compounds in cells while at the same time increasing the serum stability of these compounds and minimizing the potential for activating interferon activity in humans upon administration of the oligonucleotide construct.

[0065] In some embodiments, the MMP7 RNAi agent contains one or more modified nucleotides. As used herein, a "modified nucleotide" is a nucleotide other than a ribonucleotide (2'-hydroxyl nucleotide). In some embodiments, at least 50% (e.g., at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100%) of the nucleotides are modified nucleotides. As used herein, modified nucleotides can include, but are not limited to, deoxyribonucleotides, nucleotide mimetics, abasic nucleotides, 2'-modified nucleotides, inverted nucleotides, nucleotides containing modified nucleobases, bridged nucleic acids, peptide nucleic acids (PNAs), 2',3'-seco nucleotide mimetics (unlocked nucleobase analogs), locked nucleotides, 3'-O-methoxy (2' internucleoside linkage) nucleotides, 2'-F-arabinonucleotides, 5'-methyl-2'-fluoro nucleotides, morpholino nucleotides (modified nucleotides having a morpholine ring), nucleotides in which the typical five-membered sugar ring of a nucleotide has been modified, vinyl phosphonate deoxyribonucleotides, vinyl phosphonate-containing nucleotides, and cyclopropyl phosphonate-containing nucleotides. 2'-modified nucleotides (i.e., nucleotides with a 5-membered sugar ring with a group other than a hydroxyl group at the 2' position) can include, but are not limited to, 2'-O-methyl nucleotides (also referred to as 2'-methoxy nucleotides), 2'-fluoro nucleotides (also referred to herein as 2'-deoxy-2'-fluoro nucleotides), 2'-deoxy nucleotides, 2'-methoxyethyl (2'-O-2-methoxylethyl) nucleotides (also referred to as 2'-MOE), 2'-amino nucleotides, 2'-halo nucleotides, and 2'-alkyl nucleotides. Not all positions of a given compound need to be uniformly modified. Conversely, multiple modifications can be incorporated into a single MMP7 RNAi agent, or even into only one nucleotide thereof.The sense and antisense strands of the MMP7 RNAi agent can be synthesized and / or modified by methods known in the art, where a modification at one nucleotide is independent of a modification at another nucleotide.

[0066] Modified nucleobases include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines (e.g., 2-aminopropyladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, inosine, xanthine, hypoxanthine, 2-aminoadenine, 6-alkyl (e.g., 6-methyl, 6-ethyl, 6-isopropyl, or 6-n-butyl) derivatives of adenine and guanine, 2-alkyl (e.g., 2-methyl, 2-ethyl, 2-isopropyl, or 2-n-butyl) and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine, 2-thiocytosine ... These include synthetic and natural nucleobases such as uracil, 5-halouracil, cytosine, 5-propynyluracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-azothymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-sulfhydryl, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo (e.g., 5-bromo), 5-trifluoromethyl, and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine and 3-deazaadenine.

[0067] In some embodiments, the 5' and / or 3' end of the antisense strand can include an abasic residue (Ab) (which may also be referred to as an "abasic site" or "abasic nucleotide"). An abasic residue (Ab) is a nucleotide or nucleoside that lacks a nucleobase at the 1' position of the sugar moiety (see, e.g., U.S. Pat. No. 5,998,203). In some embodiments, the abasic residue can be located internally in the nucleotide sequence. In some embodiments, Ab or AbAb can be added to the 3' end of the antisense strand. In some embodiments, the 5' end of the sense strand can include one or more additional abasic residues (e.g., (Ab) or (AbAb)). In some embodiments, UUAb, UAb, or Ab can be added to the 3' end of the sense strand. In some embodiments, the abasic (deoxyribose) residue can be replaced with a ribitol (abasic ribose) residue.

[0068] In some embodiments, all or substantially all of the nucleotides of the RNAi agent are modified nucleotides. As used herein, an RNAi agent in which substantially all of the nucleotides present are modified nucleotides is an RNAi agent in which no more than four (i.e., 0, 1, 2, 3, or 4) nucleotides of both the sense strand and the antisense strand are ribonucleotides (i.e., unmodified). As used herein, a sense strand in which substantially all of the nucleotides present are modified nucleotides is a sense strand in which no more than two (i.e., 0, 1, or 2) nucleotides of the sense strand are unmodified ribonucleotides. As used herein, an antisense sense strand in which substantially all of the nucleotides present are modified nucleotides is an antisense strand in which no more than two (i.e., 0, 1, or 2) nucleotides of the antisense strand are unmodified ribonucleotides. In some embodiments, one or more nucleotides of the RNAi agent are unmodified ribonucleotides. The chemical structures of certain modified nucleotides are listed in Table 11 herein.

[0069] Modified Internucleoside Linkages In some embodiments, one or more nucleotides of an MMP7 RNAi agent are linked by a non-canonical bond or backbone (ie, a modified internucleoside bond or modified backbone). Modified internucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein as a lower case "s"), chiral phosphorothioates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3'-amino phosphoramidates, aminoalkyl phosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkyl phosphotriesters, morpholino linkages, boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates with reverse polarity in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2-5' to 5'-2'. In some embodiments, the modified internucleoside linkage or backbone lacks a phosphorus atom. Modified internucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl intersugar linkages, mixed heteroatom and alkyl or cycloalkyl intersugar linkages, or one or more short chain heteroatom or heterocyclic intersugar linkages. In some embodiments, modified internucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamic acid backbones, methyleneimino and methylenehydrazino backbones, sulfonic acid and sulfonamide backbones, amide backbones, and other backbones with mixed N, O, S, and CH2 components.

[0070] In some embodiments, the sense strand of an MMP7 RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, the antisense strand of an MMP7 RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, or both the sense strand and the antisense strand can independently contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages. In some embodiments, the sense strand of an MMP7 RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, the antisense strand of an MMP7 RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand can independently contain 1, 2, 3, or 4 phosphorothioate linkages.

[0071] In some embodiments, the sense strand of the MMP7 RNAi agent contains at least two phosphorothioate internucleoside linkages. In some embodiments, the phosphorothioate internucleoside linkages are between nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate internucleoside linkage is at the 5' end of the sense strand nucleotide sequence and another phosphorothioate linkage is at the 3' end of the sense strand nucleotide sequence. In some embodiments, two phosphorothioate internucleoside linkages are located at the 5' end of the sense strand and another phosphorothioate linkage is at the 3' end of the sense strand. In some embodiments, the sense strand does not contain any phosphorothioate internucleoside linkages between nucleotides, but does contain one, two, or three phosphorothioate linkages between the terminal nucleotides at both the 5' and 3' ends and the optionally present inverted abasic residue end cap. In some embodiments, the targeting ligand is linked to the sense strand via a phosphorothioate linkage.

[0072] In some embodiments, the antisense strand of the MMP7 RNAi agent contains four phosphorothioate internucleoside linkages. In some embodiments, the four phosphorothioate internucleoside linkages are between nucleotide positions 1-3 from the 5' end of the antisense strand and between nucleotide positions 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end of the antisense strand. In some embodiments, three phosphorothioate internucleoside linkages are located between positions 1-4 from the 5' end of the antisense strand and a fourth phosphorothioate internucleoside linkage is located between positions 20-21 from the 5' end of the antisense strand. In some embodiments, the MMP7 RNAi agent contains at least three or four phosphorothioate internucleoside linkages in the antisense strand.

[0073] Capping residues or moieties In some embodiments, the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as "caps," "terminal caps," or "capping residues." As used herein, a "capping residue" is a non-nucleotide compound or other moiety that may be incorporated at one or more ends of a nucleotide sequence of an RNAi agent disclosed herein. Capping residues may, in some cases, provide certain beneficial properties to the RNAi agent, such as, for example, protection against exonuclease degradation. In some embodiments, an inverted abasic residue (invAb) (also referred to in the art as an "inverted abasic site") is added as a capping residue (see Table 11) (see, for example, F. Czaudema, Nucleic Acids Res., 2003, 31(11), 2705-16). Capping residues are generally known in the art and include, for example, inverted abasic residues, and terminal C3H7 (propyl), C6H 13 (hexyl), or C 12 H 25(dodecyl) group. In some embodiments, the capping residue is present at either the 5'-end, the 3'-end, or both the 5'- and 3'-ends of the sense strand. In some embodiments, the 5'-end and / or the 3'-end of the sense strand can include multiple inverted abasic deoxyribose moieties as capping residues.

[0074] In some embodiments, one or more inverted abasic residues (invAb) are added to the 3' end of the sense strand. In some embodiments, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted abasic residues or inverted abasic sites are inserted between the targeting ligand and the sense strand nucleotide sequence of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic residues or inverted abasic sites at or near the end or ends of the sense strand of the RNAi agent allows for enhanced activity or other desired properties of the RNAi agent.

[0075] In some embodiments, one or more inverted abasic residues (invAb) are added to the 5' end of the sense strand. In some embodiments, one or more inverted abasic residues can be inserted between the targeting ligand and the sense strand nucleotide sequence of the RNAi agent. The inverted abasic residues can be linked via phosphate, phosphorothioate (e.g., shown herein as (invAb)), or other internucleoside linkages. In some embodiments, the inclusion of one or more inverted abasic residues at or near the terminus or termini of the sense strand of the RNAi agent allows for enhanced activity or other desired properties of the RNAi agent. In some embodiments, the inverted abasic (deoxyribose) residues can be replaced with inverted ribitol (abasic ribose) residues. In some embodiments, the 3' end of the antisense strand core stretch sequence or the 3' end of the antisense strand sequence can include an inverted abasic residue. The chemical structure of an inverted abasic deoxyribose residue is shown in Table 11 below.

[0076] MMP7 RNAi Agents The MMP7 RNAi agent disclosed herein is designed to target a specific position on the MMP7 gene (e.g., SEQ ID NO: 1 (NM_002423.5)). As defined herein, the antisense strand sequence is designed to target the MMP7 gene at a given position on the gene when the 5'-terminal nucleobase of the antisense strand is aligned with a position that is 21 nucleotides downstream (towards the 3' end) from the position on the gene when base-pairing with the gene. For example, as illustrated in Tables 1 and 2 herein, an antisense strand sequence designed to target the MMP7 gene at position 303 requires that the 5'-terminal nucleobase of the antisense strand is aligned with position 323 of the MMP7 gene when base-pairing with the gene.

[0077] As provided herein, MMP7 RNAi agents do not require that the nucleobase at position 1 (5'→3') of the antisense strand be complementary to the gene, provided that there is at least 85% complementarity (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) between the antisense strand and the gene over a core stretch sequence of at least 16 contiguous nucleotides. For example, for an MMP7 RNAi agent disclosed herein designed to target position 303 of the MMP7 gene, the 5' terminal nucleobase of the antisense strand of the MMP7 RNAi agent must align with position 323 of the gene. However, the 5'-terminal nucleobase of the antisense strand may be complementary to position 323 of the MMP7 gene, but need not be complementary, provided that there is at least 85% complementarity between the antisense strand and the gene transcript (e.g., at least 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100% complementarity) over a core stretch sequence of at least 16 contiguous nucleotides. In particular, as demonstrated by various examples disclosed herein, the specific binding site of the gene by the antisense strand of the MMP7 RNAi agent (e.g., whether the MMP7 RNAi agent is designed to target the MMP7 gene at position 303, 418, 971, or some other position) is an important factor for the level of inhibition achieved by the MMP7 RNAi agent (see, e.g., Kamola et al., The siRNA Non-seed Region and Its Target Sequences are Auxiliary Determinants of Off-Target Effects, PLOS Computational Biology, 11(12), Figure 1 (2015)).

[0078] In some embodiments, the MMP7 RNAi agents disclosed herein target the MMP7 gene at or near the location of the MMP7 sequence shown in Table 1. In some embodiments, the antisense strand of the MMP7 RNAi agents disclosed herein comprises a core stretch sequence that is fully, substantially, or at least partially complementary to the target MMP7 19-mer sequence disclosed in Table 1. [Table 1]

[0079] Homo sapiens matrix metallopeptidase 7 (MMP7), GenBank NM_002423.5 (SEQ ID NO: 1), gene transcript (1119 bases): [ka]

[0080] In some embodiments, the MMP7 RNAi agent comprises an antisense strand, and position 19 of the antisense strand (5'→3') can base pair to position 1 of a 19-mer target sequence disclosed in Table 1. In some embodiments, the MMP7 agent comprises an antisense strand, and position 1 of the antisense strand (5'→3') can base pair to position 19 of a 19-mer target sequence disclosed in Table 1.

[0081] In some embodiments, the MMP7 agent comprises an antisense strand, and position 2 of the antisense strand (5'→3') can base pair with position 18 of a 19-mer target sequence disclosed in Table 1. In some embodiments, the MMP7 agent comprises an antisense strand, and positions 2 through 18 of the antisense strand (5'→3') can base pair with each of the respective complementary bases located at positions 18 through 2 of a 19-mer target sequence disclosed in Table 1.

[0082] In the RNAi agent disclosed herein, the nucleotide at position 1 of the antisense strand (5'->3'-end) can be completely complementary to the MMP7 gene or can be non-complementary to the MMP7 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (5'->3'-end) is U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (5'->3'-end) forms an A:U or U:A base pair with the sense strand.

[0083] In some embodiments, the antisense strand of the MMP7 RNAi agent comprises the sequence of nucleotides 2-18 or 2-19 (5' to 3' end) of any of the antisense strand sequences in Table 2 or Table 3. In some embodiments, the sense strand of the MMP7 RNAi agent comprises the sequence of nucleotides 1-17, 1-18, or 2-18 (5' to 3' end) of any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6.

[0084] In some embodiments, the MMP7 RNAi agent is composed of (i) an antisense strand comprising a sequence of 2 to 18 or 2 to 19 nucleotides (5' to 3' end) of any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand comprising a sequence of 1 to 17 or 1 to 18 nucleotides (5' to 3' end) of any of the sense strand sequences in Table 2, Table 4, Table 5, or Table 6.

[0085] In some embodiments, the MMP7 RNAi agent comprises the core 19-mer nucleotide sequence shown in Table 2 below. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7]

[0086] The sense and antisense strands of an MMP7 RNAi agent that comprises or consists of a nucleotide sequence in Table 2 can be modified or unmodified nucleotides. In some embodiments, an MMP7 RNAi agent having a sense and antisense strand sequence that comprises or consists of any of the nucleotide sequences in Table 2 is all or substantially all modified nucleotides.

[0087] In some embodiments, the antisense strand of an MMP7 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 2 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of an MMP7 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 2 by 0, 1, 2, or 3 nucleotides.

[0088] As used herein, each N listed in the sequences disclosed in Table 2 can be independently selected from any and all nucleobases (including those found in both modified and unmodified nucleotides). In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have a nucleobase that is complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have a nucleobase that is not complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have the same nucleobase as the N nucleotide at the corresponding position on the other strand. In some embodiments, the N nucleotides listed in the sequences disclosed in Table 2 have a different nucleobase than the N nucleotide at the corresponding position on the other strand.

[0089] Specific modified MMP7 RNAi agent sense and antisense strands are shown in Tables 3, 4, 5, 6, and 10. Specific modified MMP7 RNAi agent antisense strands and their underlying unmodified nucleobase sequences are shown in Table 3. Specific modified MMP7 RNAi agent sense strands and their underlying unmodified nucleobase sequences are shown in Tables 4, 5, and 6. In forming an MMP7 RNAi agent, each of the nucleotides in each of the underlying sequences listed in Tables 3, 4, 5, and 6, and Table 2 above, can be a modified nucleotide.

[0090] The MMP7 RNAi agent described herein is formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2, Table 4, Table 5, or Table 6 can hybridize to any antisense strand containing a sequence listed in Table 2 or Table 3, provided that the two sequences have a region of at least 85% complementarity over a sequence of 16, 17, 18, 19, 20, or 21 consecutive nucleotides.

[0091] In some embodiments, the antisense strand of the MMP7 RNAi agent comprises a nucleotide sequence of any of the sequences in Table 2 or Table 3.

[0092] In some embodiments, the MMP7 RNAi agent comprises or consists of a duplex having sense and antisense strand nucleobase sequences of any of the sequences in Table 2, Table 3, Table 4, Table 5, Table 6, or Table 10.

[0093] Examples of antisense strands containing modified nucleotides are shown in Table 3. Examples of sense strands containing modified nucleotides are shown in Tables 4, 5 and 6.

[0094] As used in Tables 3, 4, 5, 6, and 10, the following notation is used to indicate modified nucleotides, targeting groups, and linking groups: A = adenosine 3'-phosphate C=Cytidine-3'-phosphate G=guanosine-3'-phosphate U = uridine-3'-phosphate I = inosine-3'-phosphate a = 2'-O-methyladenosine-3'-phosphate as = 2'-O-methyladenosine-3'-phosphorothioate c = 2'-O-methylcytidine-3'-phosphate cs = 2'-O-methylcytidine-3'-phosphorothioate g = 2'-O-methylguanosine-3'-phosphate gs = 2'-O-methylguanosine-3'-phosphorothioate i = 2'-O-methylinosine-3'-phosphate is = 2'-O-methylinosine-3'-phosphorothioate t=2'-O-methyl-5-methyluridine-3'-phosphate ts = 2'-O-methyl-5-methyluridine-3'-phosphorothioate u = 2'-O-methyluridine-3'-phosphate us = 2'-O-methyluridine-3'-phosphorothioate Af = 2'-fluoroadenosine-3'-phosphate Afs = 2'-fluoroadenosine-3'-phosphorothioate Cf = 2'-fluorocytidine-3'-phosphate Cfs = 2'-fluorocytidine-3'-phosphorothioate Gf = 2'-fluoroguanosine-3'-phosphate Gfs = 2'-fluoroguanosine-3'-phosphorothioate Tf = 2'-fluoro-5'-methyluridine-3'-phosphate Tfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioate Uf = 2'-fluorouridine-3'-phosphate Ufs = 2'-fluorouridine-3'-phosphorothioate dT = 2'-deoxythymidine-3'-phosphate A UNA= 2',3'-seco-adenosine-3'-phosphate A UNAS = 2',3'-seco-adenosine-3'-phosphorothioate C UNA = 2',3'-seco-cytidine-3'-phosphate C UNAS = 2',3'-seco-cytidine-3'-phosphorothioate G UNA = 2',3'-seco-guanosine-3'-phosphate G UNAS = 2',3'-seco-guanosine-3'-phosphorothioate U UNA = 2',3'-seco-uridine-3'-phosphate U UNAS = 2',3'-seco-uridine-3'-phosphorothioate a_2N=See Table 11 a_2Ns = See Table 11 (invAb) = inverted abasic deoxyribonucleotide-5'-phosphate, see Table 11 (invAb)s = inverted abasic deoxyribonucleotide-5'-phosphorothioate, see Table 11 s = phosphorothioate bond p = terminal phosphate (as synthesized) vpdN = vinylphosphonate deoxyribonucleotide cPrpa = cyclopropyl-2'-O-methyladenosine-3'-phosphate 5'-phosphonate (see Table 11) cPrpas = 5'-phosphonic acid cyclopropyl-2'-O-methyladenosine-3'-phosphorothioate (see Table 11) cPrpu = 5'-phosphonic acid cyclopropyl-2'-O-methyluridine-3'-phosphate (see Table 11) cPrpus = 5'-phosphonic acid cyclopropyl-2'-O-methyluridine-3'-phosphorothioate (see Table 11) (Alk-SS-C6)=See Table 11 (C6-SS-Alk)=See Table 11 (C6-SS-C6)=See Table 11 (6-SS-6) = See Table 11 (C6-SS-Alk-Me)=See Table 11 (NH2-C6) = See Table 11 (TriAlk14) = See Table 11 (TriAlk14)s = See Table 11 -C6-=See Table 11 -C6s- = See Table 11 -L6-C6-=See Table 11 -L6-C6s-=See Table 11 -Alk-cyHex-=See Table 11 -Alk-cyHexs- = See Table 11 (TA14) = See Table 11 (structure of (TriAlk14)s after conjugation) (TA14)s = See Table 11 (structure of (TriAlk14)s after conjugation)

[0095] As one of ordinary skill in the art would readily understand, unless otherwise indicated by the sequence (e.g., by a phosphorothioate linkage "s"), the nucleotide monomers, when present in an oligonucleotide, are linked to one another by a 5'-3'-phosphodiester linkage. As one of ordinary skill in the art would clearly understand, the inclusion of a phosphorothioate linkage as shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester linkage typically present in an oligonucleotide. Furthermore, one of ordinary skill in the art would readily understand that the terminal nucleotide at the 3' end of a given oligonucleotide sequence will typically have a hydroxyl (-OH) group at the 3' position of each of the given monomers, instead of an in vitro phosphate moiety. Additionally, in the embodiments disclosed herein, when each strand is viewed from 5'→3', an inverted abasic residue is inserted such that the 3' position of the deoxyribose is linked to the 3' end of the previous monomer on each strand (see, e.g., Table 11). Moreover, as one of ordinary skill in the art would readily understand and appreciate, while the chemical structures of phosphorothioates depicted herein typically show an anion on the sulfur atom, the invention disclosed herein encompasses all phosphorothioate tautomers (e.g., when the sulfur atom bears a double bond and the anion is on the oxygen atom). Such understanding of the artisan is used in describing the MMP7 RNAi agents and compositions of MMP7 RNAi agents disclosed herein, unless otherwise expressly indicated herein.

[0096] Specific examples of targeting and linking groups for use in the MMP7 RNAi agents disclosed herein are included in the chemical structures shown below in Table 11. The sense and / or antisense strands can each have any of the targeting or linking groups listed herein, as well as other targeting or linking groups, conjugated to the 5' and / or 3' ends of the sequence.

[0097] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4]

[0098] [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0099] [Table 5-1] [Table 5-2] [Table 5-3]

[0100] [Table 6]

[0101] The MMP7 RNAi agent disclosed herein is formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2, Table 4, Table 5, or Table 6 can hybridize to any antisense strand containing a sequence listed in Table 2 or Table 3, provided that the two sequences have a region of at least 85% complementarity over a sequence of 16, 17, 18, 19, 20, or 21 consecutive nucleotides.

[0102] As shown in Table 5 above, certain of the exemplary MMP7 RNAi agent nucleotide sequences are shown to further include a reactive linking group at one or both of the 5' and 3' ends of the sense strand. For example, many of the MMP7 RNAi agent sense strand sequences shown in Table 5 above have a (TriAlk14) linking group at the 5' end of the nucleotide sequence. Other linking groups, such as (NH2-C6) linking groups or (6-SS-6) or (C6-SS-C6) linking groups, can also or alternatively be present in certain embodiments. Such reactive linking groups are positioned to facilitate the linking of targeting ligands, targeting groups, and / or PK / PD modulators to the MMP7 RNAi agents disclosed herein. Ligation or conjugation reactions are well known in the art and result in the formation of a covalent bond between two molecules or reactants. Conjugation reactions suitable for use within the scope of the invention herein include, but are not limited to, amide coupling reactions, Michael addition reactions, hydrazone formation reactions, inverse-demand Diels-Alder cycloaddition reactions, oxime ligation, and copper(I)-catalyzed or strain-promoted azide-alkyne cycloaddition reactions.

[0103] In some embodiments, targeting ligands, such as the integrin targeting ligands shown in the examples and figures disclosed herein, are synthesized as activated esters, such as tetrafluorophenyl (TFP) esters, that can be substituted with reactive amino groups (e.g., NH2-C6) to allow the targeting ligands to be attached to MMP7 RNAi agents disclosed herein. In some embodiments, targeting ligands are synthesized as azides and can be conjugated to propargyl (e.g., TriAlk14) or DBCO groups, for example, via copper(I)-catalyzed or strain-promoted azide-alkyne cycloaddition reactions.

[0104] Additionally, certain of the nucleotide sequences can be synthesized with a dT nucleotide at the 3' end of the sense strand followed by a (3'→5') linker (e.g., C6-SS-C6). The linker can, in some embodiments, facilitate linking to additional components, such as, for example, a PK / PD modulator and one or more targeting ligands. As described herein, the C6-SS-C6 disulfide bond can first be reduced to remove the dT from the molecule, which can then facilitate conjugation of the desired PK / PD modulator. Thus, the terminal dT nucleotide is not part of the fully conjugated construct.

[0105] In some embodiments, the antisense strand of an MMP7 RNAi agent disclosed herein differs from any of the antisense strand sequences in Table 3 or Table 10 by 0, 1, 2, or 3 nucleotides. In some embodiments, the sense strand of an MMP7 RNAi agent disclosed herein differs from any of the sense strand sequences in Table 4, Table 5, Table 6, or Table 10 by 0, 1, 2, or 3 nucleotides.

[0106] In some embodiments, the antisense strand of the MMP7 RNAi agent comprises the nucleotide sequence of any of the sequences in Table 2 or Table 3. In some embodiments, the antisense strand of the MMP7 RNAi agent comprises the sequence of nucleotides (5' to 3') 1-17, 2-17, 1-18, 2-18, 1-19, 2-19, 1-20, 2-20, 1-21, 2-21, 1-22, 2-22, 1-23, 2-23, 1-24, or 2-24 of any of the sequences in Table 2, Table 3, or Table 10. In certain embodiments, the antisense strand of the MMP7 RNAi agent comprises or consists of a modified sequence of any one of the modified sequences in Table 3 or Table 10.

[0107] In some embodiments, the sense strand of the MMP7 RNAi agent comprises the nucleotide sequence of any of the sequences in Table 2 or Table 4. In some embodiments, the sense strand of the MMP7 RNAi agent comprises the sequence of nucleotides (5' to 3') 1-17, 2-17, 3-17, 4-17, 1-18, 2-18, 3-18, 4-18, 1-19, 2-19, 3-19, 4-19, 1-20, 2-20, 3-20, 4-20, 1-21, 2-21, 3-21, 4-21, 1-22, 2-22, 3-22, 4-22, 1-23, 2-23, 3-23, 4-23, 1-24, 2-24, 3-24, or 4-24 of any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 10. In certain embodiments, the sense strand of the MMP7 RNAi agent comprises or consists of the modified sequence of any one of the modified sequences of Table 3 or Table 10.

[0108] In the RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (5'->3'-end) can be fully complementary to the MMP7 gene or can be non-complementary to the MMP7 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (5'->3'-end) is U, A, or dT (or modified versions of U, A, or dT). In some embodiments, the nucleotide at position 1 of the antisense strand (5'->3'-end) forms an A:U or U:A base pair with the sense strand.

[0109] In some embodiments, the antisense strand of the MMP7 RNAi agent comprises the sequence of nucleotides 2-18 or 2-19 (5' end to 3' end) of any of the antisense strand sequences in Table 2, Table 3, or Table 10. In some embodiments, the sense strand of the MMP7 RNAi agent comprises the sequence of nucleotides 1-17 or 1-18 (5' end to 3' end) of any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 10.

[0110] In some embodiments, the MMP7 RNAi agent comprises (i) an antisense strand comprising a sequence of 2 to 18 or 2 to 19 nucleotides (5' end to 3' end) of any of the antisense strand sequences in Table 2, Table 3, or Table 10, and (ii) a sense strand comprising a sequence of 1 to 17 or 1 to 18 nucleotides (5' end to 3' end) of any of the sense strand sequences in Table 2, Table 4, Table 5, Table 6, or Table 10.

[0111] A sense strand containing a sequence listed in Table 2 or Table 4 can hybridize to any antisense strand containing a sequence listed in Table 2 or Table 3, provided that the two sequences have a region of at least 85% complementarity over a sequence of 16, 17, 18, 19, 20, or 21 contiguous nucleotides. In some embodiments, an MMP7 RNAi agent has a sense strand consisting of any of the modified sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand consisting of any of the modified sequences in Table 3 or Table 10. Certain representative sequence pairs are illustrated by the duplex ID numbers shown in Tables 7A, 7B, 8, and 9.

[0112] In some embodiments, the MMP7 RNAi agent comprises, consists of, or consists essentially of a duplex represented by any one of the duplex ID numbers presented herein. In some embodiments, the MMP7 RNAi agent consists of any of the duplex ID numbers presented herein. In some embodiments, the MMP7 RNAi agent comprises a sense strand and an antisense strand nucleotide sequence of any of the duplex ID numbers presented herein. In some embodiments, the MMP7 RNAi agent comprises a sense strand and an antisense strand nucleotide sequence of any of the duplex ID numbers presented herein and a targeting group, a linking group, and / or other non-nucleotide group, where the targeting group, the linking group, and / or other non-nucleotide group is covalently linked (i.e., conjugated) to the sense strand or the antisense strand. In some embodiments, the MMP7 RNAi agent comprises a sense strand and an antisense strand modified nucleotide sequence of any of the duplex ID numbers presented herein. In some embodiments, the MMP7 RNAi agent comprises a sense strand and an antisense strand modified nucleotide sequence of any of the duplex ID numbers presented herein and a targeting group, linking group, and / or other non-nucleotide group, wherein the targeting group, linking group, and / or other non-nucleotide group is covalently linked to the sense strand or the antisense strand.

[0113] In some embodiments, an MMP7 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense strand / sense strand duplexes in Table 2, 7A, 7B, 8, 9, or 10, and comprises a targeting group. In some embodiments, an MMP7 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense strand / sense strand duplexes in Table 2, 7A, 7B, 8, 9, or 10, and comprises one or more αvβ6 integrin targeting ligands.

[0114] In some embodiments, the MMP7 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense strand / sense strand duplexes in Table 2, 7A, 7B, 8, 9, or 10, and comprises a targeting group that is an integrin targeting ligand. In some embodiments, the MMP7 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequence of any of the antisense strand / sense strand duplexes in Table 2, 7A, 7B, 8, 9, or 10, and comprises one or more αvβ6 integrin targeting ligands or clusters of αvβ6 integrin targeting ligands (e.g., tridentate αvβ6 integrin targeting ligands).

[0115] In some embodiments, an MMP7 RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of any of the antisense strand / sense strand duplexes in Tables 7A, 7B, 8, 9, and 10.

[0116] In some embodiments, the MMP7 RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of any of the antisense strand / sense strand duplexes in Tables 7A, 7B, 8, 9, and 10, and comprises an integrin targeting ligand.

[0117] In some embodiments, the MMP7 RNAi agent comprises, consists of, or consists essentially of any of the duplexes in Tables 7A, 7B, 8, 9, and 10.

[0118] [Table 7-1] [Table 7-2] [Table 7-3]

[0119] [Table 8-1] [Table 8-2] [Table 8-3]

[0120] [Table 9]

[0121] [Table 10]

[0122] [Table 11-1] [Table 11-2]

[0123] In some embodiments, the MMP7 RNAi agent is prepared or provided as a salt, mixed salt, or free acid. In some embodiments, the MMP7 RNAi agent is prepared or provided as a pharma- ceutically acceptable salt. In some embodiments, the MMP7 RNAi agent is prepared or provided as a pharma- ceutically acceptable sodium or potassium salt. The RNAi agents described herein, when delivered to cells expressing the MMP7 gene, inhibit or knock down the expression of one or more MMP7 genes in vivo and / or in vitro.

[0124] Targeting Groups, Linking Groups, Pharmacokinetic / Pharmacodynamic (PK / PD) Modulators, and Delivery Vehicles In some embodiments, the MMP7 RNAi agent contains or is conjugated to one or more non-nucleotide groups, including but not limited to targeting groups, linking groups, pharmacokinetic / pharmacodynamic (PK / PD) modulators, delivery polymers, or delivery vehicles. The non-nucleotide groups can enhance targeting, delivery, or attachment of the RNAi agent. The non-nucleotide groups can be covalently linked to the 3' and / or 5' end of either the sense strand and / or the antisense strand. In some embodiments, the MMP7 RNAi agent contains a non-nucleotide group linked to the 3' and / or 5' end of the sense strand. In some embodiments, the non-nucleotide group is linked to the 5' end of the sense strand of the MMP7 RNAi agent. The non-nucleotide group can be linked to the RNAi agent directly or indirectly via a linker / linking group. In some embodiments, the non-nucleotide group is linked to the RNAi agent via a labile, cleavable, or reversible bond or linker.

[0125] In some embodiments, the non-nucleotide group enhances the pharmacokinetics or biodistribution properties of the RNAi agent or conjugate to which it is attached, improving cell- or tissue-specific distribution and cell-specific uptake of the conjugate, hi some embodiments, the non-nucleotide group enhances endocytosis of the RNAi agent.

[0126] A targeting group or moiety enhances the pharmacokinetics or biodistribution properties of the conjugate or RNAi agent to which it is attached, improving cell-specific (including organ-specific, in some cases) distribution and cell-specific (or organ-specific) uptake of the conjugate or RNAi agent. A targeting group may be monovalent, divalent, trivalent, tetravalent, or may have higher valency to the target to which it is directed. Exemplary targeting groups include, but are not limited to, compounds with affinity for cell surface molecules, cell receptor ligands, haptens, antibodies, monoclonal antibodies, antibody fragments, and antibody mimetics with affinity for cell surface molecules. In some embodiments, the targeting group is linked to the RNAi agent using a linker, such as a PEG linker or one, two, or three abasic and / or ribitol (abasic ribose) residues that may optionally serve as a linker.

[0127] A targeting group, with or without a linker, can be attached to the 5' or 3' end of any of the sense strands and / or antisense strands disclosed in Tables 2, 3, 4, 5, 6, and 10. A linker, with or without a targeting group, can be attached to the 5' or 3' end of any of the sense strands and / or antisense strands disclosed in Tables 2, 3, 4, 5, 6, and 10.

[0128] The MMP7 RNAi agents described herein can be synthesized with reactive groups, such as amino groups (also referred to herein as amines), at the 5' and / or 3' termini, which can be used to subsequently attach targeting moieties using methods typical in the art.

[0129] For example, in some embodiments, the MMP7 RNAi agent disclosed herein is synthesized with an NH2-C6 group at the 5' end of the sense strand of the RNAi agent. The terminal amino group can then be reacted to form a conjugate with a group that includes, for example, an αvβ6 integrin targeting ligand. In some embodiments, the MMP7 RNAi agent disclosed herein is synthesized with one or more alkyne groups at the 5' end of the sense strand of the RNAi agent. The terminal alkyne group(s) can then be reacted to form a conjugate with a group that includes, for example, an αvβ6 integrin targeting ligand.

[0130] In some embodiments, the targeting group comprises an integrin targeting ligand. In some embodiments, the integrin targeting ligand is an αvβ6 integrin targeting ligand. The use of an αvβ6 integrin targeting ligand facilitates cell-specific targeting to cells that have αvβ6 on their respective surfaces, and the binding of the integrin targeting ligand facilitates the entry of a therapeutic agent, such as an RNAi agent, to which it is linked, into cells, such as epithelial cells, including lung epithelial cells and renal epithelial cells. The integrin targeting ligand can be monomeric or monovalent (e.g., with a single integrin targeting moiety) or multimeric or multivalent (e.g., with multiple integrin targeting moieties). The targeting group can be attached to the 3' and / or 5' end of the RNAi oligonucleotide using methods known in the art. The preparation of targeting groups such as αvβ6 integrin targeting ligands is described, for example, in International Patent Application Publication No. WO 2018 / 085415 and International Patent Application Publication No. WO 2019 / 089765, the contents of each of which are incorporated herein by reference in their entirety.

[0131] In some embodiments, targeting group is linked to MMP7 RNAi agent without using additional linker. In some embodiments, targeting group is designed to have a readily existing linker, so that it is easy to link to MMP7 RNAi agent. In some embodiments, when two or more RNAi agents are included in the composition, two or more RNAi agents can be linked to their respective targeting groups using the same linker. In some embodiments, when two or more RNAi agents are included in the composition, two or more RNAi agents are linked to their respective targeting groups using different linkers.

[0132] In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent attachment of the agent to a targeting group, a pharmacokinetic modulator, a delivery polymer, or a delivery vehicle. The linking group can be attached to the 3' and / or 5' end of the sense or antisense strand of the RNAi agent. In some embodiments, the linking group is attached to the sense strand of the RNAi agent. In some embodiments, the linking group is conjugated to the 5' or 3' end of the sense strand of the RNAi agent. In some embodiments, the linking group is conjugated to the 5' end of the sense strand of the RNAi agent. Examples of linking groups include, but are not limited to, C6-SS-C6, 6-SS-6, reactive groups such as primary amines (e.g., NH2-C6) and alkynes, alkyl groups, abasic residues / nucleotides, amino acids, groups functionalized with trialkynes, ribitol, and / or PEG groups. Examples of specific linking groups are shown in 11.

[0133] A linker or linking group is a connection between two atoms that connects one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group, a pharmacokinetic modulator, or a delivery polymer) or segment of interest through one or more covalent bonds. A labile linkage includes a labile bond. A linkage can optionally include a spacer that increases the distance between the two linking atoms. A spacer can further add flexibility and / or length to the linkage. Spacers include, but are not limited to, alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, and aralkynyl groups, each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and carbohydrates. Spacer groups are well known in the art, and the foregoing list is not meant to limit the scope of this specification. In some embodiments, the MMP7 RNAi agent is conjugated to a polyethylene glycol (PEG) moiety or a hydrophobic group having 12 or more carbon atoms, such as a cholesterol or palmitoyl group.

[0134] In some embodiments, the MMP7 RNAi agent is linked to one or more pharmacokinetic / pharmacodynamic (PK / PD) modulators. The PK / PD modulators can increase the circulation time of the conjugate and / or increase the activity of the RNAi agent through improved cell receptor binding, improved cellular uptake, and / or other means. A variety of PK / PD modulators suitable for use with RNAi agents are known in the art. In some embodiments, the PK / PD modulator can be a cholesterol or cholesteryl derivative, or in some circumstances, the PK / PD modulator can be comprised of an alkyl group, an alkenyl group, an alkynyl group, an aryl group, an aralkyl group, an aralkenyl group, or an aralkynyl group, each of which can be linear, branched, cyclic, and / or substituted or unsubstituted. In some embodiments, the point of attachment of these moieties is at the 5' or 3' end of the sense strand, the 2' position of the ribose ring of any given nucleotide of the sense strand, and / or is attached to the phosphate or phosphorothioate backbone at any position in the sense strand.

[0135] Any of the nucleotide sequences of the MMP7 RNAi agents listed in Tables 2, 3, 4, 5, 6, and 10 can contain 3' and / or 5' targeting group(s), linking group(s), and / or PK / PD modulator(s), whether modified or unmodified. Any of the sequences of the MMP7 RNAi agents listed in Tables 3, 4, 5, 6, and 10 or otherwise described herein that contain a 3' or 5' targeting group, linking group, and / or PK / PD modulator can alternatively not contain a 3' or 5' targeting group, linking group, or PK / PD modulator, or can contain a different 3' or 5' targeting group, linking group, or pharmacokinetic modulator, including but not limited to those depicted in Table 11. Any of the MMP7 RNAi agent duplexes listed in Tables 7A, 7B, 8, 9, and 10, whether modified or unmodified, can further include a targeting or linking group, including but not limited to those depicted in Table 11, which can be attached to the 3' or 5' end of either the sense or antisense strand of the MMP7 RNAi agent duplex.

[0136] Examples of specific modified nucleotides, capping sites, and linking groups are provided in Table 11.

[0137] [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6] [Table 12-7]

[0138] Alternatively, other linking groups known in the art can be used. Often, the linking groups are commercially available or are incorporated into commercially available nucleotide phosphoramidites (see, e.g., International Patent Application Publication No. WO 2019 / 161213, which is incorporated by reference in its entirety).

[0139] In some embodiments, the MMP7 RNAi agent is delivered without being conjugated to a targeting ligand or a pharmacokinetic / pharmacodynamic (PK / PD) modulator (is "naked" or is referred to as a "naked RNAi agent").

[0140] In some embodiments, the MMP7 RNAi agent is conjugated to a targeting group, a linking group, a PK modulator, and / or another non-nucleotide group to facilitate delivery of the MMP7 RNAi agent to a selected cell or tissue, such as an epithelial cell, in vivo. In some embodiments, the MMP7 RNAi agent is conjugated to a targeting group, and the targeting group comprises an integrin targeting ligand. In some embodiments, the integrin targeting ligand is an αvβ6 integrin targeting ligand. In some embodiments, the targeting group comprises one or more αvβ6 integrin targeting ligands.

[0141] In some embodiments, a delivery vehicle can be used to deliver an RNAi agent to a cell or tissue. A delivery vehicle is a compound that improves the delivery of an RNAi agent to a cell or tissue. A delivery vehicle can include or consist of a polymer, such as, but not limited to, an amphipathic polymer, a membrane active polymer, a peptide, a melittin peptide, a melittin-like peptide (MLP), a lipid, a reversibly modified polymer or peptide, or a reversibly modified membrane active polyamine.

[0142] In some embodiments, the RNAi agent can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs, or other delivery systems available in the art for nucleic acid delivery. The RNAi agent can also be chemically conjugated to targeting groups, lipids (including but not limited to cholesteryl and cholesteryl derivatives), encapsulated in nanoparticles, liposomes, micelles, conjugated to polymers or DPCs (see, for example, WO 2000 / 053722, WO 2008 / 022309, WO 2011 / 104169, and WO 2012 / 083185, WO 2013 / 032829, WO 2013 / 158141, each of which is incorporated herein by reference) by iontophoresis or other delivery vehicles or systems available in the art, such as hydrogels, cyclodextrins, biodegradable nanocapsules, bioadhesive microspheres, or proteinaceous vectors. In some embodiments, the RNAi agent can be conjugated to an antibody with affinity for lung epithelial cells. In some embodiments, the RNAi agent can be linked to a targeting ligand that has affinity for pulmonary epithelial cells or receptors present on pulmonary epithelial cells.

[0143] Pharmaceutical Compositions and Formulations The MMP7 RNAi agents disclosed herein can be prepared as pharmaceutical compositions (alternatively referred to as pharmaceutical formulations or medicaments). The pharmaceutical compositions disclosed herein include at least one MMP7 RNAi agent. These pharmaceutical compositions are particularly useful for suppressing expression of MMP7 mRNA in a target cell, cell group, tissue, or organism. The pharmaceutical compositions can be used to treat a subject having a disease, disorder, or condition that would benefit from a reduction in the level of a target mRNA or suppression of expression of a target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease or disorder that would benefit from a reduction in the level of a target mRNA or suppression of expression of a target gene. In one embodiment, the method of the present invention includes administering to a subject to be treated an MMP7 RNAi agent linked to a targeting ligand as described herein. In some embodiments, one or more pharma- ceutical acceptable excipients (including vehicles, carriers, diluents, and / or delivery polymers) can be added to a pharmaceutical composition including an MMP7 RNAi agent, thereby forming a pharmaceutical formulation or medicament suitable for in vivo delivery to a subject, including a human.

[0144] Pharmaceutical compositions and methods comprising the MMP7 RNAi agents disclosed herein reduce the level of target mRNA in a cell, group of cells, group of cells, tissue, organ, or subject, such as by administering to a subject a therapeutically effective amount of the MMP7 RNAi agent described herein, thereby suppressing expression of MMP7 mRNA in the subject. In some embodiments, the subject has previously been identified or diagnosed as having a disease or disorder that may be mediated, at least in part, by reduced MMP7 expression. In some embodiments, the subject has previously been diagnosed as having one or more lung diseases, such as idiopathic pulmonary fibrosis (IPF), asthma (including severe asthma), acute respiratory distress syndrome, lung cancer, chronic inflammation, interstitial lung disease (ILD), or another type of fibrosis. In some embodiments, the subject has previously been diagnosed as having IPF.

[0145] The embodiments of the present disclosure include pharmaceutical compositions for delivering MMP7 RNAi agents to lung epithelial cells in vivo.Such pharmaceutical compositions can include, for example, MMP7 RNAi agents conjugated to targeting groups that include integrin targeting ligands.In some embodiments, the integrin targeting ligands are comprised of αvβ6 integrin ligands.

[0146] In some embodiments, pharmaceutical compositions comprising the disclosed MMP7 RNAi agents are used to treat or manage clinical conditions in subjects who would benefit from suppression of MMP7 expression. In some embodiments, a therapeutically or prophylactically effective amount of one or more pharmaceutical compositions is administered to a subject in need of such treatment. In some embodiments, administration of any of the disclosed MMP7 RNAi agents can be used to reduce the number, severity, and / or frequency of disease symptoms in a subject.

[0147] In some embodiments, the MMP7 RNAi agent described is optionally combined with one or more additional (i.e., second, third, etc.) therapeutic agents. The second therapeutic agent can be another MMP7 RNAi agent (e.g., an MMP7 RNAi agent that targets a different sequence in the MMP7 gene). In some embodiments, the second therapeutic agent can be an RNAi agent that targets the MMP7 gene. The additional therapeutic agent can also be a small molecule drug, an antibody, an antibody fragment, and / or an aptamer. The MMP7 RNAi agent can be combined with one or more excipients, with or without one or more additional therapeutic agents, to form a pharmaceutical composition.

[0148] The pharmaceutical compositions comprising the MMP7 RNAi agents described herein can be used to treat at least one symptom of a subject with a disease or disorder that would benefit from reduced or suppressed expression of MMP7 mRNA. In some embodiments, the subject is administered a therapeutically effective amount of a pharmaceutical composition comprising one or more MMP7 RNAi agents, thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more MMP7 RNAi agents, thereby preventing or suppressing at least one symptom.

[0149] In some embodiments, one or more of the described MMP7 RNAi agents are administered to a mammal in a pharma- ceutically acceptable carrier or diluent. In some embodiments, the mammal is a human.

[0150] The route of administration is the route by which the MMP7 RNAi agent is brought into contact with the body. In general, methods of administering drugs, oligonucleotides, and nucleic acids for the treatment of mammals are well known in the art and can be applied to the administration of the compositions described herein. The MMP7 RNAi agents disclosed herein can be administered via any suitable route with formulations appropriately tailored to suit the particular route. Thus, in some embodiments, the pharmaceutical compositions described herein are administered via inhalation, intranasal administration, intratracheal administration, or oropharyngeal aspiration administration. In some embodiments, the pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intradermally, subcutaneously, intraarticularly, intraocularly, or intraperitoneally, or topically.

[0151] Pharmaceutical compositions comprising the MMP7 RNAi agents described herein can be delivered to a cell, group of cells, tissue, or subject using oligonucleotide delivery techniques known in the art. In general, any suitable art-recognized method for delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the compositions described herein. For example, delivery can be by local administration (e.g., direct injection, implantation, or topical administration), systemic administration, or by subcutaneous, intravenous, intraperitoneal, or parenteral routes (including intracranial (e.g., intraventricular, intraparenchymal, and intrathecal)), intramuscular, transdermal, airway (aerosol), nasal, oral, rectal, or topical (including buccal and sublingual) administration. In some embodiments, the compositions are administered by inhalation, intranasal administration, oropharyngeal aspiration, or intratracheal administration.

[0152] For example, in some embodiments, the MMP7 RNAi agents described herein suppress expression of the MMP7 gene in pulmonary epithelium, for which administration by inhalation (e.g., by an inhaler device such as a metered dose inhaler, or a nebulizer, such as a jet or vibrating mesh nebulizer, or a soft mist inhaler) is particularly suitable and advantageous.

[0153] In some embodiments, the pharmaceutical compositions described herein include one or more pharma- ceutical acceptable excipients. The pharmaceutical compositions described herein are formulated for administration to a subject.

[0154] As used herein, a pharmaceutical composition comprises a pharmacologically effective amount of at least one of the therapeutic compounds described and one or more pharma- ceutically acceptable excipients. A pharma-ceutically acceptable excipient (excipient) is a substance other than an active pharmaceutical ingredient (API, therapeutic product, e.g., MMP7 RNAi agent) that is intentionally included in a drug delivery system. The excipient does not exert, or is not intended to exert, a therapeutic effect at the intended dose. The excipient may act to a) aid in the processing of the drug delivery system during manufacture, b) protect, support, or enhance the stability, bioavailability, or patient acceptability of the API, c) aid in product identification, and / or d) enhance any other attribute of the overall safety, efficacy, or delivery of the API during storage or use. A pharma-ceutically acceptable excipient may or may not be an inert substance.

[0155] Excipients include, but are not limited to, absorption enhancers, anti-adherents, antifoaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavorings, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickeners, tonicity agents, vehicles, water repellents, and wetting agents.

[0156] Pharmaceutical compositions suitable for use in injection include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0157] Sterile injection solution can be prepared by incorporating active compound in the required amount into suitable solvent with one or combination of the above-listed components, and then sterilizing by filtration if necessary.Generally, dispersion is prepared by incorporating active compound into a sterile vehicle that contains basic dispersion medium and other necessary components from above-listed components.In the case of sterile powder for preparing sterile injection solution, the preparation method includes vacuum drying and freeze-drying, which obtains powder of active ingredient and any additional desired ingredients from the solution that has been previously sterilized and filtered.

[0158] Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug, which can be in microcrystalline form, for example an aqueous microcrystalline suspension. Liposomal formulations or biodegradable polymer systems can also be used to present the drug for both intra-articular and ophthalmic administration.

[0159] The formulation suitable for inhalation administration can be prepared by incorporating the active compound in a desired amount in a suitable solvent, followed by sterile filtration. In general, the formulation suitable for inhalation administration is a sterile solution at physiological pH and has low viscosity (<5 cP). Salts can be added to balance tonicity. In some cases, surfactants or co-solvents can be added to increase the solubility of the active compound and improve aerosol properties. In some cases, excipients can be added to adjust viscosity to ensure the size and distribution of nebulized droplets.

[0160] In some embodiments, pharmaceutical formulations comprising an MMP7 RNAi agent disclosed herein suitable for inhaled administration can be prepared in water for injection (sterile water) or aqueous sodium phosphate buffer (e.g., an MMP7 RNAi agent formulated in 0.5 mM sodium phosphate monobasic, 0.5 mM sodium phosphate dibasic in water).

[0161] The active compound can be prepared with a carrier that will protect the compound from rapid excretion from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, etc. can be used. Methods for preparing such formulations will be clear to those skilled in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

[0162] MMP7 RNAi agent can be formulated into a composition in the form of dosage unit so that it can be easily administered and the dosage is uniform.Dosage unit form means a physically separate unit suitable for single dosage to the subject to be treated, each unit containing a predetermined amount of active compound calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form of the present disclosure is determined and directly depends on the inherent characteristics of active compound and therapeutic effect to be achieved, and the inherent limitation of the technology of compounding such active compound for individual treatment.

[0163] The pharmaceutical composition may contain other additional ingredients commonly found in pharmaceutical compositions. Such additional ingredients include, but are not limited to, antipruritic agents, astringents, local anesthetics, or anti-inflammatory agents (e.g., antihistamines, diphenhydramine, etc.). It is also envisioned that cells, tissues, or isolated organs expressing or containing the RNAi agent defined herein may be used as a "pharmaceutical composition". As used herein, "pharmacologically effective amount", "therapeutically effective amount", or simply "effective amount" refers to the amount of RNAi agent to produce a pharmacological, therapeutic, or preventive result.

[0164] In some embodiments, the methods disclosed herein further comprise administering a second therapeutic agent or treatment in addition to administering an RNAi agent disclosed herein. In some embodiments, the second therapeutic agent is another MMP7 RNAi agent (e.g., an MMP7 RNAi agent that targets a different sequence within the MMP7 target). In other embodiments, the second therapeutic agent can be a small molecule drug, an antibody, an antibody fragment, and / or an aptamer.

[0165] In some embodiments, compositions are described herein that include a combination or cocktail of at least two MMP7 RNAi agents with different sequences. In some embodiments, each of the two or more MMP7 RNAi agents is linked separately and independently to a targeting group. In some embodiments, each of the two or more MMP7 RNAi agents is linked to a targeting group that includes or consists of an integrin targeting ligand. In some embodiments, each of the two or more MMP7 RNAi agents is linked to a targeting group that includes or consists of an αvβ6 integrin targeting ligand.

[0166] Described herein are compositions for delivering MMP7 RNAi agents to lung epithelial cells.Furthermore, generally described herein are compositions for delivering MMP7 RNAi agents in vivo to cells, including renal epithelial cells and / or epithelial cells of the GI or genital tract and / or ocular surface epithelial cells of the eye.

[0167] In general, an effective amount of the MMP7 RNAi agent disclosed herein will be in the range of about 0.0001 to about 20 mg / kg body weight / deposition dose, for example, about 0.001 to about 5 mg / kg body weight / deposition dose. In some embodiments, an effective amount of the MMP7 RNAi agent will be in the range of about 0.01 mg / kg to about 3.0 mg / kg body weight / deposition dose. In some embodiments, an effective amount of the MMP7 RNAi agent will be in the range of about 0.03 mg / kg to about 2.0 mg / kg body weight / deposition dose. In some embodiments, an effective amount of the MMP7 RNAi agent will be in the range of about 0.01 to about 1.0 mg / kg body weight / deposition dose. In some embodiments, an effective amount of the MMP7 RNAi agent will be in the range of about 0.50 to about 1.0 mg / kg body weight / deposition dose. Calculation of the lung deposited dose (PDD) is performed according to methods known in the art (see Wolff RK, Dorato MA, Toxicologic Testing of Inhaled Pharmaceutical Aerosols, Crit Rev Toxicol., 1993; 23(4):343-369; Tepper et al., International J. Toxicology, 2016, vol. 35(4):376-392). The amount administered will also likely depend on variables such as the patient's overall health, the relative biological potency of the compound being delivered, the drug formulation, the presence and type of excipients in the formulation, and the route of administration. It should also be understood that the initial dose administered can be increased beyond the upper levels listed above to rapidly achieve the desired blood or tissue levels, or the initial dose can be less than the optimal dose. In some embodiments, administration is administered daily. In some embodiments, administration is administered weekly. In further embodiments, administration is administered once every two weeks, once every three weeks, once a month, or once a quarter (ie, once every three months).

[0168] For the treatment of a disease or to form a medicament or composition for the treatment of a disease, the pharmaceutical compositions described herein comprising an MMP7 RNAi agent can be combined with an excipient or with a second therapeutic agent or treatment, including, but not limited to, a second or other RNAi agent, a small molecule drug, an antibody, an antibody fragment, a peptide, and / or an aptamer.

[0169] The described MMP7 RNAi agents, when added to a pharma- ceutically acceptable excipient or adjuvant, can be packaged in a kit, container, pack, or dispenser. The pharmaceutical compositions described herein can be packaged in a dry powder or aerosol inhaler, other metered dose inhaler, nebulizer, pre-filled syringe, or vial.

[0170] Methods of Treatment and Inhibition of MMP7 Expression The MMP7 RNAi agents disclosed herein can be used to treat a subject (e.g., a human or other mammal) having a disease or disorder that would benefit from administration of the RNAi agent. In some embodiments, the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) that would benefit from a reduction and / or inhibition of MMP7 mRNA expression and / or a reduction in MMP7 enzyme levels.

[0171] In some embodiments, the RNAi agents disclosed herein can be used to treat a subject (e.g., a human) having a disease or disorder in which the subject would benefit from a reduction in MMP7 enzyme levels, including, but not limited to, idiopathic pulmonary fibrosis (IPF), asthma, various other types of fibrosis, chronic inflammation, interstitial lung disease (ILD), infectious diseases (e.g., SARS-COV-2), acute lung injury (e.g., acute respiratory distress syndrome (ARDS)), pulmonary hypertension, various cancers, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), fatty liver disease, biliary atresia, and chronic kidney disease (CKD). In some embodiments, the disease is IPF. In some embodiments, the subject has previously been diagnosed with IPF, asthma, ILD, ARDS, or another type of fibrosis. Treatment of the subject can include curative and / or prophylactic treatment. The subject is administered a therapeutically effective amount of any one or more of the MMP7 RNAi agents described herein. The subject can be a human, a patient, or a human patient. The subject can be an adult, an adolescent, a child, or an infant. Administration of the pharmaceutical compositions described herein can be to a human or an animal.

[0172] Increased membrane MMP7 enzyme levels are known to contribute to abnormalities in the function of epithelial cells, fibroblasts, and immune cells, and are particularly associated with fibrosis of lung tissue and cells. In some embodiments, the described MMP7 RNAi agents are used to treat at least one condition in a subject that is at least partially mediated by a decrease in MMP7 enzyme levels. The subject is administered a therapeutically effective amount of any one or more of the described MMP7 RNAi agents. In some embodiments, the subject is administered a prophylactically effective amount of any one or more of the described RNAi agents, thereby treating the subject by preventing or suppressing at least one condition.

[0173] In certain embodiments, the disclosure provides methods for treating a disease, disorder, condition, or pathological state mediated at least in part by MMP7 gene expression in a patient in need thereof, comprising administering to the patient any of the MMP7 RNAi agents described herein.

[0174] In some embodiments, the MMP7 RNAi agent is used to treat or manage a clinical condition or pathological condition in a subject, the clinical condition or pathological condition being mediated, at least in part, by reduced MMP7 expression. The subject is administered a therapeutically effective amount of one or more MMP7 RNAi agents or compositions comprising an MMP7 RNAi agent described herein. In some embodiments, the method of the invention comprises administering a composition comprising an MMP7 RNAi agent described herein to the subject to be treated.

[0175] In a further aspect, the disclosure features a method of treating (including prophylactic or preventative treatment) a disease or condition that can be addressed by reducing MMP7 enzyme levels, comprising administering to a subject in need thereof an MMP7 RNAi agent that includes an antisense strand that includes any of the sequences in Table 2, Table 3, or Table 10. Additionally, compositions for use in such methods are described herein.

[0176] The described MMP7 RNAi agents and / or compositions comprising the MMP7 RNAi agents can be used in methods for the curative treatment of diseases or conditions caused by enhanced or elevated levels of the MMP7 enzyme. Such methods include administration of the MMP7 RNAi agents described herein to a subject, e.g., a human or animal subject.

[0177] In another aspect, the disclosure provides a method for the treatment (including prophylactic treatment) of a pathological condition (such as a pathology or disease) mediated at least in part by MMP7 expression, the method comprising administering to a subject a therapeutically effective amount of an RNAi agent comprising an antisense strand comprising any of the sequences of Table 2, Table 3, or Table 10.

[0178] In some embodiments, disclosed herein is a method for inhibiting expression of the MMP7 gene, comprising administering to a cell an RNAi agent comprising an antisense strand comprising any of the sequences in Table 2, Table 3, or Table 10.

[0179] In some embodiments, disclosed herein is a method for the treatment (including prophylactic treatment) of a pathological condition mediated at least in part by MMP7 expression, the method comprising administering to a subject a therapeutically effective amount of an RNAi agent comprising a sense strand comprising any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 10.

[0180] In some embodiments, disclosed herein is a method for inhibiting expression of the MMP7 gene, comprising administering to a cell an RNAi agent comprising a sense strand comprising any of the sequences in Table 2, Table 4, Table 5, Table 6, or Table 10.

[0181] In some embodiments, disclosed herein is a method for the treatment (including prophylactic treatment) of a pathological condition mediated at least in part by MMP7 expression, the method comprising administering to a subject a therapeutically effective amount of an RNAi agent comprising a sense strand comprising any of the sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand comprising any of the sequences in Table 3 or Table 10.

[0182] In some embodiments, disclosed herein is a method for inhibiting expression of the MMP7 gene, comprising administering to a cell an RNAi agent comprising a sense strand comprising any of the sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand comprising any of the sequences in Table 3 or Table 10.

[0183] In some embodiments, disclosed herein are methods of inhibiting expression of the MMP7 gene, comprising administering to a subject an MMP7 RNAi agent comprising a sense strand consisting of a nucleobase sequence consisting of any of the sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand consisting of a nucleobase sequence consisting of any of the sequences in Table 3 or Table 10. In other embodiments, disclosed herein are methods of inhibiting expression of the MMP7 gene, comprising administering to a subject an MMP7 RNAi agent comprising a sense strand consisting of a modified sequence consisting of any of the modified sequences in Table 4, Table 5, Table 6, or Table 10, and an antisense strand consisting of a modified sequence consisting of any of the modified sequences in Table 3 or Table 10.

[0184] In some embodiments, disclosed herein are methods for suppressing expression of the MMP7 gene in a cell, comprising administering one or more MMP7 RNAi agents comprising one of the duplex structures listed in Tables 7A, 7B, 8, 9, and 10.

[0185] In some embodiments, MMP7 gene expression levels and / or MMP7 mRNA levels in certain lung epithelial cells of a subject to which a described MMP7 RNAi agent is administered are reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% compared to the respective levels in the subject prior to administration of the MMP7 RNAi agent or another subject not administered the MMP7 RNAi agent. In some embodiments, the MMP7 enzyme level or circulating MMP7 enzyme level in a particular epithelial cell of a subject to which a described MMP7 RNAi agent is administered is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% compared to the subject prior to administration of the MMP7 RNAi agent or another subject not administered the MMP7 RNAi agent. The subject's gene expression level, enzyme or protein level, and / or mRNA level may be reduced in a cell, group of cells, serum, and / or tissue of the subject. In some embodiments, MMP7 enzyme levels in a particular subject administered a described MMP7 RNAi agent are reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 98% compared to the subject prior to administration of the MMP7 RNAi agent or to another subject not administered the MMP7 RNAi agent.

[0186] The reduction of gene expression, mRNA, and enzyme or protein levels can be evaluated by any method known in the art. The reduction or decrease of MMP7 enzyme levels or MMP7 mRNA levels may be collectively referred to as the reduction, decrease, or suppression of MMP7 gene expression. The examples described herein illustrate known methods for evaluating MMP7 suppression.

[0187] Cells, tissues, organs, and non-human organisms Contemplated are cells, tissues, organs, and non-human organisms that contain at least one of the MMP7 RNAi agents described herein. The cells, tissues, organs, or non-human organisms are produced by delivering the RNAi agent to the cells, tissues, organs, or non-human organisms.

[0188] Further exemplary embodiments Certain further exemplary embodiments of the disclosed technology are provided herein, which are merely exemplary and are not intended to limit the scope of the disclosure or the claims appended hereto.

[0189] Embodiment 1. An RNAi agent for suppressing expression of matrix metallopeptidase 7 gene, comprising: an antisense strand comprising at least 17 contiguous nucleotides that differ from any one of the sequences shown in Table 2 or Table 3 by 0 or 1 nucleotide; and A sense strand that contains a nucleotide sequence that is at least partially complementary to the antisense strand. 16. An RNAi agent comprising:

[0190] Embodiment 2. The RNAi agent of embodiment 1, wherein the antisense strand comprises nucleotides 2 to 18 of any one of the sequences shown in Table 2 or Table 3.

[0191] Embodiment 3. The RNAi agent of embodiment 1 or embodiment 2, wherein the sense strand comprises a nucleotide sequence of at least 17 contiguous nucleotides that differs by 0 or 1 nucleotide from any one of the sequences set forth in Table 2 or Table 4, and the sense strand has a region of at least 85% complementarity over the 17 contiguous nucleotides to the antisense strand.

[0192] Embodiment 4. The RNAi agent of any one of embodiments 1-3, wherein at least one nucleotide of the MMP7 RNAi agent is a modified nucleotide or comprises a modified internucleoside linkage.

[0193] Embodiment 5. The RNAi agent of any one of embodiments 1-4, wherein all or substantially all of the nucleotides are modified nucleotides.

[0194] Embodiment 6. The RNAi agent of any one of embodiments 4-5, wherein the modified nucleotide is selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-seco-nucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted 2'-O-methyl nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, vinyl phosphonate-containing nucleotides, cyclopropyl phosphonate-containing nucleotides, and 3'-O-methyl nucleotides.

[0195] Embodiment 7. The RNAi agent of embodiment 5, wherein all or substantially all of the nucleotides are modified with 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or a combination thereof.

[0196] Embodiment 8 The RNAi agent of any one of embodiments 1 to 7, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences shown in Table 3.

[0197] Embodiment 9 The RNAi agent of any one of embodiments 1 to 8, wherein the sense strand comprises a nucleotide sequence of any one of the modified sequences shown in Table 4.

[0198] Embodiment 10. The RNAi agent of embodiment 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences shown in Table 3, and the sense strand comprises the nucleotide sequence of any one of the modified sequences shown in Table 4.

[0199] Embodiment 11 The RNAi agent of any one of embodiments 1 to 10, wherein the sense strand is 18 to 30 nucleotides in length and the antisense strand is 18 to 30 nucleotides in length.

[0200] Embodiment 12. The RNAi agent of embodiment 11, wherein the sense strand and the antisense strand are each 18 to 27 nucleotides in length.

[0201] Embodiment 13. The RNAi agent of embodiment 12, wherein the sense strand and the antisense strand are each 18 to 24 nucleotides in length.

[0202] Embodiment 14 The RNAi agent of embodiment 13, wherein the sense strand and the antisense strand are each 21 nucleotides in length.

[0203] Embodiment 15 The RNAi agent of embodiment 14, wherein the RNAi agent has two blunt ends.

[0204] Embodiment 16 The RNAi agent of any one of embodiments 1 to 15, wherein the sense strand comprises one or two terminal caps.

[0205] Embodiment 17 The RNAi agent of any one of embodiments 1 to 16, wherein the sense strand comprises one or two inverted abasic residues.

[0206] Embodiment 18 The RNAi agent of embodiment 1, which is composed of a sense strand and an antisense strand that form a duplex having the structure of any one of the duplexes in Table 7A, Table 7B, Table 8, Table 9, or Table 10.

[0207] Embodiment 19. The RNAi agent of embodiment 18, wherein all or substantially all of the nucleotides are modified nucleotides.

[0208] Embodiment 20. The following nucleotide sequence (5' to 3'):

[0209] [ka]

[0210] The RNAi agent of embodiment 1, comprising an antisense strand consisting of a nucleotide sequence that differs by 0 or 1 nucleotide from one of the above, an antisense strand consisting essentially of a nucleotide sequence that differs by 0 or 1 nucleotide, or an antisense strand that includes a nucleotide sequence that differs by 0 or 1 nucleotide.

[0211] Embodiment 21. The sense strand has the following nucleotide sequence (5'→3'):

[0212] [ka]

[0213] 21. The RNAi agent of embodiment 20, which consists of, consists essentially of, or comprises a nucleotide sequence which differs by 0 or 1 nucleotide from one of:

[0214] Embodiment 22 The RNAi agent of embodiment 20 or 21, wherein all or substantially all of the nucleotides are modified nucleotides.

[0215] Embodiment 23. The following nucleotide sequence (5' to 3'):

[0216] [ka]

[0217] an antisense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the above, an antisense strand consisting of a modified nucleotide sequence that differs by 0 or 1 nucleotide, or an antisense strand that essentially consists of a modified nucleotide sequence that differs by 0 or 1 nucleotide, The RNAi agent of embodiment 1, wherein in the sequences a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; cPrpu represents 5'-phosphonate cyclopropyl-2'-O-methyl uridine; s represents a phosphorothioate linkage; and all or substantially all of the nucleotides of the sense strand are modified nucleotides.

[0218] Embodiment 24. The sense strand has the following nucleotide sequence (5'→3'): [ka]

[0219] a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of The RNAi agent of embodiment 1, wherein in the sequences a, c, g, i, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, 2'-O-methyl inosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate linkage; and all or substantially all of the nucleotides of the antisense strand are modified nucleotides.

[0220] Embodiment 25 The RNAi agent of any one of embodiments 20 to 24, wherein the sense strand further comprises an inverted abasic residue at the 3' end of the nucleotide sequence, at the 5' end of the nucleotide sequence, or both.

[0221] Embodiment 26 The RNAi agent of any one of embodiments 1 to 25, wherein the RNAi agent is linked to a targeting ligand.

[0222] Embodiment 27 The RNAi agent of embodiment 26, wherein the targeting ligand has affinity for a cellular receptor expressed on epithelial cells.

[0223] Embodiment 28 The RNAi agent of embodiment 27, wherein the targeting ligand comprises an integrin targeting ligand.

[0224] Embodiment 29 The RNAi agent of embodiment 28, wherein the integrin targeting ligand is an αvβ6 integrin targeting ligand.

[0225] Embodiment 30. The targeting ligand has the structure:

[0226] [ka]

[0227] or a pharma- ceutically acceptable salt thereof, or

[0228] [ka]

[0229] or a pharma- ceutically acceptable salt thereof Including, During the ceremony,

[0230] [ka]

[0231] 30. The RNAi agent of embodiment 29, wherein indicates a point of attachment to the RNAi agent.

[0232] Embodiment 31. The targeting ligand comprises:

[0233] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0234] having a structure selected from the group consisting of During the ceremony,

[0235] [ka]

[0236] 30. The RNAi agent of any one of embodiments 26 to 29, wherein indicates a point of attachment to the RNAi agent.

[0237] Embodiment 32. The following structure:

[0238] [ka]

[0239] 32. The RNAi agent of embodiment 31, wherein the RNAi agent is conjugated to a targeting ligand having the following structure:

[0240] Embodiment 33. The targeting ligand has the structure:

[0241] [ka]

[0242] 30. The RNAi agent of any one of embodiments 26 to 29, comprising:

[0243] Embodiment 34 The RNAi agent of any one of embodiments 26 to 33, wherein the targeting ligand is conjugated to the sense strand.

[0244] Embodiment 35 The RNAi agent of embodiment 34, wherein the targeting ligand is conjugated to the 5' end of the sense strand.

[0245] Embodiment 36 A composition comprising the RNAi agent of any one of embodiments 1 to 35, further comprising a pharma- ceutically acceptable excipient.

[0246] Embodiment 37 The composition of embodiment 36, further comprising a second RNAi agent capable of suppressing expression of matrix metallopeptidase 7 gene expression.

[0247] Embodiment 38. The composition of any one of embodiments 36-37, further comprising one or more additional therapeutic agents.

[0248] Embodiment 39. The composition of any one of embodiments 36-38, which is formulated for administration by inhalation.

[0249] Embodiment 40. The composition of embodiment 39, delivered by a metered dose inhaler, a jet nebulizer, a vibrating mesh nebulizer, or a soft mist inhaler.

[0250] Embodiment 41 The composition of any of embodiments 36-40, wherein the RNAi agent is a sodium salt.

[0251] Embodiment 42. The composition of any of embodiments 36-41, wherein the pharma- ceutically acceptable excipient is water for injection.

[0252] Embodiment 43 The composition of any of embodiments 36-41, wherein the pharma- ceutically acceptable excipient is buffered saline.

[0253] Embodiment 44. A method for suppressing expression of the MMP7 gene in a cell, comprising introducing into the cell an effective amount of an RNAi agent of any one of embodiments 1 to 33 or a composition of any one of embodiments 36 to 43.

[0254] Embodiment 45 The method of embodiment 44, wherein the cell is present in a subject.

[0255] Embodiment 46 The method of embodiment 45, wherein the subject is a human subject.

[0256] Embodiment 47. The method of any one of embodiments 44-46, wherein after administration of the RNAi agent, matrix metallopeptidase 7 gene expression is suppressed by at least about 30%.

[0257] Embodiment 48. A method for treating one or more symptoms or diseases associated with enhanced or elevated membrane MMP7 activity levels, comprising administering a therapeutically effective amount of the composition of any one of embodiments 36 to 43 to a human subject in need thereof.

[0258] Embodiment 49. The method of embodiment 48, wherein the disease is a respiratory or pulmonary disease.

[0259] Embodiment 50. The method of embodiment 48, wherein the disease is selected from the group consisting of idiopathic pulmonary fibrosis (IPF), another type of pulmonary fibrosis, asthma, chronic inflammation, interstitial lung disease (ILD), SARS-COV-2 or another type of infection of the airways, acute respiratory distress syndrome (ARDS) or another type of acute lung injury, pulmonary hypertension, lung cancer, nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), fatty liver disease, biliary atresia, and chronic kidney disease (CKD).

[0260] Embodiment 51. The method of embodiment 50, wherein the disease is idiopathic pulmonary fibrosis (IPF).

[0261] Embodiment 54. The method of any one of embodiments 44-51, wherein the RNAi agent is administered at a deposition dose of about 0.01 mg / kg to about 5.0 mg / kg of the subject's body weight.

[0262] Embodiment 55. The method of any one of embodiments 44-54, wherein the RNAi agent is administered at a deposition dose of about 0.03 mg / kg to about 2.0 mg / kg of the subject's body weight.

[0263] Embodiment 56 The method of any one of embodiments 44-55, wherein the RNAi agent is administered in two or more doses.

[0264] Embodiment 57. Use of the RNAi agent of any one of embodiments 1 to 35 for the treatment of a disease, disorder, or condition mediated at least in part by membrane MMP7 activity and / or MMP7 gene expression.

[0265] Embodiment 58. Use of a composition according to any one of embodiments 36 to 43 for the treatment of a disease, disorder or condition mediated at least in part by matrix metallopeptidase 7 activity and / or matrix metallopeptidase 7 gene expression.

[0266] Embodiment 59. Use of a composition described in any one of embodiments 36 to 43 for the manufacture of a medicament for the treatment of a disease, disorder, or condition mediated at least in part by matrix metallopeptidase 7 and / or matrix metallopeptidase 7 gene expression.

[0267] Embodiment 60. The use of any one of embodiments 57 to 59, wherein the disease is pulmonary inflammation.

[0268] Embodiment 61 A method of making an RNAi agent of any one of embodiments 1 to 35, comprising annealing a sense strand and an antisense strand to form a double-stranded ribonucleic acid molecule.

[0269] Embodiment 62 The method of embodiment 61, wherein the sense strand comprises a targeting ligand.

[0270] Embodiment 63 The method of embodiment 62, comprising conjugating a targeting ligand to the sense strand.

[0271] The above-illustrated embodiments and items will now be illustrated by the following non-limiting examples. EXAMPLES

[0272] Example 1. Synthesis of MMP7 RNAi Agents The MMP7 RNAi agent duplexes disclosed herein were synthesized as follows.

[0273] A. Synthesis The sense and antisense strands of the MMP7 RNAi agents were synthesized according to the solid-phase phosphoramidite technique used for oligonucleotide synthesis. Depending on the scale, MerMade96E® (Bioautomation), MerMadel2® (Bioautomation), or OP Pilot100 (GE Healthcare) were used. Synthesis was performed on a solid support made of controlled pore glass (CPG, 500 Å or 600 Å, obtained from Prime Synthesis, Aston, PA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA). Specifically, the 2'-O-methyl phosphoramidites used included the following: (5'-O-dimethoxytrityl-N 6 -(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, 5'-O-dimethoxy-trityl-N 4-(Acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N,N-diisopropyl-amino) phosphoramidite, (5'-O-dimethoxytrityl-N 2 -(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite, and 5'-O-dimethoxytrityl-2'-O-methyl-uridine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluoro-phosphoramidite had the same protecting groups as the 2'-O-methyl RNA amidite. The 5'-dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from Glen Research (VA). Inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N,N-diisopropylamino) phosphoramidite was purchased from ChemGenes (Wilmington, MA, USA). The following UNA phosphoramidite was used: 5'-(4,4'-dimethoxytrityl)-N 6-(Benzoyl)-2',3'-seco-adenosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4'-dimethoxytrityl)-N-acetyl-2',3'-seco-cytosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, 5'-(4,4' The following linkers were used: 5'-(4,4'-dimethoxytrityl)-N-isobutyryl-2',3'-seco-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-dimethoxytrityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N,N-diisopropyl)]-phosphoramidite. TFA AminoLink phosphoramidites were also purchased commercially (Thermo Fisher). Linker L6 was purchased from BroadPharm as propargyl-PEG5-NHS (catalog no. BP-20907) and coupled to the NH2-C6 group of AminoLink phosphoramidite to form -L6-C6- using standard coupling conditions. The linker Alk-cyHex was also purchased commercially (alkyne phosphoramidite, 5' end) as a propargyl-containing compound phosphoramidite compound from Lumiprobe to form the linker-Alk-cyHex-. In both cases, the phosphorothioate bond was introduced as specified using the conditions described herein. The phosphonic acid cyclopropyl phosphoramidite was synthesized according to International Patent Application Publication No. WO 2017 / 214112 (see also Altenhofer et. al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)).

[0274] Trialkine-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while other amidites were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as activator solutions. Coupling times were 10 min (RNA), 90 s (2'O-Me), and 60 s (2'F). To introduce phosphorothioate bonds, a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, Massachusetts, USA) in anhydrous acetonitrile was used.

[0275] Alternatively, the trialkyne moiety was introduced post-synthetically (see section E below). In this route, the sense strand was functionalized with 5'- and / or 3'-terminal nucleotides containing primary amines. TFA Aminolink phosphoramidites were dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 Å) were added. 5-benzylthio-1H-tetrazole (BTT, 250 mM in acetonitrile) or 5-ethylthio-1H-tetrazole (ETT, 250 mM in acetonitrile) were used as activator solutions. Coupling times were 10 min (RNA), 90 s (2'O-Me), and 60 s (2'F). To introduce phosphorothioate bonds, a 100 mM solution of 3-phenyl-1,2,4-dithiazolin-5-one (POS, obtained from PolyOrg, Inc., Leominster, Massachusetts, USA) in anhydrous acetonitrile was used.

[0276] B. Cleavage and Deprotection of Support-Bound Oligomers After completion of solid-phase synthesis, the dried solid support was treated with a 1:1 volumetric solution of 40 wt% methylamine and 28%-31% ammonium hydroxide solution (Aldrich) in water for 1.5 h at 30° C. The solution was evaporated and the solid residue was dissolved in water (see below).

[0277] C. Purification Crude oligomers were purified by anion-exchange HPLC using a TSKgel SuperQ-5PW 13 μm column and a Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0, containing 20% ​​acetonitrile, and buffer B was the same as buffer A plus 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled and subjected to size-exclusion HPLC using a GE Healthcare XK 16 / 40 column packed with Sephadex G-25 fine, with a running buffer of 100 mM ammonium bicarbonate, pH 6.7, 20% acetonitrile or filtered water. Alternatively, pooled fractions were desalted and exchanged into the appropriate buffer or solvent system via tangential flow filtration.

[0278] D. Annealing Complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in 1x PBS (phosphate buffered saline, 1x, Corning, Cellgro) to form RNAi agents. Some RNAi agents were lyophilized and stored at -15 to -25°C. The concentration of the duplex was determined by measuring the absorbance of the solution in 1x PBS with a UV-Vis spectrometer. The absorbance of the solution at 260 nm was then multiplied by a conversion factor (0.050 mg / (mL-cm)) and the dilution factor to determine the concentration of the duplex.

[0279] E. Conjugation of Trialkine Linkers In some embodiments, the trialkine linker is conjugated to the sense strand of the RNAi agent on the resin as a phosphoramidite (see Example 1G for synthesis of an exemplary trialkine linker phosphoramidite, and Example 1A for conjugation of the phosphoramidite). In other embodiments, the trialkine linker may be conjugated to the sense strand after cleavage from the resin, as described below: Either before or after annealing, in some embodiments, the 5' or 3' amine-functionalized sense strand is conjugated to the trialkine linker. Exemplary trialkine linker structures that can be used to form the constructs disclosed herein are as follows:

[0280] [ka]

[0281] To conjugate the trialkyne linker to the annealed duplexes, the amine-functionalized duplexes were dissolved in 90% DMSO / 10% HO at ~50-70mg / mL. 40 equivalents of triethylamine were added, followed by 3 equivalents of trialkyne-PNP. Once complete, the conjugate was precipitated twice with a solvent system of 1x phosphate buffered saline / acetonitrile (1:14 ratio) and dried.

[0282] F. Synthesis of Targeting Ligand SM6.1 ((S)-3-(4-(4-(14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-1-yl)phenyl)-3-(2-(4-((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid)

[0283] [ka]

[0284] Compound 5 (tert-butyl(4-methylpyridin-2yl)carbamate) (0.501 g, 2.406 mmol, 1 equiv) was dissolved in DMF (17 mL). To the mixture was added NaH (0.116 mg, 3.01 mmol, 1.25 equiv, 60% dispersion in oil) and the mixture was stirred for 10 min before compound 20 (ethyl 4-bromobutyrate (0.745 g, 3.82 mmol, 0.547 mL)) (Sigma 167118) was added. After 3 h, the reaction was quenched with ethanol (18 mL) and concentrated. The concentrate was dissolved in DCM (50 mL), washed with saturated aqueous NaCl (1×50 mL), dried over Na2SO4, filtered, and concentrated. The product was purified on a silica column with a gradient of 0-5% methanol in DCM.

[0285] [ka]

[0286] Compound 21 (0.80 g, 2.378 mmol) was dissolved in 100 mL of acetone:0.1 M NaOH [1:1]. The reaction was monitored by TLC (5% ethyl acetate in hexanes). The organics were concentrated off and the residue was acidified to pH 3-4 with 0.3 M citric acid (40 mL). The product was extracted with DCM (3 x 75 mL). The organics were pooled, dried over Na2SO4, filtered and concentrated. The product was used without further purification.

[0287] [ka]

[0288] To a solution of compound 22 (1.1 g, 3.95 mmol, 1 equiv.), compound 45 (595 mg, 4.74 mmol, 1.2 equiv.) and TBTU (1.52 g, 4.74 mmol, 1.2 equiv.) in anhydrous DMF (10 mL) was added diisopropylethylamine (2.06 mL, 11.85 mmol, 3 equiv.) at 0° C. The reaction mixture was warmed to room temperature and stirred for 3 h. The reaction was quenched with saturated NaHCO3 solution (10 mL). The aqueous phase was extracted with ethyl acetate (3×10 mL) and the organic phases were combined, dried over anhydrous Na2SO4 and concentrated. The products were separated by CombiFlash® using silica gel as stationary phase. LC-MS: calculated [M+H]+ 366.20, found 367.

[0289] [ka]

[0290] To a solution of compound 61 (2 g, 8.96 mmol, 1 eq.) and compound 62 (2.13 mL, 17.93 mmol, 2 eq.) in anhydrous DMF (10 mL) was added K2CO3 (2.48 g, 17.93 mmol, 2 eq.) at 0° C. The reaction mixture was warmed to room temperature and stirred overnight. The reaction was quenched with water (10 mL). The aqueous phase was extracted with ethyl acetate (3×10 mL) and the organic phases were combined, dried over anhydrous Na2SO4 and concentrated. The products were separated by CombiFlash® using silica gel as stationary phase.

[0291] [ka]

[0292] To a solution of compound 60 (1.77 g, 4.84 mmol, 1 equiv.) in THF (5 mL) and H2O (5 mL) was added lithium hydroxide monohydrate (0.61 g, 14.53 mmol, 3 equiv.) portionwise at 0° C. The reaction mixture was allowed to warm to room temperature. After stirring at room temperature for 3 h, the reaction mixture was acidified to pH 3.0 with HCl (6N). The aqueous phase was extracted with ethyl acetate (3×20 mL) and the organic layers were combined, dried over Na2SO4 and concentrated. LC-MS: calculated [M+H]+ 352.18, found 352.

[0293] [ka]

[0294] To a solution of compound 63 (1.88 g, 6.0 mmol, 1.0 equiv) in anhydrous THF (20 mL) was added n-BuLi (3.6 mL, 9.0 mmol, 1.5 equiv) in hexanes dropwise at -78 °C. The reaction was kept at -78 °C for an additional 1 h. Triisopropyl borate (2.08 mL, 9.0 mmol, 1.5 equiv) was then added to the mixture at -78 °C. The reaction was then warmed to room temperature and stirred for an additional 1 h. The reaction was quenched with saturated NH4Cl solution (20 mL) and the pH was adjusted to 3. The aqueous phase was extracted with EtOAc (3 x 20 mL) and the organic phases were combined, dried over Na2SO4 and concentrated.

[0295] [ka]

[0296] Compound 12 (300 mg, 0.837 mmol, 1.0 equiv), compound 65 (349 mg, 1.256 mmol, 1.5 equiv), XPhos Pd G2 (13 mg, 0.0167 mmol, 0.02 equiv), and K3PO4 (355 mg, 1.675 mmol, 2.0 equiv) were mixed in a round bottom flask. The flask was sealed with a screw cap septum, then evacuated and backfilled with nitrogen (this process was repeated a total of three times). THF (8 mL) and water (2 mL) were then added via syringe. The mixture was bubbled with nitrogen for 20 min and the reaction was kept at room temperature overnight. The reaction was quenched with water (10 mL) and the aqueous phase was extracted with ethyl acetate (3 x 10 mL). The organic phase was dried over Na2SO4, concentrated and purified by CombiFlash® using silica gel as stationary phase and eluted with 15% EtOAc in hexane. LC-MS: calculated [M+H]+ 512.24, found 512.56.

[0297] [ka]

[0298] Compound 66 (858 mg, 1.677 mmol, 1.0 equiv) was cooled in an ice bath. HCl in dioxane (8.4 mL, 33.54 mmol, 20 equiv) was added to the flask. The reaction was allowed to warm to room temperature and stirred for an additional hour. The solvent was removed on a rotary evaporator and the product was used directly without further purification. LC-MS: calcd [M+H]+ 412.18, found 412.46

[0299] [ka]

[0300] To a solution of compound 64 (500 mg, 1.423 mmol, 1 equiv.), compound 67 (669 mg, 1.494 mmol, 1.05 equiv.), and TBTU (548 mg, 0.492 mmol, 1.2 equiv.) in anhydrous DMF (15 mL) was added diisopropylethylamine (0.744 mL, 4.268 mmol, 3 equiv.) at 0° C. The reaction mixture was warmed to room temperature and stirred for an additional hour. The reaction was quenched with saturated aqueous NaHCO3 (10 mL) and the product was extracted with ethyl acetate (3×20 mL). The organic phases were combined, dried over Na2SO4, and concentrated. The product was purified by CombiFlash® using silica gel as stationary phase and eluted with 3-4% methanol in DCM. The yield was 96.23%. LC-MS: Calculated value [M+H]+745.35, Found value 746.08

[0301] [ka]

[0302] To a solution of compound 68 (1.02 g, 1.369 mmol, 1 equiv) in ethyl acetate (10 mL) was added 10% Pd / C (0.15 g, 50% H2O) at room temperature. The reaction mixture was allowed to warm to room temperature and the reaction was monitored by LC-MS. The reaction was kept at room temperature overnight. The solid was filtered through Celite® and the solvent was removed on a rotary evaporator. The product was used directly without further purification. LC-MS: [M+H]+ 655.31, found 655.87

[0303] [ka]

[0304] To a solution of compound 69 (100 mg, 0.152 mmol, 1 equiv.) and azido-PEG5-OTs (128 mg, 0.305 mmol, 2 equiv.) in anhydrous DMF (2 mL) was added K2CO3 (42 mg, 0.305 mmol, 2 equiv.) at 0° C. The reaction mixture was stirred at 80° C. for 6 h. The reaction was quenched with saturated NaHCO3 solution and the aqueous layer was extracted with ethyl acetate (3×10 mL). The organic phases were combined, dried over Na2SO4 and concentrated. LC-MS: calculated [M+H]+ 900.40, found 901.46.

[0305] [ka]

[0306] To a solution of compound 72 (59 mg, 0.0656 mmol, 1.0 equiv) in THF (2 mL) and water (2 mL) was added lithium hydroxide (5 mg, 0.197 mmol, 3.0 equiv) at room temperature. The mixture was stirred for an additional 1 h at room temperature. The pH was acidified to 3.0 with HCl (6N) and the aqueous phase was extracted with EtOAc (3×10 mL). The organic phases were combined, dried over Na2SO4 and concentrated. TFA (0.5 mL) and DCM (0.5 mL) were added to the residue and the mixture was stirred for an additional 3 h at room temperature. The solvent was removed on a rotary evaporator. LC-MS: calculated [M+H]+ 786.37, found 786.95.

[0307] Synthesis of G.TriAik 14

[0308] TriAlk14 and (TriAlk14)s as shown in Table 11 above can be synthesized using the synthetic route shown below. Compound 14 can be added to the sense strand as a phosphoramidite using standard oligonucleotide synthesis techniques, or compound 22 can be conjugated to an amine-containing sense strand in an amide coupling reaction. [ka]

[0309] To a 3 L jacketed reactor was added 500 mL of DCM and 4 (75.0 g, 0.16 mol). The reaction was cooled to an internal temperature of 0° C. and TBTU (170.0 g, 0.53 mol) was added. The suspension was then treated dropwise with amine 5 (75.5 g, 0.53 mol) while maintaining the internal temperature below 5° C. The reaction was then slowly treated with DIPEA (72.3 g, 0.56 mol) while maintaining the internal temperature below 5° C. After the addition was complete, the reaction was warmed to 23° C. over 1 h and stirred for 3 h. A 10% kicker charge of all three reagents was added and stirred for an additional 3 h. The reaction was deemed complete when <1% of 4 remained. The reaction mixture was washed with saturated ammonium chloride solution (2×500 mL) and once with saturated aqueous sodium bicarbonate solution (500 mL). The organic layer was then dried over sodium sulfate and concentrated to an oil. The crude oil weighed 188 g and contained 72% 6 by QNMR. The crude oil was carried on to the next step. 46 H 60 N4O 11 Calculated mass = 845.0 m / z. Measured [M+H] = 846.0

[0310] [ka]

[0311] 121.2 g of the crude oil containing 72 wt% compound 6 (86.0 g, 0.10 mol) was dissolved in DMF (344 mL) and treated with TEA (86 mL, 20% v / v) while maintaining the internal temperature below 23 °C. The formation of dibenzofulvene (DBF) versus consumption of Fmoc-amine 6 was monitored by HPLC method 1 (Figure 2) and the reaction was complete within 10 h. Glutaric anhydride (12.8 g, 0.11 mol) was added to the solution and the intermediate amine 7 was converted to compound 8 within 2 h. Upon completion, DMF and TEA were removed under reduced pressure at 30 °C, resulting in 100 g of crude oil. Due to the high solubility of compound 7 in water, an aqueous workup could not be used. Chromatography was the only method to remove DBF, TMU, and glutaric anhydride. The crude oil (75 g) was purified in three runs on a Teledyne ISCO Combi-flash® purification system. The crude oil (25 g) was loaded onto a 330 g silica column and eluted with 0-20% methanol / DCM over 30 min, resulting in 42 g of compound 8 (54% yield over three steps). 36 H 55 N4O 12 Calculated mass = 736.4 m / z. Measured [M+H] = 737.0

[0312] [ka]

[0313] Compound 8 (42.0 g, 0.057 mol) was co-stripped with 10 volumes of acetonitrile to remove residual methanol from the chromatography solvent prior to use. The oil was redissolved in DMF (210 mL) and cooled to 0° C. The solution was treated with 4-nitrophenol (8.7 g, 0.063 mol) followed by EDC-hydrochloride (12.0 g, 0.063 mol) and found to reach completion within 10 hours. The solution was cooled to 0° C. and 10 volumes of ethyl acetate was added followed by 10 volumes of saturated ammonium chloride solution while maintaining the internal temperature below 15° C. The layers were separated and the ethyl acetate layer was washed with brine. The combined aqueous layers were extracted twice with 5 volumes of ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to an oil. The crude oil (55 g) was purified in three portions on a Teledyne ISCO Combi-flash® purification system. The crude oil (25 g) was loaded onto a 330 g silica column and eluted with 0-10% methanol / DCM over 30 min, resulting in 22 g of pure 9 (compound 22) (50% yield). 42 H 59 N5O 14 Calculated mass = 857.4 m / z. Measured [M+H] = 858.0

[0314] [ka]

[0315] A solution of ester 9 (49.0 g, 57.1 mmol) and 6-amino-1-hexanol (7.36 g, 6.28 mmol) in dichloromethane (3 volumes) was treated dropwise with triethylamine (11.56 g, 111.4 mmol). The reaction was monitored by observing the disappearance of compound 9 by HPLC method 1 and was found to be complete in 10 minutes. The crude reaction mixture was diluted with 5 volumes of dichloromethane and washed with saturated ammonium chloride (5 volumes) and brine (5 volumes). The organic layer was dried over sodium sulfate and concentrated to an oil. The crude oil was purified on a Teledyne ISCO Combi-flash® purification system using a 330 g silica column. 4-Nitrophenol was eluted with 100% ethyl acetate and 10 was flushed from the column with 20% methanol / DCM resulting in a colorless oil (39 g, 81% yield). C 42 H 69 N5O 12 Calculated mass = 836.0 m / z. Measured [M+H] = 837.0

[0316] [ka]

[0317] Alcohol 10 was co-stripped twice with 10 volumes of acetonitrile to remove residual methanol from the chromatography solvent and once with dry dichloromethane (KF<60 ppm) to remove traces of water. Alcohol 10 (2.30 g, 2.8 mmol) was dissolved in 5 volumes of dry dichloromethane (KF<50 ppm) and treated with diisopropylammonium tetrazolide (188 mg, 1.1 mmol). The solution was cooled to 0 °C and treated dropwise with 2-cyanoethyl N,N,N',N'-tetraisopropylphosphoramidite (1.00 g, 3.3 mmol). The solution was removed from the ice bath and stirred at 20 °C. The reaction was found to be complete within 3-6 h. The reaction mixture was cooled to 0° C. and treated with 10 volumes of a 1:1 solution of saturated ammonium bicarbonate / brine, then allowed to warm to ambient temperature over 1 min and stirred at 20° C. for an additional 3 min. The biphasic mixture was transferred to a separatory funnel and 10 volumes of dichloromethane were added. The organic layer was separated and washed with 10 volumes of saturated aqueous sodium bicarbonate to hydrolyze any unreacted bisphosphonate reagent. The organic layer was dried over sodium sulfate and concentrated to an oil, resulting in 3.08 g of compound 14, 94% by weight. C 51 H 86 N7O 13 Calculated mass of P = 1035.6 m / z. Measured [M+H] = 1036

[0318] H. Conjugation of Targeting Ligand Either before or after annealing, the 5' or 3' tridentate alkyne functionalized sense strand is conjugated to a targeting ligand. The following example illustrates the conjugation of a targeting ligand to an annealed duplex: Stock solutions of 0.5 M tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5 M copper(II) sulfate pentahydrate (Cu(II)SO4·5H2O), and 2 M sodium ascorbate solution were prepared in deionized water. A 75 mg / mL solution of targeting ligand in DMSO was made. To a 1.5 mL centrifuge tube containing the trialkyne functionalized duplex (3 mg, 75 μL, 40 mg / mL in deionized water, ~15,000 g / mol), add 25 μL of 1 M Hepes pH 8.5 buffer. After vortexing, add 35 μL of DMSO. Vortex the solution. The targeting ligand was added to the reaction (6 equiv / duplex, 2 equiv / alkyne, ~15 μL). The solution was vortexed. The pH was checked using pH paper to ensure it was pH ~8. In a separate 1.5 mL centrifuge tube, 50 μL of 0.5 M THPTA was mixed with 10 uL of 0.5 M Cu(II)SO4·5H2O, vortexed, and incubated at room temperature for 5 min. After 5 min, THPTA / Cu solution (7.2 μL, 6 equiv, 5:1 THPTA:Cu) was added to the reaction vial and vortexed. Immediately after, 2 M ascorbate (5 μL, 50 equiv per duplex, 16.7 equiv per alkyne) was added to the reaction vial and vortexed. Once the reaction was complete (typically 0.5-1 h), the reaction was immediately purified by non-denaturing anion exchange chromatography.

[0319] Example 2. MMP7-SEAP Mouse Model To evaluate the efficacy of RNAi agents, the MMP7-SEAP mouse model was used. Female C57BL / 6 albino mice aged 6-8 weeks were transiently transfected in vivo with plasmids by hydrodynamic tail vein injection at least 15 days prior to administration of MMP7 RNAi agents or controls. The plasmid contains the MMP7 cDNA sequence (Genbank NM_002423.5 (SEQ ID NO: 1)) inserted into the 3'UTR of the SEAP (secreted human placental alkaline phosphatase) reporter gene. MMP7-SEAP model mice were generated by injecting mice with 50 μg of the plasmid containing the MMP7 cDNA sequence in Ringer's solution via the tail vein, in a total volume of 10% of the animal's body weight. As previously described (Zhang G et al., “High levels of foreign gene expression in hepatocytes after tail vein injection of naked plasmid DNA.” Human Gene Therapy 1999 Vol.10, p1735-1737), the solution was injected for 5-7 seconds through a 27-gauge needle. Suppression of MMP7 expression by MMP7 RNAi agents resulted in concomitant suppression of SEAP expression, which was measured with the Phospha-Light™ SEAP Reporter Gene Assay System (Invitrogen). Prior to administration, SEAP expression levels were measured in serum, and mice were grouped according to the average SEAP levels. Analysis: SEAP levels can be measured at various time points, both before and after administration of MMP7 RNAi agents. i) Serum collection: Mice were anesthetized with 2-3% isoflurane and blood samples were collected from the submandibular region into serum separator tubes (Sarstedt AG & Co., Nümbrecht, Germany). Blood was allowed to clot for 20 min at ambient temperature. The tubes were centrifuged at 8,000 × g for 3 min to separate serum and stored at 4 °C. ii) Serum SEAP levels: serum was collected and measured with Phospha-Light™ SEAP Reporter Gene Assay System (Invitrogen) according to the manufacturer's instructions. To account for non-treatment-related reductions in MMP7 expression in this model, serum SEAP levels for each animal were normalized to a group of saline-injected control mice. First, the SEAP level for each animal at a given time point was divided by the pre-dose expression level for that animal ("pre-dose") to obtain a "pre-dose normalized" expression ratio. Expression at a particular time point was then normalized to the control group by dividing the "pre-dose normalized" ratio for each individual animal by the average "pre-dose normalized" ratio for all mice in the normal saline control group. Alternatively, in some examples described herein, serum SEAP levels for each animal were assessed only by normalizing to pre-dose levels.

[0320] Example 3. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 3.0 mg / kg (mpk) of an MMP7 RNAi agent or saline without an MMP7 RNAi agent (used as a control) according to Table 12 below.

[0321] [Table 13]

[0322] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 13 below. Mean SEAP indicates the normalized average value of SEAP.

[0323] [Table 14-1] [Table 14-2]

[0324] In each of the treatment groups (i.e., Groups 2-11), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0325] Example 4. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 3.0 mg / kg (mpk) of an MMP7 RNAi agent or saline without an MMP7 RNAi agent (used as a control) according to Table 14 below.

[0326] [Table 15]

[0327] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 15 below. Mean SEAP indicates the normalized average value of SEAP.

[0328] [Table 16]

[0329] In each of the treatment groups (i.e., Groups 2-10), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0330] Example 5. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 3.0 mg / kg (mpk) MMP7 RNAi agent, 1 mpk MMP7 RNAi agent, 0.3 mpk MMP7 RNAi agent, or saline without MMP7 RNAi agent (used as a control) according to Table 16 below.

[0331] [Table 17]

[0332] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 17 below. Mean SEAP indicates the normalized average value of SEAP.

[0333] [Table 18]

[0334] In each of the treatment groups (i.e., Groups 2-11), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0335] Example 6. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 2.0 mg / kg (mpk) of an MMP7 RNAi agent, 1.0 mpk of an MMP7 RNAi agent, or saline without an MMP7 RNAi agent (used as a control) according to Table 18 below.

[0336] [Table 19]

[0337] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Groups 1-6 tested 4 mice (n=4) per group, and Group 7 tested 3 mice (n=3). Serum was collected on days 8, 15, 22, and 29 and SEAP expression levels were measured according to the procedures described in Example 2 above. Data from the experiment are shown in Table 19 below. Mean SEAP indicates the normalized average value of SEAP.

[0338] [Table 20]

[0339] In each of the treatment groups (i.e., Groups 2-7), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0340] Example 7. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 2.0 mg / kg (mpk) MMP7 RNAi agent, 1.0 mpk MMP7 RNAi agent, 0.5 mpk MMP7 RNAi agent, or saline without MMP7 RNAi agent (used as a control) according to Table 20 below.

[0341] [Table 21]

[0342] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 21 below. Mean SEAP indicates the normalized average value of SEAP.

[0343] [Table 22]

[0344] In each of the treatment groups (i.e., Groups 2-7), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0345] Example 8. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 2.0 mg / kg (mpk) of an MMP7 RNAi agent, 1 mpk of an MMP7 RNAi agent, or saline without an MMP7 RNAi agent (used as a control) according to Table 22 below.

[0346] [Table 23]

[0347] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 23 below. Mean SEAP indicates the normalized average value of SEAP.

[0348] [Table 24-1] [Table 24-2]

[0349] In each of the treatment groups (i.e., Groups 2-11), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0350] Example 9. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 1.5 mg / kg (mpk) of an MMP7 RNAi agent or saline without an MMP7 RNAi agent (used as a control) according to Table 24 below.

[0351] [Table 25]

[0352] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 25 below. Mean SEAP indicates the normalized average value of SEAP. [Table 26]

[0353] In each of the treatment groups (i.e., Groups 2-8), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to saline controls (Group 1) across all measurement time points (except for Group 8 on Day 8), demonstrating the suppression of MMP7 in the MMP7-SEAP mouse model as described herein.

[0354] Example 10. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 2.0 mg / kg (mpk) of an MMP7 RNAi agent, 1.0 mpk of an MMP7 RNAi agent, or saline without an MMP7 RNAi agent (used as a control) according to Table 26 below.

[0355] [Table 27]

[0356] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 27 below. Mean SEAP indicates the normalized average value of SEAP. [Table 28]

[0357] In each of the treatment groups (i.e., Groups 2-9), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0358] Example 11. In vivo testing of MMP7 RNAi agents in MMP7SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 1.0 mpk of an MMP7 RNAi agent or saline without an MMP7 RNAi agent (used as a control) according to Table 28 below.

[0359] [Table 29]

[0360] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 29 below. Mean SEAP indicates the normalized average value of SEAP. [Table 30]

[0361] In each of the treatment groups (i.e., Groups 2-11), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0362] Example 12. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 1.0 mpk of an MMP7 RNAi agent or saline without an MMP7 RNAi agent (used as a control) according to Table 30 below.

[0363] [Table 31]

[0364] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, and 22, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 31 below. Mean SEAP indicates the normalized average value of SEAP. [Table 32-1] [Table 32-2]

[0365] In each of the treatment groups except for Group 10 (i.e., Groups 2-9 and 11-12), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0366] Example 13. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 1.0 mpk of an MMP7 RNAi agent or saline without an MMP7 RNAi agent (used as a control) according to Table 32 below.

[0367] [Table 33]

[0368] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 33 below. Mean SEAP indicates the normalized average value of SEAP. [Table 34-1] [Table 34-2]

[0369] In each of the treatment groups (i.e., Groups 2, 3, and 5-11) except for Group 4 on day 22, the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0370] Example 14. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 3.0 mg / kg (mpk) MMP7 RNAi agent, 1.0 mpk MMP7 RNAi agent, 0.3 mpk MMP7 RNAi agent, or saline without MMP7 RNAi agent (used as a control) according to Table 34 below.

[0371] [Table 35]

[0372] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, and 22, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 35 below. Mean SEAP indicates the normalized average value of SEAP. [Table 36-1] [Table 36-2]

[0373] In each of the treatment groups (i.e., Groups 2-10), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0374] Example 15. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 3.0 mg / kg (mpk) MMP7 RNAi agent, 1.0 mpk MMP7 RNAi agent, 0.3 mpk MMP7 RNAi agent, or saline without MMP7 RNAi agent (used as a control) according to Table 36 below.

[0375] [Table 37]

[0376] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 37 below. Mean SEAP indicates the normalized average value of SEAP. [Table 38-1] [Table 38-2]

[0377] In each of the treatment groups, except for Groups 2 and 5 on day 22, each of the MMP7 RNAi agents showed a reduction in SEAP compared to the saline control (Group 1) across all time points measured, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model, as described herein.

[0378] Example 16. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 1.0 mg / kg (mpk) of an MMP7 RNAi agent, 0.5 mpk of an MMP7 RNAi agent, or saline without an MMP7 RNAi agent (used as a control) according to Table 38 below.

[0379] [Table 39]

[0380] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, and 22, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 39 below. Mean SEAP indicates the normalized average value of SEAP. [Table 40-1] [Table 40-2]

[0381] In each of treatment groups 4-8, the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to saline controls (group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0382] Example 17. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 1.0 mpk of an MMP7 RNAi agent or saline without an MMP7 RNAi agent (used as a control) according to Table 40 below.

[0383] [Table 41-1] [Table 41-2]

[0384] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 41 below. Mean SEAP indicates the normalized average value of SEAP. [Table 42-1] [Table 42-2] [Table 42-3]

[0385] In each of the treatment groups (i.e., Groups 2-19), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0386] Example 18. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 1.0 mpk of an MMP7 RNAi agent or saline without an MMP7 RNAi agent (used as a control) according to Table 42 below.

[0387] [Table 43]

[0388] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 43 below. Mean SEAP indicates the normalized average value of SEAP. [Table 44]

[0389] In each of the treatment groups (i.e., Groups 2-10), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0390] Example 19. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 0.75 mpk of an MMP7 RNAi agent or saline without an MMP7 RNAi agent (used as a control) according to Table 44 below.

[0391] [Table 45]

[0392] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, 22, and 29, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 45 below. Mean SEAP indicates the normalized average value of SEAP. [Table 46-1] [Table 46-2]

[0393] In each of the treatment groups (i.e., Groups 2-12), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to the saline control (Group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0394] Example 20. In vivo testing of MMP7 RNAi agents in MMP7-SEAP mice The MMP7-SEAP mouse model described in Example 2 above was used. On day 1, each mouse received a single subcutaneous injection of 200 μl per 20 g body weight containing either 1.0 mg / kg (mpk) of an MMP7 RNAi agent, 0.5 mpk of an MMP7 RNAi agent, or saline without an MMP7 RNAi agent used as a control, according to Table 46 below.

[0395] [Table 47]

[0396] As shown in Tables 5 and 7B, each MMP7 RNAi agent contained an N-acetyl-galactosamine targeting ligand conjugated to the 5' end of the sense strand. Injections were made between the skin and muscle into the loose skin over the neck and shoulder area (i.e., subcutaneous injection). Four mice were tested per group (n=4). Serum was collected on days 8, 15, and 22, and SEAP expression levels were measured according to the procedure described in Example 2 above. Data from the experiment are shown in Table 47 below. Mean SEAP indicates the normalized average value of SEAP. [Table 48-1] [Table 48-2]

[0397] In all treatment groups (i.e., groups 2-9), the MMP7 RNAi agents each demonstrated a reduction in SEAP compared to saline controls (group 1) across all measurement time points, demonstrating inhibition of MMP7 in the MMP7-SEAP mouse model as described herein.

[0398] Example 21. In vivo inhalation aerosol administration of MMP7 RNAi agents in cynomolgus monkeys On study day 1, male cynomolgus monkeys were administered a single dose of MMP7 RNAi agents AC001514, AC001651, or AC001516 (see Tables 8, 6, and 3 for structural information) at a deposition dose level of 1 mg / kg. Twelve anesthetized male non-human primates (NHPs) were exposed to either aerosolized isotonic saline (control agent), AC001514, AC001516, or AC001651 (test agent) using an endotracheal inhalation delivery system. All animals received a single inhalation exposure. In this study, aerosols generated using an Aeroneb Solo nebulizer were delivered using a Harvard ventilation pump. The exposure duration for each of these tests was 8 minutes. In vivo exposure was estimated by collecting aerosols on a filter at the end of an endotracheal tube in a separate in vitro study. One filter test was performed before and after each in vivo exposure. The filters were analyzed gravimetrically and chemically. UV spectroscopy using a SpectraMax i3x was used for chemical analysis of the filters to determine the amount of test article administered throughout the exposure period.

[0399] The mean deposited doses of saline control, AC001514, AC001516 and AC001651 were 0.0, 1.04, 1.12 and 1.21 mg / kg, respectively. The target deposited dose of each of the three test articles in this study was 1.0 mg / kg. The MMP7 RNAi agents were conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1, see Table 11) at the 5' end of the sense strand and formulated in isotonic saline. The dose groups were as follows:

[0400] [Table 49]

[0401] Three monkeys were dosed per group. Bronchoalveolar lavage (BAL) and endobronchial scrapings were collected at baseline and 2 weeks after a single inhalation challenge. All animals were euthanized immediately after blood and BAL samples were collected to collect tissues of interest. Monkeys were sacrificed on study day 15, and lung samples were collected and homogenized, followed by isolation of total RNA. The data in Table 49 show mRNA expression sampled from the right anterior lobe, right middle lobe, and right posterior lobe. Cynomolgus monkey MMP7 mRNA expression was quantified by probe-based quantitative PCR, normalized to cynomolgus monkey ARL1 expression, and expressed as a percentage of vehicle control group (geometric mean, + / - 95% confidence interval).

[0402] [Table 50]

[0403] As shown in the data in Table 49 above, RNAi agents AC001514, AC001651 and AC001516 demonstrated substantial inhibition across various regions and lobes of the lung, demonstrating their ability to potently silence MMP7 expression in non-human primates.

[0404] Cynomolgus monkey MMP7 protein expression in lung tissue and BAL was quantified by Western blot using an iBright imaging system (Thermo Fisher) and is shown in Table 50 below.

[0405] [Table 51]

[0406] As shown in the data in Table 50 above, the RNAi agent AC001651 silences MMP7 expression, showing a greater than 80% reduction in protein expression in both non-human primate lung tissue and BAL.

[0407] Example 22. AAV6.2FF-CAG-hMMP7.UTRs AAV Mouse Model The following procedure was used to evaluate MMP7 RNAi agents in the AAV mouse model. To evaluate specific MMP7 RNAi agents, the AAV6.2FF-CAG-hMMP7.UTRs (adeno-associated virus) mouse model was used. The transgenic sequence contained the human MMP7 CDS with the 3'UTR. Female C57BL / 6 mice aged 6-8 weeks were transduced with human MMP7 using AAV with serotype 6.2FF. Mice received intratracheal infusions at least 10 days prior to multiple intratracheal infusions of MMP7 RNAi agents or controls. Two types of AAV were used: AAV6.2FF-CAG-hMMP7.UTRs and AAV6.2FF-CAG-eGFP. The genome of the AAV6.2FF-CAG-hMMP7.UTRs construct contains the 17-1119 region of the human MMP7 cDNA sequence (Genbank NM_002423.5). AAV6.2FF-CAG-eGFP was co-administered with AAV6.2FF-CAG-hMMP7.UTR. eGFP was used as an endogenous control to normalize human MMP7 mRNA expression by qPCR. 2E10–4E10 GC of each virus mixed in PBS in a total volume of 50 μL were delivered intratracheally (IT) to mice to generate AAV-hMMP7 model mice. Lung tissues and bronchoalveolar lavage fluid (BALF) were collected 2–3 weeks after RNAi agent administration.

[0408] Human MMP7 mRNA and protein level expression was measured in lung tissues by qPCR and Western blot. Human MMP7 protein expression in bronchoalveolar lavage fluid (BALF) was measured by ELISA.

[0409] Each mouse was given 50 μL of AAV solution containing 1 GC (genomic copy) of AAV6.2FF-CAG-eGFP and 2 GC of AAV6.2FF-CAG-hMMP7.UTRs in PBS or vehicle control (PBS) intratracheally (IT) on days 1 and 3. On days 13, 15, and 17, each mouse was given 50 μL of various dose levels of MMP7 RNAi agents formulated in isotonic saline according to Table 51 below, or vehicle control (isotonic saline without RNAi agent). [Table 52-1] [Table 52-2]

[0410] Each MMP7 RNAi agent contained a modified nucleotide conjugated to the 5' end of the sense strand with an αvβ6 integrin targeting ligand having a modified sequence as set forth in the double-stranded structure herein (see Tables 3, 4, 5, 6, 7A, 7B, 8, 9, 10, and 11 for specific modification and structural information for MMP7 RNAi agents including Tri-SM6.1-αvβ6). Each of the MMP7 RNAi agents in groups 3-8 contained a nucleotide sequence designed to suppress expression of the MMP7 gene by targeting a specific position of the MMP7 mRNA as set forth in Table 50 above (see, e.g., SEQ ID NO: 1 and Table 2 for reference MMP7 mRNA sequences).

[0411] Four mice per group were tested (n=4). MMP7 expression levels were measured according to the procedure described above. A dose response of the MMP7 RNAi agent AC001516 was observed in the AAV6.2FF-CAG-hMMP7.UTRs mouse model. Data from the experiment is shown in Table 52 below. [Table 53]

[0412] As shown in Table 52 above, the RNAi agents of Group 3 (target position 971) were active, showing an approximately 68% reduction in mRNA levels in lung tissue (0.159), an 83% reduction in secreted human MMP7 protein, and a 64% reduction in human MMP7 protein in lung tissue at day 31.

[0413] Example 23. Proof-of-concept efficacy study using rodent-specific MMP7 RNAi agents in a rat bleomycin-induced injury model MMP7 knockout mice have been shown to be protected from bleomycin-mediated lung injury (Proc Natl Acad Set USA. 2002;99:6292-629T). To evaluate MMP7 RNAi agents, several proof-of-concept efficacy studies were developed using rodent-specific MMP7 RNAi agents in a rat bleomycin-induced injury model. MMP7 expression was evaluated in a mouse bleomycin injury model and found that MMP7 mRNA levels were not increased in lung tissue following bleomycin-induced injury. Furthermore, MMP7 expression was transiently increased in rat lung tissue following bleomycin-induced injury. MMP7 expression peaked 7-10 days after bleomycin-induced injury and declined to baseline levels over 4 weeks after injury.

[0414] After screening and optimization in a rat bleomycin-induced injury model, one rat-specific RNAi agent was selected. Bleomycin was used to induce injury in rats. After injury, the RNAi agent was administered to rats by single inhalation at 1.4 mg / kg or multiple intratracheal administration at 3.0 mg / kg. Two to four weeks after bleomycin-induced injury, the RNAi agent achieved 60-90% gene silencing of MMP7. Silencing of MMP7 in the lung significantly attenuated lung injury in the rat bleomycin model, along with reduced pulmonary fibrosis Ashcroft histology scores, reduced collagen deposition, reduced inflammatory responses with reduced eosinophils and neutrophils in the lavage fluid, and reduced expression of translatable fibrotic genes, including Colla2, Col5al, Greml, Cthrcl, and Mucl6. Furthermore, silencing of MMP7 significantly improved lung function, improved functional compliance, maintained blood oxygen levels, reduced weight loss, and reduced mortality. Silencing of MMP7 by rodent-specific RNAi agents effectively protects the lung from the development of fibrosis in a rat intratracheal bleomycin injury model. Example 24. Passive uptake of MMP7 RNAi agents in human precision cut lung slices (PCLS) Precision-cut tissue slices (PCLS) are an ex vivo model and tool to study lung structure and function in a native 3D environment, allowing the natural interactions between cells, molecules, and extracellular matrix (ECM) to be examined ex vivo (Alsafadi HN et al, Am J Respir Cell Mol Biol 62(6): 681-691 (2020)). PCLS can be generated from various anatomical sites of the lung (distal and proximal) and from various species including rodents, pigs, monkeys, and humans. However, the efficacy of passive uptake of RNAi agents in human PCLS remains unclear. To validate the efficacy of RNAi agents for silencing human MMP7 mRNA, we used fresh agarose-expanded lung slices from healthy human donors.

[0415] Saline or MMP7 RNAi agents were added to cell culture medium and the medium was changed daily. PCLS were cultured in culture medium from day 1 to day 7 and harvested on day 8. mRNA expression of MMP7 and potential off-target genes MAP3K9, MTF2, and NUP107 was quantified by qPCR normalized to the endogenous reference PPIA. Efficient passive uptake of MMP7 RNAi agents was observed. Little off-target effects were observed. MMP7 RNAi agents were administered according to Table 53 below. Data from the qPCR experiments are shown in Table 54 below.

[0416] Test groups 11 and 12 were administered 1 pM of AC002026. AC002026 is an RNAi agent duplex with the same modified antisense and sense strand sequences as AC001514. However, the AC002026 sense strand is conjugated to an inactive enantiomer of an αvβ6 integrin targeting ligand. Due to the difference in stereochemistry, this chemically modified analog of the αvβ6 integrin targeting ligand cannot effectively bind to αvβ6 integrin and therefore cannot effectively support the cellular uptake of the AC002026 RNAi agent.

[0417] [Table 54]

[0418] [Table 55]

[0419] Passive uptake of MMP7 RNAi agents was observed. As shown above in Table 54, uptake of MMP7 RNAi agents resulted in up to 82-90% MMP7 silencing from the most potent RNAi agent, AC001651. This potency result is ranked similarly to the in vivo SEAP study.

[0420] At the same time, little off-target effects were observed. Upon administration, the RNAi agents caused only slight cytotoxic effects even at the highest dose concentration. Such cytotoxicity and cell viability were demonstrated by MTT colorimetric assays for cell metabolism and mitochondrial activity. The MTT assay showed that the MMP7 RNAi agents exhibited optical density (OD) equivalent to that of the control at 168 hours after administration, as shown in Figure 3.

[0421] Example 25. In vivo inhalation aerosol administration of MMP7 RNAi agents in cynomolgus monkeys On study day 1, male cynomolgus monkeys were administered a single dose of the MMP7 RNAi agent AC001651 (see Tables 8, 6, and 3 for structural information) or isotonic saline at deposition dose levels of 0.24 mg / kg, 0.66 mg / kg, 1.10 mg / kg, or 1.71 mg / kg. On day 1, three animals per group (n=3), anesthetized male non-human primates (NHPs), were exposed to either aerosolized isotonic saline (control article) or AC001651 (test article) by inhalation using a facemask inhalation exposure system. In this study, aerosols were generated using a Hudson Updraft II compressed air jet nebulizer and delivered to the animals by facemask inhalation. Concentrations in the test atmosphere were determined by gravimetric analysis of filter samples (47 mm fiber membrane filter, 0.5 micron, GF / A) taken during the entire exposure at nominal flow rate. After harvesting, the filters were removed from the filter holder and weighed. Additionally, the filter contents were chemically analyzed by absorbance measurement on a SpectraMax i3x spectrophotometer. AC001651 formulation samples were analyzed for concentration during filter analysis.

[0422] The target deposited doses for each of the three test articles in this study were 0.25 mg / kg, 0.5 mg / kg, 1.0 mg / kg, and 2.0 mg / kg, respectively. The mean deposited doses for saline control and AC001651 were 0.0, 0.24, 0.66, 1.10, and 1.71 mg / kg, respectively. The MMP7 RNAi agent was conjugated to a tridentate small molecule αvβ6 epithelial cell targeting ligand (Tri-SM6.1, see Table 11) at the 5' end of the sense strand and formulated in isotonic saline. The dose groups were as follows:

[0423] [Table 56]

[0424] Three monkeys were administered per group. Bronchoalveolar lavage (BAL) fluid samples were collected at baseline on day 1 and 2 weeks after a single inhalation challenge on day 14. All animals were euthanized immediately after blood and BAL samples were collected and tissues of interest were harvested. Monkeys were sacrificed on study day 14, and total RNA was isolated from lung samples after collection and homogenization. The data in Table 56 show mRNA expression sampled from the right posterior lobe, right anterior lobe, right middle lobe, and left posterior lobe. Cynomolgus monkey MMP7 mRNA expression was quantified by probe-based quantitative PCR and normalized to cynomolgus monkey GAPDH mRNA expression and vehicle control group (geometric mean, + / - geometric SD).

[0425] [Table 57-1] [Table 57-2] [Table 57-3]

[0426] As shown in the data in Table 56 above, the RNAi agent AC001651 demonstrated substantial inhibition across various regions and lobes of the lung, demonstrating its ability to potently silence MMP7 expression in non-human primates.

[0427] MMP7 protein expression in lung tissue was quantified by Western blot using an iBright imaging system (Thermo Fisher) and shown in Table 57 below.

[0428] [Table 58]

[0429] As shown in the data in Table 57 above, the RNAi agent AC001651 silences MMP7 expression, showing an average of -69% reduction in protein expression in lung tissue of non-human primates at a deposited dose of 1.10 mg / kg. PDD = lung deposited dose.

[0430] MMP7 mRNA expression was quantified by qPCR in BAL exosomes from cynomolgus monkeys administered a single deposition dose of 0.24 mg / kg, 0.66 mg / kg, 1.10 mg / kg, or 1.71 mg / kg PDD of the RNAi agent AC001651. Data are normalized to baseline and GAPDH and vehicle control groups on day 7 (GMEAN+ / -geometric standard deviation). Data are shown in Table 58.

[0431] [Table 59]

[0432] As shown in the data in Table 58 above, the RNAi agent AC001651 potently silences MMP7 expression, demonstrating a mean reduction of MMP7 mRNA in BAL exosomes of 64% at a deposition dose of 1.10 mg / kg in non-human primates.

[0433] MMP7 protein expression in BAL was quantified by Western blot using the iBright imaging system (Thermo Fisher) and is shown in Table 59 below.

[0434] [Table 60]

[0435] As shown in the data in Table 59 above, the RNAi agent AC001651 potently silences MMP7 expression, showing an average of -78% reduction in protein expression in BAL at a deposition dose of 0.66 mg / kg in non-human primates.

[0436] Other embodiments Although the invention has been described in conjunction with its detailed description, it should be understood that the foregoing description is intended to be illustrative, and not limiting, of the scope of the invention as defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. An RNAi agent for suppressing expression of matrix metallopeptidase 7 gene, comprising: an antisense strand comprising at least 17 consecutive nucleotides that differ from any one of the sequences shown in Table 2 or Table 3 by 0 or 1 nucleotide; and a sense strand comprising a nucleotide sequence that is at least partially complementary to the antisense strand.

1. An RNAi agent comprising:

2. The RNAi agent of claim 1, wherein the antisense strand comprises nucleotides 2 to 18 of any one of the sequences shown in Table 2 or Table 3.

3. 3. The RNAi agent of claim 1 or claim 2, wherein the sense strand comprises a nucleotide sequence of at least 17 contiguous nucleotides that differs by 0 or 1 nucleotide from any one of the sequences shown in Table 2 or Table 4, and the sense strand has a region of at least 85% complementarity over the 17 contiguous nucleotides to the antisense strand.

4. The RNAi agent of claim 1 , wherein at least one nucleotide of the MMP7 RNAi agent is a modified nucleotide or comprises a modified internucleoside linkage.

5. 2. The RNAi agent of claim 1, wherein all or substantially all of the nucleotides are modified nucleotides.

6. 5. The RNAi agent of claim 4, wherein the modified nucleotide is selected from the group consisting of 2'-O-methyl nucleotides, 2'-fluoro nucleotides, 2'-deoxy nucleotides, 2',3'-seco-nucleotide mimics, locked nucleotides, 2'-F-arabino nucleotides, 2'-methoxyethyl nucleotides, abasic nucleotides, ribitol, inverted nucleotides, inverted 2'-O-methyl nucleotides, inverted 2'-deoxy nucleotides, 2'-amino modified nucleotides, 2'-alkyl modified nucleotides, morpholino nucleotides, vinyl phosphonate-containing nucleotides, cyclopropyl phosphonate-containing nucleotides, and 3'-O-methyl nucleotides.

7. The RNAi agent of claim 5, wherein all or substantially all of the nucleotides are modified with 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or a combination thereof.

8. 2. The RNAi agent of claim 1, wherein the antisense strand comprises a nucleotide sequence of any one of the modified sequences shown in Table 3.

9. 2. The RNAi agent of claim 1, wherein the sense strand comprises a nucleotide sequence of any one of the modified sequences shown in Table 4.

10. 2. The RNAi agent of claim 1, wherein the antisense strand comprises the nucleotide sequence of any one of the modified sequences shown in Table 3, and the sense strand comprises the nucleotide sequence of any one of the modified sequences shown in Table 4.

11. The RNAi agent of claim 1, wherein the sense strand is 18 to 30 nucleotides in length and the antisense strand is 18 to 30 nucleotides in length.

12. The RNAi agent of claim 11, wherein the sense strand and the antisense strand are each 18 to 27 nucleotides in length.

13. The RNAi agent of claim 12, wherein the sense strand and the antisense strand are each 18 to 24 nucleotides in length.

14. The RNAi agent of claim 13 , wherein the sense strand and the antisense strand are each 21 nucleotides in length.

15. The RNAi agent of claim 14, wherein the RNAi agent has two blunt ends.

16. The RNAi agent of claim 1 , wherein the sense strand comprises one or two terminal caps.

17. The RNAi agent of claim 1 , wherein the sense strand comprises one or two inverted abasic residues.

18. The RNAi agent of claim 1, which is composed of a sense strand and an antisense strand that form a duplex having a duplex structure of any one of Table 7A, Table 7B, Table 8, Table 9, or Table 10.

19. 19. The RNAi agent of claim 18, wherein all or substantially all of the nucleotides are modified nucleotides.

20. The following nucleotide sequence (5' to 3'): 【Chemistry 1】 2. The RNAi agent of claim 1, comprising an antisense strand consisting of a nucleotide sequence that differs by 0 or 1 nucleotide from one of the above, an antisense strand consisting essentially of a nucleotide sequence that differs by 0 or 1 nucleotide, or an antisense strand that includes a nucleotide sequence that differs by 0 or 1 nucleotide.

21. The sense strand has the following nucleotide sequence (5' to 3'): 【Chemistry 2】 21. The RNAi agent of claim 20, wherein the RNAi agent consists of, consists essentially of, or comprises a nucleotide sequence that differs by 0 or 1 nucleotide from one of:

22. 21. The RNAi agent of claim 20, wherein all or substantially all of the nucleotides are modified nucleotides.

23. The following nucleotide sequence (5' to 3'): 【Transformation 3】 an antisense strand comprising a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of the above, an antisense strand consisting of a modified nucleotide sequence that differs by 0 or 1 nucleotide, or an antisense strand that consists essentially of a modified nucleotide sequence that differs by 0 or 1 nucleotide, wherein a, c, g, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; cPrpu represents 5'-phosphonate cyclopropyl-2'-O-methyl uridine; s represents a phosphorothioate linkage; and all or substantially all of the nucleotides of the sense strand are modified nucleotides. The RNAi agent of claim 1.

24. The sense strand has the following nucleotide sequence (5' to 3'): 【Chemistry 4】 a modified nucleotide sequence that differs by 0 or 1 nucleotide from one of: wherein a, c, g, i, and u represent 2'-O-methyl adenosine, 2'-O-methyl cytidine, 2'-O-methyl guanosine, 2'-O-methyl inosine, and 2'-O-methyl uridine, respectively; Af, Cf, Gf, and Uf represent 2'-fluoro adenosine, 2'-fluoro cytidine, 2'-fluoro guanosine, and 2'-fluoro uridine, respectively; s represents a phosphorothioate linkage; and all or substantially all of the nucleotides of the antisense strand are modified nucleotides. The RNAi agent of claim 1.

25. 21. The RNAi agent of claim 20, wherein the sense strand further comprises an inverted abasic residue at the 3' end of the nucleotide sequence, at the 5' end of the nucleotide sequence, or both.

26. The RNAi agent of claim 1 , which is linked to a targeting ligand.

27. 27. The RNAi agent of claim 26, wherein the targeting ligand has affinity for a cellular receptor expressed on epithelial cells.

28. 28. The RNAi agent of claim 27, wherein the targeting ligand comprises an integrin targeting ligand.

29. 29. The RNAi agent of claim 28, wherein the integrin targeting ligand is an αvβ6 integrin targeting ligand.

30. 10. The method of claim 1, wherein the targeting ligand has the structure: 【Transformation 5】 or a pharmaceutically acceptable salt thereof, or 【Transformation 6】 or a pharmaceutically acceptable salt thereof Including, During the ceremony, 【Transformation 7】 30. The RNAi agent of claim 29, wherein: indicates a point of attachment to the RNAi agent.

31. The targeting ligand is 【Chemistry 8-1】 【Chemistry 8-2】 【Chemistry 8-3】 【Chemistry 8-4】 【Chemistry 8-5】 【Chemistry 8-6】 【Chemistry 8-7】 【Chemistry 8-8】 [Chemistry 8-9] having a structure selected from the group consisting of During the ceremony, 【Chemistry 9】 27. The RNAi agent of claim 26, wherein: indicates a point of attachment to the RNAi agent.

32. The following structure: 【Chemistry 10】 32. The RNAi agent of claim 31 , conjugated to a targeting ligand having the formula:

33. The targeting ligand has the following structure: 【Chemistry 11】 27. The RNAi agent of claim 26, having the following structure:

34. 27. The RNAi agent of claim 26, wherein the targeting ligand is conjugated to the sense strand.

35. 35. The RNAi agent of claim 34, wherein the targeting ligand is conjugated to the 5' end of the sense strand.

36. 10. A composition comprising the RNAi agent of claim 1, further comprising a pharmaceutically acceptable excipient.

37. 37. The composition of claim 36, further comprising a second RNAi agent capable of suppressing expression of matrix metallopeptidase 7 gene expression.

38. 37. The composition of claim 36, further comprising one or more additional therapeutic agents.

39. 37. The composition of claim 36, formulated for administration by inhalation.

40. 40. The composition of claim 39, delivered by a metered dose inhaler, a jet nebulizer, a vibrating mesh nebulizer, or a soft mist inhaler.

41. 37. The composition of claim 36, wherein the RNAi agent is a sodium salt.

42. 37. The composition of claim 36, wherein the pharmaceutically acceptable excipient is water for injection.

43. 37. The composition of claim 36, wherein the pharmaceutically acceptable excipient is buffered saline.

44. A method for suppressing expression of the MMP7 gene in a cell, comprising introducing into the cell an effective amount of the RNAi agent of claim 1 or the composition of claim 36.

45. 45. The method of claim 44, wherein the cell is present in a subject.

46. 46. ​​The method of claim 45, wherein the subject is a human subject.

47. 47. The method of any one of claims 44-46, wherein after said administration of said RNAi agent, said matrix metallopeptidase 7 gene expression is suppressed by at least about 30%.

48. 44. A method for treating one or more conditions or diseases associated with enhanced or elevated membrane MMP7 activity levels, comprising administering a therapeutically effective amount of the composition of any one of claims 36 to 43 to a human subject in need thereof.

49. 49. The method of claim 48, wherein the disease is idiopathic pulmonary fibrosis (IPF), asthma, another type of fibrosis, chronic inflammation, interstitial lung disease (ILD), SARS-COV-2 or another type of infection, acute respiratory distress syndrome (ARDS) or another type of acute lung injury, pulmonary hypertension, cancer, non-alcoholic fatty liver disease (NAFUD), non-alcoholic steatohepatitis (NASH), fatty liver disease, biliary atresia, and chronic kidney disease (CKD).

50. 50. The method of claim 49, wherein the disease is idiopathic pulmonary fibrosis (IPF).

51. 45. The method of claim 44, wherein the RNAi agent is administered at a deposition dose of about 0.01 mg / kg to about 5.0 mg / kg of body weight of the subject.

52. 45. The method of claim 44, wherein the RNAi agent is administered at a deposition dose of about 0.03 mg / kg to about 2.0 mg / kg of body weight of the subject.

53. 45. The method of claim 44, wherein the RNAi agent is administered in two or more administrations.

54. 10. Use of the RNAi agent of claim 1 for the treatment of a disease, disorder, or condition mediated at least in part by membrane MMP7 activity and / or MMP7 gene expression.

55. 44. Use of the composition of any one of claims 36 to 43 for the treatment of a disease, disorder, or condition mediated at least in part by matrix metallopeptidase 7 activity and / or matrix metallopeptidase 7 gene expression.

56. Use of the composition of any one of claims 36 to 43 for the manufacture of a medicament for the treatment of a disease, disorder, or condition mediated at least in part by matrix metallopeptidase 7 and / or matrix metallopeptidase 7 gene expression.

57. 55. The use of claim 54, wherein the disease is pulmonary inflammation.

58. 10. A method for making the RNAi agent of claim 1, comprising annealing a sense strand and an antisense strand to form a double-stranded ribonucleic acid molecule.

59. 59. The method of claim 58, wherein the sense strand comprises a targeting ligand.

60. 60. The method of claim 59, comprising conjugating a targeting ligand to the sense strand.

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