RNAi AGENTS FOR DUAL INHIBITION OF EXPRESSION OF THYMIC STROMAL LYMPHOPOIETIN (TSLP) AND INTERLEUKIN 33 (IL33), COMPOSITIONS THEREOF, AND METHODS OF USE

Abstract

Described are RNAi agents, compositions that include RNAi agents, and methods for dual inhibition of a Thymic Stromal Lymphopoietin (TSLP) and Interleukin 33 (IL33) gene. The TSLP-IL33 RNAi agents disclosed herein inhibit the expression of a TSLP and an IL33 gene. Pharmaceutical compositions that include one or more TSLP-IL33 RNAi agents, optionally with one or more additional therapeutics, are also described. Delivery of the described TSLP-IL33 RNAi agents to pulmonary cells, in vivo, provides for inhibition of TSLP and / or IL33 gene expression, which can provide a therapeutic benefit to subjects, including human subjects, for the treatment of various diseases including asthma or chronic obstructive pulmonary disease (COPD).

Description

RNAi Agents for Dual Inhibition of Expression of Thymic Stromal Lymphopoietin (TSLP) and Interleukin 33 (IL33)CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to United States Provisional Patent Application Serial No. 63 / 715,782, filed on November 4, 2024, and United States Provisional Patent Application Serial No. 63 / 800,747, filed on May 6, 2025, the contents of each of which are incorporated herein by reference in their entirety.FIELD OF THE INVENTION

[0002] The present disclosure relates to RNA interference (RNAi) agents, e.g.. multimeric (for example, “dimer”) double stranded RNAi agents such as small interfering RNAs (siRNAs), for dual and concurrent inhibition of Thymic Stromal Lymphopoietin (TSLP) and Interleukin 33 (IL33) gene expression, compositions that include TSLP-IL33 RNAi agents, and methods of use thereof.SEQUENCE LISTING

[0003] This application contains a Sequence Listing (in compliance with Standard ST26), which has been submitted in xml format and is hereby incorporated by reference in its entirety.The xml sequence listing file is named 30738-WO_SeqListing.xml, created October 28, 2025, and is 417,568 bytes in size.BACKGROUND

[0004] Thymic Stromal Lymphopoietin (“TSLP”) is an epithelial cell-derived cytokine implicated in the initiation and persistence of inflammatory pathways in asthma (Pames. et al.2022). TSLP is a member of the 4-helix-bundle cytokine family and a distant paralog of interleukin (IL)-7, which is expressed by human epithelial cells in the thymus, lung, intestine, skin, and stroma, as well as in tonsils and mast cells (Hu, et al. 2017). TSLP affects various cell types through a heterodimeric receptor consisting of the IL-7 receptor chain (IL-7Ra) and a specific subunit, TSLP-specific receptor (TSLPR) (Pandey, et al. 2000).

[0005] Interleukin-33 (IL-33) is a member of the IL-1 cytokine family that includes IL- la, IL-ip, and IL- 18 and constitutively expressed in structural and lining cells including fibroblasts, endothelial, and epithelial cells of skin, gastrointestinal tract, and lungs that are exposed to the environment. IL-33 is a Th2-oriented cytokine which enhances the production of Th2 cytokines, particularly IL-5 and IL-13. Chan BCL, Lam CWK, Tam L-S and Wong CK (2019) IL33: Roles in Allergic Inflammation and Therapeutic Perspectives. Front. Immunol. 10:364.

[0006] Transcriptome-wide association study (TWAS) identified TSLP and IL-33 as strongly associated with adult- and childhood-onset asthma. TSLP and IL33 signaling reciprocally enhanced each other’s protein release and expression in the lung following Altemaria challenge, and improve anti-inflammatory efficacy by targeting multiple immune pathways. Nature Communications. 2022; 13:1632, A llergy. 2020;75:1606-1617.

[0007] Accordingly, it would be desirable to develop RNAi therapeutic(s) simultaneously targeting both TSLP and IL33 to provide suitable treatment for diseases and conditions that benefit from synergistic interactions of TSLP and IL33 signaling. Single delivery via multimeric RNAi agents utilizing a single targeting ligand interacting with an mtegrin receptor can provide efficiencies in administration.

[0008] However, obtaining suitable delivery' of oligonucleotide-based therapeutics such as RNAi agents is and still remains the most pressing challenge to overcome in discovering and identifying viable RNAi therapeutics. While developments over the past few decades have led to an understanding of how to better deliver oligonucleotides, further improvements are needed and desired. Improving delivery can potentially permit less drug to be administered to the patient or subject, which can provide the benefit of reducing the likelihood of toxicological side-effects and potentially lower the costs of the therapeutic as less material will be required to be manufactured.

[0009] One such long-proposed concept for potential delivery' improvement has been to link two or more RNAi agents together, and to further link the multimeric (e.g., “dimer”) complex to a single targeting ligand, thereby forming a multimeric RNAi agent conjugate. (See, e.g., US Patent Application Publication No. 2007 / 0173473, at Figures 22-24). In theory, this could allow a single delivery targeting ligand to carry' double (in the case of two RNAi agents, or an RNAi agent “dimer”), or potentially even more oligonucleotide-based therapeutic cargo payloads per each targeting ligand. However, it has been a long-existing challenge to turn this relatively straightforward concept into a reality’ that sufficiently delivers an RNAi therapeutic in vivo that provides any advantage over monomeric conjugates.SUMMARY

[0010] Disclosed herein are multimeric RNAi agents for inhibiting expression a Thymic Stromal Lymphopoietin (TSLP) and Interleukin 33 (IL33) gene.

[0011] In some embodiments, the RNAi agents are multimeric oligonucleotide-based therapeutics including RNA interference (RNAi) agents (also herein termed RNAi agent, RNAi trigger, or trigger; e.g., double-stranded RNAi agents or small (or short) interfering RNA (siRNAs)), for inhibiting gene expression in hepatic cells in vivo. The delivery' of multimeric RNAi agents facilitates the selective and efficient inhibition of the expression of genes present in the lung, and specifically the inhibition or silencing of genes expressed in pulmonary cells.

[0012] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill 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. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0013] Other objects, features, aspects, and advantages of the invention will be apparent from the following detailed description, accompanying figures, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] FIG. 1. Chemical structure representation of the tridentate av|36 epithelial cell targeting ligand referred to herein as Tri-SM6.1-avb6-(TA14).

[0015] FIG. 2. Graph plot showing rat BALF total protein levels (in pg / mL); statistical significance level is denoted as * = p<0.05 and ** = p<0.001 (see also Example 4).

[0016] FIG. 3A. Graph plot showing rat BALF soluble collagen levels (in pg / mL); statistical significance level is denoted as * = p<0.05 and ** = p<0.001 (see also Example 4).

[0017] FIG. 3B. Graph plot showing rat BALF IL5 levels (in pg / mL); statistical significance level is denoted as * = p<0.05 and ** = p<0.001 (see also Example 4).

[0018] FIG. 3C. Graph plot showing rat BALF IL10 levels (in pg / mL); statistical significance level is denoted as * = p<0.05 and ** = p<0.001 (see also Example 4).

[0019] FIG. 3D. Graph plot showing rat BALF IL 13 levels (in pg / mL); statistical significance level is denoted as * = p<0.05 (see also Example 4).

[0020] FIG. 3E. Graph plot showing rat BALF leptin levels (in pg / mL); statistical significance level is denoted as * = p<0.05 and ** = p<0.001 (see also Example 4).

[0021] FIG. 4. Graph plot showing rat BALF total protein levels (in pg / mL); statistical significance level is denoted as * = p<0.05 and ** = p<0.001 (see also Example 7).

[0022] FIG. 5. Graph plot showing rat BALF soluble collagen levels (in pg / mL); statistical significance level is denoted as * = p<0.05 and ** = p<0.001 (see also Example 7).DETAILED DESCRIPTION

[0023] The disclosed RNAi agents, compositions thereof, and methods of use may be understood more readily by reference to the following detailed description, which form a part of this disclosure. It is to be understood that the disclosure is not limited to what is specifically described and / or shown herein, and that the terminology used herein is for the purpose of describing particular embodiments by way of example only and is not intended to be limiting.

[0024] It is to be appreciated that while certain features of the disclosures included herein are. for clarity, described herein in the context of separate embodiments, they may also be provided in combination in a single embodiment. Conversely, various features of the disclosed methods that are, for brevity, described in the context of a single embodiment, may also be provided separately or in any subcombination.Definitions

[0025] As used herein, the terms “oligonucleotide” and “polynucleotide” mean a polymer of linked nucleosides each of which can be independently modified or unmodified.

[0026] As used herein, an “RNAi agent” (also referred to as an “RNAi trigger”) means a chemical composition of matter that contains an RNA or RNA-like (e.g., chemically modified RNA) oligonucleotide molecule that is capable of degrading or inhibiting (e.g., degrades or inhibits under appropriate conditions) translation of messenger RNA (mRNA) transcripts of a target mRNA in a sequence specific manner. As used herein. RNAi agents may operate through the RNA interference mechanism (i.e., inducing 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). While it is believed that RNAi agents, as that term is used herein, operate primarily through the RNA interferencemechanism, the disclosed RNAi agents are not bound by or limited to any particular pathway or mechanism of action. RNAi agents disclosed herein are comprised of a sense strand and an antisense strand, and include, but are not limited to: short (or small) interfering RNAs (siRNAs), double stranded RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), and dicer substrates. The antisense strand of the RNAi agents described herein is at least partially complementary to the mRNA being targeted. RNAi agents can include one or more modified nucleotides and / or one or more non-phosphodiester linkages.

[0027] As used herein, the term “antisense strand” and “guide strand” are consistent with how those term are used in the art, and can be used to refer to either a first antisense strand or a second antisense strand of a multimeric RNAi agent.

[0028] As used herein, the terms “sense strand” and “passenger strand” are consistent with how those terms are used in the art, and refers to either a first sense strand or a second sense strand of a multimeric RNAi agent.

[0029] As used herein, the term “monomeric RNAi agent” refers to an RNAi agent comprised of one antisense strand and one sense strand. A monomeric RNAi agent is t pically designed to have an antisense strand sequence that is designed to inhibit gene expression of a single gene.

[0030] As used herein, the terms “multimeric RNAi agent complex”, “multimeric RNAi agents”, and “multimeric complex” refer to RNAi agents comprising one or more oligonucleotides that are, independently, at least partially complementary to the mRNA of one or more target genes. For example, multimeric complexes, as described herein, may comprise one or more antisense strands which are hybridized (i.e., have formed base pair hydrogen bonds to form a double helical structure) to a single sense strand. The antisense strands of the multimeric RNAi agent complex may initiate the RNA-induced silencing complex (RISC), to silence expression of the respective targeted gene or genes. In some embodiments, the one or more antisense strands are at least partially complementary to the mRNA of the same gene (e.g., two antisense strands of a single multimeric complex may be at least partially complementary to the mRNA of a single gene at different positions of the gene). In some embodiments, the one or more antisense strands are at least partially complementary to the mRNA of different genes (e g., two antisense strands of a single multimeric complex may be at least partially complementary to the mRNA of two different genes).

[0031] Exemplary multimeric RNAi complexes can be referred to as “[Gene l]-[Gene 2] RNAi agents.” For example, an “TSLP-IL33 RNAi agent” describes a multimeric RNAi complex having an antisense strand for silencing TSLP gene expression and an antisense strandfor silencing IL33 gene expression. In some embodiments, a multimeric RNAi agent complex comprising two RNAi agent antisense strands, and can be referred to as a “dimer.”

[0032] As used herein, the terms “silence,” “reduce,” “inhibit,” “down-regulate,” or "knockdow n" when referring to expression of a given gene, mean that the expression of the gene, as measured by the level of RNA transcribed from the gene or the level of polypeptide, protein, or protein subunit translated from the mRNA in a cell, group of cells, tissue, organ, or subject in which the gene is transcribed, is reduced when the cell, group of cells, tissue, organ, or subject is treated with the RNAi agents described herein as compared to a second cell, group of cells, tissue, organ, or subject that has not or have not been so treated.

[0033] As used herein, the terms “sequence” and “nucleotide sequence” mean a succession or order of nucleobases or nucleotides, described with a succession of letters using standard nomenclature.

[0034] As used herein, a “base,” “nucleotide base,” or “nucleobase,” is a heterocyclic pyrimidine or purine compound that is a component of a nucleotide, and includes the primary purine bases adenine and guanine, and the primary pyrimidine bases cytosine, thymine, and uracil. A nucleobase may further be modified to include, without limitation, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. (See, e.g., 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.

[0035] As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleobase or nucleotide sequence (e.g., RNAi agent sense strand or targeted mRNA) in relation to a second nucleobase or nucleotide sequence (e.g., RNAi agent antisense strand or a single-stranded antisense oligonucleotide), means the ability of an oligonucleotide or polynucleotide including the first nucleotide sequence to hybridize (form base pair hydrogen bonds under mammalian physiological conditions (or similar conditions in vitro)) and form a complex or double helical structure under certain standard conditions w ith an oligonucleotide or polynucleotide including the second nucleotide sequence. Complementary sequences include Watson-Crick base pairs or non-Watson-Crick base pairs and include natural or modified nucleotides or nucleotide mimics, at least to the extent that the above hybridization requirements are fulfilled. Sequence identity' or complementarity' is independent of modification. For example, a and Af, as defined herein, are complementary' to U (or T) and identical to A for the purposes of determining identity or complementarity.

[0036] As used herein, “perfectly complementary" or “fully complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, all (100%) of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.

[0037] As used herein, “partially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 70%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.

[0038] As used herein, “substantially complementary” means that in a hybridized pair of nucleobase or nucleotide sequence molecules, at least 85%, but not all, of the bases in a contiguous sequence of a first oligonucleotide will hybridize with the same number of bases in a contiguous sequence of a second oligonucleotide. The contiguous sequence may comprise all or a part of a first or second nucleotide sequence.

[0039] As used herein, the terms “complementary,” “fully complementary,” "partially complementary,” and “substantially complementary” are used with respect to the nucleobase or nucleotide matching between the sense strand and the antisense strand of an RNAi agent, or between the antisense strand of an RNAi agent and a sequence of a target mRNA.

[0040] As used herein, an “oligonucleotide-based agent” is a nucleotide sequence containing about 10-50 (e g., 10 to 48, 10 to 46, 10 to 44, 10 to 42, 10 to 40, 10 to 38, 10 to 36, 10 to 34, 10 to 32, 10 to 30, 10 to 28, 10 to 26, 10 to 24, 10 to 22, 10 to 20, 10 to 18, 10 to 16, 10 to 14, 10 to 12, 12 to 50, 12 to 48, 12 to 46, 12 to 44, 12 to 42, 12 to 40, 12 to 38, 12 to 36, 12 to 34, 12 to 32, 12 to 30, 12 to 28, 12 to 26, 12 to 24, 12 to 22. 12 to 20. 12 to 18, 12 to 16, 12 to 14, 14 to 50, 14 to 48, 14 to 46, 14 to 44, 14 to 42, 14 to 40, 14 to 38, 14 to 36, 14 to 34, 14 to 32, 14 to 30, 14 to 28, 14 to 26, 14 to 24, 14 to 22, 14 to 20, 14 to 18, 14 to 16, 16 to 50, 16 to 48, 16 to 46, 16 to 44, 16 to 42, 16 to 40, 16 to 38, 16 to 36, 16 to 34, 16 to 32, 16 to 30, 16 to 28, 16 to 26, 16 to 24, 16 to 22, 16 to 20, 16 to 18, 18 to 50. 18 to 48. 18 to 46, 18 to 44, 18 to 42, 18 to 40, 18 to 38, 18 to 36, 18 to 34, 18 to 32, 18 to 30. 18 to 28, 18 to 26, 18 to 24, 18 to 22, 18 to 20, 20 to 50, 20 to 48, 20 to 46, 20 to 44, 20 to 42, 20 to 40, 20 to 38, 20 to 36, 20 to 34, 20 to 32, 20 to 30, 20 to 28, 20 to 26, 20 to 24, 20 to 22, 22 to 50, 22 to 48, 22 to 46, 22 to 44, 22 to 42, 22 to 40, 22 to 38, 22 to 36, 22 to 34, 22 to 32. 22 to 30, 22 to 28, 22 to 26, 22 to 24, 24 to 50, 24 to 48, 24 to 46, 24 to 44, 24 to 42, 24 to 40. 24 to 38. 24 to 36. 24 to 34, 24 to 32, 24 to 30, 24 to 28, 24 to 26, 26 to 50, 26 to 48, 26 to 46, 26 to 44, 26 to 42, 26 to 40,26 to 38, 26 to 36, 26 to 34, 26 to 32, 26 to 30, 26 to 28, 28 to 50, 28 to 48, 28 to 46, 28 to 44, 28 to 42, 28 to 40, 28 to 38, 28 to 36, 28 to 34, 28 to 32, to 28 to 30, 30 to 50, 30 to 48, 30 to 46, 30 to 44, 30 to 42, 30 to 40, 30 to 38, 30 to 36, 30 to 34, 30 to 32, 32 to 50, 32 to 48, 32 to 46, 32 to 44, 32 to 42, 32 to 40, 32 to 38, 32 to 36, 32 to 34, 34 to 50, 34 to 48, 34 to 46, 34 to 44, 34 to 42, 34 to 40, 34 to 38, 34 to 36, 36 to 50, 36 to 48, 36 to 46, 36 to 44, 36 to 42, 36 to 40, 36 to 38, 38 to 50, 38 to 48, 38 to 46, 38 to 44, 38 to 42, 38 to 40. 40 to 50, 40 to 48, 40 to 46, 40 to 44, 40 to 42, 42 to 50, 42 to 48, 42 to 46, 42 to 44, 44 to 50, 44 to 48, 44 to 46, 46 to 50, 46 to 48, or 48 to 50) nucleotides or nucleotide base pairs. In some embodiments, an oligonucleotide-based agent has anucleobase sequence that is at least partially complementary to a coding sequence in an expressed target nucleic acid or target gene within a cell. In some embodiments, the oligonucleotide-based agent, upon delivery to a cell expressing a gene, are able to inhibit the expression of the underlying gene, and are referred to herein as “expressioninhibiting oligonucleotide-based agents.” The gene expression can be inhibited in vitro or in vivo.

[0041] “Oligonucleotide-based agents” include, but are not limited to: single-stranded oligonucleotides, single-stranded antisense oligonucleotides, short interfering RNAs (siRNAs), double-strand RNAs (dsRNA), micro RNAs (miRNAs), short hairpin RNAs (shRNA), ribozymes, interfering RNA molecules, and dicer substrates. In some embodiments, an oligonucleotide-based agent is a single-stranded oligonucleotide, such as an antisense oligonucleotide. In some embodiments, an oligonucleotide-based agent is a double-stranded oligonucleotide. In some embodiments, an oligonucleotide-based agent is a double-stranded oligonucleotide that is an RNAi agent.

[0042] As used herein, the term “substantially identical” or “substantial identity,” as applied to a nucleic acid sequence means the nucleotide sequence (or a portion of a nucleotide sequence) has at least about 85% sequence identity or more, e.g., at least 90%, at least 95%, or at least 99% identity, compared to a reference sequence. 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 at which the same type of nucleic acid base occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity7.

[0043] As used herein, the terms “treat,” “treatment,” and the like, mean the methods or steps taken to provide relief from 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” mayinclude the preventative treatment, management, prophylactic treatment, and / or inhibition or reduction of the number, severity, and / or frequency of one or more symptoms of a disease in a subject.

[0044] As used herein, the phrase ‘'introducing into a cell,” when referring to an RNAi agent, means functionally delivering the RNAi agent into a cell. The phrase “functional delivery,” means delivering the RNAi agent to the cell in a manner that enables the RNAi agent to have the expected biological activity, e.g., sequence-specific inhibition of gene expression.

[0045] As used herein, the term “isomers” refers to compounds that have identical molecular formulae, but that differ in the nature or the sequence of bonding of their atoms or in the arrangement of their atoms in space. Isomers that differ in the arrangement of their atoms in space are termed “stereoisomers.” Stereoisomers that are not mirror images of one another are termed '‘diastereoisomers,” and stereoisomers that are non-superimposable mirror images are termed “enantiomers,” or sometimes optical isomers. A carbon atom bonded to four nonidentical substituents is termed a “chiral center.”

[0046] As used herein, unless specifically identified in a structure as having a particular conformation, for each structure in which asymmetric centers are present 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 mixtures of diastereomers as well as single stereoisomers.

[0047] As used in a claim herein, the phrase “consisting of’ excludes any element, step, or ingredient not specified in the claim. When used in a claim herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.

[0048] The person of ordinary skill in the art would 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 upon the environment in which the compound or composition is placed. Accordingly, as used herein, the structures disclosed herein envisage that certain functional groups, such as, for example, OH, SH, or NH, may be protonated or deprotonated. The disclosure herein is intended to cover the disclosed compounds and compositions regardless of their state of protonation based on the environment (such as pH), as would be readily understood by the person of ordinary' skill in the art.

[0049] As used herein, the term “linked” or “conjugated” when referring to the connection between two compounds or molecules means that two molecules are joined by a covalent bondor are associated via noncovalent bonds (e.g., hydrogen bonds or ionic bonds). In some examples, where the term "‘linked” or “conjugated” refers to the association between two molecules via noncovalent bonds, the association between the two different molecules has a KD of less than 1 x 10'4M (e.g., less than 1 x 10'5M, less than 1 x 10'6M, or less than 1 x 10'7M) in physiologically acceptable buffer (e.g., buffered saline). Unless stated, the terms “linked” and “conjugated” as used herein may refer to the connection between a first compound and a second compound either with or without any intervening atoms or groups of atoms.

[0050] As used herein, a linking group is one or more atoms that connects one molecule or portion of a molecule to another to second molecule or second portion of a molecule. Similarly, as used in the art, the term scaffold is sometimes used interchangeably with a linking group. Linking groups may comprise any number of atoms or functional groups. In some embodiments, linking groups may not facilitate any biological or pharmaceutical response, and merely serve to link two biologically active molecules.

[0051] Unless stated otherwise, the symbolas used herein means that any group or groups may be linked thereto that is in accordance with the scope of the inventions described herein.

[0052] As used herein, the term “including” is used to herein 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 the context clearly indicates otherwise.

[0053] As used in a claim herein, the phrase “consisting of' excludes any element, step, or ingredient not specified in the claim. When used in a claim herein, the phrase “consisting essentially of’ limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s) of the claimed invention.Modified Nucleotides

[0054] In some embodiments, an 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 mimics, abasic nucleotides (represented herein as Ab), 2'-modified nucleotides, 3' to 3' linkages (inverted) nucleotides (represented herein as invdN,invN, invn), modified nucleobase-comprising nucleotides, bridged nucleotides, peptide nucleic acids (PNAs), 2', 3 '-seco nucleotide mimics (unlocked nucleobase analogues, represented herein as NUNA or NUNA), locked nucleotides (represented herein as NLNA or NLNA), 3'-O-methoxy (2' intemucleoside linked) nucleotides (represented herein as 3'-0Men), 2-F-Arabino nucleotides (represented herein as NfANA orNfANA), 5'-Me, 2'-fluoro nucleotide (represented herein as 5Me-Nf), morpholino nucleotides, vinyl phosphonate deoxyribonucleotides (represented herein as vpdN), vinyl phosphonate containing nucleotides, and cyclopropyl phosphonate containing nucleotides. 2'-modified nucleotides (i.e., a nucleotide with a group other than a hydroxyl group at the 2' position of the five-membered sugar ring) include, but are not limited to, 2'-O-methyl nucleotides (represented herein as a lower case letter ‘n‘ in a nucleotide sequence), 2'-deoxy-2'-fluoro nucleotides (also referred to herein as 2’-fluoro nucleotide, and represented herein as Nf), 2'-deoxy nucleotides (represented herein as dN), 2'-methoxyethyl (2'-O-2-methoxylethyl) nucleotides (also referred to herein as 2'-M0E, and represented herein as NM), 2'-amino nucleotides, and 2'-alkyl nucleotides. It is not necessary for all positions in a given compound to be uniformly modified. Conversely, more than one modification can be incorporated in a single RNAi agent or even in a single nucleotide thereof. The RNAi agent sense strands and antisense strands can be synthesized and / or modified by methods known in the art. Modification at one nucleotide is independent of modification at another nucleotide.

[0055] Modified nucleobases include synthetic and natural nucleobases. such as 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, (e.g., 2-aminopropvladenine, 5-propynyluracil, or 5-propynylcytosine), 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, 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, 5-halouracil, cytosine, 5-propynyl uracil, 5-propynyl cytosine, 6-azo uracil, 6-azo cytosine, 6-azo thymine, 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 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine, 7 -deazaadenine, 3 -deazaguanine, and 3 -deazaadenine.

[0056] In some embodiments, all or substantially all of the nucleotides of an RNAi agent are modified nucleotides. As used herein, an RNAi agent wherein substantially all of the nucleotides present are modified nucleotides is an RNAi agent having four or fewer (i.e., 0, 1,2, 3, or 4) nucleotides in both the sense strand and the antisense strand being ribonucleotides (i. e., unmodified). As used herein, a sense strand wherein substantially all of the nucleotides present are modified nucleotides is a sense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. As used herein, an antisense sense strand wherein substantially all of the nucleotides present are modified nucleotides is an antisense strand having two or fewer (i.e., 0, 1, or 2) nucleotides in the sense strand being unmodified ribonucleotides. In some embodiments, one or more nucleotides of an RNAi agent is an unmodified ribonucleotide.Modified Intemucleoside Linkages

[0057] In some embodiments, one or more nucleotides of an RNAi agent are linked by non-standard linkages or backbones (i.e., modified intemucleoside linkages or modified backbones). Modified intemucleoside linkages or backbones include, but are not limited to, phosphorothioate groups (represented herein as a lower casec's”), chiral phosphorothi oates, thiophosphates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, alkyl phosphonates (e.g., methyl phosphonates or 3'-alkylene phosphonates), chiral phosphonates, phosphinates, phosphoramidates (e.g., 3 '-amino phosphorami date, aminoalkylphosphoramidates, or thionophosphoramidates), thionoalkyl-phosphonates, thionoalkylphosphotriesters, morpholino linkages, boranophosphates having normal 3'-5' linkages, 2'-5' linked analogs of boranophosphates, or boranophosphates having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3 '-5' to 5'-3' or 2'-5' to 5'-2'. In some embodiments, a modified intemucleoside linkage or backbone lacks a phosphorus atom. Modified intemucleoside linkages lacking a phosphorus atom include, but are not limited to, short chain alkyl or cycloalkyl inter-sugar linkages, mixed heteroatom and alkyl or cycloalkyl inter-sugar linkages, or one or more short chain heteroatomic or heterocyclic inter-sugar linkages. In some embodiments, modified intemucleoside backbones include, but are not limited to, siloxane backbones, sulfide backbones, sulfoxide backbones, sulfone backbones, formacetyl and thioformacetyl backbones, methylene formacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and other backbones having mixed N, O, S, and CH2 components.

[0058] In some embodiments, a sense strand of an RNAi agent can contain 1, 2, 3, 4, 5, or 6 phosphorothioate linkages, an antisense strand of an RNAi agent can contain 1, 2, 3, 4, 5, or6 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3. 4, 5, or 6 phosphorothioate linkages. In some embodiments, a sense strand of an RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, an antisense strand of an RNAi agent can contain 1, 2, 3, or 4 phosphorothioate linkages, or both the sense strand and the antisense strand independently can contain 1, 2, 3, or 4 phosphorothioate linkages.

[0059] In some embodiments, an RNAi agent sense strand contains at least two phosphorothioate intemucleoside linkages. In some embodiments, the at least two phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 3' end of the sense strand. In some embodiments, one phosphorothioate intemucleoside linkage is at the 5' end of the sense strand, and another phosphorothioate linkage is at the 3’ end of the sense strand. In some embodiments, two phosphorothioate intemucleoside linkage 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 include any phosphorothioate intemucleoside linkages between the nucleotides, but contains one, two, or three phosphorothioate linkages between the terminal nucleotides on both the 5’ and 3' ends and the optionally present inverted abasic residue terminal caps. In some embodiments, the targeting ligand is linked to the sense strand via a phosphorothioate linkage.

[0060] In some embodiments, an RNAi agent antisense strand contains four phosphorothioate intemucleoside linkages. In some embodiments, the four phosphorothioate intemucleoside linkages are between the nucleotides at positions 1-3 from the 5' end of the antisense strand and between the nucleotides at positions 17-19, 18-20, 19-21, 20-22, 21-23, 22-24, 23-25, or 24-26 from the 5' end. In some embodiments, three phosphorothioate intemucleoside linkages are located between positions 1-4 from the 5’ end of the antisense strand, and a fourth phosphorothioate intemucleoside linkage is located between positions 20-21 from the 5’ end of the antisense strand. In some embodiments, an RNAi agent contains at least three or four phosphorothioate intemucleoside linkages in the antisense strand.

[0061] In some embodiments, an RNAi agent contains one or more modified nucleotides and one or more modified intemucleoside linkages. In some embodiments, a 2'-modified nucleoside is combined with modified intemucleoside linkage.TSLP-IL33 RNAi Agents

[0062] The TSLP-IL33 RNAi agents disclosed herein are designed to target specific positions on a TSLP or an IL33 gene (e.g., SEQ ID N Os: 1 and 2).NM_033035.5 Homo sapiens thymic stromal lymphopoietin (TSLP), mRNA transcript (SEQ ID NO: 1), 2610 bases:1 atcagggaga ctccaactta aggcaacagc atgggtgaat aagggcttcc tgtggactgg61 caatgagagg caaaacctgg tgcttgagca ctggccccta aggcaggcct tacagatctc121 ttacactcgt ggtgggaaga gtttagtgtg aaactggggt ggaattgggt gtccacgtat181 gttccctttt gccttactat atgttctgtc agtttctttc aggaaaatct tcatcttaca241 acttgtaggg ctggtgttaa cttacgactt cactaactgt gactttgaga agattaaagc301 agcctatctc agtactattt ctaaagacct gattacatat atgagtggga ccaaaagtac361 cgagttcaac aacaccgtct cttgtagcaa tcggccacat tgccttactg aaatccagag421 cctaaccttc aatcccaccg ccggctgcgc gtcgctcgcc aaagaaatgt tcgccatgaa481 aactaaggct gccttagcta tctggtgccc aggctattcg gaaactcaga taaatgctac541 tcaggcaatg aagaagagga gaaaaaggaa agtcacaacc aataaatgtc tggaacaagt601 gtcacaatta caaggattgt ggcgtcgctt caatcgacct ttactgaaac aacagtaaac661 catctttatt atggtcatat ttcacagcac caaaataaat catctttatt aagtagatga721 aacattaact ctaactgtga caaagaagac cacaaatagt tatcttttaa ttacagaaga781 gtttcttaac ttacttttgt aagtttttat tgtgtaagtt tataatgcag gggaagtact841 actcctcaaa tgttgaggga agcttccata acattgatga ctggcttcat ggcagtaatt901 ctcggctgta gttgcataag cattgctcaa gaggaaaatc caaaagtgca gcaggagaac961 tcttttccct gaaaaaggaa aaatattgaa ctcaatgata gcacctaaac ttacatttaa1021 aagacagaca ttccttctac atgtaatgac acttcttgtg ttaaactaaa aatttacaag1081 agaagaaagt gaaagcaaat ggggtttcac aaatagttgt aaatatagtg aagcaatttg1141 aaataatttt caagcaaagt attgtgaaag tattctaagc caagttttaa atattatcta1201 acagacaaga gtggtatata caagtagatc ctgagaagta cctttgttac agctactata1261 aatatacata taaattatag aatctacttt aatttatttt gtgaacactt ttgaaaatgt1321 acatgttcct ttgtaattga cactatatat ttcttaataa aataattctc aaatttgttt1381 cttatgaatc atctctcaaa tctagttaga caatttgcac acatactttt ctaagggaca1441 ttatcttcct tcaggttttt acctccactc atccttagag cccactgact gctccccttt1501 atacctgttg gccctgccta taggagagaa tatttggaga taggcagctt caggatgcat1561 tgcaatcatc cttttcttaa attatgtcac tagtctttta ttttttcccc tcttgaactt1621 tcctcacacc tggaagaaac aaagtaggaa aaagtgaaca ggggatgtca aatcgattct1681 tgaattcccg ctgcaagcta gagccgcagg caccctctca ctcaatttcc actcagaacc1741 ctataaacac cagtgggaag ggcaacccac tgcacgtggg aatgcactga tttttcctag1801 gagtagacat gttcctctaa ttactccctg agggttagtt ggggctaaac catgacagaa1861 gtggggaagt tcaatgtcct taaatccatc ttacttgcca acaggtaaga ggaagcttac1921 attacatgtc cagtccacat taaagagca cttactgtgg aacaagcct cagccaaaca 1981 atggggatag aaaagtaggt aagactcagc ctttgtccag agaagctcag ggtatagctg2041 aataggcagt ttcttttgtc ctgaggaaaa tcaggacatg cctgcttct aaaaatcttc2101 ctctgaagac ctgacccaag ctcttaaatg ctattgtaag agaaatttct ttgtctatta2161 actccatttt agtagggatt cactgactag attttactga actatgaaaa taaatacaca2221 taatttttca caaaattttg ggcccaattc ccctaaaaga attgaggat agggagaaag2281 gagacaactc aaagtcatcc cataagtgc agtttctttg aatcttctgc tttatctta2341 aaaatttgta taatttatat atttatct atgtgttcca tagatatctt aatgtaaaat2401 tagtcattta aattacactg tcaattaaaa gtaatgggca agagattgca tcatactaat2461 tagtaagaa cgtcccaaa tgttgtaaca atgtggatca tacatctctg gtttttaaa2521 tgtatgagg cttcttggt ggactagtat agtatacggt cagttatgtc aatgttcat2581 ggtcaataaa aaggaagtg caaattgtgaNM_033439.4. Homo sapiens, interleukin 33 (IL33), mRNA transcript (SEQ ID NO: 2), 2685 bases:1 acagagctgc agctcttcag ggaagaaatc aaaacaagat cacaagaata ctgaaaaatg61 aagcctaaaa tgaagtattc aaccaacaaa atttccacag caaagtggaa gaacacagca121 agcaaagcct tgtgtttcaa gctgggaaaa tcccaacaga aggccaaaga agtttgcccc181 atgtacttta tgaagctccg ctctggcctt atgataaaaa aggaggcctg ttactttagg241 agagaaacca ccaaaaggcc ttcactgaaa acaggtagaa agcacaaaag acatctggta301 ctcgctgcct gtcaacagca gtctactgtg gagtgctttg cctttggtat atcaggggtc361 cagaaatata ctagagcact tcatgattca agtatcacag gaatttcacc tattacagag421 tatcttgctt ctctaagcac atacaatgat caatccatta cttttgcttt ggaggatgaa481 agttatgaga tatatgttga agacttgaaa aaagatgaaa agaaagataa ggtgttactg541 agttactatg agtctcaaca cccctcaaat gaatcaggtg acggtgttga tggtaagatg601 ttaatggtaa ccctgagtcc tacaaaagac ttctggttgc atgccaacaa caaggaacac661 tctgtggagc tccataagtg tgaaaaacca ctgccagacc aggccttctt tgtccttcat721 aatatgcact ccaactgtgt ttcatttgaa tgcaagactg atcctggagt gtttataggt781 gtaaaggata atcatcttgc tctgattaaa gtagactctt ctgagaattt gtgtactgaa841 aatatcttgt ttaagctctc tgaaacttag ttgatggaaa cctgtgagtc ttgggttgag901 tacccaaatg ctaccactgg agaaggaatg agagataaag aaagagacag gtgacatcta961 agggaaatga agagtgctta gcatgtgtgg aatgttttcc atattatgta taaaaatatt1021 ttttctaatc ctccagttat tcttttattt ccctctgtat aactgcatct tcaatacaag1081 tatcagtata ttaaataggg tatggtaaa gaaacggtca acatctaaa gagatacagt 1141 ctgaccttta cttttctcta gtttcagtcc agaaagaact tcatattag agctaaggcc1201 actgaggaaa gagccatagc taagtctct atgtagacag ggatccatt taaagagcta1261 cttagagaaa taattttcca cagttccaaa cgataggctc aaacactaga gctgctagta1321 aaaagaagac cagatgcttc acagaattat cattttttca actggaataa aacaccaggt1381 ttgtttgtag atgtcttagg caacactcag agcagatctc ccttactgtc aggggatatg1441 gaactcaaa ggcccacatg gcaagccagg taacataaat gtgtgaaaaa gtaaagataa1501 ctaaaaaatt tagaaaaata aatccagtat tgtaaagtg aataacttca tttctaattg1561 tttaattttt aaaattctga tttttatata ttgagtttaa gcaaggcatt cttacacgag1621 gaagtgaagt aaatttagt tcagacataa aatttcactt attaggaata tgtaacatgc1681 taaaacttt tttttttaa agagtactga gtcacaacat gtttagagc atccaagtac1741 catataatcc aactatcatg gtaaggccag aaatctcta acctaccaga gcctagatga1801 gacaccgaat taacattaaa atttcagtaa ctgactgtcc ctcatgtcca tggcctacca1861 tcccttctga ccctggcttc cagggaccta tgtcttttaa tactcactgt cacattgggc1921 aaagtgct ctaatccta tttcccatgt gcacaagtct tttgtatc cagctcctg1981 ataacactgc ttactgtgga atattcattt gacatctgtc tcttttcat tctttaact2041 accatgccct tgatatatct tttgcacctg ctgaacttca tttctgtatc acctgacctc2101 tggatgccaa aacgtttatt ctgctttgtc tgtgtagaa ttttagataa agctattaat2161 ggcaatattt ttttgctaaa cgtttttgtt tttactgtc actagggcaa taaaattat2221 actcaaccat ataataacat tttaacta ctaaaggagt agtttttatt taaagtct2281 agcaatttct atacaactt ttcttagact taacactat gataaatgac taacatagta2341 acagaatctt tatgaaatat gaccttttct gaaaatacat acttttacat ttctacttta2401 ttgagaccta ttagatgtaa gtgctagtag aatataagat aaaagaggct gagaattacc2461 atacaagggt atacaactg taaaacaat tatcttgt tcatgtct gtcaataat2521 gttaccaaag agataaaaat aaaagcagaa tgtatatcat cccatctgaa aaacactaat2581 tattgacatg tgcatctgta caataaactt aaaatgatta ttaaataatc aaatatatct2641 actacattgt ttatattatt gaataaagta tattttccaa atgta

[0063] As defined herein, an antisense strand sequence is designed to target a TSLP gene or an IL33 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 to the gene. For example, as illustrated in Tables 1A, IB, 2 A, and 2B herein, an antisense strand sequence designed to target an IL33 gene atposition 1322 requires that when base pairing to the gene, the 5' terminal nucleobase of the antisense strand is aligned with position 1342 of the IL33 gene.

[0064] As provided herein, a TSLP-IL33 agent does 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) of the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides. For example, for a TSLP-IL33 RNAi agent disclosed herein that is designed to target position 1322 of an IL33 gene, the 5' terminal nucleobase of the antisense strand of the of the IL33 RNAi agent is aligned with position 1342 of the gene; however, the 5' terminal nucleobase of the antisense strand may be, but is not required to be, complementary to position 1342 of an IL33 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) of the antisense strand and the gene across a core stretch sequence of at least 16 consecutive nucleotides. As shown by, among other things, the various examples disclosed herein, the specific site of binding of the gene by the antisense strand of the TSLP- IL33 RNAi agent (e.g.. the position where the TSLP-IL33 RNAi agent is designed to target the IL33 gene and the position where the TSLP-IL33 RNAi agent is designed to target the TSLP gene) is important to the level of inhibition achieved by the TSLP-IL33 RNAi agent.

[0065] In some embodiments, the TSLP-IL33 RNAi agents disclosed herein target a TSLP or IL33 gene at or near the positions of the TSLP or IL33 gene sequence shown in Table 1 A or Table IB. In some embodiments, the antisense strands of aTSLP-IL33 RNAi agent disclosed herein include a core stretch sequence that is fully, substantially, or at least partially complementary to a target TSLP or IL33 19-mer sequence disclosed in Table 1A and Table IB.Table 1. TSLP 19-mer mRNA Target Sequences (taken from Homo sapiens thymic stromal lymphopoietin (TSLP), mRNA, GenBankNM_033035.5 (SEQ ID NO:1)).Table IB. IL33 19-mer mRNA Target Sequences (taken from Homo sapiens interleukin 33 (IL33), mRNA, GenBank NM_033439.4 (SEQ ID N0:2)).

[0066] In some embodiments, a TSLP-IL33 RNAi agent includes an antisense strand wherein position 19 of the antisense strand (5 — >3') is capable of forming a base pair with position 1 of a 19-mer target sequence disclosed in Table 1A or Table IB. In some embodiments, a TSLP-IL33 RNAi agent includes an antisense strand wherein position 1 of the antisense strand (5'— >3') is capable of forming a base pair with position 19 of the 19-mer target sequence disclosed in Table 1A or Table IB.Capping Residues or Moieties

[0067] In some embodiments, the sense strand may include one or more capping residues or moieties, sometimes referred to in the art as a “cap,’" a “terminal cap,” or a “capping residue.” As used herein, a “capping residue” is a non-nucleotide compound or other moiety that can be incorporated at one or more termini of a nucleotide sequence of an RNAi agent disclosed herein. A capping residue can provide the RNAi agent, in some instances, with certain beneficial properties, such as, for example, protection against nuclease degradation. In some embodiments, inverted abasic residues (invAb) (also referred to in the art as “inverted abasic sites”) are added as capping residues (see Table 6). (See, e.g., 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 as well as carbon chains such as a terminal C3H7 (propyl), CeHi3 (hexyl), or C12H25 (dodecyl) groups. In some embodiments, a capping residue is present at either the 5' terminal end, the 3' terminal end, or both the 5' and 3' terminal ends of the sense strand. In some embodiments, the 5 ’ end and / or the 3' end of the sense strand may include more than one inverted abasic deoxyribose moiety as a capping residue.

[0068] 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 invertedabasic residues or inverted abasic sites are inserted between the targeting ligand and the nucleotide sequence of the sense strand of the RNAi agent. In some embodiments, the inclusion of one or more inverted abasic residues or inverted abasic sites at or near the terminal end or terminal ends of the sense strand of an RNAi agent allows for enhanced activity or other desired properties of an RNAi agent.

[0069] 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 nucleotide sequence of the sense strand of the RNAi agent. The inverted abasic residues may be linked via phosphate, phosphorothioate (e.g., shown herein as (invAb)s)), or other intemucleoside linkages. In some embodiments, the inclusion of one or more inverted abasic residues at or near the terminal end or terminal ends of the sense strand of an RNAi agent may allow for enhanced activity or other desired properties of an RNAi agent. In some embodiments, an inverted abasic (deoxyribose) residue can be replaced with an inverted ribitol (abasic ribose) residue. In some embodiments, the 3' end of the antisense strand core stretch sequence, or the 3' end of the antisense strand sequence, may include an inverted abasic residue. The chemical structures for inverted abasic deoxyribose residues are shown in Table 6 below.

[0070] In some embodiments, a TSLP-IL33 RNAi agent includes an antisense strand wherein position 2 of the antisense strand (5'^3') is capable of forming a base pair with position 18 of the 19-mer target sequence disclosed in Table 1A or Table IB. In some embodiments, a TSLP-IL33 RNAi agent includes an antisense strand wherein positions 2 through 18 of the antisense strand (5 ' — >3') are capable of forming base pairs with each of the respective complementary bases located at positions 18 through 2 of the 19-mer target sequence disclosed in Table 1 A or Table IB.

[0071] For the RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (from 5' end 3' end) can be perfectly complementary to the TSLP or IL33 gene, or can be non-complementary to the TSLP or IL33 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end — 3' end) is a U, A, or dT. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end — > 3' end) forms an A: U or U: A base pair with the sense strand.

[0072] In some embodiments, a TSLP-IL33 RNAi agent antisense strand comprises the sequence of nucleotides (from 5' end —> 3' end) at positions 2-18, 2-19, 2-20, or 2-21 of any of the antisense strand sequences in Table 2A, Table 2B, Table 3A, Table 3B, or Table 5C. Insome embodiments, a TSLP-IL33 RNAi sense strand comprises the sequence of nucleotides (from 5' end 3' end) at positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences in Table 2A, Table 2B, Table 4A, Table 4B, Table 4C, or Table 5C.

[0073] In some embodiments, a TSLP-IL33 RNAi agent antisense strand comprises the sequence of nucleotides (from 5' end 3' end) at positions 2-18, 2-19, 2-20, or 2-21 of any of the antisense strand sequences of Table 2A, Table 2B, Table 3A, Table 3B, or Table 5C. In some embodiments, a TSLP-IL33 RNAi sense strand comprises the sequence of nucleotides (from 5' end 3' end) at positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18, 2-18, or 1-18 of any of the sense strand sequences of Table 2A, Table 2B, Table 4A, Table 4B, Table 4C, or Table 5C.

[0074] In some embodiments, a TSLP-IL33 RNAi agent is comprised of (i) an antisense strand comprising the sequence of nucleotides (from 5' end 3' end) at positions 2-18 or 2-19 of any of the antisense strand sequences in Table 2 or Table 3, and (ii) a sense strand comprising the sequence of nucleotides (from 5' end — > 3' end) at positions 3-21, 2-21, 1-21, 3-20, 2-20, 1-20, 3-19, 2-19, 1-19, 3-18. 2-18, or 1-18 of any of the sense strand sequences in Table 2A, Table 2B, Table 4A, Table 4B, or Table 4C.

[0075] In some embodiments, the TSLP-IL33 RNAi agents include core 19-mer nucleotide sequences shown in the following Table 2A or Table 2B.Table 2A. TSLP-IL33 RNAi Agent Antisense Strand and Sense Strand Core Stretch Base Sequences (N=any nucleobase; I = hypoxanthine (inosine nucleotide); (A2N) = 2-aminoadenine nucleotide), targeting TSLPTable 2B. TSLP-IL33 RNAi Agent Antisense Strand and Sense Strand Core Stretch Base Sequences (N=any nucleobase; I = hypoxanthine (inosine nucleotide); (A2N) = 2-aminoadenine nucleotide), targeting IL33

[0076] The TSLP-IL33 RNAi agent sense strands and antisense strands that comprise or consist of the sequences in Table 2A or Table 2B can be modified nucleotides or unmodified nucleotides. In some embodiments, the TSLP-IL33 RNAi agents having the sense and antisense strand sequences that comprise or consist of the sequences in Table 2A or Table 2B are all or substantially all modified nucleotides.

[0077] In some embodiments, the antisense strand of aTSLP-IL33 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 2A or Table 2B. In some embodiments, the sense strand of a TSLP-IL33 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 2A or Table 2B.

[0078] As used herein, each N listed in a sequence disclosed in Table 2A or Table 2B may be independently selected from any and all nucleobases (including those found on both modified and unmodified nucleotides). In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2A or Table 2B has a nucleobase that is complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2A or Table 2B has a nucleobase that is not complementary to the N nucleotide at the corresponding position on the other strand. In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2A or Table 2B has a nucleobase that is the same as the N nucleotide at the corresponding position on the other strand. In some embodiments, an N nucleotide listed in a sequence disclosed in Table 2A or Table 2B has a nucleobase that is different from the N nucleotide at the corresponding position on the other strand.

[0079] Certain modified TSLP-IL33 RNAi agent antisense strands, as well as their underlying unmodified nucleobase sequences, are provided in Table 3A or Table 3B. Certain modified TSLP-IL33 RNAi agent sense strands, as well as their underlying unmodified nucleobase sequences, are provided in Table 4A, Table 4B, or Table 4C. In forming TSLP-IL33 RNAi agents, each of the nucleotides in each of the underlying base sequences listed in Tables 3A, 3B, 4A, 4B, and 4C, as well as in Tables 2A and 2B, above, can be a modified nucleotide.

[0080] The TSLP-IL33 RNAi agents described herein are formed by annealing an antisense strand with a sense strand. A sense strand containing a sequence listed in Table 2A, Table 2B, Table 4A, Table 4B, or Table 4C, can be hybridized to any antisense strand containing asequence listed in Table 2A, Table 2B, Table 3A, or Table 3B, provided the two sequences have a region of at least 85% complementarity over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence.

[0081] In some embodiments, a TSLP-IL33 RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2A, Table 2B, Table 3A, or Table 3B.

[0082] In some embodiments, a TSLP-IL33 RNAi agent comprises or consists of a complex having the nucleobase sequences of the sense strand and the antisense strand of any of the sequences in Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, or Table 4C.

[0083] Examples of antisense strands containing modified nucleotides are provided in Table 3A, Table 3B, and Table 5C. Examples of sense strands containing modified nucleotides are provided in Table 4A, Table 4B, Table 4C, and Table 5C.

[0084] As used in Tables 3A, Table 3B, Table 4A, Table 4B, Table 4C, and Table 5C the following notations are used to indicate modified nucleotides and linking groups:A = adenosine-3'-phosphate;C = cytidine-3 '-phosphate;G = guanosine-3'-phosphate;U = uridine-3 '-phosphateI = inosine-3 '-phosphatea = 2'-O-methyladenosine-3 '-phosphateas = 2'-O-methyladenosine-3'-phosphorothioatec = 2'-O-methylcytidine-3 '-phosphatecs = 2'-O-methylcytidine-3'-phosphorothioateg = 2'-O-methylguanosine-3'-phosphategs = 2'-O-methylguanosine-3'-phosphorothioatet = 2'-O-methyl-5-methyluridine-3'-phosphatets = 2'-O-methyl-5-methyluridine-3'-phosphorothioateu = 2'-O-methyluridine-3'-phosphateus = 2'-O-methyluridine-3'-phosphorothioatei = 2'-O-methylinosine-3'-phosphateis = 2'-O-methylinosine-3'-phosphorothioateAf = 2'-fluoroadenosine-3'-phosphateAfs = 2'-fluoroadenosine-3'-phosporothioateCf = 2'-fluorocytidine-3'-phosphateCfs = 2'-fluorocytidine-3'-phosphorothioateGf = 2'-fluoroguanosine-3'-phosphateGfs = 2'-fluoroguanosine-3'-phosphorothioateTf = 2'-fluoro-5'-methyluridine-3'-phosphateTfs = 2'-fluoro-5'-methyluridine-3'-phosphorothioateUf = 2'-fluorouridine-3'-phosphateUfs = 2'-fluorouridine-3'-phosphorothioateAUNA = 2',3'-seco-adenosine-3'-phosphate (see Table 6)AUNAS = 2',3'-seco-adenosine-3'-phosphorothioate (see Table 6)CUNA = 2',3'-seco-cytidine-3'-phosphate (see Table 6)CUNAS = 2',3'-seco-cytidine-3'-phosphorothioate (see Table 6)GUNA = 2',3'-seco-guanosine-3'-phosphate (see Table 6)GUNAS = 2',3'-seco-guanosine-3'-phosphorothioate (see Table 6)UUNA = 2'.3'-seco-uridine-3’-phosphate (see Table 6)UUNAS = 2',3'-seco-uridine-3'-phosphorothioate (see Table 6)a_2N = 2'-O-methyl-2-aminoadenosine-3'-phosphate (see Table 6) a_2Ns = 2'-O-methyl-2-aminoadenosine-3'-phosphorothioate (see Table 6) (invAb) = inverted abasic deoxyribonucleotide (see Table 6)(invAb)s = inverted abasic deoxyribonucleotide-5 '- phosphorothioate (see Table 6)cPrpa = 5 ’-cyclopropyl phosphonate-2'-O-methyladenosine-3'-phosphate (see Table 6)cPrpas = 5 ’-cyclopropyl phosphonate-2'-O-methyladenosine-3'- phosphorothioate (see Table 6)cPrpu = 5 ’-cyclopropyl phosphonate-2'-O-methyluridine-3'-phosphate (see Table 6)cPrpus = 5 ’-cyclopropyl phosphonate-2'-O-methyluridine-3'- phosphorothioate (see Table 6)dT = 2 ’-deoxythymidine-3’ -phosphatedTs = 2 ’-deoxythymidine-3 ’-phosphorothioatedTss = 2 ’-deoxythymidine-3’ -phosphorodithioatedU = 2 ' -deoxy uridine-3' -phosphatedUs = 2’-deoxyuridine-3’-phosphorothioatedUss = 2’ -deoxyuridine-3’ -phosphorodithioatedC = 2’-deoxycytidine-3 ’-phosphatedCs = 2’-deoxycytidine-3’-phosphorothioatedG = 2 ’-deoxyguanosine-3’ -phosphatedGs = 2’ -deoxyguanosine-3 ’-phosphorothioatedA = 2 '-deoxy adenosine-3' -phosphatedAs = 2 ’-deoxy adenosine-3' -phosphorothioatedAss = 2 ’-deoxy adenosine-3 ’-phosphorodithioateSpl8 = see Table 6Spl8s = see Table 6Tri-SM6.1-avb6-(TA14) =.see FIG. 1

[0085] As the person of ordinary skill in the art would readily understand, unless otherwise indicated by the sequence (such as. for example, by a phosphorothioate linkagec’s" or phosphorodithioate linkage “ss”), when present in an oligonucleotide, the nucleotide monomers are mutually linked by 5’-3’-phosphodiester bonds. As the person of ordinary skill in the art would clearly understand, the inclusion of a phosphorothioate or phosphorodithioate linkage as shown in the modified nucleotide sequences disclosed herein replaces the phosphodiester linkage typically present in oligonucleotides. Further, the person of ordinary skill in the art would readily understand that the terminal nucleotide at the 3’ end of a given oligonucleotide sequence would typically have a hydroxyl (-OH) group at the respective 3’ position of the given monomer instead of a phosphate moiety ex vivo. Additionally, for the various embodiments disclosed herein, when viewing the respective strand 5’ 3’, the inverted abasic residues are inserted such that the 3’ position of the deoxyribose is linked at the 3’ end of the preceding monomer on the respective strand (see, e.g., Table 6). Moreover, as the person of ordinary skill would readily understand and appreciate, while the phosphorothioate chemical structures depicted herein typically show the anion on the sulfur atom, the inventions disclosed herein encompass all phosphorothioate tautomers and resonance structures (e.g., where the sulfur atom has a double-bond and the anion is on an oxygen atom). Unless expressly indicated otherwise herein, such understandings of the person of ordinaryskill in the art are used when describing the TSLP-IL33 RNAi agents and compositions of TSLP-IL33 RNAi agents disclosed herein.

[0086] Certain examples of targeting ligands, targeting groups, and linking groups used with the TSLP-IL33 RNAi agents disclosed herein are provided below in Table 6. More specifically, targeting groups and linking groups (which together can form a targeting ligand) include Tri-SM6.1-avb6-(TA14), for which their chemical structures are provided below in Figure 1. Each sense strand and / or antisense strand can have any targeting ligands, targeting groups, or linking groups listed herein, as well as other groups, conjugated to the 5' and / or 3' end of the sequence.Table 3A. TSLP-IL33 RNAi Agent Antisense Strand Sequences, targeting TSLP(A2N) = 2-aminoadenine nucleotide; I = hypoxanthine (inosine) nucleotideTable 3B. TSLP-IL33 RNAi Agent Antisense Strand Sequences, targeting IL335 (A2N) = 2-aminoadenine nucleotide; I = hypoxanthine (inosine) nucleotideTable 4A. TSLP-IL33 RNAi Agent Sense Strand Sequences, complementary to an antisense strand targeting TSLP(A2N) = 2-aminoadenine nucleotide; I = hypoxanthine (inosine) nucleotideTable 4B. TSLP-IL33 RNAi Agent Sense Strand Sequences, complementary to an antisense strand targeting IL335 (A2N) = 2-aminoadenine nucleotide; I = hypoxanthine (inosine) nucleotideTable 4C. TSLP-IL33 RNAi Agent Sense Strand Sequences(A2N) = 2-aminoadenine nucleotide; I = hypoxanthine (inosine) nucleotide

[0087] The TSLP-IL33 RNAi agents described herein are formed by annealing each antisense strand to a sense strand sequence having sufficient complementarity to the antisense strand. A sense strand sequence listed in Table 2A, Table 2B, Table 4A, Table 4B, Table 4C, or Table 5C can be hybridized to any antisense strand containing a sequence listed in Table 2A, Table 2B, Table 3A, Table 3B, or Table 5C provided the two sequences have a region of at least 85% complementarity over a contiguous 15, 16, 17, 18. 19, 20, or 21 nucleotide sequence.

[0088] In some embodiments, an antisense strand of a TSLP-1L33 RNAi agent disclosed herein differs by 0, 1, 2, or 3 nucleotides from any of the antisense strand sequences in Table 3A, Table 3B, or Table 5C. In some embodiments, a sense strand sequence of a TSLP-IL33 RNAi agent disclosed herein differs by 0. 1, 2, or 3 nucleotides from any of the sense strand sequences in Table 4A, Table 4B, Table 4C or Table 5C. In some embodiments, a sense strand complementary to an antisense strand targeting TSLP is covalently linked to a sense strand complementary to an antisense strand targeting IL33, and each antisense strand is then annealed to the TSLP-IL33 RNAi agent sense strand.

[0089] In some embodiments, a TSLP-IL33 RNAi agent antisense strand comprises a nucleotide sequence of any of the sequences in Table 2A, Table 2B, Table 3A, Table 3B, or Table 5C. In some embodiments, a TSLP-IL33 RNAi agent antisense strand comprises the sequence of nucleotides (from 5' end —>■ 3' end) at positions 1-17, 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 Table 2A, Table 2B. Table 3A, Table 3B, or Table 5C. In certain embodiments, a TSLP-IL33 RNAi agent antisense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 3A, Table 3B, or Table 5C.

[0090] In some embodiments, a TSLP-IL33 RNAi agent sense strand comprises the nucleotide sequence of any of the sequences in Table 2A, Table 2B, Table 4A, Table 4B, Table 4C, or Table 5C. In some embodiments, a TSLP-IL33 RNAi agent sense strand comprises the sequence of nucleotides (from 5' end — > 3' end) at positions 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, or 4-21, of any of the sequences in Table 2A, Table 2B, Table 4A, Table 4B, Table 4C, or Table 5C. In certain embodiments, a TSLP-IL33 RNAi agent sense strand comprises or consists of a modified sequence of any one of the modified sequences in Table 4A, Table 4B, Table 4C, or Table 5C.

[0091] For the TSLP-IL33 RNAi agents disclosed herein, the nucleotide at position 1 of the antisense strand (from 5' end —> 3' end) can be perfectly complementary to a TSLP gene ora IL33 gene, or can be non-complementary to a TSLP gene or a IL33 gene. In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end —> 3' end) is a U, A, or dT (or a modified version thereof). In some embodiments, the nucleotide at position 1 of the antisense strand (from 5' end — > 3' end) forms an A: U or U: A base pair with the sense strand.

[0092] A sense strand containing a sequence listed in Table 2A, Table 2B, Table 4A, Table 4B, Table 4C, or Table 5C can be hybridized to any antisense strand containing a sequence listed in Table 2A, Table 2B, Table 3A, Table 3B, or Table 5C, provided the two sequences have a region of at least 85% complementarity' over a contiguous 16, 17, 18, 19, 20, or 21 nucleotide sequence. In some embodiments, the TSLP-IL33 RNAi agent has a sense strand consisting of the modified sequence of any of the modified sequences in Table 4A, Table 4B, Table 4C, or Table 5C, and an antisense strand consisting of the modified sequence of any of the modified sequences in Table 3A, Table 3B, or Table 5C. Certain representative sequence pairings are exemplified by the Complex ID Nos. shown in Tables 5A, 5B, and 5C.

[0093] In some embodiments, a TSLP-IL33 RNAi agent comprises, consists of. or consists essentially of a complex represented by any one of the Complex ID Nos. presented herein. In some embodiments, a TSLP-IL33 RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the complexes represented by any of the Complex ID NOs. presented herein. In some embodiments, a TSLP-IL33 RNAi agent comprises the sense strand and antisense strand nucleotide sequences of any of the complexes represented by any of the Complex ID NOs. presented herein and a targeting group and / or linking group wherein the targeting group and / or linking group is covalently linked (i.e., conjugated) to the sense strand or the antisense strand. In some embodiments, a TSLP-IL33 RNAi agent includes the sense strand and antisense strand modified nucleotide sequences of any of the Complex ID NOs. presented herein. In some embodiments, a TSLP-IL33 RNAi agent comprises the sense strand and antisense strand modified nucleotide sequences of any of the Complex ID NOs. presented herein and a targeting group and / or linking group, wherein the targeting group and / or linking group is covalently linked to the sense strand or the antisense strand.

[0094] In some embodiments, a TSLP-IL33 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand complexes of Table 2A, Table 2B, Table 5A, Table 5B, or 5C, and further comprises atargeting group or targeting ligand. In some embodiments, a TSLP-IL33 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisensestrand / sense strand complexes of Table 2A, Table 2B, Table 5A, Table 5B, Table 5C, and further comprises an integrin targeting ligand targeting group.

[0095] A targeting group, with or without a linker, can be linked to the 5' or 3' end of any of the sense and / or antisense strands disclosed in Table 2A, Table 2B, Table 3A, Table 3B, Table 4A, Table 4B, Table 4C, or Table 5C. A linker, with or without a targeting group, can be attached to the 5' or 3' end of any of the sense and / or antisense strands disclosed in Tables Table 2A, Table 2B. Table 3A, Table 3B, Table 4A, Table 4B, Table 4C. or Table 5C.

[0096] In some embodiments, a TSLP-IL33 RNAi agent comprises an antisense strand and a sense strand having the nucleotide sequences of any of the antisense strand / sense strand complexes of Table 2A, Table 2B, Table 5 A, Table 5B, or Table 5C, and further comprises an integrin targeting ligand.

[0097] In some embodiments, aTSLP-IL33 RNAi agent comprises an antisense strand and a sense strand having the modified nucleotide sequence of any of the antisense strand and / or sense strand nucleotide sequences in Table 3A, Table 3B, Table 4A, Table 4B, or Table 4C.

[0098] In some embodiments, a TSLP-IL33 RNAi agent comprises an antisense strand and a sense strand having a modified nucleotide sequence of any of the antisense strand and / or sense strand nucleotide sequences of any of the complexes Tables 5A, 5B, and 5C, and further comprises an integrin targeting ligand.

[0099] In some embodiments, a TSLP-IL33 RNAi agent comprises, consists of. or consists essentially of any of the complexes of Tables 5A, 5B, and 5C.Table 5A. TSLP-IL33 RN Ai Agents Complexes with Corresponding Sense and Antisense Strand ID Numbers and Sequence ID numbers for the modified and unmodified nucleotide sequences.Table 5B. TSLP-IL33 RNAi Agents Complexes with Corresponding Sense and Antisense Strand ID Numbers Referencing Position Targeted on TSLP or IL33 Gene (SEQ ID NOs: 1 and 2).Table 5C. TSLP-IL33 RNAi Agent Complexes Showing Chemically Modified Antisense Strand and Sense Strand Sequences

[0100] In some embodiments, a TSLP-IL33 RNAi agent is prepared or provided as a salt, mixed salt, or a free-acid. The multimeric RNAi agents described herein, upon deliver}' to a cell expressing a TSLP and / or IL33 gene, inhibit or knockdown expression of one or more TSLP and / or IL33 genes in vivo and / or in vitro.Targeting Ligands and Targeting Groups

[0101] As disclosed herein, the multimeric RNAi agent conjugate delivery' platform is comprised of one or more targeting groups. Targeting groups or targeting moieties enhance the pharmacokinetic or biodistribution properties of a conjugate or RNAi agent to which they are attached to improve cell-specific (including, in some cases, organ specific) distribution and cell-specific (or organ specific) uptake of the conjugate or RNAi agent. A targeting group can be monovalent, divalent, trivalent, tetravalent, or have higher valency for the target to which it is directed. Representative targeting groups include, without limitation, compounds with affinity to cell surface molecule, cell receptor ligands, hapten, antibodies, monoclonal antibodies, antibody fragments, and antibody mimics with affinity’ to cell surface molecules. In some embodiments, a targeting group is linked to an RNAi agent using a linker, such as a PEG linker or one, two, or three abasic and / or ribitol (abasic ribose) residues, which in some instances can serve as linkers.

[0102] In some embodiments, a targeting group is covalently linked to the 3' and / or 5' end of either the sense strand and / or the antisense strand of an RNAi agent. In some embodiments, atargeting ligand is linked to the 3' and / or 5' end of the sense strand of one of the RNAi agents. In some embodiments, atargeting group is linked to the 5' end of an RNAi agent sense strand of one RNAi agent. In some embodiments, a targeting group is linked internally to one or more nucleotides of an RNAi agent sense strand. In some embodiments, a targeting ligand is positioned between two RNAi agents in the multimeric RNAi agent conjugate. A targeting group may be linked directly or indirectly to the RNAi agent via a linker / linking group. In some embodiments, a targeting group is linked to the RNAi agent via a metabolically stabilized bond or linkage.

[0103] In some embodiments, a targeting group comprises an integrin targeting ligand. In some embodiments, an integrin targeting ligand is an av(36 integrin targeting ligand. The use of an av(36 integrin targeting ligand facilitates cell-specific targeting to cells having av|36 on its respective surface, and binding of the integrin targeting ligand can facilitate entry' of the therapeutic agent, such as an RNAi agent, to which it is linked, into cells such as epithelialcells, including pulmonary epithelial cells and renal epithelial cells. Integrin targeting ligands can be monomeric or monovalent (e.g., having a single integrin targeting moiety) or multimeric or multivalent (e.g., having 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 avP6 integrin targeting ligands, is described, for example, in International Patent Application Publication No. WO 2018 / 085415 and in International Patent Application Publication No. WO 2019 / 089765, the contents of each of which are incorporated herein in its entirety.

[0104] In some embodiments, targeting groups are linked to the TSLP-IL33 RNAi agents without the use of an additional linker. In some embodiments, the targeting group is designed having a linker readily present to facilitate the linkage to a TSLP-IL33 RNAi agent. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents can be linked to their respective targeting groups using the same linkers. In some embodiments, when two or more RNAi agents are included in a composition, the two or more RNAi agents are linked to their respective targeting groups using different linkers.

[0105] In some embodiments, a linking group is conjugated to the RNAi agent. The linking group facilitates covalent linkage of the agent to a targeting group, pharmacokinetic modulator, delivery polymer, or delivery vehicle. The linking group can be linked to the 3' and / or the 5' end of the RNAi agent sense strand or antisense strand. In some embodiments, the linking group is linked to the RNAi agent sense strand. In some embodiments, the linking group is conjugated to the 5' or 3' end of an RNAi agent sense strand. In some embodiments, a linking group is conjugated to the 5' end of an RNAi agent sense strand. Examples of linking groups, include but are not limited to: C6-SS-C6, 6-SS-6, reactive groups such a primary amines (e.g., NH2-C6) and alkynes, alkyl groups, abasic residues / nucleotides, amino acids, tri-alkyne functionalized groups, ribitol, and / or PEG groups. Examples of certain linking groups are provided in Table 6.

[0106] A linker or linking group is a connection between two atoms that links one chemical group (such as an RNAi agent) or segment of interest to another chemical group (such as a targeting group, pharmacokinetic modulator, or delivery polymer) or segment of interest via one or more covalent bonds. A labile linkage contains a labile bond. A linkage can optionally include a spacer that increases the distance between the two joined atoms. A spacer may further add flexibility and / or length to the linkage. Spacers include, but are not be limited to, alky l groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, andaralkynyl groups; each of which can contain one or more heteroatoms, heterocycles, amino acids, nucleotides, and saccharides. Spacer groups are well known in the art and the preceding list is not meant to limit the scope of the description. In some embodiments, a TSLP RNAi agent is conjugated to a polyethylene glycol (PEG) moiety, or to a hydrophobic group having 12 or more carbon atoms, such as a cholesterol or palmitoyl group.

[0107] In some embodiments, a TSLP-IL33 RNAi agent is linked to one or more pharmacokinetic / pharmacodynamic (PK / PD) modulators. PK / PD modulators can increase circulation time of the conjugated drug and / or increase the activity of the RNAi agent through improved cell receptor binding, improved cellular uptake, and / or other means. Various PK / PD modulators suitable for use with RNAi agents are known in the art. In some embodiments, the PK / PD modulators can be cholesterol or cholesteryl derivatives, or in some circumstances a PK / PD modulator can be comprised of alkyl groups, alkenyl groups, alkynyl groups, aryl groups, aralkyl groups, aralkenyl groups, or aralkynyl groups, each of which may be linear, branched, cyclic, and / or substituted or unsubstituted. In some embodiments, the location of attachment for these moieties is at the 5’ or 3’ end of the sense strand, at the 2' position of the ribose ring of any given nucleotide of the sense strand, and / or attached to the phosphate or phosphorothioate backbone at any position of the sense strand.

[0108] Any of the TSLP-IL33 RNAi agent nucleotide sequences listed in Tables 2A, 2B, 3 A, 3B, 4A, 4B, 4C, and 5C, whether modified or unmodified, can contain 3' and / or 5' targeting group(s). linking group(s). and / or PK / PD modulator(s). Any of the TSLP-IL33 RNAi agent sequences listed in Tables 3A, 3B, 4A, 4B, 4C, and 5C, or are otherwise described herein, which contain a 3' or 5' targeting group, linking group, and / or PK / PD modulator can alternatively contain no 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 6. Any of the TSLP-IL33 RNAi agent duplexes listed in Tables 5A, 5B, and 5C, whether modified or unmodified, can further comprise a targeting group or linking group, including, but not limited to, those depicted in Table 6, and the targeting group or linking group can be attached to the 3' or 5' terminus of either the sense strand or the antisense strand of the TSLP-IL33 RNAi agent duplex.

[0109] Examples of certain modified nucleotides, capping moieties, and linking groups are provided in Table 6.

[0110] In some embodiments, compounds that may be conjugated to RNAi agents to synthesize a delivery platform for an RNAi agent are shown in Table 6 below, or a pharmaceutically acceptable salt thereof.

[0111] Table 6. Structures Representing Various Modified Nucleotides and Linking Groups

[0112] Alternatively, other linking groups known in the art may be used.

[0113] In addition or alternatively to linking an RNAi agent to one or more targeting ligands, targeting groups, and / or PK / PD modulators, in some embodiments, a delivery vehicle may be used to deliver an RNAi agent to a cell or tissue. A delivery vehicle is a compound that can improve delivery of the RNAi agent to a cell or tissue, and can include, or consist of, but is not limited to: a polymer, such as 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.

[0114] In some embodiments, the RNAi agents can be combined with lipids, nanoparticles, polymers, liposomes, micelles, DPCs or other delivery systems available in the art. The RNAi agents can also be chemically conjugated to targeting groups, lipids (including, but not limited to cholesterol and cholesteryl derivatives), nanoparticles, polymers, liposomes, micelles, 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), or other delivery systems available in the art.Pharmaceutical Compositions and Formulations

[0115] The TSLP-IL33 RNAi agents disclosed herein can be prepared as pharmaceutical compositions or formulations (also referred to herein as ‘'medicaments”). In some embodiments, pharmaceutical compositions include at least one TSLP-IL33 RNAi agent. These pharmaceutical compositions are particularly useful in the inhibition of the expression of the target mRNA in a target cell, a group of cells, a tissue, or an organism.

[0116] The pharmaceutical compositions can be used to treat a subject having a disease, disorder, or condition that would benefit from reduction in the level of the target TSLP and / or IL33 mRN A, or inhibition in expression of the target gene. The pharmaceutical compositions can be used to treat a subject at risk of developing a disease, disorder, symptom, or condition that would benefit from reduction of the level of the target mRNA or an inhibition in expression the target gene. In one embodiment, the method includes administering a TSLP-IL33 RNAi agent linked to a targeting ligand as described herein, to a subject to be treated. In some embodiments, one or more pharmaceutically acceptable excipients (including vehicles, carriers, diluents, and / or delivery’ polymers) are added to the pharmaceutical compositions that include a TSLP-IL33 RNAi agent, thereby forming a pharmaceutical formulation or medicament suitable for in vivo delivery to a subject, including a human.

[0117] The pharmaceutical compositions that include a TSLP-IL33 RNAi agent and methods disclosed herein decrease the level of the target mRNA in a cell, group of cells, group of cells, tissue, organ, or subject, including by administering to the subject a therapeutically effective amount of a herein described TSLP-IL33 RNAi agent, thereby inhibiting the expression of TSLP and / or IL33 mRNA in the subject. In some embodiments, the subject has been previously identified as having a pathogenic upregulation of the target gene in pulmonary cells. In some embodiments, the subject has been previously identified or diagnosed as having asthma or chronic obstructive pulmonary disease (COPD). In some embodiments, the subject has been suffering from symptoms associated yvith diseases such as asthma or chronic obstructive pulmonary disease (COPD). In some embodiments, the subject yvould benefit from a reduction of TSLP and / or IL33 gene expression in the subject’s lung.

[0118] In some embodiments, the described pharmaceutical compositions including a TSLP-1L33 RNAi agent are used for treating or managing clinical presentations associated yvith asthma, chronic obstructive pulmonary’ disease (COPD), other TSLP-related disease, or other IL33-related disease.. In some embodiments, a therapeutically (including prophylactically) effective amount of one or more of pharmaceutical compositions is administered to a subjectin need of such treatment. In some embodiments, administration of any of the disclosed TSLP-IL33 RNAi agents can be used to decrease the number, severity, and / or frequency of symptoms of a disease in a subject.

[0119] In some embodiments, the subject is administered a therapeutically effective amount of one or more pharmaceutical compositions that include a TSLP-IL33 RNAi agent thereby treating the symptom. In other embodiments, the subject is administered a prophylactically effective amount of one or more TSLP-IL33 RNAi agents, thereby preventing or inhibiting the at least one symptom.

[0120] The route of administration is the path by which a TSLP-IL33 RNAi agent is brought into contact with the body. In general, methods of administering drugs and oligonucleotides and nucleic acids for treatment of a mammal are well known in the art and can be applied to administration of the compositions described herein. The TSLP-IL33 RN Ai agents disclosed herein can be administered via any suitable route in a preparation appropriately tailored to the particular route. Thus, herein described pharmaceutical compositions can be administered by injection, for example, intravenously, intramuscularly, intracutaneously, subcutaneously, intraarticularly, or intraperitoneally.

[0121] In some embodiments, the herein described pharmaceutical compositions are administered via subcutaneous injection.

[0122] The pharmaceutical compositions including a TSLP-IL33 RNAi agent described herein can be delivered to a cell, group of cells, tissue, or subj ect using oligonucleotide delivery technologies known in the art. In general, any suitable method recognized in the art for delivering a nucleic acid molecule (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 administering), systemic administration, or 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 certain embodiments, the compositions are administered by subcutaneous or intravenous infusion or injection.

[0123] In some embodiments, the pharmaceutical compositions described herein comprise one or more pharmaceutically acceptable excipients. The pharmaceutical compositions described herein are formulated for administration to a subj ect.

[0124] As used herein, a pharmaceutical composition or medicament includes a pharmacologically effective amount of at least one of the described therapeutic compounds and one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients (excipients) are substances other than the Active Pharmaceutical Ingredient (API, therapeutic product, e.g, TSLP-IL33 RNAi agent) that are intentionally included in the drug delivery system. Excipients do not exert or are not intended to exert a therapeutic effect at the intended dosage. Excipients can act to a) aid in processing of the drug delivery system during manufacture, b) protect, support or enhance stability, bioavailability or patient acceptability of the API, c) assist in product identification, and / or d) enhance any other attribute of the overall safety, effectiveness, of delivery of the API during storage or use. A pharmaceutically acceptable excipient may or may not be an inert substance.

[0125] Excipients include, but are not limited to: absorption enhancers, anti-adherents, anti-foaming agents, anti-oxidants, binders, buffering agents, carriers, coating agents, colors, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, extenders, fillers, flavors, glidants, humectants, lubricants, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained release matrices, sweeteners, thickening agents, tonicity agents, vehicles, water-repelling agents, and wetting agents.

[0126] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor® ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). Suitable carriers 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 a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, 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 dispersion 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.

[0127] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle, which contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0128] In some embodiments, pharmaceutical formulations that include the TSLP-IL33 RNAi agents disclosed herein suitable for subcutaneous administration can be prepared in an aqueous sodium phosphate buffer (e.g., the TSLP-IL33 RNAi agent formulated in 0.5 mM sodium phosphate monobasic, 0.5 mM sodium phosphate dibasic, in water). In some embodiments, pharmaceutical formulations that include the TSLP-IL33 RNAi agents disclosed herein suitable for subcutaneous administration can be prepared in water for injection (sterile water). TSLP-IL33 RNAi agents disclosed herein suitable for subcutaneous administration can be prepared in isotonic saline (0.9%).

[0129] Formulations suitable for intra-articular administration can be in the form of a sterile aqueous preparation of the drug that can be in microcrystallinc form, for example, in the form of 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.

[0130] Formulations suitable for oral administration of the TSLP-IL33 RNAi agents disclosed herein can also be prepared. In some embodiments, the TSLP-IL33 RNAi agents disclosed herein are administered orally. In some embodiments, the TSLP-IL33 RNAi agents disclosed herein are formulated in a capsule for oral administration.

[0131] The active compounds can be prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent 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. Patent No. 4,522,811.

[0132] The TSLP-IL33 RNAi agents can be formulated in compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the disclosure are dictated by and directly dependent on the unique characteristics of the active compound and the therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.

[0133] A pharmaceutical composition can contain other additional components commonly found in pharmaceutical compositions. Such additional components include, but are not limited to: anti-pruritics, astringents, local anesthetics, analgesics, antihistamines, or antiinflammatory agents (e.g., acetaminophen, NSAIDs, diphenhydramine, etc.). It is also envisioned that cells, tissues, or isolated organs that express or comprise the herein defined RNAi agents may be used as ‘‘pharmaceutical compositions.” As used herein, “pharmacologically effective amount,” “therapeutically effective amount,” or simply “effective amount” refers to that amount of an RNAi agent to produce a pharmacological, therapeutic, or preventive result.

[0134] In some embodiments, the methods disclosed herein further comprise the step of administering a second therapeutic or treatment in addition to administering an RNAi agent disclosed herein. In some embodiments, the second therapeutic is another TSLP-IL33 RNAi agent (e g., aTSLP-IL33 RNAi agent that targets a different sequence within the TSLP and / or IL33 target). In other embodiments, the second therapeutic can be a small molecule drug, an antibody, an antibody fragment, or an aptamer.

[0135] In some embodiments, the described TSLP-IL33 RNAi agent(s) are optionally combined with one or more additional therapeutics. The TSLP-IL33 RNAi agent and additional therapeutic(s) can be administered in a single composition or they can be administered separately. In some embodiments, the one or more additional therapeutics is administered separately in separate dosage forms from the RNAi agent (e.g., the TSLP-IL33 RNAi agent is administered by subcutaneous injection, while the additional therapeutic involved in the method of treatment dosing regimen is administered orally). In some embodiments, the described TSLP-IL33 RNAi agent(s) are administered to a subject in need thereof via subcutaneous injection, and the one or more optional additional therapeutics are administered orally, which together provide for a treatment regimen for diseases and conditionsassociated with obesity, diabetes, liver inflammation, dyslipidemia, or metabolic disease. In some embodiments, the described TSLP-IL33 RNAi agent(s) are administered to a subject in need thereof via subcutaneous injection, and the one or more optional additional therapeutics are administered via a separate subcutaneous injection. In some embodiments, the TSLP-IL33 RNAi agent and one or more additional therapeutics are combined into a single dosage form (e.g., a “cocktail” formulated into a single composition for subcutaneous injection). The TSLP-IL33 RNAi agents, with or without the one or more additional therapeutics, can be combined with one or more excipients to form pharmaceutical compositions.

[0136] Generally, an effective amount of a TSLP-IL33 RNAi agent will be in the range of from about 0.1 to about 100 mg / kg of body weight / dose. e g., from about 1.0 to about 50 mg / kg of body weight / dose. In some embodiments, an effective amount of an active compound will be in the range of from about 0.25 to about 5 mg / kg of body weight per dose. In some embodiments, an effective amount of an active ingredient will be in the range of from about 0.5 to about 4 mg / kg of body weight per dose. In some embodiments, an effective amount of a TSLP-IL33 RNAi agent may be a fixed dose. In some embodiments, the fixed dose is in the range of from about 5 mg to about 1,000 mg of TSLP-IL33 RNAi agent. In some embodiments, the fixed does is in the range of 50 to 400 mg of TSLP-IL33 RNAi agent. Dosing may be weekly, bi-weekly, monthly, quarterly, or at any other interval depending on the dose of TSLP-IL33 RNAi agent administered, the activity level of the particular TSLP-IL33 RNAi agent, and the desired level of inhibition for the particular subject. The Examples herein show suitable levels for inhibition in certain animal species. The amount administered will depend on such variables as the overall health status of the patient or subject, the relative biological efficacy of the compound delivered, the formulation of the drug, the presence and types of excipients in the formulation, and the route of administration. Also, it is to be understood that the initial dosage administered can be increased beyond the above upper level to rapidly achieve the desired blood-level or tissue level, or the initial dosage can be smaller than the optimum.

[0137] For treatment of disease or for formation of a medicament or composition for treatment of a disease, the pharmaceutical compositions described herein including a TSLP-IL33 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, peptide and / or an aptamer.

[0138] The described TSLP-IL33 RNAi agents, when added to pharmaceutically acceptable excipients or adjuvants, can be packaged into kits, containers, packs, or dispensers.The pharmaceutical compositions described herein may be packaged in pre-filled syringes, pen injectors, autoinjectors, infusion bags / devices. or vials.Methods of Treatment and Inhibition of Expression

[0139] The TSLP-IL33 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 multimeric RNAi agents disclosed herein can be used to treat a subject (e.g., a human) that would benefit from reduction and / or inhibition in expression of TSLP and / or IL33 mRNA and / or TSLP and / or IL33 protein levels, a subject that has been diagnosed with or is suffering from symptoms related to diseases such as obesity, diabetes, liver inflammation, dyslipidemia, or metabolic disease.

[0140] In some embodiments, the subject is administered a therapeutically effective amount of any one or more TSLP-IL33 RNAi agents. Treatment of a subject can include therapeutic and / or prophylactic treatment. The subject is administered a therapeutically effective amount of any one or more TSLP-IL33 RNAi agents described herein. The subject can be a human, patient, or human patient. The subject may be an adult, adolescent, child, or infant. Administration of a pharmaceutical composition described herein can be to a human being or animal.

[0141] The TSLP-IL33 RNAi agents described herein can be used to treat at least one symptom in a subject having a TSLP- and / or IL33-related disease or disorder, or having a disease or disorder that is mediated at least in part by TSLP and / or IL33 gene expression. In some embodiments, the TSLP-IL33 RNAi agents are used to treat or manage a clinical presentation of a subject with a disease or disorder that would benefit from or be mediated at least in part by a reduction in TSLP and / or IL33 mRNA. The subject is administered a therapeutically effective amount of one or more of the TSLP-IL33 RNAi agents or TSLP-IL33 RNAi agent-containing compositions described herein. In some embodiments, the methods disclosed herein comprise administering a composition comprising a TSLP-IL33 RNAi agent described herein to a subject to be treated. In some embodiments, the subject is administered a prophylactically effective amount of any one or more of the described TSLP-IL33 RNAi agents, thereby treating the subject by preventing or inhibiting the at least one symptom.

[0142] In certain embodiments, the present disclosure provides methods for treatment of diseases, disorders, conditions, or pathological states mediated at least in part by TSLP and / orIL33 gene expression, in a patient in need thereof, wherein the methods include administering to the patient any of the TSLP-IL33 RNAi agents described herein.

[0143] In some embodiments, the 5 ’ end of the sense strand is coupled to a targeting ligand comprising the structure of Tri-SM6.1-avb6-(TA14).

[0144] In some embodiments, the gene expression level and / or mRNA level of a TSLP and / or IL33 gene in a subject to whom a described TSLP-IL33 RNAi agent is administered is reduced by at least about 30%. 35%. 40%. 45%. 50%. 55%. 60%. 65%. 70%. 75%. 80%. 85%.95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the TSLP-IL33 RNAi agent or to a subject not receiving the TSLP-IL33 RNAi agent. The gene expression level and / or mRNA level in the subject may be reduced in a cell, group of cells, and / or tissue of the subject. In some embodiments, the TSLP and / or IL33 gene expression is inhibited by at least about 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, or greater than 65% in the cytoplasm of pulmonary cells relative to the subject prior to being administered the TSLP-IL33 RNAi agent or to a subject not receiving the TSLP-IL33 RNAi agent.

[0145] In some embodiments, the TSLP and / or IL33 protein expression level in a subject to whom a described TSLP-IL33 RNAi agent has been administered is reduced by at least about 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than 99% relative to the subject prior to being administered the TSLP-IL33 RNAi agent or to a subject not receiving the TSLP-IL33 RNAi agent. The protein expression level in the subject may be reduced in a cell, group of cells, tissue, blood, and / or other fluid of the subject.

[0146] A reduction in TSLP and / or IL33 mRNA expression levels and TSLP and / or IL33 protein expression levels can be assessed by any methods known in the art. As used herein, a reduction or decrease in TSLP and / or IL33 mRNA level and / or protein level are collectively referred to herein as a reduction or decrease in TSLP and / or IL33 or inhibiting or reducing the gene expression of TSLP and / or IL33. The Examples set forth herein illustrate known methods for assessing inhibition of TSLP and / or IL33 gene expression. The person of ordinary skill in the art would further know suitable methods for assessing inhibition of TSLP and / or IL33 gene expression in vivo and / or in vitro.

[0147] In some embodiments, disclosed herein are methods of treatment (including prophylactic or preventative treatment) of diseases, disorders, or symptoms caused by diseases such as asthma, chronic obstructive pulmonary disease (COPD), other TSLP-related disease, or other IL33-related disease, wherein the methods include administering to a subject in needthereof a therapeutically effective amount of a TSLP-IL33 RNAi agent that includes an antisense strand that is at least partially complementary to the portion of the TSLP and / or IL33 mRNA having the sequence in Table 1 A or Table IB. In some embodiments, disclosed herein are methods of treatment (including prophylactic or preventative treatment) of diseases or symptoms caused by diseases such as asthma, chronic obstructive pulmonary disease (COPD), other TSLP-related disease, or other IL33-related disease, wherein the methods include administering to a subject in need thereof a therapeutically effective amount of a TSLP-IL33 RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2A, Table 2B, Table 3A, Table 3B or 5C, and a sense strand that comprises any of the sequences in Tables 2A, Table 2B, Table 4A, Table 4B, Table 4C, or Table 5C that is at least partially complementary to the antisense strand. In some embodiments, disclosed herein are methods of treatment (including prophylactic or preventative treatment) of diseases or symptoms caused by diseases such as obesity, diabetes, liver inflammation, dyslipidemia, or metabolic disease, wherein the methods include administering to a subject in need thereof a therapeutically effective amount of a TSLP-IL33 RNAi agent that includes a sense strand that comprises any of the sequences in Table 2A, Table 2B, Table 4A, Table 4B, Table 4C, or Table 5C, and an antisense strand comprising the sequence of any of the sequences in Table 2A, Table 2B, Table 3A, Table 3B, or Table 5C that is at least partially complementary' to the sense strand.

[0148] In some embodiments, the 5 ‘ end of the sense strand is coupled to a targeting ligand comprising the structure of Tri-SM6. l-otvb6-(TA14).

[0149] In some embodiments, disclosed herein are methods for inhibiting expression of a TSLP and / or IL33 gene in a cell, wherein the methods include administering to the cell a TSLP-IL33 RNAi agent that includes an antisense strand that is at least partially complementary' to the portion of the TSLP and / or IL33 mRNA having the sequence in Table 1 A or Table IB. In some embodiments, disclosed herein are methods of inhibiting expression of a TSLP and / or IL33 gene in a cell, wherein the methods include administering to a cell a TSLP-IL33 RNAi agent that includes an antisense strand comprising the sequence of any of the sequences in Table 2A, Table 2B, Table 3A, Table 3B. or Table 5C and a sense strand that comprises any of the sequences in Table 2A, Table 2B. Table 4A, Table 4B, Table 4C, or Table 5C that is at least partially complementary to the antisense strand. In some embodiments, disclosed herein are methods of inhibiting expression of a TSLP and IL33 gene in a cell, wherein the methods include administering a TSLP-IL33 RNAi agent that includes a sense strand that comprises any of the sequences in Table 2A, Table 2B, Table 4A. Table 4B, Table 4C. or Table 5C. and anantisense strand that includes the sequence of any of the sequences in Table 2A, Table 2B, Table 3A, Table 3B, or Table 5C that is at least partially complementary to the sense strand.

[0150] In some embodiments, the TSLP-IL33 RNAi agents are administered to a subject in need thereof as a first line therapy. In some embodiments, the TSLP-IL33 RNAi agents are administered to a subject in need thereof as a second line therapy. In certain embodiments, the TSLP-IL33 RNAi agents are administered as a second line therapy to patients who have failed one or more first line standard of care therapies. In certain embodiments, the TSLP-IL33 RNAi agents are administered as a maintenance therapy following the administration of one or more prior therapies. In certain embodiments, the TSLP-IL33 RNAi agents administered as a maintenance therapy following the administration of one or more standard of care therapies. In some embodiments, the TSLP-IL33 RNAi agents administered in combination with one or more additional therapies. In some embodiments, the one or more additional therapies is a standard of care therapy. In some embodiments, the one or more additional therapies is an oral therapy.

[0151] The use of TSLP-IL33 RNAi agents provides methods for therapeutic (including prophylactic) treatment of diseases / disorders associated with diseases such as asthma, chronic obstructive pulmonary disease (COPD), other TSLP-related disease, or other IL33-related disease. The described TSLP-IL33 RNAi agents mediate RNA interference to inhibit the expression of one or more genes necessary for production of TSLP protein and / or IL33 protein. TSLP-IL33 RNAi agents can also be used to treat or prevent various diseases, disorders, or conditions, including diseases such as asthma, chronic obstructive pulmonary disease (COPD), other TSLP-related disease, or other IL33-related disease. Furthermore, compositions for delivery of TSLP-IL33 RNAi agents to pulmonary cells, and specifically to pulmonary cells, in vivo, are described.Cells, Tissues, Organs, and Non-Human Organisms

[0152] Cells, tissues, organs, and non-human organisms that include at least one of the TSLP-IL33 RNAi agents described herein are contemplated. The cell, tissue, organ, or non-human organism is made by delivering the RNAi agent to the cell, tissue, organ or non-human organism.Illustrative Embodiments

[0153] Provided here are illustrative embodiments of the disclosed technology. These embodiments are illustrative only and do not limit the scope of the present disclosure or of the claims attached hereto.1. An RNAi agent, for inhibiting the expression of a Thymic Stromal Lymphopoietin (TSLP) gene and an Interleukin 33 (IL33) gene, comprising:(i) a first antisense strand that is complementary to part of an mRNA encoding a TSLP gene or an IL33 gene, and(ii) a second antisense strand that is complementary to part of an mRNA encoding a TSLP gene or an IL33 gene, and(iii) one or more sense strands,wherein the one or more sense strands comprise a nucleotide sequence that is at least partially complementary to the first antisense strand and the second antisense strand.2. The RNAi agent of embodiment 1, whereinthe first antisense strand comprises at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any of the sequences of Table 2A, or Table 2B, Table 3A, Table 3B, or Table 5C, andthe second antisense strand comprises at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any of the sequences of Table 2A, Table 2B, Table 3A, Table 3B, or Table 5C3. The RNAi agent of embodiment 1 or embodiment 2. wherein the one or more sense strands comprise a nucleotide sequence of any one of the sequences of Table 4A, Table 4B, or Table 4C.4. The RNAi agent of any of embodiments 1-3, wherein the RNAi agent comprises a first sense strand sequence of any one of the sequences of Table 4A, Table 4B, or Table 4C and a second sense strand sequence of any one of the sequences of Table 4A, Table 4B, or Table 4C.The RNAi agent of any one of embodiments 1-4, wherein the RNAi agent comprises a first antisense strand sequence of any one of the sequences of Table 2A, Table 3A, or Table 5C, and a second sense strand sequence of any one of the sequences of Table 2B, Table 3B, or Table 5C.The RNAi agent of any one of embodiments 1-4, wherein the RNAi agent comprises a first antisense strand sequence of any one of the sequences of Table 2B, Table 3B, or Table 5C, and a second sense strand sequence of any one of the sequences of Table 2A, Table 3A, or Table 5C.The RNAi agent of any one of embodiments 1-6, wherein at least one nucleotide of the RNAi agent is a modified nucleotide or includes a modified intemucleoside linkage.The RNAi agent of any one of embodiments 1-7, wherein all or substantially all of the nucleotides of the sense and / or antisense strand of the RNAi agent are modified nucleotides.The RNAi agent of any one of embodiments 1-8, wherein the modified nucleotide is selected from the group consisting of: 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2'-deoxy nucleotide, 2'.3'-seco nucleotide mimic, locked nucleotide. 2'-F-arabino nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2'-O-methyl nucleotide, inverted 2'-deoxy nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholine nucleotide, vinyl phosphonate containing nucleotide, cyclopropyl phosphonate containing nucleotide, and 3'-O-methyl nucleotide.The RNAi agent of embodiment 9, wherein all or substantially all of the modified nucleotides are 2’-O-methyl nucleotides, 2'-fluoro nucleotides, or combinations thereof.The RNAi agent of any one of embodiments 1-10, wherein the first antisense strand sequence is selected from any one of the sequences of Table 3 A, and the second antisense strand sequence is selected from any one of the sequences of Table 3B.The RNAi agent of any one of embodiments 1-10, wherein the first antisense strand sequence is selected from any one of the sequences of Table 3B, and the second antisense strand sequence is selected from any one of the sequences of Table 3 A.The RNAi agent of any one of embodiments 1-12, wherein:(a) the one or more sense strands comprise between 30 and 60 nucleotides, or (b) the first antisense strand is between 18 and 30 nucleotides in length, or(c) the second antisense strand is between 18 and 30 nucleotides in length, or (d) any combination of (a) through (c) above.The RNAi agent of embodiment 13, wherein:(a) the one or more sense strands comprise between 36 and 54 nucleotides, or(b) the first antisense strand is between 18 and 27 nucleotides in length, or(c) the second antisense strand is between 18 and 27 nucleotides in length, or(d) any combination of (a) through (c) above.The RNAi agent of embodiment 14, wherein:(a) the one or more sense strands comprise between 36 and 48 nucleotides, or(b) the first antisense strand is between 18 and 24 nucleotides in length, or(c) the second antisense strand is between 18 and 24 nucleotides in length, or(d) any combination of (a) through (c) above.The RNAi agent of embodiment 15, wherein:(a) the one or more sense strands comprise between 38 and 42 nucleotides, or(b) the first antisense strand is between 19 and 21 nucleotides in length, or(c) the second antisense strand is between 19 and 21 nucleotides in length, or(d) any combination of (a) through (c) above.The RNAi agent of embodiment 16, wherein:(a) the one or more sense strands comprises 38 or 42 nucleotides, or(b) the second antisense strand is 19 nucleotides in length, or(c) the second antisense strand is 21 nucleotides in length, or(d) any combination of (a) through (c) above.The RNAi agent of embodiment 1, wherein the one or more sense strands comprises the structure:SS1- L - SS2wherein SSi comprises a first sense strand sequence;552 comprises a second sense strand sequence; andL is a linker, or a bond.The RNAi agent of embodiment 18, wherein L is a nucleotide linker.The RNAi agent of embodiment 18, wherein L is a non-nucleotide linker.The RNAi agent of embodiment 20, wherein L is polyethylene glycol (PEG).The RNAi agent of any one of embodiments 18-21, wherein SSi comprises any one of the sense strand sequences listed in Table 4A or Table 4B.The RNAi agent of any one of embodiments 18-22, wherein SS2 comprises any one of the sense strand sequences listed in Table 4A or Table 4B.The RNAi agent of embodiment 18, wherein SSi and SS2 each independently comprise any one of the sense strand sequences listed in Table 4A or Table 4B, wherein at least one sense strand sequence is selected from Table 4A and the other sense strand sequence is selected from Table 4B.The RNAi agent of any one of embodiments 18-24, wherein L comprises the structure:The RNAi agent of embodiment 18, whereinjsselected from any one of the sense strands listed in Table 4C.The RNAi agent of any one of embodiments 18-26, wherein the first antisense strand consists of. consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3A, and the second antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3B.The RNAi agent of any one of embodiments 18-27, wherein the first antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3B, and the second antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3 A.The RNAi agent of any one of embodiments 1-28, wherein the one or more sense strands consists of, consists essentially of, or comprises the nucleotide sequence of any of the modified sense strand sequences of Table 4A. Table 4B, Table 4C, or Table 5C.The RNAi agent of any one of embodiments 1-29, wherein the RNAi agent is linked to a targeting ligand.The RNAi agent of embodiment 30, wherein the targeting ligand has affinity for a cell receptor expressed on an epithelial cell.The RNAi agent of embodiment 31, wherein the targeting ligand comprises an integrin targeting ligand.The RNAi agent of embodiment 32, wherein the integrin targeting ligand is an avf>6 integrin targeting ligand.The RNAi agent of embodiment 33, wherein the targeting ligand comprises the structure:salt thereof.wherein? indicates the point of connection to the RNAi agent.The RNAi agent of any one of embodiments 30-33, wherein the targeting ligand has a structure selected from the group consisting of:PHI, wherein « indicates the point of connection to the RNAi agent.The RNAi agent of embodiment 35, wherein RNAi agent is conjugated to a targeting ligand having the following structure:The RNAi agent of any one of embodiments 30-36, wherein the targeting ligand is conjugated to the one or more sense strands.The RNAi agent of embodiment 37, wherein the targeting ligand is conjugated to the 5' terminal of the one or more sense strands.The RNAi agent of any of embodiments 1-38, wherein the RNAi agent has two blunt ends.The RNAi agent of any of embodiments 1-39, wherein the one or more sense strands comprise one or two terminal caps.The RNAi agent of any of embodiments 1-40, wherein the one or more sense strands comprise one or two inverted abasic residues.An RNAi agent of any of the complexes set forth in Table 5A, Table 5B, or Table 5C.The RNAi agent of any one of embodiments 1-42, wherein the one or more sense strands comprises inverted abasic residues at the 3’ terminal end of the nucleotide sequence, at the 5’ end of the nucleotide sequence, or at both.The RNAi agent of any one of embodiments 1-43, wherein the RNAi agent is a pharmaceutically acceptable salt.A composition comprising the RNAi agent of any one of embodiments 1-44, wherein the composition comprises a pharmaceutically acceptable excipient.The composition of embodiment 45, wherein the pharmaceutically acceptable excipient is sodium phosphate buffer.The composition of embodiment 45, wherein the pharmaceutically acceptable excipient is isotonic saline or water for injection.A method for inhibiting expression of a TSLP and an IL33 gene in a cell, the method comprising introducing into a cell an effective amount of an RNAi agent of any one of embodiments 1-44 or the composition of any one of embodiments 45-47.The method of embodiment 48, wherein the cell is within a subject.The method of embodiment 49, wherein the subject is a human subject.The method of any one of embodiments 48-50, wherein TSLP gene expression is inhibited by at least about 30% and IL33 gene expression of the subject is inhibited by at least about 30%. as measured by reductions in mRNA and / or protein levels.The method of any one of embodiments 48-51, wherein TSLP protein levels are reduced by about 50% and IL33 protein levels are reduced by about 50%.A method of treating a TSLP -related and / or IL33-related disease, disorder, or symptom, the method comprising administering to a human subject in need thereof a therapeutically effective amount of the composition of any one of embodiments 45-47.The method of embodiment 53, wherein the disease is asthma or chronic obstructive pulmonary disease (COPD).The method of any one of embodiments 48-54, wherein the RNAi agent is administered at a dose of about 0.05 mg / kg to about 5.0 mg / kg of body weight of the human subject.56. The method of any one of embodiments 48-55, wherein the RNAi agent is administered in two or more doses.57. Use of the RNAi agent of any one of embodiments 1-44 or the composition of any one of embodiments 45-47, for the treatment of a disease, disorder, or symptom that is mediated at least in part by a reduction in TSLP gene expression and / or IL33 gene expression.58. The use of embodiment 57, wherein the disease is asthma or chronic obstructive pulmonary disease (COPD).59. Use of the RNAi agent of any one of embodiments 1-44 or the composition of any one of embodiments 45-47, for the preparation of a pharmaceutical composition for treating a disease, disorder, or symptom that is mediated at least in part by a reduction in TSLP mRNA and / or protein levels and / or a reduction in IL33 mRNA and / or protein levels.60. The use according to any one of embodiments 57-59, wherein the RNAi agent is administered at a dose of about 0.05 mg / kg to about 10.0 mg / kg of body weight of the human subject.

[0154] The above provided embodiments and items are now illustrated with the following, non-limiting examples.EXAMPLESExample 1. Synthesis of RNAi agents and Multimeric RNAi agents conjugates.

[0156] The following describes the general procedures for the syntheses of certain RNAi agents, and conjugates thereof, including the multimeric RNAi conjugates that are illustrated in the non-limiting Examples set forth herein.

[0157] A. Synthesis of RNAi Agents. RNAi agents can be synthesized using methods generally known in the art. For the synthesis of the RNAi agents illustrated in the Examples set forth herein, the sense and antisense strands of the RNAi agents were synthesized according to phosphorami dite technology on solid phase used in oligonucleotide synthesis. Depending on the scale, a MerMade96E® (Bioautomation), a MerMadel2® (Bioautomation), or an Oligopilot 100 (GE Healthcare) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 500 A or 600A, obtained from Prime Synthesis, Aston, PA, USA) or polystyrene (obtained from Kinovate, Oceanside, CA, USA). All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA), ChemGenes (Wilmington, MA, USA), or Hongene Biotech (Morrisville, NC, USA). Specifically, the 2'-O-methyl phosphoramidites that were used include the following: (5'-O-dimethoxytrityl-N6-(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N, N-diisopropylamino) phosphoramidite. 5'-O-dimethoxy-trityl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N, N-diisopropyl-amino) phosphoramidite, (5'-O-dimethoxytrityl-N2-(isobutyryl)-2'-O-methyl-guanosine-3'-O-(2-cyanoethyl-N, N-diisopropylamino) phosphoramidite, and 5 '-O-dimcthoxy tri t l-2'-O-methy l-uridinc-3'-O-(2-cy anoethyl -N, N-diisopropylamino) phosphoramidite. The 2'-deoxy-2'-fluoro-phosphoramidites and 2'-O-propargyl phosphoramidites carried the same protecting groups as the 2'-O-methyl phosphoramidites. 5'-dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N, N-diisopropylamino) phosphoramidites were purchased from Glen Research (Virginia). The inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N, N-diisopropylamino) phosphoramidites were purchased from ChemGenes. The following UNA phosphoramidites that were used included the following: 5 '-(4, 4'-Dimethoxytrityl)-N6-(benzoyl)-2', 3 '-secoadenosine, 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-diiso-propyl)] -phosphoramidite, 5'-(4,4'-Dimethoxytrityl)-N-isobutyryl-2',3'-seco-guanosine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N, N-diisopropyl)]-phosphoramidite, and 5'-(4,4'-Dimethoxy-trityl)-2', 3 '-seco-uridine, 2'-benzoyl-3'-[(2-cyanoethyl)-(N, N- diiso-propyl)] -phosphoramidite.In order to introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl 1,2,4-dithiazoline-5-one (POS, obtained from PolyOrg. Inc., Leominster, MA, USA) in anhydrous acetonitrile or a 200mM solution of xanthane hydride (TCI America, Portland, OR, USA) in pyridine was employed.

[0158] TFA aminolink phosphoramidites were also commercially purchased (ThermoFisher) to introduce the (NH2-C6) reactive group linkers. TFA aminolink phosphoramidite was dissolved in anhydrous acetonitrile (50 mM) and molecular sieves (3 A) were added. 5 -Benzylthio- IH-tetrazole (BTT, 250 mM in acetonitrile) or 5 -Ethylthio- 1 H-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 min (RNA), 90 sec (2' O-Me), and 60 sec (2' F). Trialkyne-containing phosphoramidites were synthesized to introduce the respective (TriAik#) linkers. When used in connection with the RNAi agents presented in certain Examples herein, trialkyne-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM), and molecular sieves (3A) were added. 5-Benzylthio-lH-tetrazole (BTT, 250 mM in acetonitrile) or 5 -Ethylthio- 1H-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 10 min (RNA), 90 sec (2' O-Me), and 60 sec (2' F).

[0159] For some RNAi agents, a linker, such as a C6-SS-C6 or a 6-SS-6 group, C6-SS(Me)-C5 was introduced at the 3’ terminal end of the sense strand. Pre-loaded resin was commercially acquired with the respective linker. Alternatively, for some sense strands, a dT resin was used and the respectively linker was then added via standard phosphoramidite synthesis.

[0160] B. Cleavage and deprotection of support bound oligomer. After finalization of the solid phase synthesis, the dried solid support was treated with a 1: 1 volume solution of 40 wt. % methylamine in water and 28% to 1% ammonium hydroxide solution (Aldrich) for 1 5 hours at 30 °C. The solution was evaporated and the solid residue was reconstituted in water (see below).

[0161] C. Purification. Crude oligomers were purified by anionic exchange HPLC using a TSKgel SuperQ-5PW 1 pm column and Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 M EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC using a GE Healthcare XK 16 / 40column packed with Sephadex G25 fine with a running buffer of lOOmM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile or filtered water.

[0162] D. Annealing. Complementary strands were mixed by combining equimolar RNA solutions (sense and antisense) in lx PBS (Phosphate-Buffered Saline, lx, Coming, Cellgro) to form the RNAi agents. Some RNAi agents were lyophilized and stored at -15 to -25 °C. Complex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in lx PBS. The solution absorbance at 260 nm was then multiplied by a conversion factor and the dilution factor to determine the complex concentration. The conversion factor used was either 0.050 mg / (mL-cm) or was calculated from an experimentally determined extinction coefficient.

[0163] E. Conjugation of Tri-alkyne linker. In some embodiments a tri-alkyne linker is conjugated to the sense strand of the RNAi agent on resin as a phosphorami dite (see Example 1 for the synthesis of an example tri-alkyne linker phosphorami dite and Example 1A for the conjugation of the phosphoramidite.). In other embodiments, a tri-alkyne linker may be conjugated to the sense strand following cleavage from the resin, described as follows: either prior to or after annealing, in some embodiments, the 5' or 3' amine functionalized sense strand is conjugated to a tri-alkyne linker. An example tri-alkyne linker structure that can be used in forming the constructs disclosed herein is as follows:To conjugate the tri-alkyne linker to the annealed duplex, amine-functionalized duplex was dissolved in 90% DMSO / 10% H2O, at -SOO mg / mL. 40 equivalents triethylamine was added, followed by 3 equivalents tri-alkyne- PNP. Once complete, the conjugate was precipitated twice in a solvent system of lx phosphate buffered saline / acetonitrile (1:14 ratio), and dried.

[0164] F. Synthesis of Targeting Ligand SM6.1((S)-3-(4-(4-((14-azido-3,6,9,12-tetraoxatetradecyl)oxy)naphthalen-l-yl)phenyl)-3-(2-(4- ((4-methylpyridin-2-yl)amino)butanamido)acetamido)propanoic acid)

[0165] Compound 5 (tert-Butyl(4-methylpyridin-2-yl)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 eq, 60 % dispersion in oil) The mixture stirred for 10 min before adding Compound 20 (Ethyl 4-Bromobutyrate (0.745 g, 3.82 mmol, 0.547 mL)) (Sigma 167118). After 3 hours the reaction was quenched with ethanol (18 mL) and concentrated. The concentrate was dissolved in DCM (50 mL) and washed with saturated aq. NaCl solution (1 x 50 mL), dried overNa2SO4, filtered and concentrated. The product was purified on silica column, gradient 0-5% Methanol in DCM.

[0166] Compound 21 was dissolved (0.80 g, 2.378 mmol) in 100 mL of Acetone: 0.1 M NaOH [1:1]. The reaction was monitored by TLC (5% ethyl acetate in hexane). The organics were concentrated away, 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.

[0167] 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 reactionmixture was warmed to room temperature and stirred 3 hours. The reaction was quenched by saturated NaHCCh solution (10 mL). The aqueous phase was extracted with ethyl acetate (3 x 10 mL) and the organic phase was combined, dried over anhydrous Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase. LC-MS: calculated [M+H]+ 366.20, found 367.

[0168] To a solution of compound 61 (2 g, 8.96 mmol, 1 equiv.). and compound 62 (2.13 mL, 17.93 mmol, 2 equiv.) in anhydrous DMF (10 mL) was added K.2CO3 (2.48 g, 17.93 mmol, 2 equiv.) at 0 °C. The reaction mixture was warmed to room temperature and stirred overnight. The reaction was quenched by water (10 mL). The aqueous phase was extracted with ethyl acetate (3 x 10 mL) and the organic phase was combined, dried over anhydrous Na2SO4, and concentrated. The product was separated by CombiFlash® using silica gel as the stationary phase.

[0169] 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 warmed to room temperature. After stirring at room temperature for 3 hours, the reaction mixture w as acidified by HC1 (6 N) to pH 3.0. The aqueous phase was extracted with ethyl acetate (3 x 20 mL) and the organic layer was combined, dried overNa2SO4, and concentrated. LC-MS: calculated [M+H]+ 352.18, found 352.

[0170] To a solution of compound 63 (1.88 g, 6.0 mmol, 1.0 equiv.) in anhydrous THF (20 mL) was added n-BuLi in hexane (3.6 mL, 9.0 mmol, 1.5 equiv.) drop-wise at -78 °C. The reaction was kept at -78 °C for another 1 hour. Triisopropylborate (2.08 mL, 9.0 mmol, 1.5 equiv.) was then added into the mixture at -78 °C. The reaction was then warmed up to room temperature and stirred for another 1 hour. The reaction was quenched by saturated NFLCl solution (20 mL) and the pH was adjusted to 3. The aqueous phase was extracted with EtOAc (3 x 20 mL) and the organic phase was combined, dried over Na2SO4, and concentrated.

[0171] 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 K.3PO4 (355 mg, 1,675mmol, 2.0 equiv.) were mixed in a round-bottom flask. The flask was sealed with a screwcap septum, and then evacuated and backfilled with nitrogen (this process was repeated a total of 3 times). Then, THF (8 mL) and water (2 mL) were added via syringe. The mixture was bubbled with nitrogen for 20 min and the reaction was kept at room temperature for 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 Na2SC>4, concentrated, and purified via CombiFlash® using silica gel as the stationary phase and was eluted with 15% EtOAc in hexane. LC-MS: calculated [M+H]+ 512.24, found 512.56.

[0172] Compound 66 (858 mg, 1.677 mmol, 1.0 equiv.) was cooled by ice bath. HC1 in dioxane (8.4 mL, 33.54 mmol, 20 equiv.) was added into the flask. The reaction was warmed to room temperature and stirred for another 1 hr. The solvent was removed by rotary' evaporator and the product was directly used without further purification. LC-MS: calculated [M+H]+ 412.18. found 412.46.

[0173] 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 another 1 hr. The reaction was quenched by saturated NaHCCL aqueous solution (10 mL) and the product was extracted with ethyl acetate (3 x 20 mL). The organic phase was combined, dried over NazSC, and concentrated. The product was purified by CombiFlash® using silica gel as the stationary phase and was eluted with 3-4% methanol in DCM. The yield was 96.23%. LC-MS: calculated [M+H]+ 745.35, found 746.08.

[0174] 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 warmed to room temperature and the reaction was monitored by LC-MS. The reaction was kept at room temperature overnight. The solids were filtered through Celite® and the solvent was removed by rotary evaporator. The product was directly used without further purification. LC- MS: [M+H]+ 655.31, found 655.87.

[0175] To a solution of compound 69 (100 mg, 0.152 mmol, 1 equiv.) and azido-PEGs-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 for 6 hours at 80 °C. The reaction was quenched by saturated NaHCCh solution and the aqueous layer was extracted with ethyl acetate (3 x 10 mL). The organic phase was combined, dried over Na2SO4, and concentrated. LC-MS: calculated [M+H]+ 900.40, found 901.46.

[0176] 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 at room temperature for another 1 hr. The pH was adjusted to 3.0 by HC1 (6N) and the aqueous phase was extracted with EtOAc (3 x 10 mL). The organic phase was combined, dried overNa2SO4, and concentrated. TFA (0.5 mL) and DCM (0.5 mL) was added into the residue and the mixture was stirred at room temperature for another 3 hr. The solvent was removed by rotary evaporator. LC-MS: calculated [M+H]+ 786.37. found 786.95.

[0177] G. Synthesis fTriAlkl4

[0178] TriAlkl4 and (TriAlkl4)s as shown in Table 11, above, may be synthesized using the synthetic route shown below. Compound 14 may be added to the sense strand as a phosphoramidite using standard oligonucleotide synthesis techniques, or compound 22 may be conjugated to the sense strand comprising an amine in an amide coupling reaction.

[0179] To a 3-L jacketed reactor was added 500 mL DCM and 4 (75.0 g, 0.16 mol). The internal temperature of the reaction was cooled to 0 °C and TBTU (170.0 g, 0.53 mol) was added. The suspension was then treated with the amine 5 (75.5 g, 0.53 mol) dropwise keeping the internal temperature less than 5 °C. The reaction was then treated with DIPEA (72.3 g, 0.56 mol) slowly, keeping the internal temperature less than 5 °C. After the addition was complete, the reaction w as warmed up to 23 °C over 1 hour, and allowed to stir for 3 hours. A 10% kickercharge of all three reagents were added and allowed to stir an additional 3 hours. The reaction was deemed complete when <1% of 4 remained. The reaction mixture was washed with saturated ammonium chloride solution (2 x 500 mL) and once with saturated sodium bicarbonate solution (500 mL). The organic layer was then dried over sodium sulfate and concentrated to an oil. The mass of the crude oil was 188 g which contained 72% 6 by QNMR. The crude oil was carried to the next step. Calculated mass for C46H60N4O11 = 845.0 m / z. Found [M+H] = 846.0.

[0180] The 121.2 g of 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%), keeping the internal temperature below 23 °C. The formation of dibenzofulvene (DBF) relative to the consumption of Fmoc-amine 6 was monitored viaHPLC method 1 (Figure 2) and the reaction was complete within 10 hours. To the solution was added glutaric anhydride (12.8 g, 0.11 mol) and the intermediate amine 7 was converted to compound 8 within 2 hours. Upon completion, the DMF and TEA were removed at 30 °C under reduced pressure resulting in 100 g of a crude oil. Due to the high solubility of compound 7 in water, an aqueous workup could not be used, and chromatography is the only way to remove DBF, TMU, and glutaric anhydride. The crude oil (75 g) was purified on a Teledyne ISCO Combi-flash® purification system in three portions. The crude oil (25 g) was loaded onto a 330 g silica column and eluted from 0 - 20%methanol / DCM over 30 minutes resulting in 42 g of compound 8 (54% yield over 3 steps). Calculated mass for C36H55N4O12 = 736.4 m / z. Found [M+H] = 737.0.Compound 22

[0181] Compound 8 (42.0 g, 0.057 mol) was co-stripped with 10 volumes of acetonitrile prior to use to remove any residual methanol from chromatography solvents. 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 ethyl acetate was added followed by 10 volumes saturated ammonium chloride solution, keeping the internal temperature below 15 °C. The layers were allowed to separate and the ethyl acetate layer was washed with brine. The combined aqueous layers were extracted twice with 5 volumes ethyl acetate. The combined organic layers were dried over sodium sulfate and concentrated to an oil. The crude oil (55 g) was purified on a Teledyne ISCO Combi-Flash® purification system in three portions. The crude oil (25 g) was loaded onto a 330 g silica column and eluted from 0 - 10% methanol / DCM over 30 minutes resulting in 22 g of pure 9 (Compound 22) (50% yield). Calculated mass for C42H59N5O14 = 857.4 m / z. Found [M+H] = 858.0.

[0182] 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 with triethylamine (11.56g, 111.4 mmol) dropwise. The reaction was monitored by observing the disappearance of compound 9 onHPLC Method 1 and was found to be complete in 10 minutes. The crude reaction mixture was diluted with 5 volumes 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. The 4-nitrophenol was eluted with 100% ethyl acetate and 10 was flushed from the column using 20% methanol / DCM resulting in a colorless oil (39 g, 81% yield). Calculated mass for C42H69N5O12 = 836.0 m / z. Found [M+H] = 837.0.

[0183] Alcohol 10 was co-stripped twice with 10 volumes of acetonitrile to remove any residual methanol from chromatography solvents and once more with dry dichloromethane (KF < 60 ppm) to remove trace water. The alcohol 10 (2.30 g, 2.8 mmol) was dissolved in 5 volumes dry dichloromethane (KF < 50 ppm) and treated with diisopropylammonium tetrazolide (188 mg, 1.1 mmol). The solution was cooled to 0 °C and treated with 2-cy anoethyl N, N, N', N’-tetraisopropylphosphoramidite (1.00 g, 3.3 mmol) dropwise. The solution was removed from ice-bath and stirred at 20 °C. The reaction was found to be complete within 3 -6 hours. The reaction mixture was cooled to 0 °C and treated with 10 volumes of a 1: 1 solution of saturated ammonium bicarbonate / brine and then warmed to ambient over 1 minute and allowed to stir an additional 3 minutes at 20 °C. The biphasic mixture was transferred to a separatory funnel and 10 volumes of di chloromethane was added. The organic layer was separated and washed with 10 volumes of saturated sodium bicarbonate solution to hydrolyze unreacted bis-phosphorous reagent. The organic layer was dried over sodium sulfate and concentrated to an oil resulting in 3.08 g of 94 wt% Compound 14. Calculated mass for C51H86N7O13P = 1035.6 m / z. Found [M+H] = 1036.

[0184] H. Conjugation of Targeting Ligands. Either prior to or after annealing, the 5' or 3' tridentate alkyne functionalized sense strand is conjugated to targeting ligands. The following example describes the conjugation of targeting ligands to the annealed duplex: Stock solutions of 0.5M Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5M of Cu(II) sulfate pentahydrate (Cu(II)SO4 • 5H2O) and 2M solution of sodium ascorbate were prepared in deionized water. A 75 mg / mL solution in DMSO of targeting ligand was made. In a 1.5 mLcentrifuge tube containing tri-alkyne functionalized duplex (3mg, 75pL.40mg / mL in deionized water, -15.000 g / mol). 25 pL of IM Hepes pH 8.5 buffer is added. After vortexing, 35 pL of DMSO was added and the solution is vortexed. Targeting ligand was added to the reaction (6 equivalents / duplex, 2 equivalents / alkyne, ~15pL) and the solution is vortexed. Using pH paper, pH was checked and confirmed to be pH -8. In a separate 1.5 mL centrifuge tube, 50 pL of 0.5M THPTA was mixed with lOuL of 0.5M Cu(II)SO4 • 5H2O, vortexed, and incubated at room temp for 5 min. After 5 min, THPTA / Cu solution (7.2 pL, 6 equivalents 5:1 THPTA: Cu) was added to the reaction vial, and vortexed. Immediately afterwards, 2M ascorbate (5 pL, 50 equivalents per duplex, 16.7 per alkyne) was added to the reaction vial and vortexed. Once the reaction was complete (typically complete in 0.5-lh), the reaction was immediately purified by non-denaturing anion exchange chromatography.Example 2. Conjugation of Linkers and Targeting Ligands to RNAi agents

[0185] A. Conjugation of Activated Ester Linkers

[0186] One potential method for conjugation of linkers is by the coupling of activated esters. In some embodiments, the following procedures may be used to conjugate linking groups having terminal propargyl groups to an RNAi agent with an amine-functionalized sense strand, such as C6-NH2, NH2-C6, or (NH2-C6)s, as shown in Table 2, above. An annealed RNAi Agent dried by lyophilization is dissolved in DMSO and 10% water (v / v%) at 25 rng / mL. Then 50-100 equivalents of TEA and 3 equivalents of activated ester linker are added to the solution. The solution is allowed to react for 1-2 hours, while monitored by RP-HPLC-MS (mobile phase A 100 mM HFIP, 14 mM TEA; mobile phase B: acetonitrile on an XBridge Cl 8 column, Waters Corp.)

[0187] The product can then be precipitated by adding 12 mL acetonitrile and 0.4 mL PBS and centrifuging the solid to a pellet. The pellet is then re-dissolved in 0.4 mL of 1XPBS and 12 mL of acetonitrile. The resulting pellet is dried on high vacuum for one hour.

[0188] B. Conjugation of Targeting Ligands to Propargyl Linkers

[0189] Similarly, another acceptable method to couple targeting ligands of the disclosed compounds herein is through their conjugation to propargyl linkers. In some embodiments, either prior to or after annealing, a 5' or 3' tri dentate alkyne functionalized sense strand can be conjugated to the NAG ligand. The following describes one possible method for the conjugation of a / p-anomeric metabolically stabilized NAG to an annealed complex: Stock solutions of 0.5M Tris(3-hydroxypropyltriazolylmethyl)amine (THPTA), 0.5M of Cu(II) sulfate pentahydrate (Cu(II)SO4 • 5 H2O) and 2M solution of sodium ascorbate are prepared indeionized water. A 75 mg / mL solution in DMSO of NAG ligand azide is made. In a 1.5 mL centrifuge tube containing tri-alkyne functionalized complex (3mg, 75pL, 40mg / mL in deionized water, approximately 15,000 g / mol), 25 pL of IM Hepes pH 8.5 buffer is added. After vortexing, 35 pL of DMSO is added and the solution is vortexed. The ligand can then be added to the reaction (e.g., 6 eq / complex, 2 eq / alkyne, approximately 15pL) and the solution is vortexed. Using pH paper, pH is checked and confirmed to be pH approximately 8. In a separate 1.5 mL centrifuge tube, 50 pL of0.5MTHPTAis mixed with lOuL of0.5M Cu(II)SO4 • 5 H2O, vortexed, and incubated at room temp for 5 min. After 5 min, THPTA / Cu solution (7.2 pL, 6 eq 5:1 THPTA: Cu) is added to the reaction vial, and vortexed. Immediately afterwards, 2M ascorbate (5 pL, 50 eq per complex, 16.7 per alkyne) is added to the reaction vial and vortexed. Once the reaction was complete (typically complete in 0.5-lh), the reaction mixture is immediately purified by non-denaturing anion exchange chromatography.

[0190] C. Conjugation of Targeting Ligands to Amine-Functionalized Sense Strand

[0191] In some embodiments, the following procedure may be used to conjugate an activated ester-functionalized targeting ligand such as a metabolically stabilized carbohydrate ligand to an amine functionalized RNAi agent comprising an amine, such as C6-NH2, NH2-C6, or (NH2-C6)s, as shown in Table 2: An annealed, lyophilized RNAi agent is dissolved in DMSO and 10% water (v / v%) at 25 mg / mL. Then 50-100 equivalents TEA and three equivalents of activated ester targeting ligand are added to the mixture. The reaction mixture is allowed to stir for 1-2 hours while monitored by RP-HPLC-MS (mobile phase A: 100 mM HFIP, 14 mM TEA; mobile phase B: Acetonitrile; column: XBridge Cl 8). After the reaction mixture is complete, 12 mL of acetonitrile was added followed by 0.4 mL of PBS and then the mixture is centrifuged. The solid pellet is collected and dissolved in 0.4 mL of IxPBS and then 12 mL of acetonitrile is added. The resulting pellet is collected and dried under vacuum for 1 hour.

[0192] D. Addition of Targeting Ligands by Phosphoramidite Synthesis or On Resin.

[0193] Other acceptable methods to couple targeting ligands are to prepare the desired ligand as a phosphoramidite compound, which may be added to the 5’ end of the strand using standard solid phase synthesis, or to prepare the targeting ligand on resin which can be placed at the 3’ end of the strand after cleavage, again using standard solid phase oligonucleotide synthesis.Example 3. Synthesis of TSLP-IL33 RNAi Agents

[0194] TSLP-IL33 RNAi agent complexes shown in Tables 5A, 5B, and 5C, above, were synthesized in accordance with the following general procedures:A. Synthesis.

[0195] The sense and antisense strands of the RNAi agents were synthesized according to phosphoramidite technology on solid phase used in oligonucleotide synthesis. Such standard synthesis is generally known in the art. Depending on the scale, either a MerMade96E® (Bioautomation), a MerMadel2® (Bioautomation), or an OP Pilot 100 (GE Elealthcare) was used. Syntheses were performed on a solid support made of controlled pore glass (CPG, 500 A or 600A, obtained from Prime Synthesis, Aston, PA, USA). The monomer positioned at the 3’ end of the respective strand was attached to the solid support as a starting point for synthesis. All RNA and 2'-modified RNA phosphoramidites were purchased from Thermo Fisher Scientific (Milwaukee, WI, USA) or Flongene Biotech (Shanghai, PRC). The 2'-O-methyl phosphoramidites included the following: (5'-O-dimethoxytrityl-N6-(benzoyl)-2'-O-methyl-adenosine-3'-O-(2-cyanoethyl-N, N-diisopropylamino) phosphoramidite, 5'-O-dimethoxy-trityl-N4-(acetyl)-2'-O-methyl-cytidine-3'-O-(2-cyanoethyl-N, N-diisopropyl-amino) phosphoramidite, (5'-O-dimethoxytrityl-N2-(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-phosphoramidites carried the same protecting groups as the 2’-O-methyl amidites. 5'-(4,4'-Dimethoxytrityl)-2',3'-seco-uridine, 2'-benzoyl-3'-[(2- cyanoethyl)-(N, N- diisopropyl)]-phosphoramidite was also purchased from Thermo Fisher Scientific or Hongene Biotech. 5'-dimethoxytrityl-2'-O-methyl-inosine-3'-O-(2-cyanoethyl-N, N-diisopropylamino) phosphoramidites were purchased from Glen Research (Virginia) or Hongene Biotech. The cyclopropyl phosphonate phosphoramidites were synthesized in accordance with International Patent Application Publication No. WO 2017 / 214112 (see also Altenhofer et. al., Chem. Communications (Royal Soc. Chem.), 57(55):6808-6811 (July 2021)). The inverted abasic (3'-O-dimethoxytrityl-2'-deoxyribose-5'-O-(2-cyanoethyl-N. N-diisopropylamino) phosphoramidites were purchased from ChemGenes (Wilmington, MA, USA) or SAFC (St Louis, MO, USA).5’-O-dimethoxytrityl-N2, N6-(phenoxyacetate)-2’-O-methyl-diaminopurine-3 -O-(2-cyanoethyl-N. N-diisopropylamino) phosphoramidites were obtained from ChemGenes or Hongene Biotech.

[0196] Targeting ligand-containing phosphoramidites were dissolved in anhydrous dichloromethane or anhydrous acetonitrile (50 mM), while all other amidites were dissolved in anhydrous acetonitrile (50 mM), or anhydrous dimethylformamide and molecular sieves (3A) were added. 5-Benzylthio-lH-tetrazole (BTT, 250 mM in acetonitrile) or 5-Ethylthio-lH-tetrazole (ETT, 250 mM in acetonitrile) was used as activator solution. Coupling times were 12 min (RNA), 15 min (targeting ligand), 90 sec (2'-OMe), and 60 sec (2'-F). In order to introduce phosphorothioate linkages, a 100 mM solution of 3-phenyl l,2,4-dithiazoline-5-one (POS, obtained from PolyOrg, Inc., Leominster, MA, USA) in anhydrous Acetonitrile was employed. Unless specifically identified as a “naked” RNAi agent having no targeting ligand present, each of the TSLP-IL33 RNAi agent complexes synthesized and tested in the following Examples utilized N-acetyl-galactosamine as “NAG” in the targeting ligand chemical structures represented in Table 6.B. Cleavage and deprotection of support bound oligomer.

[0197] After finalization of the solid phase synthesis, the dried solid support was treated with a 1: 1 volume solution of 40 wt. % methylamine in water and 28% ammonium hydroxide solution (Aldrich) for 1.5 hours at 30°C. The solution was evaporated and the solid residue was reconstituted in water (see below).C. Purification.

[0198] Crude oligomers were purified by anionic exchange HPLC using aTSKgel SuperQ-5PW Bpm column and Shimadzu LC-8 system. Buffer A was 20 mM Tris, 5 mM EDTA, pH 9.0 and contained 20% Acetonitrile and buffer B was the same as buffer A with the addition of 1.5 M sodium chloride. UV traces at 260 nm were recorded. Appropriate fractions were pooled then run on size exclusion HPLC using a GE Healthcare XK 26 / 40 column packed with Sephadex G-25 fine with a running buffer of filtered DI water or lOOmM ammonium bicarbonate, pH 6.7 and 20% Acetonitrile.D. Annealing.

[0199] Complementary' strands were mixed by combining equimolar RNA solutions (sense and antisense) in IxPhosphate-Buffered Saline (Coming, Cellgro) to form the RNAi agents. Some RNAi agents were lyophilized and stored at -15 to -25°C. Complex concentration was determined by measuring the solution absorbance on a UV-Vis spectrometer in 1 x Phosphate-Buffered Saline. The solution absorbance at 260 nm was then multiplied by a conversion factor and the dilution factor to determine the complex concentration. The conversion factor used waseither 0.050 mg / (mL cm) or was calculated from an experimentally determined extinction coefficient.Example 4. In vivo administration of monomeric TSLP and IL33 RNAi agents in rat.

[0200] Monomeric TSLP and IL33 RNAi agents were evaluated in vivo in rat. On Day 1 and Day 3, six (n=6) Brown Norway rats were administered either PBS, monomeric TSLP RNAi agent (formulated in PBS at 5.0 mg / kg), monomeric IL33 RNAi agent (formulated in PBS at 5.0 mg / kg), or co-dosed with monomeric TSLP and monomeric IL33 RNAi agent (each monomeric RNAi agent formulated in PBS at 5.0 mg / kg), via intratracheal (IT) administration via microsprayer, at 200 pL dose volume.

[0201] On Day 15, the rats were administered and challenged, via intratracheal IT administration via microsprayer, with a single dose of 150 pg per rat of Alternaria alternata prepared in PBS. Rats in Group 1 were administered with PBS and no Alternaria alternata, as control.

[0202] Dosing was in accordance with Table 7 below.

[0203] Table 7. RNAi Agent and Dosing for Example 4.

[0204] AC001714 includes a rat-specific sequence designed to target the rat TSLP transcript (NCBI GenBank XM_008772052.2) and does not have homology with the huma TSLP gene, and was chemically modified as follows:Tri-SM6.1-avb6-(TA14)-gsa_2NaucaaaCfCfUfcacaaauucus(invAb) (SEQ ID NO: 104) Modified Antisense Strand (5’ 3’):cPrpasGfsasAfuUfuGfuGfaGfgUfuUfgAfuUfsc (SEQ ID NO: 105)

[0205] AC005889 includes a rat-specific sequence designed to target the rat IL33 transcript (NCBI GenBank NM_001014166.2) and does not have homology with the human IL33 gene, and was chemically modified as follows:Modified Sense Strand (5’ -^ 3):Tri-SM6.1-avb6-(TA14)-uscugacuuCfGfUfuucauucgaas(invAb) (SEQ ID NO: 106) Modified Antisense Strand (5’ 3):cPrpusUfscgAfaugaaaCfgAfaGfucagsa (SEQ ID NO: 107)

[0206] On Day 16, the test animals were sacrificed. From the test animals, bronchoalveolar lavage fluid (BALF) and whole lungs were collected and harvested.

[0207] From the rat lungs, rTSLP and rIL33 mRNA transcript expression was quantified by qPCR, using rB2M as endogenous control gene. Groups 1 and 3-5 were normalized to Group 2 animals dosed with Saline with Altemaria challenge. qPCR data is shown in the following Table 8.

[0208] Table 8. Relative rTSLP and rIL33 mRNA transcript levels in rats, of Example 4.

[0209] Groups 4 and 5 showed reduction in rTSLP transcript levels out to at least Day 16. Group 4 rats dosed with monomeric TSLP RNAi agent AC001714 achieved -58% rTSLP reduction (0.422), and Group 5 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -67% rTSLP reduction (0.326).

[0210] Groups 3 and 5 showed reduction in rIL33 transcript levels out to at least Day 16. Group 3 rats dosed with monomeric IL33 RNAi agent AC005889 achieved -77% rIL33 reduction (0.132), and Group 5 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -72% rIL33 reduction (0.178).

[0211] From the rat lungs, rIL33 protein levels were quantified by Jess assay, using GAPDH as endogenous control gene. Groups 3-5 were normalized to Group 2 animals dosed with Saline with Altemaria challenge. rIL33 protein expression data is shown in the following Table 9.

[0212] Table 9. rIL33 protein expression levels in rats of Example 4.

[0213] Groups 3 and 5 showed reduction in rIL33 protein levels out to at least Day 16. Most notably, Group 5 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -64% rIL33 reduction (0.362).

[0214] From the rat lungs, rIL5 and rIL13 mRNA transcript expression was quantified by qPCR, using rB2M as endogenous control gene. Groups 1 and 3-5 were normalized to Group 2 animals dosed with Saline with Altemaria challenge. qPCR data is shown in the following Table 10.

[0215] Type 2 immune responses are defined by cytokines interleukin IL4, IL5, IL-9, and IL- 13, having either host protective or pathogenic activity. Wynn, T. Type 2 cytokines: mechanisms and therapeutic strategies. Nat Rev Immunol 15, 271-282 (2015). https: / / doi.org / 10.1038 / nri3831. Type 2 immunity induces a complex inflammatory response that are crucial to the pathogenesis of many allergic and fibrotic disorders.

[0216] Table 10. Relative rIL5 and rIL13 mRNA transcript levels in rats, of Example 4.

[0217] In comparison to the control Group 2, Group 5 rats co-dosed with both monomeric TSLP RNAi agent AC001714 and monomeric IL33 RNAi agent AC005889 showed decreased mRNA transcript levels of both rIL5 (-88%, 0.120) and rIL13 (-88%, 0.122) in comparison to Groups 3 and 4 dosed with only one monomeric RNAi agent (targeting either IL33 or TSLP, respectively). This suggests dual inhibition of TSLP and IL33 results in additive antiinflammatory effects in comparison to singular inhibition of TSLP or IL33.

[0218] Figure 2 shows the total protein of the bronchoalveolar lavage fluid (BALF) of the test animals. Figure 3A shows the soluble collagen of the bronchoalveolar lavage fluid (BALF) of the test animals. Analysis of variance ANOVA (one-way) was performed between the testGroups, and statistical significance level is denoted as * = p<0.05 and ** = p<0.001. Elevated total protein levels are indicative of inflammation. Elevated soluble collagen levels are indicative of fibrosis. As seen in Figure 2, Group 5 rats co-dosed with both monomeric TSLP and IL33 RNAi agents showed statistically significant reduction in total protein compared to the Group 2 control and Group 3 dosed with only the monomeric IL33 AC005889 RNAi agent. As seen in Figure 3A, Group 5 rats co-dosed with both monomeric TSLP and IL33 RNAi agents showed statistically significant reduction in soluble collagen compared to the Group 2 control and Group 3 dosed with only the monomeric IL33 AC005889 RNAi agent.

[0219] Figure 3B shows the IL5 levels in the bronchoalveolar lavage fluid (BALF) of the test animals. Figure 3C shows the IL10 levels in the BALF of the test animals. Figure 3D shows the IL13 levels in the BALF of the test animals. Figure 3E shows the leptin levels in the BALF of the test animals. Analysis of variance ANOVA (one-way) was performed between the test Groups, and statistical significance level is denoted as * = p<0.05 and ** = p<0.001. Elevated levels ofIL5, IL10, IL13, or leptin are indicative of inflammation. As seen in Figures 3B, 3C, 3D, and 3E. greater reductions of ILS, IL10, IL13, and leptin were observed in rats co-dosed with both IL33 and TSLP RNAi agents, compared to in rats dosed with either IL33 RNAi agent or TSLP RNAi agent alone.

[0220] From the BALF samples, total and differential cells were counted, and the number of inflammatory cells were derived. The total cell count of eosinophils were counted, both in total numbers of eosinophil cells and the cell ratio of eosinophil to total cell counts. Total cell counts included epithelial cells, eosinophils, neutrophils, lymphocytes, and monocytes / macrophages. Eosinophil protein X (EPX) levels were quantified via ELISA. Total eosinophils, eosinophil cell ratio, and EPX data are presented in Table 11 below.

[0221] Eosinophils are known to play a role in response to immune-mediated inflammatory responses. Eosinophil protein X (EPX) is a basic cellular protein with potent cytotoxic and neurotoxic properties, and is a marker of eosinophil activation and degranulation. Joseph Katzinger ND, Textbook of Natural Medicine (Fifth Edition). 2020.

[0222] Table 11. Eosinophil cell count, eosinophil cell ratio, and EPX levels of rats, of Example 4.

[0223] Groups 3-5 rats showed reduction of eosinophil cell counts and cell ratio in comparison to Group 2 rats dosed with saline and challenged with Altemaria. Group 5 rats codosed with both monomeric TSLP and IL33 RNAi agents showed additional reduction in both total eosinophil cell count (3.846xl05) and cell ratio (0.059) in comparison to Group 3 (7.281xl05; 0.089) and Group 4 (8.889xl05; 0.108) dosed with only one monomeric RNAi agent (targeting either IL33 or TSLP, respectively). The EPX levels show comparable levels between Groups 3-5. This further suggests dual inhibition of TSLP and IL33 results in additive anti-inflammatory effects in comparison to singular inhibition of TSLP or IL33.Example 5. In vivo administration of monomeric TSLP and IL33 RNAi agents in rat.

[0224] Monomeric TSLP and IL33 RNAi agents were evaluated in vivo in rat. On Day 1 and Day 3, six (n=6) Brown Norway rats were administered either PBS, monomeric TSLP RNAi agent (formulated in PBS at 5.0 mg / kg). monomeric IL33 RNAi agent (formulated in PBS at 5.0 mg / kg), AC002666 '‘RISC-blocked” construct (formulated in PBS at 10 mg / kg) that includes chemical modifications designed to prevent the loading of the antisense strand into RISC (serving as a negative control) or co-dosed with monomeric TSLP and monomeric IL33 RNAi agent (each monomeric RNAi agent formulated in PBS at 5.0 mg / kg). via intratracheal (IT) administration via microsprayer, at 200 pL dose volume.

[0225] On Day 11 and Day 15. the rats were administered and challenged, via intratracheal IT administration via microsprayer, with a single dose of 150 pg per rat of Alternaria alternata prepared in PBS. Rats in Group 1 were administered with PBS and no Alternaria alternata, as control.

[0226] Dosing was in accordance with Table 12 below.

[0227] Table 12. RNAi Agent and Dosing for Example 5.

[0228] AC001714 includes a rat-specific sequence designed to target the rat TSLP transcript (NCBI GenBank XM_008772052.2) and does not have homology with the huma TSLP gene, and was chemically modified as follows:Modified Sense Strand (5’ 3)t(invAb) (SEQ ID NO: 104)cPrpasGfsasAfuUfuGfuGfaGfgUfuUfgAfuUfsc (SEQ ID NO: 105)

[0229] AC005889 includes a rat-specific sequence designed to target the rat IL33 transcript (NCBI GenBank NM_001014166.2) and does not have homology7with the human IL33 gene, and was chemically modified as follows:Modified Sense Strand (5’ 3’):Tri-SM6.1-avb6-(TA14)-uscugacuuCfGfUfuucauucgaas(invAb) (SEQ ID NO: 106) Modified Antisense Strand (5’ 3’):cPrpusUfscgAfaugaaaCfgAfaGfucagsa (SEQ ID NO: 107)

[0230] On Day 16, the test animals were sacrificed. From the test animals, bronchoalveolar lavage fluid (BALF) and whole lungs were collected and harvested.

[0231] From the rat lungs, rTSLP and rIL33 mRNA transcript expression was quantified by qPCR, using rB2M as endogenous control gene. Groups 1 and 3-6 were normalized to Group 2 animals dosed with Saline with Altemaria challenge. qPCR data is shown in the following Table 13.

[0232] Table 13. Relative rTSLP and rIL33 mRNA transcript levels in rats, of Example 5.

[0233] Groups 5 and 6 showed reduction in rTSLP transcript levels out to at least Day 16. Group 5 rats dosed with monomeric TSLP RNAi agent AC001714 achieved -52% rTSLPreduction (0.481), and Group 5 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -63% rTSLP reduction (0.369).

[0234] Groups 4 and 6 showed reduction in rIL33 transcript levels out to at least Day 16. Group 4 rats dosed with monomeric IL33 RNAi agent AC005889 achieved -64% rIL33 reduction (0.362), and Group 6 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -70% rIL33 reduction (0.299).

[0235] From the rat lungs, rIL33 protein levels were quantified by Jess assay, using GAPDH as endogenous control gene. Groups 3-5 were normalized to Group 2 animals dosed with Saline with Altemaria challenge. rIL33 protein expression data is shown in the following Table 14.

[0236] Table 14. rIL33 protein expression levels in rats of Example 5.

[0237] Groups 3 and 5 showed reduction in rIL33 protein levels out to at least Day 16. Most notably. Group 5 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -46% rIL33 reduction (0.535).

[0238] From the rat lungs, rIL5 and rIL13 mRNA transcript expression was quantified by qPCR, using rB2M as endogenous control gene. Groups 1 and 3-5 were normalized to Group 2 animals dosed with Saline with Altemaria challenge. qPCR data is shown in the following Table 15.

[0239] Type 2 immune responses are defined by cytokines interleukin IL4. IL5, IL-9, and IL- 13, having either host protective or pathogenic activity. Wynn, T. Type 2 cytokines: mechanisms and therapeutic strategies. Nat Rev Immunol 15, 271-282 (2015). https: / / doi.org / 10.1038 / nri3831. Type 2 immunity induces a complex inflammatory response that are crucial to the pathogenesis of many allergic and fibrotic disorders.

[0240] Table 15. Relative rIL5 and rIL13 mRNA transcript levels in rats, of Example 5.

[0241] In comparison to the control Group 2, Group 6 rats co-dosed with both monomeric TSLP RNAi agent AC001714 and monomeric IL33 RNAi agent AC005889 showed decreased mRNA transcript levels of both rIL5 (-81% less, 0.189) and rIL13 (-82% less, 0.176). Group 6 rats further show decreased rIL5 and rIL13 in comparison to Groups 4 and 5 dosed with only one monomeric RNAi agent (targeting either IL33 or TSLP, respectively). This suggests dual inhibition of TSLP and IL33 results in additive anti-inflammatory' effects in comparison to singular inhibition of TSLP or IL33.

[0242] From the bronchoalveolar lavage fluid (BALF) of the test animals, the total protein and soluble collagen were quantified. Elevated total protein levels are indicative of inflammation. Elevated soluble collagen levels are indicative of fibrosis. The BALF total protein and soluble collagen levels are shown in the following Table 16.

[0243] Table 16. Total protein and soluble collagen in rat BALF, of Example 5.

[0244] Group 6 rats co-dosed with both monomeric TSLP RNAi agent AC001714 and monomeric IL33 RNAi agent AC005889 showed lower levels of total protein (2610 pg / mL) and soluble collagen (249.8 pg / mL) in comparison to Group 4 rats dosed with monomeric IL33 RNAi agent (4626 pg / mL; 622.9 pg / mL) and to Group 5 rats dosed with monomeric TSLP RNAi agent (3982 pg / mL; 508.7 pg / mL). This further suggests dual inhibition of TSLP and IL33 results in additive anti-inflammatory effects in comparison to singular inhibition of TSLP or IL33.

[0245] From the BALF samples, total and differential cells were counted, and the number of inflammatory cells were derived. The total cell count of eosinophils were counted, both in total numbers of eosinophil cells and the cell ratio of eosinophil to total cell counts. Total cell counts included epithelial cells, eosinophils, neutrophils, lymphocytes, and monocytes / macrophages. Total eosinophils and eosinophil cell ratio data are presented in Table 17 below.

[0246] Eosinophils are known to play a role in response to immune-mediated inflammatory responses. Eosinophil protein X (EPX) is a basic cellular protein with potent cytotoxic and neurotoxic properties, and is a marker of eosinophil activation and degranulation. Joseph Katzinger ND, Textbook of Natural Medicine (Fifth Edition), 2020.

[0247] Table 17. Eosinophil cell count and cell ratio of rats, of Example 5.

[0248] Groups 4-6 rats showed reduction of eosinophil cell counts and cell ratio in comparison to Group 2 rats dosed with saline and challenged with Altemaria. Group 6 rats codosed with both monomeric TSLP and IL33 RNAi agents showed additional reduction in both total eosinophil cell count (4.571xl05) and cell ratio (0.064) in comparison to Group 4 (15.729xl05; 0.235) and Group 5 (23.548xl05; 0.255) dosed with only one monomeric RNAi agent (targeting either IL33 or TSLP, respectively). This further suggests dual inhibition of TSLP and IL33 results in additive anti-inflammatory effects in comparison to singular inhibition of TSLP or IL33.Example 6. In vivo administration of monomeric TSLP and IL33 RNAi agents in rat.

[0249] Monomeric TSLP and IL33 RNAi agents were evaluated in vivo in rat. On Day 1 and Day 3, six (n=6) Brown Norway rats were administered either PBS, monomeric TSLP RNAi agent (formulated in PBS at 5.0 mg / kg), monomeric IL33 RNAi agent (formulated in PBS at 5.0 mg / kg), AC002666 "RISC-blocked’’ construct (formulated in PBS at 5.0 mg / kg) that includes chemical modifications designed to prevent the loading of the antisense strand into RISC (serving as a negative control), or co-dosed with monomeric TSLP and monomeric IL33 RNAi agent (each monomeric RNAi agent formulated in PBS at 2.5 mg / kg), via intratracheal (IT) administration via microsprayer, at 200 pL dose volume.

[0250] On Day 11 and Day 15. the rats were administered and challenged, via intratracheal IT administration via microsprayer, with a single dose of 150 pg per rat of Alternaria alternata prepared in PBS. Rats in Group 1 were administered with PBS and no Alternaria alternata, as control.

[0251] Dosing was in accordance with Table 18 below.

[0252] Table 18. RNAi Agent and Dosing for Example 6.

[0253] AC001714 includes a rat-specific sequence designed to target the rat TSLP transcript (NCBI GenBank XM_008772052.2) and does not have homology with the huma TSLP gene, and was chemically modified as follows:Modified Sense Strand (5’ 3)t(invAb) (SEQ ID NO: 104)cPrpasGfsasAfuUfuGfuGfaGfgUfuUfgAfuUfsc (SEQ ID NO: 105)

[0254] AC005889 includes a rat-specific sequence designed to target the rat IL33 transcript (NCBI GenBank NM_001014166.2) and does not have homology7with the human IL33 gene, and was chemically modified as follows:Modified Sense Strand (5’ 3’):Tri-SM6.1-avb6-(TA14)-uscugacuuCfGfUfuucauucgaas(invAb) (SEQ ID NO: 106) Modified Antisense Strand (5’ 3’):cPrpusUfscgAfaugaaaCfgAfaGfucagsa (SEQ ID NO: 107)

[0255] On Day 16, the test animals were sacrificed. From the test animals, bronchoalveolar lavage fluid (BALF) and whole lungs were collected and harvested.

[0256] From the rat lungs, rTSLP and rIL33 mRNA transcript expression was quantified by qPCR, using rB2M as endogenous control gene. Groups 1, 2, and 4-6 were normalized to Group 3 animals dosed with the AC002666 RISC-blocked construct. qPCR data is show n in the following Table 19.

[0257] Table 19. Relative rTSLP and rIL33 mRNA transcript levels in rats, of Example 6.

[0258] Groups 5 and 6 showed reduction in rTSLP transcript levels out to at least Day 16. Group 5 rats dosed with monomeric TSLP RNAi agent AC001714 achieved -20% rTSLPreduction (0.795), and Group 6 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -18% rTSLP reduction (0.815).

[0259] Groups 4 and 6 showed reduction in rIL33 transcript levels out to at least Day 16. Group 4 rats dosed with monomeric IL33 RNAi agent AC005889 achieved -70% rIL33 reduction (0.296), and Group 6 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -62% rIL33 reduction (0.381).

[0260] From the rat lungs, rIL33 protein levels were quantified by Jess assay, using GAPDH as endogenous control gene. Groups 4-6 were normalized to Group 3 animals dosed with AC002666 RISC-blocked construct. rIL33 protein expression data is shown in the following Table 20.

[0261] Table 20. rIL33 protein expression levels in rats of Example 6.

[0262] Groups 4 and 6 showed reduction in rIL33 protein levels out to at least Day 16. Most notably. Group 5 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -34% rIL33 reduction (0.664).

[0263] From the rat lungs, rIL5 and rIL13 mRNA transcript expression was quantified by qPCR, using rB2M as endogenous control gene. Groups 1, 2, and 4-6 were normalized to Group 3 animals dosed AC002666 RISC-blocked construct. qPCR data is shown in the following Table 21.

[0264] Type 2 immune responses are defined by cytokines interleukin IL4. IL5, IL-9, and IL- 13, having either host protective or pathogenic activity. Wynn, T. Type 2 cytokines: mechanisms and therapeutic strategies. Nat Rev Immunol 15, 271-282 (2015). https: / / doi.org / 10.1038 / nri3831. Type 2 immunity induces a complex inflammatory response that are crucial to the pathogenesis of many allergic and fibrotic disorders.

[0265] Table 21. Relative rIL5 and rIL13 mRNA transcript levels in rats, of Example 6.

[0266] In comparison to the control Group 3, Group 6 rats co-dosed with both monomeric TSLP RNAi agent AC001714 and monomeric IL33 RNAi agent AC005889 showed decreased mRNA transcript levels of both rIL5 (-72% less, 0.277) and rIL13 (-66% less, 0.339). Group 6 rats show further decreased rIL5 and rIL13 in comparison to Groups 4 and 5 dosed with only one monomeric RNAi agent (targeting either IL33 or TSLP, respectively). Furthermore, Group 6 rats were dosed at half (2.5 mg / kg) of the dose levels of Groups 4 and 5 (5.0 mg / kg). This suggests dual inhibition of TSLP and IL33 results in additive anti-inflammatory effects in comparison to singular inhibition of TSLP or IL33.

[0267] From the bronchoalveolar lavage fluid (BALF) of the test animals, the total protein and soluble collagen were quantified. Elevated total protein levels are indicative of inflammation. Elevated soluble collagen levels are indicative of fibrosis. The BALF total protein and soluble collagen levels are shown in the following Table 22.

[0268] Table 22. Total protein and soluble collagen in rat BALF, of Example 6.

[0269] Group 6 rats co-dosed with both monomeric TSLP RNAi agent AC001714 and monomeric IL33 RNAi agent AC005889 showed comparable levels of total protein (2666 pg / mL) and soluble collagen (350.1 pg / rnL) in comparison to Group 4 rats dosed with monomeric 1L33 RNAi agent (2761 pg / mL; 391.6 pg / mL) and to Group 5 rats dosed with monomeric TSLP RNAi agent (2522 pg / mL; 348.2 pg / mL). This comparable level of total protein and soluble collagen is despite Group 6 rats dosed at half (2.5 mg / kg) of the dose levels of Groups 4 and 5 (5.0 mg / kg). This further suggests dual inhibition of TSLP and IL33 results in additive anti-inflammatory effects in comparison to singular inhibition of TSLP or IL33.Example 7. In vivo administration of monomeric TSLP and IL33 RNAi agents in rat.

[0270] Monomeric TSLP and IL33 RNAi agents were evaluated in vivo in rat. On Day 1 and Day 3, six (n=6) Brown Norway rats were administered either PBS. monomeric TSLP RNAi agent (formulated in PBS at 5.0 mg / kg), monomeric IL33 RNAi agent (formulated in PBS at 5.0 mg / kg), AC002666 “RISC-blocked’’ construct (formulated in PBS at 5.0 mg / kg)that includes chemical modifications designed to prevent the loading of the antisense strand into RISC (serving as a negative control), or co-dosed with monomeric TSLP and monomeric IL33 RNAi agent (each monomeric RNAi agent formulated in PBS at 2.5 mg / kg), via intratracheal (IT) administration via microsprayer, at 200 pL dose volume.

[0271] On Day 11 and Day 15, the rats were administered and challenged, via intratracheal IT administration via microsprayer, with a single dose of 150 pg per rat of Alternaria alternata prepared in PBS.

[0272] Dosing was in accordance with Table 23 below.

[0273] Table 23. RNAi Agent and Dosing for Example 7.

[0274] AC001714 includes a rat-specific sequence designed to target the rat TSLP transcript (NCBI GenBank XM 008772052.2) and does not have homology with the huma TSLP gene, and was chemically modified as follows:Modified Sense Strand (5’ 3):(invAb) (SEQ ID NO: 104)cPrpasGfsasAfuUfuGfuGfaGfgUfuUfgAfuUfsc (SEQ ID NO: 105)

[0275] AC005889 includes a rat-specific sequence designed to target the rat IL33 transcript (NCBI GenBank NM_001014166.2) and does not have homology with the human IL33 gene, and was chemically modified as follows:Modified Sense Strand (5’ 3):Tri-SM6.1-avb6-(TA14)-uscugacuuCfGfUfuucauucgaas(invAb) (SEQ ID NO: 106) Modified Antisense Strand (5’ -^ 3):cPrpusUfscgAfaugaaaCfgAfaGfucagsa (SEQ ID NO: 107)

[0276] On Day 16, the test animals were sacrificed. From the test animals, bronchoalveolar lavage fluid (BALF) and whole lungs were collected and harvested.

[0277] From the rat lungs, rTSLP and rIL33 mRNA transcript expression was quantified by qPCR, using rB2M as endogenous control gene. Groups 1 and 3-5 were normalized to Group 2 animals dosed with the AC002666 RISC-blocked construct. qPCR data is shown in the following Table 24.

[0278] Table 24. Relative rTSLP and rIL33 mRNA transcript levels in rats, of Example 7.

[0279] Groups 4 and 5 showed reduction in rTSLP transcript levels out to at least Day 16. Group 4 rats dosed with monomeric TSLP RNAi agent AC001714 achieved -55% rTSLPreduction (0.449), and Group 5 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -51% rTSLP reduction (0.487).

[0280] Groups 3 and 5 showed reduction in rIL33 transcript levels out to at least Day 16. Group 3 rats dosed with monomeric IL33 RNAi agent AC005889 achieved -78% rIL33 reduction (0.223), and Group 5 rats co-dosed with monomeric TSLP RNAi agent AC001714 and rIL33 monomeric RNAi agent AC005889 achieved -52% rIL33 reduction (0.481).

[0281] From the rat lungs, rIL5, rIL6, rIL13, and Muc5ac mRNA transcript expression was quantified by qPCR, using rB2M as endogenous control gene. Groups 1 and 3-5 were normalized to Group 2 animals dosed AC002666 RISC-blocked construct. qPCR data is shown in the following Table 25.

[0282] Type 2 immune responses are defined by cytokines interleukin IL4, IL5, IL-9, and IL-13, having either host protective or pathogenic activity. Wynn, T. Type 2 cytokines: mechanisms and therapeutic strategies. Nat Rev Immunol 15, 271-282 (2015). https: / / doi.org / 10.1038 / nri3831. Type 2 immunity induces a complex inflammatory response that are crucial to the pathogenesis of many allergic and fibrotic disorders. Additionally, increased Mucin 5AC C‘Muc5ac”) has also been associated with allergic inflammation in asthma. Frankenthal Figueira et al., Mucus -targeting therapies of defective mucus clearance for cystic fibrosis: A short review. Current Opinion in Pharmacology, 2022.

[0283] Table 25. Relative rIL5, rIL6, rIL13, and Muc5ac mRNA transcript levels in rats, of Example 7.

[0284] In comparison to the control Group 2, Group 5 rats co-dosed with both monomeric TSLP RNAi agent AC001714 and monomeric IL33 RNAi agent AC005889 showed decreased mRNA transcript levels of both rIL5 (-65% less, 0.354), rIL6 (-24% less, 0.755), rIL13 (-63% less, 0.374), and rMuc5ac (-82% less, 0.184). These mRNA transcript levels are comparable to Groups 3 and 4 rats dosed with monomeric TSLP or IL33 RNAi agents. Furthermore, Group 5 rats were dosed at half (2.5 mg / kg) of the dose levels of Groups 3 and 4 (5.0 mg / kg). This suggests dual inhibition of TSLP and IL33 results in additive anti-inflammatory effects in comparison to singular inhibition of TSLP or IL33.

[0285] Figure 4 shows the total protein of the bronchoalveolar lavage fluid (BALF) of the test animals. Figure 5 shows the soluble collagen of the bronchoalveolar lavage fluid (BALF) of the test animals. Analysis of variance ANOVA (one-way) was performed between the test Groups, and statistical significance level is denoted as * = p<0.05 and ** = p<0.001. Elevated total protein levels are indicative of inflammation. Elevated soluble collagen levels are indicative of fibrosis. As seen in Figure 4, Group 5 rats co-dosed with both monomeric TSLP and IL33 RNAi agents showed reduction in total protein compared to the Group 2 control. As seen in Figure 5, Group 5 rats co-dosed with both monomeric TSLP and IL33 RNAi agents showed statistically significant reduction in soluble collagen compared to the Group 2 control. For both total protein and soluble collagen, Group 5 co-dosed rats showed comparable reduction in comparison to Group 3 and Group 4 rats dosed with only either IL33 or TSLP RNAi agents, respectively. This comparable reduction in total protein and soluble collagen is despite Group 5 rats co-dosed at half (2.5 mg / kg) of the dose levels of Groups 3 and 4 (5.0 mg / kg). This further suggests dual inhibition of TSLP and IL33 results in additive antiinflammatory effects in comparison to singular inhibition of TSLP or IL33.Example 8. In vivo administration of multimeric and monomeric TSLP and IL33 RNAi agents in rat.

[0286] Multimeric TSLP-IL33 RNAi agents and monomeric TSLP and IL33 RNAi agents were evaluated in vivo in rat. On Day 1 and Day 3, six (n=6) Brown Norway rats were administered either PBS, monomeric TSLP RNAi agent (formulated in PBS at 5.0 mg / kg), monomeric IL33 RNAi agent (formulated in PBS at 5.0 mg / kg), AC002666 “RISC-blocked” construct (formulated in PBS at 5.0 mg / kg) that includes chemical modifications designed to prevent the loading of the antisense strand into RISC (serving as a negative control), or multimeric TSLP-IL33 RNAi agent (formulated in PBS at 5.0 mg / kg), via intratracheal (IT) administration via microsprayer, at 200 pL dose volume.

[0287] On Day 15, the rats were administered and challenged, via intratracheal IT administration via microsprayer, with a single dose of 150 pg per rat of Alternaria alternata prepared in PBS. Rats in Group 1 were administered with PBS and no Alternaria alternata, as control.

[0288] Dosing was in accordance with Table 26 below.

[0289] Table 26. RNAi Agent and Dosing for Example 8.

[0290] AC001714 includes a rat-specific sequence designed to target the rat TSLP transcript (NCBI GenBank XM 008772052.2) and does not have homology with the huma TSLP gene, and was chemically modified as follows:Modified Sense Strand (5’ 3):(invAb) (SEQ ID NO: 104)cPrpasGfsasAfuUfuGfuGfaGfgUfuUfgAfuUfsc (SEQ ID NO: 105)

[0291] AC005889 includes a rat-specific sequence designed to target the rat IL33 transcript (NCBI GenBank NM_001014166.2) and does not have homology7with the human IL33 gene, and was chemically modified as follows:Modified Sense Strand (5’ -^ 3):Tri-SM6. l-avb6-(TA14)-uscugacuuCfGfUfuucauucgaas(invAb) (SEQ ID NO: 106) Modified Antisense Strand (5’ 3):cPrpusUfscgAfaugaaaCfgAfaGfucagsa (SEQ ID NO: 107)

[0292] AC006672 is a multimeric RNAi agent designed for dual inhibition of both TSLP and IL33. AC006672 includes a rat-specific sequence designed to target the rat IL33 transcript (NCBI GenBank NM 001014166.2) and rat TSLP transcript (NCBI GenBank XM_008772052.2). This sequence does not have homology7with the human IL33 nor TSLP gene, and was chemically modified as follows:Modified Sense Strand (5’ -^ 3):Tri-SM6.1 -avb6-(TAl 4)-gsa_2NaucaaaCfCfUfcacaaauucus(invAb)-Spl 8-uscugacuuCfGfUfuucauucgaas(invAb) (SEQ ID NO: 108)Modified Antisense Strand 1 (5’ 3):cPrpasGfsasAfuUfuGfuGfaGfgUfuUfgAfuUfsc (SEQ ID NO: 109)Modified Antisense Strand 2 (5’ -^ 3):cPrpusUfscgAfaugaaaCfgAfaGfucagsa (SEQ ID NO: 110)

[0293] On Day 16, the test animals were sacrificed. From the test animals, bronchoalveolar lavage fluid (BALF) and whole lungs were collected and harvested.

[0294] From the rat lungs, rTSLP and rIL33 mRNA transcript expression was quantified by qPCR, using rB2M as endogenous control gene. Groups 1 and 3-6 w ere normalized to Group 2 animals dosed w ith Saline and challenged with Altemaria. qPCR data is shown in the following Table 27.

[0295] Table 27. Relative rTSLP and rIL33 mRNA transcript levels in rats, of Example 8.

[0296] Groups 5 and 6 showed reduction in rTSLP transcript levels out to at least Day 16. Group 5 rats dosed with monomeric TSLP RNAi agent AC001714 achieved -57% rTSLP reduction (0.430), and Group 6 rats dosed with multimeric TSLP-IL33 RNAi agent AC006672 achieved comparable reduction of -49% rTSLP reduction (0.513).

[0297] Groups 4 and 6 showed reduction in rIL33 transcript levels out to at least Day 16. Group 4 rats dosed with monomeric IL33 RNAi agent AC005889 achieved -74% rIL33 reduction (0.259), and Group 6 rats dosed with multimeric TSLP-IL33 RNAi agent AC006672 achieved comparable reduction of -71% rIL33 reduction (0.294).

[0298] The above data suggests dosing multimeric TSLP-IL33 RNAi agent AC006672 can achieve comparable TSLP and IL33 inhibition in comparison to dosing with either monomeric TSLP or IL33 RNAi agents.OTHER EMBODIMENTS

[0299] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. Claims1. An RNAi agent, for inhibiting the expression of a Thymic Stromal Lymphopoietin (TSLP) gene and an Interleukin 33 (IL33) gene, comprising:3.(i) a first antisense strand that is complementary to part of an mRNA encoding a TSLP gene or an IL33 gene, and4.(ii) a second antisense strand that is complementary to part of an mRNA encoding a TSLP gene or an IL33 gene, and5.(iii) one or more sense strands,6.wherein the one or more sense strands comprise a nucleotide sequence that is at least partially complementary to the first antisense strand and the second antisense strand.

2. The RNAi agent of claim 1, wherein8.the first antisense strand comprises at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any of the sequences of Table 2A, or Table 2B, Table 3A, Table 3B, or Table 5C, and9.the second antisense strand comprises at least 17 contiguous nucleotides differing by 0 or 1 nucleotides from any of the sequences of Table 2A, Table 2B. Table 3A, Table 3B, or Table 5C3. The RNAi agent of claim 1 or claim 2, wherein the one or more sense strands comprise a nucleotide sequence of any one of the sequences of Table 4 A, Table 4B. or Table 4C.

4. The RNAi agent of any of claims 1-3, wherein the RNAi agent comprises a first sense strand sequence of any one of the sequences of Table 4A, Table 4B, or Table 4C and a second sense strand sequence of any one of the sequences of Table 4A, Table 4B, or Table 4C.

5. The RNAi agent of any one of claims 1-4, wherein the RNAi agent comprises a first antisense strand sequence of any one of the sequences of Table 2A, Table 3 A, or Table 5C. and a second sense strand sequence of any one of the sequences of Table 2B, Table 3B, or Table 5C.

6. The RNAi agent of any one of claims 1-4, wherein the RNAi agent comprises a first antisense strand sequence of any one of the sequences of Table 2B, Table 3B, or Table 5C, and a second sense strand sequence of any one of the sequences of Table 2A, Table 3A, or Table 5C.

7. The RNAi agent of any one of claims 1-6, wherein at least one nucleotide of the RNAi agent is a modified nucleotide or includes a modified intemucleoside linkage.

8. The RNAi agent of any one of claims 1 -7, wherein all or substantially all of the nucleotides of the sense and / or antisense strand of the RNAi agent are modified nucleotides.

9. The RNAi agent of any one of claims 1 -8, wherein the modified nucleotide is selected from the group consisting of: 2'-O-methyl nucleotide, 2'-fluoro nucleotide, 2’-deoxy nucleotide, 2',3'-seco nucleotide mimic, locked nucleotide. 2'-F-arabino nucleotide, 2'-methoxyethyl nucleotide, abasic nucleotide, ribitol, inverted nucleotide, inverted 2'-O-methyl nucleotide, inverted 2'-deoxy nucleotide, 2'-amino-modified nucleotide, 2'-alkyl-modified nucleotide, morpholine nucleotide, vinyl phosphonate containing nucleotide, cyclopropyl phosphonate containing nucleotide, and 3'-O-methyl nucleotide.

10. The RNAi agent of claim 9, wherein all or substantially all of the modified nucleotides are 2'-O-methyl nucleotides, 2'-fluoro nucleotides, or combinations thereof.

11. The RNAi agent of any one of claims 1-10. wherein the first antisense strand sequence is selected from any one of the sequences of Table 3A, and the second antisense strand sequence is selected from any one of the sequences of Table 3B.

12. The RNAi agent of any one of claims 1-10, wherein the first antisense strand sequence is selected from any one of the sequences of Table 3B, and the second antisense strand sequence is selected from any one of the sequences of Table 3 A.

13. The RNAi agent of any one of claims 1-12, wherein:20.(a) the one or more sense strands comprise between 30 and 60 nucleotides, or (b) the first antisense strand is between 18 and 30 nucleotides in length, or21.(c) the second antisense strand is between 18 and 30 nucleotides in length, or22.(d) any combination of (a) through (c) above.

14. The RNAi agent of claim 13, wherein:24.(a) the one or more sense strands comprise between 36 and 54 nucleotides, or25.(b) the first antisense strand is between 18 and 27 nucleotides in length, or26.(c) the second antisense strand is between 18 and 27 nucleotides in length, or27.(d) any combination of (a) through (c) above.

15. The RNAi agent of claim 14, wherein:29.(a) the one or more sense strands comprise between 36 and 48 nucleotides, or30.(b) the first antisense strand is between 18 and 24 nucleotides in length, or31.(c) the second antisense strand is between 18 and 24 nucleotides in length, or32.(d) any combination of (a) through (c) above.

1. The RNAi agent of claim 15, wherein:34.(a) the one or more sense strands comprise between 38 and 42 nucleotides, or35.(b) the first antisense strand is between 19 and 21 nucleotides in length, or36.(c) the second antisense strand is between 19 and 21 nucleotides in length, or37.(d) any combination of (a) through (c) above.

17. The RNAi agent of claim 16, wherein:39.(a) the one or more sense strands comprises 38 or 42 nucleotides, or40.(b) the second antisense strand is 19 nucleotides in length, or41.(c) the second antisense strand is 21 nucleotides in length, or42.(d) any combination of (a) through (c) above.

18. The RNAi agent of claim 1, wherein the one or more sense strands comprises the structure:44.SS, - L - SS245.wherein SSi comprises a first sense strand sequence;46.SS2 comprises a second sense strand sequence; and47.L is a linker, or a bond.

19. The RNAi agent of claim 18, wherein L is a nucleotide linker.

20. The RNAi agent of claim 18, wherein L is a non-nucleotide linker.

21. The RNAi agent of claim 20, wherein L is polyethylene glycol (PEG).

22. The RNAi agent of any one of claims 18-21, wherein SSi comprises any one of the sense strand sequences listed in Table 4A or Table 4B.

23. The RNAi agent of any one of claims 18-22, wherein SS2 comprises any one of the sense strand sequences listed in Table 4A or Table 4B.

24. The RNAi agent of claim 18, wherein SSi and SS2 each independently comprise anyone of the sense strand sequences listed in Table 4A or Table 4B. wherein at least one sense strand sequence is selected from Table 4A and the other sense strand sequence is selected from Table 4B.

25. The RNAi agent of any one of claims 18-24, wherein L comprises the structure:

57.

26. The RNAi agent of claim 18, wherein60.

61. selected from any one of the sense strands listed in Table 4C.

27. The RNAi agent of any one of claims 18-26, wherein the first antisense strand consists of, consists essentially of. or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3A, and the second antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3B.

28. The RNAi agent of any one of claims 18-27. wherein the first antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3B, and the second antisense strand consists of, consists essentially of, or comprises the nucleotide sequence of any one of the modified antisense strand sequences of Table 3 A.

29. The RNAi agent of any one of claims 1-28, wherein the one or more sense strands consists of, consists essentially of, or comprises the nucleotide sequence of any of the modified sense strand sequences of Table 4A, Table 4B, Table 4C, or Table 5C.

30. The RNAi agent of any one of claims 1-29, wherein the RNAi agent is linked to a targeting ligand.

31. The RNAi agent of claim 30, wherein the targeting ligand has affinity for a cell receptor expressed on an epithelial cell.

32. The RNAi agent of claim 31, wherein the targeting ligand comprises an integrin targeting ligand.

33. The RNAi agent of claim 32, wherein the integrin targeting ligand is an avP6 integrin targeting ligand.

34. The RNAi agent of claim 33, wherein the targeting ligand comprises the structure:

70.

71. a pharmaceutically acceptable salt thereof, or74.

75. pharmaceutically acceptable salt thereof,76.wherein indicates the point of connection to the RNAi agent.77.The RNAi agent of any one of claims 30-33, wherein the targeting ligand has a structure selected from the group consisting of:

78.

80.

83.

85.

87.

89.

91.

93. 95.PHI97.

99. 100., wherein? indicates the point of connection to the RNAi agent.101.The RNAi agent of claim 35, wherein RNAi agent is conjugated to a targeting ligand having the following structure:

102.

37. The RNAi agent of any one of claims 30-36, wherein the targeting ligand is conjugated to the one or more sense strands.

38. The RNAi agent of claim 37, wherein the targeting ligand is conjugated to the 5’ terminal of the one or more sense strands.

39. The RNAi agent of any of claims 1-38, wherein the RNAi agent has two blunt ends.

40. The RNAi agent of any of claims 1-39, wherein the one or more sense strands comprise one or two terminal caps.

41. The RNAi agent of any of claims 1-40. wherein the one or more sense strands comprise one or two inverted abasic residues.

42. An RNAi agent of any of the complexes set forth in Table 5A, Table 5B, or Table 5C.

43. The RNAi agent of any one of claims 1-42, wherein the one or more sense strands comprises inverted abasic residues at the 3’ terminal end of the nucleotide sequence, at the 5’ end of the nucleotide sequence, or at both.

44. The RNAi agent of any one of claims 1-43, wherein the RNAi agent is a pharmaceutically acceptable salt.

45. A composition comprising the RNAi agent of any one of claims 1-44, wherein the composition comprises a pharmaceutically acceptable excipient.

46. The composition of claim 45, wherein the pharmaceutically acceptable excipient is sodium phosphate buffer.

47. The composition of claim 45, wherein the pharmaceutically acceptable excipient is isotonic saline or water for injection.

48. A method for inhibiting expression of a TSLP and an IL33 gene in a cell, the method comprising introducing into a cell an effective amount of an RNAi agent of any one of claims 1-44 or the composition of any one of claims 45-47.

49. The method of claim 48, wherein the cell is within a subject.

50. The method of claim 49, wherein the subject is a human subject.

51. The method of any one of claims 48-50, wherein TSLP gene expression is inhibited by at least about 30% and IL33 gene expression of the subject is inhibited by at least about 30%, as measured by reductions in mRNA and / or protein levels.

52. The method of any one of claims 48-51, wherein TSLP protein levels are reduced by about 50% and IL33 protein levels are reduced by about 50%.

53. A method of treating a TSLP-related and / or IL33-related disease, disorder, or symptom, the method comprising administering to a human subject in need thereof a therapeutically effective amount of the composition of any one of claims 45-47.

54. The method of claim 53, wherein the disease is asthma or chronic obstructive pulmonary disease (COPD).

55. The method of any one of claims 48-54, wherein the RNAi agent is administered at a dose of about 0.05 mg / kg to about 5.0 mg / kg of body weight of the human subject.

56. The method of any one of claims 48-55, wherein the RNAi agent is administered in two or more doses.

57. Use of the RNAi agent of any one of claims 1-44 or the composition of any one of claims 45-47, for the treatment of a disease, disorder, or symptom that is mediated at least in part by a reduction in TSLP gene expression and / or IL33 gene expression.

58. The use of claim 57, wherein the disease is asthma or chronic obstructive pulmonary disease (COPD).

59. Use of the RNAi agent of any one of claims 1-44 or the composition of any one of claims 45-47. for the preparation of a pharmaceutical composition for treating a disease, disorder, or symptom that is mediated at least in part by a reduction in TSLP mRNA and / or protein levels and / or a reduction in IL33 mRNA and / or protein levels.

60. The use according to any one of claims 57-59, wherein the RNAi agent is administered at a dose of about 0.05 mg / kg to about 10.0 mg / kg of body weight of the human subject.

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